New Perspectives for Cancer Diagnosis and Therapy

*Tumor Progression and Metastasis*

2017;**8**:591-605

2014;**2**:904-908

therapy and current strategies—A review. Journal of Advanced Research. [45] Hossen S, Khalid Hossain M, Basher MK, Mia MNH, Rahman MT, Jalal Uddin M. Smart nanocarrier-based drug delivery systems for cancer therapy and toxicity studies: A review. Journal of Advanced Research. 2019;**15**:1-18

[46] Girish S, Gupta M, Wang B, Lu D, et al. Clinical pharmacology of transtuzumab emtansine (T-DM1): An antibody drug conjugate in development of the treatment for HER2-positive cancer. Cancer Chemotherapy and Pharmacology. 2012;**69**(5):1229-1240

[37] Bazak R, Houri M, Elachy S, Hussein W, Refaat T. Passive targeting of nanoparticles to cancer: A comprehensive review of literature. Molecular and Clinical Oncology.

[38] Narayanan E, Wakaskar R. Utilization of nanoparticulate therapy in cancer targeting. Cogent Medicine. 2018;**5**(1504504):1-9

Reviews. 2016;**99**:28-51

2017;**1**(4):346-357

2001;**19**(4):424-436

[39] Soo Suk J, Xu Q, Kim N, Hanes J, Ensign LM. PEGylation as a strategy for improving nanoparticle-based drug gene delivery. Advanced Drug Delivery

[40] Din F u, Ullah WAI, et al. Effective use of nanocarriers as drug delivery systems for the treatment of selected tumors. International Journal of Nanomedicine. 2017;**12**:7291-7309

[41] Li R, Zheng K, Yuan C, Chen Z, Huang M. Be active or Not: The relative contribution of active and passive tumor targeting of nanomaterials. Nano.

[42] Gabizon A. Pegylated liposomal doxorubicin: Metamorphosis of an old drug into a new form of chemotherapy. Cancer Investigation.

[43] Miele E, Spineli GP, Miele E, Tomao F, Tomao S. Albumin-bound formulation of paclitaxel (Abraxane ABI-007) in the treatment of breast cancer. International Journal of Nanomedicine. 2009;**4**:99-105

[44] Wang K, Kievit FM, Zhang M. Nanoparticles for cancer gene therapy: Recent advances, challenges and strategies. Pharmacological Research. 2016;**114**:56-66

**Chapter 9**

**Abstract**

Multiforme

Stereotactic Radiosurgery for

Glioblastoma multiforme (GBM) is the most aggressive intracranial tumor that primarily affects adults. Since the introduction of temozolomide in 2005, maximal resection surgery with concurrent chemoradiation has become the standard treatment method for patients with newly diagnosed GBM. Although newly discovered chemoagents have been demonstrated to improve the median survival time, GBM still recurs in most patients. Recurrent GBM is still a therapeutic challenge for clinical physicians. Surgical intervention and other conventional chemoagents have been applied to manage recurrent GBM. Stereotactic radiosurgery (SRS) provides a highly precise radiation dose to the tumor lesion and reduces the dose to the adjacent normal brain tissue. After standard treatment for newly diagnosed GBM is completed, conventional re-irradiation therapy is not suitable for patients with recurrent GBMs. Therefore, SRS may become an alternative option in the treatment of recurrent GBMs. In this review, we discuss the relevant literature regarding SRS

for recurrent GBMs and provide treatment advice for clinical physicians.

**Keywords:** stereotactic radiosurgery, recurrent glioblastoma multiforme,

Glioblastoma multiforme (GBM) is the most common primary brain neoplasm in adults [1, 2]. There are 1.6 times more males than females who develop a GBM [1]. According to the latest statistical report of the Central Brain Tumor Registry of the USA, the annual incidence of GBM has been estimated at approximately 3.22 cases per 100,000 people, and the median age is 65 years [3]. The current standard treatment of patients with a newly diagnosed GBM was established in 2005, and it consists of maximal surgical resection of the tumor followed by chemotherapy and conventional radiotherapy [4, 5]. Despite this therapy, the median overall survival

GBM is a refractory malignant and infiltrating tumor that may recur any time after initial multimodal treatments are completed [6]. Managing recurrent GBM has always been challenging, and a balance has to be achieved between significant treatment toxicity and associated morbidities and mortalities [6, 7]. Reoperation with maximal resection at recurrence remains as an independent predictor to improve overall survival [8, 9]. However, repeat gross-total resection may not be

Recurrent Glioblastoma

*Cheng-Ta Hsieh and Da-Tong Ju*

re-irradiation, survival time, prognosis

time is approximately 15–17 months [2].

**1. Introduction**

#### **Chapter 9**

## Stereotactic Radiosurgery for Recurrent Glioblastoma Multiforme

*Cheng-Ta Hsieh and Da-Tong Ju*

#### **Abstract**

Glioblastoma multiforme (GBM) is the most aggressive intracranial tumor that primarily affects adults. Since the introduction of temozolomide in 2005, maximal resection surgery with concurrent chemoradiation has become the standard treatment method for patients with newly diagnosed GBM. Although newly discovered chemoagents have been demonstrated to improve the median survival time, GBM still recurs in most patients. Recurrent GBM is still a therapeutic challenge for clinical physicians. Surgical intervention and other conventional chemoagents have been applied to manage recurrent GBM. Stereotactic radiosurgery (SRS) provides a highly precise radiation dose to the tumor lesion and reduces the dose to the adjacent normal brain tissue. After standard treatment for newly diagnosed GBM is completed, conventional re-irradiation therapy is not suitable for patients with recurrent GBMs. Therefore, SRS may become an alternative option in the treatment of recurrent GBMs. In this review, we discuss the relevant literature regarding SRS for recurrent GBMs and provide treatment advice for clinical physicians.

**Keywords:** stereotactic radiosurgery, recurrent glioblastoma multiforme, re-irradiation, survival time, prognosis

#### **1. Introduction**

Glioblastoma multiforme (GBM) is the most common primary brain neoplasm in adults [1, 2]. There are 1.6 times more males than females who develop a GBM [1]. According to the latest statistical report of the Central Brain Tumor Registry of the USA, the annual incidence of GBM has been estimated at approximately 3.22 cases per 100,000 people, and the median age is 65 years [3]. The current standard treatment of patients with a newly diagnosed GBM was established in 2005, and it consists of maximal surgical resection of the tumor followed by chemotherapy and conventional radiotherapy [4, 5]. Despite this therapy, the median overall survival time is approximately 15–17 months [2].

GBM is a refractory malignant and infiltrating tumor that may recur any time after initial multimodal treatments are completed [6]. Managing recurrent GBM has always been challenging, and a balance has to be achieved between significant treatment toxicity and associated morbidities and mortalities [6, 7]. Reoperation with maximal resection at recurrence remains as an independent predictor to improve overall survival [8, 9]. However, repeat gross-total resection may not be

easily achieved when recurrent GBM involves important eloquent brain structures, such as the brainstem or motor area. Extensive bevacizumab and temozolomide are the two main FDA-approved chemoagents used to treat patients with recurrent GBMs, but the prognosis remains poor [10].

Re-irradiation is an alternative option for managing recurrent GBMs [11]. The majority of recurrent tumors occur at the initial or adjacent regional sites [12]. Because a total dose of 60 Gy in 30 fractions has been prescribed for initial radiation therapy, re-irradiation with further dose escalation appears to produce more significant toxicity [10, 11]. Stereotactic radiosurgery (SRS) is a noninvasive treatment that provides a highly precise, targeted additional radiation boost to the tumor lesion, and it maintains an acceptable rate of adverse radiation effects while reducing the dose to adjacent normal brain tissues [13]. In this review, we searched the relevant literature and investigated the role of SRS in the management of recurrent GBMs. Our results may provide treatment information for clinical treatment.

#### **2. Search methodology**

In this study, different combinations of the keywords "recurrent glioblastoma multiple," "high-grade glioma," "stereotactic radiosurgery," and "re-irradiation" were used to search the published literature in the PubMed database until October 31, 2019. The inclusion criteria of the study were (1) patients with recurrent GBMs, (2) treatment with stereotactic radiosurgery (SRS) or a fractionated radiosurgery (less than 5 fractions), and (3) outcomes with overall survival time. Tumor progression was also accepted as a recurrent disease. Potentially relevant studies were identified from the reference lists of the studies obtained from the database search. Articles excluded from the review were those written in languages other than English and those that lacked survival response data. Finally, a total of 49 studies were included in this review.

#### **3. The summary of patients with recurrent GBMs treated with SRS**

A total of 49 studies published from 1994 to 2019 were enrolled in this review, as summarized in **Table 1** [13–61]. There were 6 prospective studies and 43 retrospective studies. About 2066 patients with recurrent glioblastomas treated with SRS, including linear accelerator (LINAC) radiosurgery, Gamma Knife radiosurgery, and Cyberknife radiosurgery, are reported. In all studies, the median age of the patients who received SRS treatment for recurrent GBM ranged from 34 to 62 years. The majority of patients were males. The median prescribed dose of SRS ranged from 6 to 30 Gy. The median targeted volume for treatment ranged from 1.35 to 21.3 cc. The overall survival time from treatment for SRS ranged from 3.9 to 17.9 months, where the progression-free survival time from the treatment of SRS ranged from 2.1 to 14.9 months. In the prognostic analysis of survival time in patients with recurrent GBMs treated with SRS, a small tumor volume, younger age, higher Karnofsky performance scale (KPS) score, lower recursive partitioning analysis (RPA) class, adjuvant bevacizumab, methylated O6-methylguanine-DNAmethyltransferase (MGMT) promoter, and longer interval between the original surgery and SRS were significantly associated with patients'survival outcomes.

#### **3.1 The effect of LINAC radiosurgery in patients with recurrent GBMs**

From 1994 to 2018, a total of 501 patients with recurrent GBMs treated with LINAC SRS were enrolled in 22 studies, including 3 prospective trials and 17

**221**

*Stereotactic Radiosurgery for Recurrent Glioblastoma Multiforme*

*DOI: http://dx.doi.org/10.5772/intechopen.91163*

#### *Stereotactic Radiosurgery for Recurrent Glioblastoma Multiforme DOI: http://dx.doi.org/10.5772/intechopen.91163*

easily achieved when recurrent GBM involves important eloquent brain structures, such as the brainstem or motor area. Extensive bevacizumab and temozolomide are the two main FDA-approved chemoagents used to treat patients with recurrent

Re-irradiation is an alternative option for managing recurrent GBMs [11]. The majority of recurrent tumors occur at the initial or adjacent regional sites [12]. Because a total dose of 60 Gy in 30 fractions has been prescribed for initial radiation therapy, re-irradiation with further dose escalation appears to produce more significant toxicity [10, 11]. Stereotactic radiosurgery (SRS) is a noninvasive treatment that provides a highly precise, targeted additional radiation boost to the tumor lesion, and it maintains an acceptable rate of adverse radiation effects while reducing the dose to adjacent normal brain tissues [13]. In this review, we searched the relevant literature and investigated the role of SRS in the management of recurrent GBMs. Our results may provide treatment information for clinical treatment.

In this study, different combinations of the keywords "recurrent glioblastoma multiple," "high-grade glioma," "stereotactic radiosurgery," and "re-irradiation" were used to search the published literature in the PubMed database until October 31, 2019. The inclusion criteria of the study were (1) patients with recurrent GBMs, (2) treatment with stereotactic radiosurgery (SRS) or a fractionated radiosurgery (less than 5 fractions), and (3) outcomes with overall survival time. Tumor progression was also accepted as a recurrent disease. Potentially relevant studies were identified from the reference lists of the studies obtained from the database search. Articles excluded from the review were those written in languages other than English and those that lacked survival response data. Finally, a total of 49 studies were included in this review.

**3. The summary of patients with recurrent GBMs treated with SRS**

**3.1 The effect of LINAC radiosurgery in patients with recurrent GBMs**

From 1994 to 2018, a total of 501 patients with recurrent GBMs treated with LINAC SRS were enrolled in 22 studies, including 3 prospective trials and 17

A total of 49 studies published from 1994 to 2019 were enrolled in this review, as summarized in **Table 1** [13–61]. There were 6 prospective studies and 43 retrospective studies. About 2066 patients with recurrent glioblastomas treated with SRS, including linear accelerator (LINAC) radiosurgery, Gamma Knife radiosurgery, and Cyberknife radiosurgery, are reported. In all studies, the median age of the patients who received SRS treatment for recurrent GBM ranged from 34 to 62 years. The majority of patients were males. The median prescribed dose of SRS ranged from 6 to 30 Gy. The median targeted volume for treatment ranged from 1.35 to 21.3 cc. The overall survival time from treatment for SRS ranged from 3.9 to 17.9 months, where the progression-free survival time from the treatment of SRS ranged from 2.1 to 14.9 months. In the prognostic analysis of survival time in patients with recurrent GBMs treated with SRS, a small tumor volume, younger age, higher Karnofsky performance scale (KPS) score, lower recursive partitioning analysis (RPA) class, adjuvant bevacizumab, methylated O6-methylguanine-DNAmethyltransferase (MGMT) promoter, and longer interval between the original surgery and SRS were significantly associated with patients'survival outcomes.

GBMs, but the prognosis remains poor [10].

*Tumor Progression and Metastasis*

**2. Search methodology**



**223**

*Stereotactic Radiosurgery for Recurrent Glioblastoma Multiforme*

*DOI: http://dx.doi.org/10.5772/intechopen.91163*

#### *Stereotactic Radiosurgery for Recurrent Glioblastoma Multiforme DOI: http://dx.doi.org/10.5772/intechopen.91163*


**222**


#### **Table 1.**

*Summary of the published literature on stereotactic radiosurgery in patients with recurrent glioblastoma multiforme.* retrospective studies [14

11 months.

–16, 19, 21, 23

*Stereotactic Radiosurgery for Recurrent Glioblastoma Multiforme*

*DOI: http://dx.doi.org/10.5772/intechopen.91163*

60, 61]. The median age ranged from 34 to 54 years. The median prescribed dose ranged from 13 to 30 Gy. The median targeted tumor volume was 4.5 to 41.3 cc. The

The first study about LINAC radiosurgery for recurrent GBMs was described by Chamerian et al. [14]. The median prescribed dose was 13.4 Gy, and the median treated tumor volume was 17 cc. The median overall survival time was only 8 months, whereas the median progression-free survival time was 4 months. After that, only one retrospective study of more than 100 patients with recurrent highgrade gliomas treated with LINAC SRS has been reported [16]. Shrieve et al. showed that the median survival time of 72 recurrent GBM patients was 10.2 months [16]. Younger age (less than 46 years) and small tumor volume (less than 10.1 cc) were the significant prognostic factors associated with survival time. There were two studies that enrolled patients with only recurrent GBMs [21, 33]. In 2005, Combs et al. reported 32 patients, including 19 males and 13 females with recurrent GBMs treated with LINAC SRS [21]. The median age was 56 years, ranging from 33 to 76 years. The median prescribed radiation dose was 15 Gy, ranging from 10 to 20 Gy. The median targeted tumor volume was 10 cc with a range of 1.2 to 59.2 cc. The median overall survival time and progression-free survival time were 10 and 5 months, respectively. However, no prognostic factor was significant enough to influence the survival time. In a retrospective study of 19 patients with recurrent

median overall survival time from the treatment of SRS ranged from 3.9 to 14.4 months, whereas the median progression-free survival time was 2.1 to

GBMs, Sirin et al. also showed that the median overall survival time and

survival time in patients with recurrent GBMs.

from 3.8 to 14.9 months.

**225**

factors associated with patients

progression-free survival time were only 9.3 and 5.7 months, respectively [33]. In the bevacizumab era, three studies reported the combination of LINAC SRS, and bevacizumab improved the overall survival time ranging from 11.2 to 14.4 months [35, 39, 40]. In a retrospective study of 48 patients with recurrent GBMs, Cuneo et al. reported that the median progression-free survival time in recurrent patients who received adjuvant bevacizumab and LINAC SRS was 5.2 months vs. 2.1 months for patients who received LINAC SRS alone. The median overall survival times for patients who received a combination of adjuvant bevacizumab/LINAC SRS and LINAC SRS alone were 11.2 and 3.9 months, respectively. The authors concluded that the combination of salvage radiosurgery and bevacizumab to treat recurrent malignant gliomas seemed to be associated with improved outcomes. Younger age and higher KPS were still significant prognostic factors associated with overall

**3.2 The effect of gamma knife radiosurgery in patients with recurrent GBMs**

From 1996 to 2019, a total of 1247 patients with recurrent GBMs in 23 published studies were treated with Gamma Knife SRS [13, 17, 18, 20, 22, 24, 27, 29, 31, 36

38, 41, 45, 47, 50, 52, 53, 55, 57–59, 61]. The median age ranged from 43 to 61 years. The median prescribed marginal dose varied from 6 to 20 Gy. The median targeted tumor volume ranged from 1.35 to 21.4 cc. The median overall survival time ranged from 7 to 30 months, whereas the median progression-free survival time ranged

In a retrospective study of 189 patients with recurrent high-grade gliomas treated with Gamma Knife SRS, Larson et al. first reported that the median overall survival time in 66 patients with recurrent GBMs was 10 months [17]. Younger age, smaller tumor volume, higher KPS, and unifocal tumors were significant prognostic

impact of a combination of Gamma Knife SRS and adjuvant chemoagents on overall

' overall survival times. Several studies reported the

–

–26, 28, 32, 33, 35, 39, 40, 43, 49, 51, 54, 56,

retrospective studies [14–16, 19, 21, 23–26, 28, 32, 33, 35, 39, 40, 43, 49, 51, 54, 56, 60, 61]. The median age ranged from 34 to 54 years. The median prescribed dose ranged from 13 to 30 Gy. The median targeted tumor volume was 4.5 to 41.3 cc. The median overall survival time from the treatment of SRS ranged from 3.9 to 14.4 months, whereas the median progression-free survival time was 2.1 to 11 months.

The first study about LINAC radiosurgery for recurrent GBMs was described by Chamerian et al. [14]. The median prescribed dose was 13.4 Gy, and the median treated tumor volume was 17 cc. The median overall survival time was only 8 months, whereas the median progression-free survival time was 4 months. After that, only one retrospective study of more than 100 patients with recurrent highgrade gliomas treated with LINAC SRS has been reported [16]. Shrieve et al. showed that the median survival time of 72 recurrent GBM patients was 10.2 months [16]. Younger age (less than 46 years) and small tumor volume (less than 10.1 cc) were the significant prognostic factors associated with survival time. There were two studies that enrolled patients with only recurrent GBMs [21, 33]. In 2005, Combs et al. reported 32 patients, including 19 males and 13 females with recurrent GBMs treated with LINAC SRS [21]. The median age was 56 years, ranging from 33 to 76 years. The median prescribed radiation dose was 15 Gy, ranging from 10 to 20 Gy. The median targeted tumor volume was 10 cc with a range of 1.2 to 59.2 cc. The median overall survival time and progression-free survival time were 10 and 5 months, respectively. However, no prognostic factor was significant enough to influence the survival time. In a retrospective study of 19 patients with recurrent GBMs, Sirin et al. also showed that the median overall survival time and progression-free survival time were only 9.3 and 5.7 months, respectively [33]. In the bevacizumab era, three studies reported the combination of LINAC SRS, and bevacizumab improved the overall survival time ranging from 11.2 to 14.4 months [35, 39, 40]. In a retrospective study of 48 patients with recurrent GBMs, Cuneo et al. reported that the median progression-free survival time in recurrent patients who received adjuvant bevacizumab and LINAC SRS was 5.2 months vs. 2.1 months for patients who received LINAC SRS alone. The median overall survival times for patients who received a combination of adjuvant bevacizumab/LINAC SRS and LINAC SRS alone were 11.2 and 3.9 months, respectively. The authors concluded that the combination of salvage radiosurgery and bevacizumab to treat recurrent malignant gliomas seemed to be associated with improved outcomes. Younger age and higher KPS were still significant prognostic factors associated with overall survival time in patients with recurrent GBMs.

#### **3.2 The effect of gamma knife radiosurgery in patients with recurrent GBMs**

From 1996 to 2019, a total of 1247 patients with recurrent GBMs in 23 published studies were treated with Gamma Knife SRS [13, 17, 18, 20, 22, 24, 27, 29, 31, 36– 38, 41, 45, 47, 50, 52, 53, 55, 57–59, 61]. The median age ranged from 43 to 61 years. The median prescribed marginal dose varied from 6 to 20 Gy. The median targeted tumor volume ranged from 1.35 to 21.4 cc. The median overall survival time ranged from 7 to 30 months, whereas the median progression-free survival time ranged from 3.8 to 14.9 months.

In a retrospective study of 189 patients with recurrent high-grade gliomas treated with Gamma Knife SRS, Larson et al. first reported that the median overall survival time in 66 patients with recurrent GBMs was 10 months [17]. Younger age, smaller tumor volume, higher KPS, and unifocal tumors were significant prognostic factors associated with patients' overall survival times. Several studies reported the impact of a combination of Gamma Knife SRS and adjuvant chemoagents on overall

**Table 1.**

**224**

*Tumor Progression and Metastasis*

*Summary of the published literature on stereotactic*

 *radiosurgery*

 *in patients with recurrent glioblastoma*

 *multiforme.* survival times [17, 37, 47, 55, 59]. In 2002, Larson et al. reported a prospective phase II study on patients who received a combination of Gamma Knife SRS, and marimastat had a median overall survival of 8.7 months, whereas the median survival time in patients who received only Gamma Knife SRS alone was 10.1 months. Marimastat did not offer an advantage for patients with recurrent GBMs. However, in a retrospective study of 57 patients with recurrent GBMs, Kim et al. showed that the combination of adjuvant temozolomide and Gamma Knife SRS significantly improved the medium overall survival time from 9.2 to 15.5 months [47]. In the bevacizumab era, two studies reported that the median survival time was approximately 13 months after the combined treatment of Gamma Knife SRS and adjuvant bevacizumab [55, 59].

alternative option for treating recurrent GBMs. In a systematic review and metaanalysis of re-irradiation with external beam radiotherapy for recurrent GBMs, Kazmi et al. showed that the 6- and 12-month overall survival times from the time of re-irradiation were 70 and 34%, respectively, whereas the 6- and 12-month progression-free survival times were 40 and 16%, respectively [11]. The overall

SRS has the ability to combine surgical and radio-oncological treatments to deliver a high dose of focused radiation on the focal tumor lesion and spare the adjacent normal anatomical structures. For recurrent GBMs, the majority of tumors tend to grow within 2 cm of the contrast-enhancing lesion border, and SRS seems to be a reasonable tool to add radiation boost for the focal lesion followed by the standard treatment of initial radiation with 60 Gy in 30 fractions [4, 13]. In our present review, despite the different SRS modalities with the median prescribed dose ranging from 6 to 30 Gy, the overall survival time from the treatment of SRS ranged from 3.9 to 17.9 months, where the progression-free survival time from the treatment of SRS ranged from 2.1 to 14.9 months. Severe prognostic factors, such as small tumor volume, younger age, higher KPS score, and lower RPA class, were mostly suggested to be significantly associated with the overall survival time in patients with recurrent GBMs treated with SRS. These results showed that reirradiation with the SRS modality are an alternative and feasible method to manage

**4.2 The impact of SRS and adjuvant temozolomide for recurrent GBMs**

Since 2005, temozolomide, which is an alkylating agent, is the most important FDA-approved chemoagent for the standard treatment of patients with newly diagnosed GBMs [1, 4]. The median overall survival time significantly improved from 12.1 to 14.6 months after the patients with newly diagnosed GBMs received combined treatments with radiotherapy and adjuvant temozolomide. However, the disease frequently progresses within 6–9 months, and the 2-year survival rate is less than 25% [62]. The failure of temozolomide treatment has been found to be associated with the expression of MGMT protein [63–65]. Among the GBM patients with a methylated MGMT promoter, the median overall survival time was 21.7 months after treatment with radiotherapy and temozolomide, whereas the median survival time was 15.3 months in the unmethylated group treated with radiotherapy alone [64].

Due to the blood-brain barrier, temozolomide rechallenge is considered to be a reasonable option in patients with recurrent GBMs. In this review, the combination of SRS and temozolomide was employed in three studies [34, 42, 47]. Cyberknife SRS was performed in two studies, and the other study used the Gamma Knife SRS. The median overall survival time ranged from 9 to 15.5 months, and the median progression-free survival time was approximately 7 months after the time of SRS treatment. In 2012, Conti et al. analyzed the effect of adjuvant temozolomide in recurrent GBM patients treated with Cyberknife SRS [34]. The median overall survival time significantly improved from 7 to 12 months, whereas the median progression-free survival time improved from 4 to 7 months. Based on 57 recurrent GBM patients, Kim et al. also showed that the improved median overall survival time and progression-free survival time were 15.5 and 6 months, respectively [47]. Otherwise, in a retrospective review of 61 patients who received Gamma Knife SRS as a salvage treatment at the time of the first progression, Kim et al. showed that the median overall survival time was 14 months in the methylated MGMT promoter group and 9 months in the unmethylated group [53]. Methylation of the MGMT promoter was significantly corrected with better overall survival times and progression-free survival times. The results mentioned above indicated that the

toxicity rate was low, ranging from 4 to 10%.

*DOI: http://dx.doi.org/10.5772/intechopen.91163*

*Stereotactic Radiosurgery for Recurrent Glioblastoma Multiforme*

patients with recurrent GBMs.

**227**

#### **3.3 The effect of Cyberknife radiosurgery in patients with recurrent GBMs**

From 2009 to 2017, a total of 318 patients with recurrent GBMs in eight published studies were treated with Cyberknife SRS [30, 34, 42, 44, 46, 48, 51, 54]. The median age ranged from 37 to 59.9 years. The median prescribed marginal dose ranged from 15 to 30 Gy. The median targeted tumor volume ranged from 2 to 24 cc. The median overall survival time after Cyberknife SRS ranged from 5.3 to 12 months, whereas the median progression-free survival time ranged from 4 to 7.9 months.

The first report on Cyberknife SRS for patients with recurrent GBMs was published by Villavicencio et al. [30]. The median overall survival time in a total of 26 patients with recurrent GBMs was 7 months. No prognostic factor associated with the overall survival time was identified. In 2015, Pinzi et al. reported a retrospective study of more than 100 patients who had recurrent high-grade glioma treated with Cyberknife SRS [48]. Among 88 patients with recurrent GBMs, the median survival time was 10 months after treatment with Cyberknife SRS. Adjuvant second-line chemotherapy and/or surgery were the significant prognostic factors associated with overall survival times. The effect of adjuvant chemoagents, including bevacizumab, temozolomide, and anti-epidermal factor (125)-mAB 425, on the overall survival times was evaluated in four studies [34, 42, 44, 46]. In 2012, Conti et al. compared the effect of a combination of temozolomide and Cyberknife SRS with that of Cyberknife SRS alone on the overall survival times of patients with recurrent GBMs [34]. The progression-free survival time and median survival time in patients who received the adjuvant temozolomide and Cyberknife SRS were 7 and 12 months, respectively. The patients who received Cyberknife SRS alone had a progression-free survival time and median survival time of only 4 and 7 months, respectively. In the bevacizumab era, Yazici et al. revealed that the median survival time in 37 patients with recurrent GBMs was 10.6 months, whereas the median progression-free survival time was 7.9 months [44]. A tumor volume less than 24 cc was the only significant prognostic factor associated with overall survival times.

#### **4. Discussion**

#### **4.1 The role of SRS for recurrent GBMs**

GBM is an incurable disease with local progression in the majority of patients. The management of recurrent GBMs is a clinically challenging problem, and treatment options are limited [7, 10]. Although reoperation with gross-total removal of the tumor has been shown to improve the overall survival time in patients with recurrent GBMs, surgery may not be preferred for patients with tumors in the eloquent area, older age, or lower performance status [8]. Re-irradiation offers an

#### *Stereotactic Radiosurgery for Recurrent Glioblastoma Multiforme DOI: http://dx.doi.org/10.5772/intechopen.91163*

survival times [17, 37, 47, 55, 59]. In 2002, Larson et al. reported a prospective phase

II study on patients who received a combination of Gamma Knife SRS, and marimastat had a median overall survival of 8.7 months, whereas the median survival time in patients who received only Gamma Knife SRS alone was 10.1 months. Marimastat did not offer an advantage for patients with recurrent GBMs. However, in a retrospective study of 57 patients with recurrent GBMs, Kim et al. showed that the combination of adjuvant temozolomide and Gamma Knife SRS significantly improved the medium overall survival time from 9.2 to 15.5 months [47]. In the bevacizumab era, two studies reported that the median survival time was approximately 13 months after the combined treatment of Gamma Knife SRS and adjuvant

**3.3 The effect of Cyberknife radiosurgery in patients with recurrent GBMs**

The first report on Cyberknife SRS for patients with recurrent GBMs was published by Villavicencio et al. [30]. The median overall survival time in a total of 26 patients with recurrent GBMs was 7 months. No prognostic factor associated with the overall survival time was identified. In 2015, Pinzi et al. reported a retrospective study of more than 100 patients who had recurrent high-grade glioma treated with Cyberknife SRS [48]. Among 88 patients with recurrent GBMs, the median survival time was 10 months after treatment with Cyberknife SRS. Adjuvant second-line chemotherapy and/or surgery were the significant prognostic factors associated with overall survival times. The effect of adjuvant chemoagents, including bevacizumab, temozolomide, and anti-epidermal factor (125)-mAB 425, on the overall survival times was evaluated in four studies [34, 42, 44, 46]. In 2012, Conti et al. compared the effect of a combination of temozolomide and Cyberknife SRS with that of Cyberknife SRS alone on the overall survival times of patients with recurrent GBMs [34]. The progression-free survival time and median survival time in patients who received the adjuvant temozolomide and Cyberknife SRS were 7 and 12 months, respectively. The patients who received Cyberknife SRS alone had a progression-free survival time and median survival time of only 4 and 7 months, respectively. In the bevacizumab era, Yazici et al. revealed that the median survival time in 37 patients with recurrent GBMs was 10.6 months, whereas the median progression-free survival time was 7.9 months [44]. A tumor volume less than 24 cc was the only significant prognostic factor associated with overall survival times.

GBM is an incurable disease with local progression in the majority of patients. The management of recurrent GBMs is a clinically challenging problem, and treatment options are limited [7, 10]. Although reoperation with gross-total removal of the tumor has been shown to improve the overall survival time in patients with recurrent GBMs, surgery may not be preferred for patients with tumors in the eloquent area, older age, or lower performance status [8]. Re-irradiation offers an

median progression-free survival time ranged from 4 to 7.9 months.

From 2009 to 2017, a total of 318 patients with recurrent GBMs in eight published studies were treated with Cyberknife SRS [30, 34, 42, 44, 46, 48, 51, 54]. The median age ranged from 37 to 59.9 years. The median prescribed marginal dose ranged from 15 to 30 Gy. The median targeted tumor volume ranged from 2 to 24 cc. The median overall survival time after Cyberknife SRS ranged from 5.3 to 12 months, whereas the

bevacizumab [55, 59].

*Tumor Progression and Metastasis*

**4. Discussion**

**226**

**4.1 The role of SRS for recurrent GBMs**

alternative option for treating recurrent GBMs. In a systematic review and metaanalysis of re-irradiation with external beam radiotherapy for recurrent GBMs, Kazmi et al. showed that the 6- and 12-month overall survival times from the time of re-irradiation were 70 and 34%, respectively, whereas the 6- and 12-month progression-free survival times were 40 and 16%, respectively [11]. The overall toxicity rate was low, ranging from 4 to 10%.

SRS has the ability to combine surgical and radio-oncological treatments to deliver a high dose of focused radiation on the focal tumor lesion and spare the adjacent normal anatomical structures. For recurrent GBMs, the majority of tumors tend to grow within 2 cm of the contrast-enhancing lesion border, and SRS seems to be a reasonable tool to add radiation boost for the focal lesion followed by the standard treatment of initial radiation with 60 Gy in 30 fractions [4, 13]. In our present review, despite the different SRS modalities with the median prescribed dose ranging from 6 to 30 Gy, the overall survival time from the treatment of SRS ranged from 3.9 to 17.9 months, where the progression-free survival time from the treatment of SRS ranged from 2.1 to 14.9 months. Severe prognostic factors, such as small tumor volume, younger age, higher KPS score, and lower RPA class, were mostly suggested to be significantly associated with the overall survival time in patients with recurrent GBMs treated with SRS. These results showed that reirradiation with the SRS modality are an alternative and feasible method to manage patients with recurrent GBMs.

#### **4.2 The impact of SRS and adjuvant temozolomide for recurrent GBMs**

Since 2005, temozolomide, which is an alkylating agent, is the most important FDA-approved chemoagent for the standard treatment of patients with newly diagnosed GBMs [1, 4]. The median overall survival time significantly improved from 12.1 to 14.6 months after the patients with newly diagnosed GBMs received combined treatments with radiotherapy and adjuvant temozolomide. However, the disease frequently progresses within 6–9 months, and the 2-year survival rate is less than 25% [62]. The failure of temozolomide treatment has been found to be associated with the expression of MGMT protein [63–65]. Among the GBM patients with a methylated MGMT promoter, the median overall survival time was 21.7 months after treatment with radiotherapy and temozolomide, whereas the median survival time was 15.3 months in the unmethylated group treated with radiotherapy alone [64].

Due to the blood-brain barrier, temozolomide rechallenge is considered to be a reasonable option in patients with recurrent GBMs. In this review, the combination of SRS and temozolomide was employed in three studies [34, 42, 47]. Cyberknife SRS was performed in two studies, and the other study used the Gamma Knife SRS. The median overall survival time ranged from 9 to 15.5 months, and the median progression-free survival time was approximately 7 months after the time of SRS treatment. In 2012, Conti et al. analyzed the effect of adjuvant temozolomide in recurrent GBM patients treated with Cyberknife SRS [34]. The median overall survival time significantly improved from 7 to 12 months, whereas the median progression-free survival time improved from 4 to 7 months. Based on 57 recurrent GBM patients, Kim et al. also showed that the improved median overall survival time and progression-free survival time were 15.5 and 6 months, respectively [47]. Otherwise, in a retrospective review of 61 patients who received Gamma Knife SRS as a salvage treatment at the time of the first progression, Kim et al. showed that the median overall survival time was 14 months in the methylated MGMT promoter group and 9 months in the unmethylated group [53]. Methylation of the MGMT promoter was significantly corrected with better overall survival times and progression-free survival times. The results mentioned above indicated that the

combination of salvage SRS and adjuvant temozolomide may offer an important treatment option to improve the overall survival times in patients with recurrent GBMs.

60 Gy typically applied in the first-line treatment [59]. Although younger age is commonly considered as an important independent prognostic factor that is associated with survival, the selected criteria of salvage SRS for better outcomes need to be investigated in further large prospective studies. In the future, individualized precise multi-modality treatment will play an important role in patients with recurrent GBMs, including the combination of cytotoxic chemotherapy, angiogenesis inhibitors, or immunotherapy [76]. Salvage SRS with a combination of other treatment modalities may offer an alternative therapeutic method to manage

*Stereotactic Radiosurgery for Recurrent Glioblastoma Multiforme*

*DOI: http://dx.doi.org/10.5772/intechopen.91163*

Our review suggests that salvage SRS is an important treatment protocol for managing patients with recurrent GBMs. The irradiation doses provided by SRS may improve the clinical outcome of patients with recurrent GBMs, which is not hampered by the standard case of 60 Gy prescribed for newly diagnosed GBMs. The dual role of salvage SRS and other cytotoxic chemoagents, such as temozolomide and bevacizumab, also seems to be effective in the management of recurrent GBMs. Further application of salvage SRS combined with other chemoagents or a new

\*

1 Division of Neurosurgery, Department of Surgery, Sijhih Cathay General

2 Department of Medicine, School of Medicine, Fu Jen Catholic University,

3 Department of Neurological Surgery, Tri-Service General Hospital, National

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

patients with recurrent GBMs.

treatment modality needs to be investigated.

The authors declare no conflict of interest.

Cheng-Ta Hsieh1,2,3 and Da-Tong Ju<sup>3</sup>

Hospital, New Taipei City, Taiwan

Defense Medical Center, Taipei City, Taiwan

provided the original work is properly cited.

\*Address all correspondence to: wxyz670628@yahoo.com.tw

New Taipei City, Taiwan

**229**

**5. Conclusion**

**Conflict of interest**

**Author details**

#### **4.3 The impact of SRS and adjuvant bevacizumab for recurrent GBMs**

Bevacizumab is a recombinant human monoclonal antibody that acts against the vascular endothelial growth factor to prevent the growth and maintenance of tumor blood vessels. In 2009, bevacizumab was approved by the USFDA for the treatment of patients with recurrent GBMs [66, 67]. The use of bevacizumab demonstrated a radiological response of up to 40% [68]. However, in a large prospective phase III trial, the use of adjuvant bevacizumab revealed only improvement in the progression-free survival times from 1.5 to 4.2 months but not in the overall survival times [69]. In a systematic review and meta-analysis, Diaz et al. showed that the survival advantage of bevacizumab at recurrence was limited to 4 months [70]. Although bevacizumab may reduce steroid requirements, there was no additional benefit in the health-related quality of life. The role of bevacizumab in combination with other cytotoxic chemoagents remains unclear.

The role of adjuvant bevacizumab in patients with recurrent GBMs treated with SRS has been reported in nine studies, which were included in our review [35, 37, 39, 40, 44, 46, 49, 55, 59]. The median overall survival time ranged from 5.3 to 17.9 months, whereas the median progression-free survival time ranged from 3.9 to 14.9 months. The comparison of SRA with or without adjuvant bevacizumab was investigated in two studies [35, 37]. Among 49 patients with recurrent GBMs, Cuneo et al. showed that the median overall survival time was 11.2 months in patients receiving SRS and adjuvant bevacizumab and 3.9 months in patients receiving SRS therapy alone [35]. The progression-free survival time also improved from 2.1 to 5.2 months. In a case-controlled study of patients with recurrent GBMs treated with SRS and adjuvant bevacizumab plus temozolomide or irinotecan, Park et al. also showed that the median overall survival time and progression-free survival time improved from 12.2 to 17.9 months and 6.7 to 14.9 months, respectively [37]. In a retrospective study and review of the literature, Morris et al. reported that the dual role of bevacizumab and radiosurgery had a benefit in the overall survival times (11.2–17.9 months) and progression-free survival times (3.9–14.9 months). These results showed the potential therapeutic effect of adjuvant bevacizumab in combination with other treatment modalities, such as cytotoxic chemoagents or salvage SRS, in patients with recurrent GBMs.

#### **4.4 The future of SRS for recurrent GBMs**

With the advance of molecular diagnostic techniques, newly diagnosed GBMs should be classified based on the mutant status of isocitrate dehydrogenase 1 defined by the updated guidelines of the World Health Organization in 2016 [71]. These molecular profiles influence the overall survival time and the possible therapeutic effects of chemoagents. Similar to the recurrent GBMs, several main molecules, such as MLH1 [72], CASP8 [73], MSH2 [74], and P53 [74], were found to be different from primary GBMs [75]. The molecular features, intra-tumor heterogeneity, immunogenicity, and microenvironment around the tumor contribute to the clinical prognostic outcomes in patients with recurrent GBMs [7, 10]. Reoperation, re-chemotherapy, and re-irradiation currently remain as the standard treatments for most patients with recurrent GBMs [2, 7, 10, 11]. A growing body of literature, including our current review, demonstrates the tolerability and efficacy of salvage SRS for recurrent GBMs, which did not inhibit re-irradiation, followed by a total of *Stereotactic Radiosurgery for Recurrent Glioblastoma Multiforme DOI: http://dx.doi.org/10.5772/intechopen.91163*

60 Gy typically applied in the first-line treatment [59]. Although younger age is commonly considered as an important independent prognostic factor that is associated with survival, the selected criteria of salvage SRS for better outcomes need to be investigated in further large prospective studies. In the future, individualized precise multi-modality treatment will play an important role in patients with recurrent GBMs, including the combination of cytotoxic chemotherapy, angiogenesis inhibitors, or immunotherapy [76]. Salvage SRS with a combination of other treatment modalities may offer an alternative therapeutic method to manage patients with recurrent GBMs.

#### **5. Conclusion**

combination of salvage SRS and adjuvant temozolomide may offer an important treatment option to improve the overall survival times in patients with recurrent

Bevacizumab is a recombinant human monoclonal antibody that acts against the vascular endothelial growth factor to prevent the growth and maintenance of tumor blood vessels. In 2009, bevacizumab was approved by the USFDA for the treatment of patients with recurrent GBMs [66, 67]. The use of bevacizumab demonstrated a radiological response of up to 40% [68]. However, in a large prospective phase III

**4.3 The impact of SRS and adjuvant bevacizumab for recurrent GBMs**

trial, the use of adjuvant bevacizumab revealed only improvement in the

with other cytotoxic chemoagents remains unclear.

salvage SRS, in patients with recurrent GBMs.

**4.4 The future of SRS for recurrent GBMs**

**228**

progression-free survival times from 1.5 to 4.2 months but not in the overall survival times [69]. In a systematic review and meta-analysis, Diaz et al. showed that the survival advantage of bevacizumab at recurrence was limited to 4 months [70]. Although bevacizumab may reduce steroid requirements, there was no additional benefit in the health-related quality of life. The role of bevacizumab in combination

The role of adjuvant bevacizumab in patients with recurrent GBMs treated with SRS has been reported in nine studies, which were included in our review [35, 37, 39, 40, 44, 46, 49, 55, 59]. The median overall survival time ranged from 5.3 to 17.9 months, whereas the median progression-free survival time ranged from 3.9 to 14.9 months. The comparison of SRA with or without adjuvant bevacizumab was investigated in two studies [35, 37]. Among 49 patients with recurrent GBMs, Cuneo et al. showed that the median overall survival time was 11.2 months in patients receiving SRS and adjuvant bevacizumab and 3.9 months in patients receiving SRS therapy alone [35]. The progression-free survival time also improved from 2.1 to 5.2 months. In a case-controlled study of patients with recurrent GBMs treated with SRS and adjuvant bevacizumab plus temozolomide or irinotecan, Park et al. also showed that the median overall survival time and progression-free survival time improved from 12.2 to 17.9 months and 6.7 to 14.9 months, respectively [37]. In a retrospective study and review of the literature, Morris et al. reported that the dual role of bevacizumab and radiosurgery had a benefit in the overall survival times (11.2–17.9 months) and progression-free survival times (3.9–14.9 months). These results showed the potential therapeutic effect of adjuvant bevacizumab in combination with other treatment modalities, such as cytotoxic chemoagents or

With the advance of molecular diagnostic techniques, newly diagnosed GBMs

should be classified based on the mutant status of isocitrate dehydrogenase 1 defined by the updated guidelines of the World Health Organization in 2016 [71]. These molecular profiles influence the overall survival time and the possible therapeutic effects of chemoagents. Similar to the recurrent GBMs, several main molecules, such as MLH1 [72], CASP8 [73], MSH2 [74], and P53 [74], were found to be different from primary GBMs [75]. The molecular features, intra-tumor heterogeneity, immunogenicity, and microenvironment around the tumor contribute to the clinical prognostic outcomes in patients with recurrent GBMs [7, 10]. Reoperation, re-chemotherapy, and re-irradiation currently remain as the standard treatments for most patients with recurrent GBMs [2, 7, 10, 11]. A growing body of literature, including our current review, demonstrates the tolerability and efficacy of salvage SRS for recurrent GBMs, which did not inhibit re-irradiation, followed by a total of

GBMs.

*Tumor Progression and Metastasis*

Our review suggests that salvage SRS is an important treatment protocol for managing patients with recurrent GBMs. The irradiation doses provided by SRS may improve the clinical outcome of patients with recurrent GBMs, which is not hampered by the standard case of 60 Gy prescribed for newly diagnosed GBMs. The dual role of salvage SRS and other cytotoxic chemoagents, such as temozolomide and bevacizumab, also seems to be effective in the management of recurrent GBMs. Further application of salvage SRS combined with other chemoagents or a new treatment modality needs to be investigated.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Cheng-Ta Hsieh1,2,3 and Da-Tong Ju<sup>3</sup> \*

1 Division of Neurosurgery, Department of Surgery, Sijhih Cathay General Hospital, New Taipei City, Taiwan

2 Department of Medicine, School of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan

3 Department of Neurological Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei City, Taiwan

\*Address all correspondence to: wxyz670628@yahoo.com.tw

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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(Suppl. 5):v1-v100

2005;**352**(10):987-996

2011;**115**(1):3-8

Focus. 2006;**20**(4):E5

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[1] Preusser M, de Ribaupierre S, Wöhrer A, Erridge SC, Hegi M, Weller M, et al. Current concepts and management of glioblastoma. Annals of

*Tumor Progression and Metastasis*

[9] Lu VM, Goyal A, Graffeo CS, Perry A, Burns TC, Parney IF, et al. Survival benefit of maximal resection for glioblastoma reoperation in the temozolomide era: A meta-analysis. World Neurosurgery. 2019;**127**:31-37

Reports. 2019;**21**(10):94

**142**(1):79-90

2018;**13**(1):133

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[11] Kazmi F, Soon YY, Leong YH, Koh WY, Vellayappan B. Re-irradiation for recurrent glioblastoma (GBM): A systematic review and meta-analysis. Journal of Neuro-Oncology. 2019;

[12] Jayamanne D, Wheeler H,

[13] Niranjan A, Kano H, Iyer A, Kondziolka D, Flickinger JC, Lunsford LD. Role of adjuvant or salvage radiosurgery in the management of unresected residual or progressive glioblastoma multiforme in the pre-bevacizumab era. Journal of Neurosurgery. 2015;**122**(4):757-765

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[15] Hall WA, Djalilian HR, Sperduto PW, Cho KH, Gerbi BJ, Gibbons JP, et al. Stereotactic

1995;**13**(7):1642-1648

radiosurgery for recurrent malignant gliomas. Journal of Clinical Oncology.

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et al. Comparison of stereotactic

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implications on radiotherapy treatment portals. Radiotherapy and Oncology.

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[4] Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. The New England Journal of Medicine.

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[8] Bloch O, Han SJ, Cha S, Sun MZ, Aghi MK, McDermott MW, et al. Impact of extent of resection for recurrent glioblastoma on overall survival: Clinical article. Journal of Neurosurgery. 2012;**117**(6):1032-1038

Institute. 2016;**28**(4):199-210

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[19] Cho KH, Hall WA, Gerbi BJ, Higgins PD, McGuire WA, Clark HB. Single dose versus fractionated stereotactic radiotherapy for recurrent high-grade gliomas. International Journal of Radiation Oncology, Biology, Physics. 1999;**45**(5):1133-1141

[20] Larson DA, Prados M, Lamborn KR, Smith V, Sneed PK, Chang S, et al. Phase II study of high central dose Gamma knife radiosurgery and marimastat in patients with recurrent malignant glioma. International Journal of Radiation Oncology, Biology, Physics. 2002;**54**(5):1397-1404

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[35] Cuneo KC, Vredenburgh JJ, Sampson JH, Reardon DA, Desjardins A, Peters KB, et al. Safety and efficacy of stereotactic radiosurgery and adjuvant bevacizumab in patients with recurrent malignant gliomas. International Journal of Radiation Oncology, Biology, Physics. 2012;**82**(5):2018-2024

[36] Koga T, Maruyama K, Tanaka M, Ino Y, Saito N, Nakagawa K, et al. Extended field stereotactic radiosurgery for recurrent glioblastoma. Cancer. 2012;**118**(17):4193-4200

[37] Park KJ, Kano H, Iyer A, Liu X, Niranjan A, Flickinger JC, et al. Salvage gamma knife stereotactic radiosurgery followed by bevacizumab for recurrent glioblastoma multiforme: A case-control study. Journal of Neuro-Oncology. 2012; **107**(2):323-333

Ávila R, Guerrero-Tejada R, Saura-Rojas

*DOI: http://dx.doi.org/10.5772/intechopen.91163*

*Stereotactic Radiosurgery for Recurrent Glioblastoma Multiforme*

et al. Gamma knife radiosurgery in recurrent glioblastoma. Stereotactic and Functional Neurosurgery. 2016;**94**(4):

[51] Holt DE, Bernard ME, Quan K, Clump DA, Engh JA, Burton SA, et al. Salvage stereotactic radiosurgery for recurrent glioblastoma multiforme with prior radiation therapy. Journal of Cancer Research and Therapeutics.

[52] Imber BS, Kanungo I, Braunstein S, Barani IJ, Fogh SE, Nakamura JL, et al. Indications and efficacy of gamma knife stereotactic radiosurgery for recurrent glioblastoma: 2 decades of institutional experience. Neurosurgery. 2017;**80**(1):

[53] Kim BS, Kong DS, Seol HJ, Nam DH, Lee JI. MGMT promoter methylation status as a prognostic factor for the outcome of gamma knife radiosurgery for recurrent glioblastoma. Journal of Neuro-Oncology. 2017;**133**(3):615-622

[54] Sutera PA, Bernard ME, Gill BS, Quan K, Engh JA, Burton SA, et al. Salvage stereotactic radiosurgery for recurrent gliomas with prior radiation therapy. Future Oncology. 2017;**13**(29):

[55] Abbassy M, Missios S, Barnett GH,

[56] Gigliotti MJ, Hasan S, Karlovits SM, Ranjan T, Wegner RE. Re-irradiation with stereotactic radiosurgery/ radiotherapy for recurrent high-grade gliomas: Improved survival in the modern era. Stereotactic and Functional Neurosurgery. 2018;**96**(5):289-295

Simonova G, Novotny J Jr. Gamma knife

Brewer C, Peereboom DM, Ahluwalia M, et al. Phase I trial of radiosurgery dose escalation plus bevacizumab in patients with recurrent/ progressive glioblastoma. Neurosurgery.

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2016;**12**(4):1243-1248

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[48] Pinzi V, Orsi C, Marchetti M, Milanesi IM, Bianchi LC, DiMeco F, et al. Radiosurgery reirradiation for high-grade glioma recurrence: A retrospective analysis. Neurological Sciences. 2015;**36**(8):1431-1440

[49] Bokstein F, Blumenthal DT, Corn BW, Gez E, Matceyevsky D, Shtraus N, et al. Stereotactic radiosurgery (SRS) in high-grade glioma: Judicious selection of small target volumes improves results. Journal of Neuro-Oncology. 2016;**126**(3):551-557

[50] Frischer JM, Marosi C, Woehrer A, Hainfellner JA, Dieckmann KU, Eiter H,

reirradiation for recurrent glioblastoma. Journal of Neuro-Oncology. 2014;

[45] Bir SC, Connor DE Jr, Ambekar S, Wilden JA, Nanda A. Factors predictive of improved overall survival following stereotactic radiosurgery for recurrent glioblastoma. Neurosurgical Review.

[46] Hasan S, Chen E, Lanciano R, Yang J, Hanlon A, Lamond J, et al. Salvage fractionated stereotactic radiotherapy with or without

chemotherapy and immunotherapy for recurrent glioblastoma multiforme: A single institution experience. Frontiers

[38] Skeie BS, Enger PØ, Brøgger J, Ganz JC, Thorsen F, Heggdal JI, et al. Gamma knife surgery versus reoperation for recurrent glioblastoma multiforme. World Neurosurgery. 2012; **78**(6):658-669

[39] Cabrera AR, Cuneo KC, Desjardins A, Sampson JH, McSherry F, Herndon JE 2nd, et al. Concurrent stereotactic radiosurgery and bevacizumab in recurrent malignant gliomas: A prospective trial. International Journal of Radiation Oncology, Biology, Physics. 2013;**86**(5): 873-879

[40] Clark GM, McDonald AM, Nabors LB, Fathalla-Shaykh H, Han X, Willey CD, et al. Hypofractionated stereotactic radiosurgery with concurrent bevacizumab for recurrent malignant gliomas: The University of Alabama at Birmingham experience. Neurooncology Practice. 2014;**1**(4): 172-177

[41] Dodoo E, Huffmann B, Peredo I, Grinaker H, Sinclair G, Machinis T, et al. Increased survival using delayed gamma knife radiosurgery for recurrent high-grade glioma: A feasibility study. World Neurosurgery. 2014;**82**(5):e623 e632

[42] Greenspoon JN, Sharieff W, Hirte H, Overholt A, Devillers R, Gunnarsson T, et al. Fractionated stereotactic radiosurgery with concurrent temozolomide chemotherapy for locally recurrent glioblastoma multiforme: A prospective cohort study. OncoTargets and Therapy. 2014;**7**:485-490

[43] Martínez-Carrillo M, Tovar-Martín I, Zurita-Herrera M, Del Moral*Stereotactic Radiosurgery for Recurrent Glioblastoma Multiforme DOI: http://dx.doi.org/10.5772/intechopen.91163*

Ávila R, Guerrero-Tejada R, Saura-Rojas E, et al. Salvage radiosurgery for selected patients with recurrent malignant gliomas. BioMed Research International. 2014;**2014**:657953

diagnosed and recurrent glioblastoma multiforme: A multicenter experience. Neurosurgical Review. 2009;**32**(4):

*Tumor Progression and Metastasis*

gamma knife stereotactic radiosurgery followed by bevacizumab for recurrent glioblastoma multiforme: A case-control study. Journal of Neuro-Oncology. 2012;

[38] Skeie BS, Enger PØ, Brøgger J, Ganz JC, Thorsen F, Heggdal JI, et al.

reoperation for recurrent glioblastoma multiforme. World Neurosurgery. 2012;

Desjardins A, Sampson JH, McSherry F, Herndon JE 2nd, et al. Concurrent stereotactic radiosurgery and bevacizumab in recurrent malignant

Gamma knife surgery versus

[39] Cabrera AR, Cuneo KC,

gliomas: A prospective trial. International Journal of Radiation Oncology, Biology, Physics. 2013;**86**(5):

[40] Clark GM, McDonald AM,

Nabors LB, Fathalla-Shaykh H, Han X, Willey CD, et al. Hypofractionated stereotactic radiosurgery with

concurrent bevacizumab for recurrent malignant gliomas: The University of Alabama at Birmingham experience. Neurooncology Practice. 2014;**1**(4):

[41] Dodoo E, Huffmann B, Peredo I, Grinaker H, Sinclair G, Machinis T, et al. Increased survival using delayed gamma knife radiosurgery for recurrent high-grade glioma: A feasibility study. World Neurosurgery. 2014;**82**(5):e623-

[42] Greenspoon JN, Sharieff W, Hirte H, Overholt A, Devillers R, Gunnarsson T, et al. Fractionated stereotactic radiosurgery with concurrent temozolomide

chemotherapy for locally recurrent glioblastoma multiforme: A prospective cohort study. OncoTargets and Therapy.

[43] Martínez-Carrillo M, Tovar-Martín I, Zurita-Herrera M, Del Moral-

**107**(2):323-333

**78**(6):658-669

873-879

172-177

e632

2014;**7**:485-490

[31] Elliott RE, Parker EC, Rush SC, Kalhorn SP, Moshel YA, Narayana A, et al. Efficacy of gamma knife

(1–2):128-140 discussion 61-2

[33] Sirin S, Oysul K, Surenkok S, Sager O, Dincoglan F, Dirican B, et al. Linear accelerator-based stereotactic radiosurgery in recurrent glioblastoma:

Vojnosanitetski Pregled. 2011;**68**(11):

[34] Conti A, Pontoriero A, Arpa D, Siragusa C, Tomasello C, Romanelli P, et al. Efficacy and toxicity of Cyberknife

re-irradiation and "dose dense" temozolomide for recurrent gliomas. Acta Neurochirurgica. 2012;**154**(2):

[35] Cuneo KC, Vredenburgh JJ,

2012;**82**(5):2018-2024

2012;**118**(17):4193-4200

**232**

Sampson JH, Reardon DA, Desjardins A, Peters KB, et al. Safety and efficacy of stereotactic radiosurgery and adjuvant bevacizumab in patients with recurrent malignant gliomas. International Journal of Radiation Oncology, Biology, Physics.

[36] Koga T, Maruyama K, Tanaka M, Ino Y, Saito N, Nakagawa K, et al. Extended field stereotactic radiosurgery for recurrent glioblastoma. Cancer.

[37] Park KJ, Kano H, Iyer A, Liu X, Niranjan A, Flickinger JC, et al. Salvage

A single center experience.

961-966

203-209

radiosurgery for small-volume recurrent malignant gliomas after initial radical resection. World Neurosurgery. 2011;**76**

[32] Maranzano E, Anselmo P, Casale M, Trippa F, Carletti S, Principi M, et al. Treatment of recurrent glioblastoma with stereotactic radiotherapy: Longterm results of a mono-institutional trial. Tumori. 2011;**97**(1):56-61

417-424

[44] Yazici G, Cengiz M, Ozyigit G, Eren G, Yildiz F, Akyol F, et al. Hypofractionated stereotactic reirradiation for recurrent glioblastoma. Journal of Neuro-Oncology. 2014; **120**(1):117-123

[45] Bir SC, Connor DE Jr, Ambekar S, Wilden JA, Nanda A. Factors predictive of improved overall survival following stereotactic radiosurgery for recurrent glioblastoma. Neurosurgical Review. 2015;**38**(4):705-713

[46] Hasan S, Chen E, Lanciano R, Yang J, Hanlon A, Lamond J, et al. Salvage fractionated stereotactic radiotherapy with or without chemotherapy and immunotherapy for recurrent glioblastoma multiforme: A single institution experience. Frontiers in Oncology. 2015;**5**:106

[47] Kim HR, Kim KH, Kong DS, Seol HJ, Nam DH, Lim DH, et al. Outcome of salvage treatment for recurrent glioblastoma. Journal of Clinical Neuroscience. 2015;**22**(3): 468-473

[48] Pinzi V, Orsi C, Marchetti M, Milanesi IM, Bianchi LC, DiMeco F, et al. Radiosurgery reirradiation for high-grade glioma recurrence: A retrospective analysis. Neurological Sciences. 2015;**36**(8):1431-1440

[49] Bokstein F, Blumenthal DT, Corn BW, Gez E, Matceyevsky D, Shtraus N, et al. Stereotactic radiosurgery (SRS) in high-grade glioma: Judicious selection of small target volumes improves results. Journal of Neuro-Oncology. 2016;**126**(3):551-557

[50] Frischer JM, Marosi C, Woehrer A, Hainfellner JA, Dieckmann KU, Eiter H, et al. Gamma knife radiosurgery in recurrent glioblastoma. Stereotactic and Functional Neurosurgery. 2016;**94**(4): 265-272

[51] Holt DE, Bernard ME, Quan K, Clump DA, Engh JA, Burton SA, et al. Salvage stereotactic radiosurgery for recurrent glioblastoma multiforme with prior radiation therapy. Journal of Cancer Research and Therapeutics. 2016;**12**(4):1243-1248

[52] Imber BS, Kanungo I, Braunstein S, Barani IJ, Fogh SE, Nakamura JL, et al. Indications and efficacy of gamma knife stereotactic radiosurgery for recurrent glioblastoma: 2 decades of institutional experience. Neurosurgery. 2017;**80**(1): 129-139

[53] Kim BS, Kong DS, Seol HJ, Nam DH, Lee JI. MGMT promoter methylation status as a prognostic factor for the outcome of gamma knife radiosurgery for recurrent glioblastoma. Journal of Neuro-Oncology. 2017;**133**(3):615-622

[54] Sutera PA, Bernard ME, Gill BS, Quan K, Engh JA, Burton SA, et al. Salvage stereotactic radiosurgery for recurrent gliomas with prior radiation therapy. Future Oncology. 2017;**13**(29): 2681-2690

[55] Abbassy M, Missios S, Barnett GH, Brewer C, Peereboom DM, Ahluwalia M, et al. Phase I trial of radiosurgery dose escalation plus bevacizumab in patients with recurrent/ progressive glioblastoma. Neurosurgery. 2018;**83**(3):385-392

[56] Gigliotti MJ, Hasan S, Karlovits SM, Ranjan T, Wegner RE. Re-irradiation with stereotactic radiosurgery/ radiotherapy for recurrent high-grade gliomas: Improved survival in the modern era. Stereotactic and Functional Neurosurgery. 2018;**96**(5):289-295

[57] Guseynova K, Liscak R, Simonova G, Novotny J Jr. Gamma knife radiosurgery for local recurrence of glioblastoma. Neuro Endocrinology Letters. 2018;**39**(4):281-287

[58] Sharma M, Schroeder JL, Elson P, Meola A, Barnett GH, Vogelbaum MA, et al. Outcomes and prognostic stratification of patients with recurrent glioblastoma treated with salvage stereotactic radiosurgery. Journal of Neurosurgery. 2018:1-11

[59] Morris SL, Zhu P, Rao M, Martir M, Zhu JJ, Hsu S, et al. Gamma knife stereotactic radiosurgery in combination with bevacizumab for recurrent glioblastoma. World Neurosurgery. 2019;**127**:e523-e533

[60] Sanghavi S, Skrupky R, Badie B, Robins I, Tome W, Mehta MP. Recurrent malignant gliomas treated with radiosurgery. Journal of Radiosurgery. 1999;**2**(3):119-125

[61] Park JL, Suh JH, Barnett GH, Reddy CA, Peereboom DM, Stevens GHJ, et al. Survival after stereotactic radiosurgery for recurrent glioblastoma multiforme. Journal of Radiosurgery. 2000;**3**(4):169-175

[62] Wen PY, Kesari S. Malignant gliomas in adults. The New England Journal of Medicine. 2008;**359**(5): 492-507

[63] Silber JR, Bobola MS, Blank A, Chamberlain MC. O6-methylguanine-DNA methyltransferase in glioma therapy: Promise and problems. Biochimica et Biophysica Acta. 2012; **1826**(1):71-82

[64] Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. The New England Journal of Medicine. 2005;**352**(10):997-1003

[65] Hsieh CT, Su IC, Huang CT, Chang CJ, Wang JS. The prognostic value of O6-methylguanine-DNA methyltransferase gene promoter methylation detected by gel-based methylation-specific polymerase chain reaction in patients with glioblastoma multiforme: A systematic review. International Journal of Clinical and Experimental Medicine. 2016;**9**(6): 10899-10906

[72] Stark AM, Doukas A, Hugo HH, Mehdorn HM. The expression of mismatch repair proteins MLH1, MSH2 and MSH6 correlates with the Ki67 proliferation index and survival in patients with recurrent glioblastoma. Neurological Research. 2010;**32**(8):

*DOI: http://dx.doi.org/10.5772/intechopen.91163*

*Stereotactic Radiosurgery for Recurrent Glioblastoma Multiforme*

[73] Martinez R, Setien F, Voelter C, Casado S, Quesada MP, Schackert G,

hypermethylation of the pro-apoptotic gene caspase-8 is a common hallmark of relapsed glioblastoma multiforme. Carcinogenesis. 2007;**28**(6):1264-1268

[74] Stark AM, Witzel P, Strege RJ, Hugo HH, Mehdorn HM. p53, mdm2, EGFR, and msh2 expression in paired initial and recurrent glioblastoma multiforme. Journal of Neurology, Neurosurgery, and Psychiatry. 2003;

[75] Campos B, Olsen LR, Urup T, Poulsen HS. A comprehensive profile of recurrent glioblastoma. Oncogene. 2016;

[76] Seystahl K, Gramatzki D, Roth P, Weller M. Pharmacotherapies for the treatment of glioblastoma—Current evidence and perspectives. Expert Opinion on Pharmacotherapy. 2016;

et al. CpG island promoter

816-820

**74**(6):779-783

**35**(45):5819-5825

**17**(9):1259-1270

**235**

[66] Kreisl TN, Kim L, Moore K, Duic P, Royce C, Stroud I, et al. Phase II trial of single-agent bevacizumab followed by bevacizumab plus irinotecan at tumor progression in recurrent glioblastoma. Journal of Clinical Oncology. 2009; **27**(5):740-745

[67] Friedman HS, Prados MD, Wen PY, Mikkelsen T, Schiff D, Abrey LE, et al. Bevacizumab alone and in combination with irinotecan in recurrent glioblastoma. Journal of Clinical Oncology. 2009;**27**(28):4733-4740

[68] Chinot OL, Wick W, Mason W, Henriksson R, Saran F, Nishikawa R, et al. Bevacizumab plus radiotherapytemozolomide for newly diagnosed glioblastoma. The New England Journal of Medicine. 2014;**370**(8):709-722

[69] Wick W, Gorlia T, Bendszus M, Taphoorn M, Sahm F, Harting I, et al. Lomustine and bevacizumab in progressive glioblastoma. The New England Journal of Medicine. 2017; **377**(20):1954-1963

[70] Diaz RJ, Ali S, Qadir MG, De La Fuente MI, Ivan ME, Komotar RJ. The role of bevacizumab in the treatment of glioblastoma. Journal of Neuro-Oncology. 2017;**133**(3):455-467

[71] Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, et al. The 2016 World Health Organization classification of tumors of the central nervous system: A summary. Acta Neuropathologica. 2016; **131**(6):803-820

*Stereotactic Radiosurgery for Recurrent Glioblastoma Multiforme DOI: http://dx.doi.org/10.5772/intechopen.91163*

[72] Stark AM, Doukas A, Hugo HH, Mehdorn HM. The expression of mismatch repair proteins MLH1, MSH2 and MSH6 correlates with the Ki67 proliferation index and survival in patients with recurrent glioblastoma. Neurological Research. 2010;**32**(8): 816-820

radiosurgery for local recurrence of glioblastoma. Neuro Endocrinology value of O6-methylguanine-DNA methyltransferase gene promoter methylation detected by gel-based methylation-specific polymerase chain reaction in patients with glioblastoma multiforme: A systematic review. International Journal of Clinical and Experimental Medicine. 2016;**9**(6):

[66] Kreisl TN, Kim L, Moore K, Duic P, Royce C, Stroud I, et al. Phase II trial of single-agent bevacizumab followed by bevacizumab plus irinotecan at tumor progression in recurrent glioblastoma. Journal of Clinical Oncology. 2009;

[67] Friedman HS, Prados MD, Wen PY, Mikkelsen T, Schiff D, Abrey LE, et al. Bevacizumab alone and in combination

[68] Chinot OL, Wick W, Mason W, Henriksson R, Saran F, Nishikawa R, et al. Bevacizumab plus radiotherapytemozolomide for newly diagnosed glioblastoma. The New England Journal of Medicine. 2014;**370**(8):709-722

[69] Wick W, Gorlia T, Bendszus M, Taphoorn M, Sahm F, Harting I, et al. Lomustine and bevacizumab in progressive glioblastoma. The New England Journal of Medicine. 2017;

[70] Diaz RJ, Ali S, Qadir MG, De La Fuente MI, Ivan ME, Komotar RJ. The role of bevacizumab in the treatment of

[71] Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, et al. The 2016 World Health Organization classification of tumors of the central nervous system: A summary. Acta Neuropathologica. 2016;

glioblastoma. Journal of Neuro-Oncology. 2017;**133**(3):455-467

**377**(20):1954-1963

**131**(6):803-820

with irinotecan in recurrent glioblastoma. Journal of Clinical Oncology. 2009;**27**(28):4733-4740

10899-10906

**27**(5):740-745

[58] Sharma M, Schroeder JL, Elson P, Meola A, Barnett GH, Vogelbaum MA,

stratification of patients with recurrent glioblastoma treated with salvage stereotactic radiosurgery. Journal of

[59] Morris SL, Zhu P, Rao M, Martir M, Zhu JJ, Hsu S, et al. Gamma knife stereotactic radiosurgery in combination

Letters. 2018;**39**(4):281-287

*Tumor Progression and Metastasis*

et al. Outcomes and prognostic

with bevacizumab for recurrent glioblastoma. World Neurosurgery.

with radiosurgery. Journal of Radiosurgery. 1999;**2**(3):119-125

[61] Park JL, Suh JH, Barnett GH, Reddy CA, Peereboom DM, Stevens GHJ, et al. Survival after stereotactic radiosurgery for recurrent glioblastoma multiforme. Journal of Radiosurgery. 2000;**3**(4):169-175

[62] Wen PY, Kesari S. Malignant gliomas in adults. The New England Journal of Medicine. 2008;**359**(5):

[63] Silber JR, Bobola MS, Blank A, Chamberlain MC. O6-methylguanine-DNA methyltransferase in glioma therapy: Promise and problems. Biochimica et Biophysica Acta. 2012;

[64] Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. The New England Journal of Medicine.

492-507

**1826**(1):71-82

**234**

2005;**352**(10):997-1003

[65] Hsieh CT, Su IC, Huang CT, Chang CJ, Wang JS. The prognostic

[60] Sanghavi S, Skrupky R, Badie B, Robins I, Tome W, Mehta MP. Recurrent malignant gliomas treated

Neurosurgery. 2018:1-11

2019;**127**:e523-e533

[73] Martinez R, Setien F, Voelter C, Casado S, Quesada MP, Schackert G, et al. CpG island promoter hypermethylation of the pro-apoptotic gene caspase-8 is a common hallmark of relapsed glioblastoma multiforme. Carcinogenesis. 2007;**28**(6):1264-1268

[74] Stark AM, Witzel P, Strege RJ, Hugo HH, Mehdorn HM. p53, mdm2, EGFR, and msh2 expression in paired initial and recurrent glioblastoma multiforme. Journal of Neurology, Neurosurgery, and Psychiatry. 2003; **74**(6):779-783

[75] Campos B, Olsen LR, Urup T, Poulsen HS. A comprehensive profile of recurrent glioblastoma. Oncogene. 2016; **35**(45):5819-5825

[76] Seystahl K, Gramatzki D, Roth P, Weller M. Pharmacotherapies for the treatment of glioblastoma—Current evidence and perspectives. Expert Opinion on Pharmacotherapy. 2016; **17**(9):1259-1270

**237**

**Chapter 10**

Anticancer Photodynamic

Photosensitizers

contemporary anticancer therapies.

**1. Introduction**

*and Mark Roufaiel*

**Abstract**

Therapy Using Ruthenium(II)

and Os(II)-Based Complexes as

*Pavel Kaspler, Arkady Mandel, Roger Dumoulin-White* 

Photodynamic therapy (PDT) is an approved procedure using a photosensitizer (PS) activated by light to selectively destroy malignant/premalignant cells. Transition metal complexes, such as Ru(II)- and Os(II)-based PSs (Theralase Technologies Inc., Ontario. Canada), are activated in a wide range of wavelengths, are resistant to photobleaching and have a high singlet oxygen quantum yield and ability to produce cytotoxic reactive oxygen species (ROS). Their design allows fine-tuning of the photophysical and photochemical properties. They demonstrate Type I and II photoreactions, and some are activated in hypoxia. High PDT potency and activation under NIR light and even X-ray may provide an advantage over the approved PSs. Their ability to associate with transferrin (Tf) as an endogenous delivery system increases photobleaching resistance, ROS production, selective cellular uptake, and PDT efficacy in combination with a decreased systemic toxicity. This makes these PSs attractive for systemic therapy of recurrent/progressive cancers. Their PDT efficacy has been demonstrated in various in vitro and in vivo clinically relevant models. The unique properties of the mentioned PSs allow bypassing such limitations of PDT as low specific uptake ratio, insufficiently broad absorption band, and low efficacy in hypoxia. One of these PSs (TLD-1433) was successful against non-muscle invasive urinary bladder cancer unresponsive to

**Keywords:** photodynamic therapy, photosensitizer, transition metal, Ru(II), Os(II),

Photodynamic therapy (PDT) is an actively developing anticancer modality that

offers advantages compared to conventional treatments (ionizing radiation and chemotherapy). PDT utilizes two components, light and a photosensitizing compound (PS) activated by light upon photon absorption and producing in its activated state highly cytotoxic reactive oxygen species (ROS) [1]. The attractiveness of PDT is in the use of safe nonthermal doses of light and nontoxic concentrations of the PS and evoking cytotoxic and immunologic effects upon activation of the PS by

complex, transferrin, selectivity, tumor, cancer, urinary bladder

#### **Chapter 10**

## Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes as Photosensitizers

*Pavel Kaspler, Arkady Mandel, Roger Dumoulin-White and Mark Roufaiel*

#### **Abstract**

Photodynamic therapy (PDT) is an approved procedure using a photosensitizer (PS) activated by light to selectively destroy malignant/premalignant cells. Transition metal complexes, such as Ru(II)- and Os(II)-based PSs (Theralase Technologies Inc., Ontario. Canada), are activated in a wide range of wavelengths, are resistant to photobleaching and have a high singlet oxygen quantum yield and ability to produce cytotoxic reactive oxygen species (ROS). Their design allows fine-tuning of the photophysical and photochemical properties. They demonstrate Type I and II photoreactions, and some are activated in hypoxia. High PDT potency and activation under NIR light and even X-ray may provide an advantage over the approved PSs. Their ability to associate with transferrin (Tf) as an endogenous delivery system increases photobleaching resistance, ROS production, selective cellular uptake, and PDT efficacy in combination with a decreased systemic toxicity. This makes these PSs attractive for systemic therapy of recurrent/progressive cancers. Their PDT efficacy has been demonstrated in various in vitro and in vivo clinically relevant models. The unique properties of the mentioned PSs allow bypassing such limitations of PDT as low specific uptake ratio, insufficiently broad absorption band, and low efficacy in hypoxia. One of these PSs (TLD-1433) was successful against non-muscle invasive urinary bladder cancer unresponsive to contemporary anticancer therapies.

**Keywords:** photodynamic therapy, photosensitizer, transition metal, Ru(II), Os(II), complex, transferrin, selectivity, tumor, cancer, urinary bladder

#### **1. Introduction**

Photodynamic therapy (PDT) is an actively developing anticancer modality that offers advantages compared to conventional treatments (ionizing radiation and chemotherapy). PDT utilizes two components, light and a photosensitizing compound (PS) activated by light upon photon absorption and producing in its activated state highly cytotoxic reactive oxygen species (ROS) [1]. The attractiveness of PDT is in the use of safe nonthermal doses of light and nontoxic concentrations of the PS and evoking cytotoxic and immunologic effects upon activation of the PS by

light. PDT is supposed to selectively destroy unwanted and/or malignant cells while largely sparing the surrounding healthy tissue. Another desirable property is the ability to induce antigen-specific therapeutic and/or protective immune responses.

Preferential PS uptake by the tumors would make them exclusive targets for cytotoxicity while sparing normal tissues. Light delivery (both source location and emitted energy) can also be controlled more carefully (within the confines of the effective light attenuation in the tissue), which could make PDT a very efficient and safe modality. PDT effects reply upon a variety of photoreactions. The most commonly considered are the two types dependent on oxygen and associated with ROS production: electron transfer from the excited PS generating hydroxyl radical OH among other species (Type I) and energy transfer to a ground-state molecular oxygen 3O2 generating singlet oxygen 1O2 and superoxide radical anions (Type II) [2]. It is proposed that two more types are possible and are oxygen-independent: Type III as the interaction of the activated PS with native free radicals and Type IV as light-induced structural changes in PS allowing it to bind to subcellular targets [3].

PDT has been approved almost 20 years ago as an anticancer treatment. Nevertheless, despite the potential advantages, it is still underutilized clinically. Only a small number of porphyrin- and chlorine-like photosensitizers, as well as one bacteriochlorophyll-based PS, are approved. The number of indications for each photosensitizer is also very limited and includes primarily superficial cancerous/precancerous lesions and other conditions such as actinic keratosis, basal cell carcinoma, high-grade dysplasia in Barrett's esophagus, and age-related macular degeneration. As a palliative measure, PDT is approved for obstructive esophageal or lung cancer and centrally located lung cancer [4, 5]. There are several reasons for this.

One of them is a small depth of penetration of visible light into the tissues restricting PDT to superficial lesions with a thickness not exceeding few millimeters. For deeper organs/tissues, an invasive direct delivery of light is required. Light absorbance of longer wavelengths by the PSs is therefore very advantageous. Light in the range of 650–1350 nm (known as "near-infrared window") [6] has the greatest penetrating ability into biological tissues. This includes parts of red (625– 740 nm) and near-infrared (>750 nm) range that can be used for the PS activation.

Another problem is an unsatisfactory selectivity for malignant tissues resulting in PDT-associated damage of normal tissues. For example, Photofrin® is known for this [4]. Prolonged retention of many porphyrin-based PSs in healthy tissues leads to a problem of sensitivity to sunlight and potentially serious damage to the patients' skin and eye [7, 8]. This could be mitigated by delivery systems selectively targeting malignant cells. These systems employ two modes of action [9]. Passive targeting relies upon the morphological and physiological peculiarities of tumor tissue in combination with physicochemical properties of the PS carrier. Active targeting, in contrast, is based on a molecular recognition of the PS carrier by cancer cells such as binding of specific ligands or antibodies to overexpressed cancer cell receptors. Passive PS delivery systems include nanoparticles, fullerenes, and liposomes and have the advantage of protecting the PS from degradation upon injection. Active systems, on the other hand, have the advantage of improved uptake of the PS. The carriers belonging by themselves to passive targeting systems can be nevertheless supplemented with molecular recognition capacity belonging to the features of active systems, such as decoration with Tf to target Tf receptors overexpressed in malignant cells [10–12]. Nevertheless, smaller active targeting systems (such as PS-Tf conjugates discussed further in this chapter) could have an advantage of greater mobility upon intracellular uptake and potentially the advantage of the blood-brain barrier crossing.

Lastly, PDT-induced ROS production strongly relies upon oxygen availability, which is well known for the porphyrin-based PSs [13, 14]. Deep bulky tumors have

**239**

Photofrin (0.89).

PSs in PDT [27].

**2.1 Molecular structure**

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes…*

extensive hypoxic regions, which are also associated with the tumor aggressiveness [15, 16]. Although hypoxic regions still can be treated (at a slower rate) by application of fractionated exposure or inducing reperfusion [17, 18], hypoxia severely decreases PDT efficacy [19]. Together with the limited light penetration, this is another reason why PDT in its current state is usually limited to relatively superficial lesions. This problem could be bypassed by PSs employing photoreactions that

Considering the said above, an advanced PS should have the ability for targeted delivery; penetration through the blood-brain barrier (BBB) and blood-tumor cell barrier (BTCB); activation by a wide range of wavelengths, including NIR light; and employing of different types of photoreactions enabling induction of immune responses to tumor antigens. Solubility in water and/or saline is a great asset for a successful PS as it makes its delivery both easier and safer, without the use of excipi-

Metal-based coordination complexes are among the obvious candidates to satisfy these requirements. Specifically, transition metal complexes possess a wide range of metal oxidation states and the complex geometries [5, 20]. These complexes (e.g., Ru(II) polypyridyl complexes) are of increasing interest as PSs in photodynamic therapy (PDT) and, more recently, for photochemotherapy (PCT) [21]. Importantly, they can have their properties fine-tuned by choosing the central metal and organic ligands (such as bipyridine and 2,2′-biquinoline). These PSs can employ a great variety of excited states associated with the central metal, ligands, or metal-ligand interactions. This is manifested in photoreactions that are ROS-dependent (Type I/II) or ROS-independent (electron transfer to substrates other than molecular oxygen), excitation at different wavelengths, solubility, systemic toxicity, and finally PDT efficacy. Historically, Pt(IV)-, Ru(II)-, and Rh(III)-based complexes were most actively studied as PSs followed by Ir(III) and Os(II) complexes; see the review by Monro et al. [5]. The examples of the most recent studies [22–26] include a summary on the use of ruthenium complexes as

This chapter reviews the results obtained by our group and collaborators. The properties and PDT efficacy of Theralase Technologies Inc. PSs [28] and Ru(II)- and Os(II)-based complexes are discussed in the perspective of their clinical application.

**2. Physical and chemical properties of the transitional metal-based PSs**

The molecular structure of Ru(II)- and Os(II)-based PSs (later referred to as Ru- and Os-based) is shown in **Tables 1**–**3**. These are relatively small (approximately 1 kDa) complexes with the ligands involving bipyridine (bip), 2,2′-biquinoline (biq), imidazo[4,5-f ][1,10]phenanthroline, and a variable number of thiophene units. A variety of the ligands defines some of the PS properties. For example, the biq ligand is responsible for relatively good absorbance in near-infrared (NIR) light, while the number of thiophene units may be associated with the PS solubility in water [5]. Water solubility, as it was mentioned, represents a serious advantage for this group of PSs as many of the established PSs have poor water solubility [29, 30]. Ru-based PSs are characterized by <sup>1</sup>

quantum yield that is much higher (up to 99%) than for the established (FDAapproved) porphyrin-based PSs: PPIX, an active metabolite of ALA (0.56) and

O2

*DOI: http://dx.doi.org/10.5772/intechopen.88519*

have little or no dependency on oxygen.

ents with potential toxicity/side effects on their own.

#### *Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes… DOI: http://dx.doi.org/10.5772/intechopen.88519*

extensive hypoxic regions, which are also associated with the tumor aggressiveness [15, 16]. Although hypoxic regions still can be treated (at a slower rate) by application of fractionated exposure or inducing reperfusion [17, 18], hypoxia severely decreases PDT efficacy [19]. Together with the limited light penetration, this is another reason why PDT in its current state is usually limited to relatively superficial lesions. This problem could be bypassed by PSs employing photoreactions that have little or no dependency on oxygen.

Considering the said above, an advanced PS should have the ability for targeted delivery; penetration through the blood-brain barrier (BBB) and blood-tumor cell barrier (BTCB); activation by a wide range of wavelengths, including NIR light; and employing of different types of photoreactions enabling induction of immune responses to tumor antigens. Solubility in water and/or saline is a great asset for a successful PS as it makes its delivery both easier and safer, without the use of excipients with potential toxicity/side effects on their own.

Metal-based coordination complexes are among the obvious candidates to satisfy these requirements. Specifically, transition metal complexes possess a wide range of metal oxidation states and the complex geometries [5, 20]. These complexes (e.g., Ru(II) polypyridyl complexes) are of increasing interest as PSs in photodynamic therapy (PDT) and, more recently, for photochemotherapy (PCT) [21]. Importantly, they can have their properties fine-tuned by choosing the central metal and organic ligands (such as bipyridine and 2,2′-biquinoline). These PSs can employ a great variety of excited states associated with the central metal, ligands, or metal-ligand interactions. This is manifested in photoreactions that are ROS-dependent (Type I/II) or ROS-independent (electron transfer to substrates other than molecular oxygen), excitation at different wavelengths, solubility, systemic toxicity, and finally PDT efficacy. Historically, Pt(IV)-, Ru(II)-, and Rh(III)-based complexes were most actively studied as PSs followed by Ir(III) and Os(II) complexes; see the review by Monro et al. [5]. The examples of the most recent studies [22–26] include a summary on the use of ruthenium complexes as PSs in PDT [27].

This chapter reviews the results obtained by our group and collaborators. The properties and PDT efficacy of Theralase Technologies Inc. PSs [28] and Ru(II)- and Os(II)-based complexes are discussed in the perspective of their clinical application.

#### **2. Physical and chemical properties of the transitional metal-based PSs**

#### **2.1 Molecular structure**

The molecular structure of Ru(II)- and Os(II)-based PSs (later referred to as Ru- and Os-based) is shown in **Tables 1**–**3**. These are relatively small (approximately 1 kDa) complexes with the ligands involving bipyridine (bip), 2,2′-biquinoline (biq), imidazo[4,5-f ][1,10]phenanthroline, and a variable number of thiophene units. A variety of the ligands defines some of the PS properties. For example, the biq ligand is responsible for relatively good absorbance in near-infrared (NIR) light, while the number of thiophene units may be associated with the PS solubility in water [5]. Water solubility, as it was mentioned, represents a serious advantage for this group of PSs as many of the established PSs have poor water solubility [29, 30]. Ru-based PSs are characterized by <sup>1</sup> O2 quantum yield that is much higher (up to 99%) than for the established (FDAapproved) porphyrin-based PSs: PPIX, an active metabolite of ALA (0.56) and Photofrin (0.89).

*Tumor Progression and Metastasis*

light. PDT is supposed to selectively destroy unwanted and/or malignant cells while largely sparing the surrounding healthy tissue. Another desirable property is the ability to induce antigen-specific therapeutic and/or protective immune responses. Preferential PS uptake by the tumors would make them exclusive targets for cytotoxicity while sparing normal tissues. Light delivery (both source location and emitted energy) can also be controlled more carefully (within the confines of the effective light attenuation in the tissue), which could make PDT a very efficient and safe modality. PDT effects reply upon a variety of photoreactions. The most commonly considered are the two types dependent on oxygen and associated with ROS production: electron transfer from the excited PS generating hydroxyl radical OH among other species (Type I) and energy transfer to a ground-state molecular oxygen 3O2 generating singlet oxygen 1O2 and superoxide radical anions (Type II) [2]. It is proposed that two more types are possible and are oxygen-independent: Type III as the interaction of the activated PS with native free radicals and Type IV as light-induced structural changes in PS allowing it to bind to subcellular targets [3]. PDT has been approved almost 20 years ago as an anticancer treatment. Nevertheless, despite the potential advantages, it is still underutilized clinically. Only a small number of porphyrin- and chlorine-like photosensitizers, as well as one bacteriochlorophyll-based PS, are approved. The number of indications for each photosensitizer is also very limited and includes primarily superficial cancerous/precancerous lesions and other conditions such as actinic keratosis, basal cell carcinoma, high-grade dysplasia in Barrett's esophagus, and age-related macular degeneration. As a palliative measure, PDT is approved for obstructive esophageal or lung cancer

and centrally located lung cancer [4, 5]. There are several reasons for this.

One of them is a small depth of penetration of visible light into the tissues restricting PDT to superficial lesions with a thickness not exceeding few millimeters. For deeper organs/tissues, an invasive direct delivery of light is required. Light absorbance of longer wavelengths by the PSs is therefore very advantageous. Light in the range of 650–1350 nm (known as "near-infrared window") [6] has the greatest penetrating ability into biological tissues. This includes parts of red (625– 740 nm) and near-infrared (>750 nm) range that can be used for the PS activation. Another problem is an unsatisfactory selectivity for malignant tissues resulting in PDT-associated damage of normal tissues. For example, Photofrin® is known for this [4]. Prolonged retention of many porphyrin-based PSs in healthy tissues leads to a problem of sensitivity to sunlight and potentially serious damage to the patients' skin and eye [7, 8]. This could be mitigated by delivery systems selectively targeting malignant cells. These systems employ two modes of action [9]. Passive targeting relies upon the morphological and physiological peculiarities of tumor tissue in combination with physicochemical properties of the PS carrier. Active targeting, in contrast, is based on a molecular recognition of the PS carrier by cancer cells such as binding of specific ligands or antibodies to overexpressed cancer cell receptors. Passive PS delivery systems include nanoparticles, fullerenes, and liposomes and have the advantage of protecting the PS from degradation upon injection. Active systems, on the other hand, have the advantage of improved uptake of the PS. The carriers belonging by themselves to passive targeting systems can be nevertheless supplemented with molecular recognition capacity belonging to the features of active systems, such as decoration with Tf to target Tf receptors overexpressed in malignant cells [10–12]. Nevertheless, smaller active targeting systems (such as PS-Tf conjugates discussed further in this chapter) could have an advantage of greater mobility upon intracellular uptake and potentially the advantage of the blood-brain barrier crossing. Lastly, PDT-induced ROS production strongly relies upon oxygen availability, which is well known for the porphyrin-based PSs [13, 14]. Deep bulky tumors have

**241**

TLD-OsH2B

TLD-OsH2IP

TLD-Os10H

TLD-Os14H

MW = 954.0

MW = 994.0

MW = 1158.2

MW = 1240.3

1

O2 quantum yield = 0.03

Fluorescent quantum yield =0.0009

1

O2 quantum yield = 0.035

Fluorescent quantum yield =0.0013

1

O2 quantum yield = 0.044

Fluorescent quantum yield =0.0008

1

O2 quantum yield = 0.035

Fluorescent quantum yield

= 0.0011

TLD-OsH2dppn

MW = 1106.1

1

O2 quantum yield n/d

Fluorescent quantum yield

n/d

**Table 2.**

*Molecular structures of Os(II)-based complexes. Quantum yields are measured in solution rather than in cells.*

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes…*

*DOI: http://dx.doi.org/10.5772/intechopen.88519*

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes… DOI: http://dx.doi.org/10.5772/intechopen.88519*

### 

*Molecular structures of Os(II)-based complexes. Quantum yields are measured in solution rather than in cells.*

*Tumor Progression and Metastasis*

**240**

TLD-1011

TLD-1411

TLD-1611

MW = 868.8

MW = 950.9

MW = 1033.1

1

n/d

Fluorescent quantum yield

n/d

TLD-1633

O2 quantum yield

1

O2 quantum yield

≈ 0.99

Fluorescent quantum yield

< 0.01

TLD-1433

MW = 1007.1

MW = 1089.2

1

n/d

Fluorescent quantum yield

n/d

O2 quantum yield

1

O2 quantum yield

≈ 0.99

Fluorescent quantum yield

< 0.01

**Table 1.**

*Ru(II)-based complexes. Quantum yields are measured in solution rather than in cells.*

1

O2 quantum yield

= 0.74

Fluorescent quantum yield

= 0.001

#### **Table 3.** *Ligands involved in the PSs' molecular structure.*

#### **2.2 Absorbance spectra**

The absorbance spectra of the Ru- and Os-based PSs are shown in **Figure 1**. Among the Ru-based PSs, methylation of bidentate ligands (bip) decreases absorbance. An increase in the number of thiophene rings redshifts the main absorbance peak and eventually results in a considerable increase in absorbance at longer wavelengths (see TLD-1633).

Os-based PSs having biq ligands, in contrast to the Ru-based PSs, have similar spectra. They demonstrate rather uniformly located strong main peak at approximately 340 nm attributed to ligand-centered transitions and a characteristic secondary peak at ≈550 nm attributed to metal-to-ligand charge transfer (MLCT) centered on the non-biq ligands. Importantly, these PSs demonstrate consistent

**243**

1

hυ−<sup>1</sup>

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes…*

absorbance at longer wavelengths (red to NIR range). The NIR absorbance is attributed to MLCT that involves biq ligands. Altogether, the spectra similarity suggests similar accessible electronic transitions and ground and excited states. Broad absorption band of the Os-based PSs allows for a wider range of photon attenuation coefficients. Considerable absorbance in a clinically important PDT window of 700–900 nm suggests a capacity for one-photon absorption [31]. This asset is emphasized by a good solubility of these PSs in water. Poor solubility in water may hamper PDT potential of PSs even with a good absorbance in this range of the spectrum, as in the case of porphyrin- and phthalocyanine-based PSs [32]. The inclusion of thiophenes and the increase in their number in the ligands (from TLD-OsH2B to TLD-Os14H) not only decreases the main absorbance peak but also markedly redshifts its shape, with a minimal effect on the secondary peak and

If the PS is resistant to photobleaching, this allows less PS being destroyed by the light exposure. In turn, this makes ROS production and subsequent cytotoxic action more efficient, because the process of conversion of photons to cytotoxicity becomes catalytic without stoichiometric consumption of the PS. This allows making the efficacy of PDT treatment independent on the availability of the PS during the treatment. The bleaching resistance is hence a very valuable property, especially if the delivered light energy must be increased to achieve the desired PDT efficacy. This could be a drawback though in the case of bleaching-based dosimetry during the

Ru-based PSs show notable bleaching under exposure to green light (525 nm). TLD-1433 is slightly more bleaching resistant than TLD-1411 although they have almost identical absorbance at 525 nm. Nevertheless, more than 50% of each PS

the 416–417 nm UV peak, TLD-1433 absorbance in clinically useful range rapidly increases (1.7-fold at 525 nm, 2.0-fold at 625 nm, 1.8-fold at 800 nm) and remains

for TLD-OsH2IP, and 1.5\*10<sup>−</sup><sup>2</sup>

[31]. For comparison, the photobleaching rate for the approved PSs can be much

Production of ROS represents a final event of the PS activation by light leading to PDT cytotoxicity. Ru-based TLD-1433 is able to generate hydroxyl

O2 production is not detected. Importantly, ROS is generated despite very low

Os-based PSs having biq ligands show variable bleaching resistance under green light (525 nm); TLD-OsH2B is the most resistant and TLD-OsH2dppn the most vulnerable. Compared to the Ru-based PS, the bleaching resistance of the Os-based PSs with biq ligands is greater in general, with at least 75% of their initial absorbance retained. The best performers, TLD-OsH2B and TLD-OsH2IP, showed no more than 10% loss of absorbance in the UV peak, with no absorbance loss in green-NIR range. This provides the photobleaching rates in aqueous solution (calculated based

of radiant exposure corresponding to 6.6x1019-

. Moreover, while bleaching results in the deterioration of

).

cm<sup>−</sup><sup>2</sup>

cm<sup>−</sup><sup>2</sup>

6 M hυ−<sup>1</sup>

for TLD-OsH2dppn,

for PPIX, and 4.8\*10<sup>−</sup>16 M

), although singlet oxygen

for TLD-OsH2B

for benzoporphyrin

cm<sup>−</sup><sup>2</sup>

cm<sup>−</sup><sup>2</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.88519*

absorbance in the longer wavelengths (**Figure 1B**).

**2.3 Photobleaching resistance**

remains intact even after 200 Jcm<sup>−</sup><sup>2</sup>

at this level up to the end of light exposure (200 Jcm<sup>−</sup><sup>2</sup>

on the incident irradiance) equal to 8.7\*10<sup>−</sup>28 M hυ−<sup>1</sup>

derivative mono acid A (BPD), 7.3\*10<sup>−</sup>23 M hυ−<sup>1</sup>

radical \*OH under red light (625 nm, 119 mWcm<sup>−</sup><sup>2</sup>

for curcumin [35–37].

higher (by several orders of magnitude): 5.6\*10<sup>−</sup>24 M hυ−<sup>1</sup>

cm<sup>−</sup><sup>2</sup>

absorbed photons per cm3

treatment [33, 34].

4.1\*10<sup>−</sup>27 M hυ−<sup>1</sup>

cm<sup>−</sup><sup>2</sup>

**2.4 ROS production**

**Figure 1.** *Absorbance spectra of the Ru(II)- and the Os(II)-based PSs (panels A and B, respectively) in water.* *Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes… DOI: http://dx.doi.org/10.5772/intechopen.88519*

absorbance at longer wavelengths (red to NIR range). The NIR absorbance is attributed to MLCT that involves biq ligands. Altogether, the spectra similarity suggests similar accessible electronic transitions and ground and excited states. Broad absorption band of the Os-based PSs allows for a wider range of photon attenuation coefficients. Considerable absorbance in a clinically important PDT window of 700–900 nm suggests a capacity for one-photon absorption [31]. This asset is emphasized by a good solubility of these PSs in water. Poor solubility in water may hamper PDT potential of PSs even with a good absorbance in this range of the spectrum, as in the case of porphyrin- and phthalocyanine-based PSs [32]. The inclusion of thiophenes and the increase in their number in the ligands (from TLD-OsH2B to TLD-Os14H) not only decreases the main absorbance peak but also markedly redshifts its shape, with a minimal effect on the secondary peak and absorbance in the longer wavelengths (**Figure 1B**).

#### **2.3 Photobleaching resistance**

*Tumor Progression and Metastasis*

Bipyridine

Thiophene

2,2′-biquinoline

Methylated bipyridine

Imidazo[4,5-f][1,10]phenanthroline

benzo[*i*]dipyrido[3,2-*a*:2′,3′-c]phenazine

*Ligands involved in the PSs' molecular structure.*

**2.2 Absorbance spectra**

TLD-1633).

**Table 3.**

The absorbance spectra of the Ru- and Os-based PSs are shown in **Figure 1**. Among the Ru-based PSs, methylation of bidentate ligands (bip) decreases absorbance. An increase in the number of thiophene rings redshifts the main absorbance peak and eventually results in a considerable increase in absorbance at longer wavelengths (see

Os-based PSs having biq ligands, in contrast to the Ru-based PSs, have similar spectra. They demonstrate rather uniformly located strong main peak at approximately 340 nm attributed to ligand-centered transitions and a characteristic secondary peak at ≈550 nm attributed to metal-to-ligand charge transfer (MLCT) centered on the non-biq ligands. Importantly, these PSs demonstrate consistent

*Absorbance spectra of the Ru(II)- and the Os(II)-based PSs (panels A and B, respectively) in water.*

**242**

**Figure 1.**

If the PS is resistant to photobleaching, this allows less PS being destroyed by the light exposure. In turn, this makes ROS production and subsequent cytotoxic action more efficient, because the process of conversion of photons to cytotoxicity becomes catalytic without stoichiometric consumption of the PS. This allows making the efficacy of PDT treatment independent on the availability of the PS during the treatment.

The bleaching resistance is hence a very valuable property, especially if the delivered light energy must be increased to achieve the desired PDT efficacy. This could be a drawback though in the case of bleaching-based dosimetry during the treatment [33, 34].

Ru-based PSs show notable bleaching under exposure to green light (525 nm). TLD-1433 is slightly more bleaching resistant than TLD-1411 although they have almost identical absorbance at 525 nm. Nevertheless, more than 50% of each PS remains intact even after 200 Jcm<sup>−</sup><sup>2</sup> of radiant exposure corresponding to 6.6x1019 absorbed photons per cm3 . Moreover, while bleaching results in the deterioration of the 416–417 nm UV peak, TLD-1433 absorbance in clinically useful range rapidly increases (1.7-fold at 525 nm, 2.0-fold at 625 nm, 1.8-fold at 800 nm) and remains at this level up to the end of light exposure (200 Jcm<sup>−</sup><sup>2</sup> ).

Os-based PSs having biq ligands show variable bleaching resistance under green light (525 nm); TLD-OsH2B is the most resistant and TLD-OsH2dppn the most vulnerable. Compared to the Ru-based PS, the bleaching resistance of the Os-based PSs with biq ligands is greater in general, with at least 75% of their initial absorbance retained. The best performers, TLD-OsH2B and TLD-OsH2IP, showed no more than 10% loss of absorbance in the UV peak, with no absorbance loss in green-NIR range. This provides the photobleaching rates in aqueous solution (calculated based on the incident irradiance) equal to 8.7\*10<sup>−</sup>28 M hυ−<sup>1</sup> cm<sup>−</sup><sup>2</sup> for TLD-OsH2dppn, 4.1\*10<sup>−</sup>27 M hυ−<sup>1</sup> cm<sup>−</sup><sup>2</sup> for TLD-OsH2IP, and 1.5\*10<sup>−</sup><sup>2</sup> 6 M hυ−<sup>1</sup> cm<sup>−</sup><sup>2</sup> for TLD-OsH2B [31]. For comparison, the photobleaching rate for the approved PSs can be much higher (by several orders of magnitude): 5.6\*10<sup>−</sup>24 M hυ−<sup>1</sup> cm<sup>−</sup><sup>2</sup> for benzoporphyrin derivative mono acid A (BPD), 7.3\*10<sup>−</sup>23 M hυ−<sup>1</sup> cm<sup>−</sup><sup>2</sup> for PPIX, and 4.8\*10<sup>−</sup>16 M hυ−<sup>1</sup> cm<sup>−</sup><sup>2</sup> for curcumin [35–37].

#### **2.4 ROS production**

Production of ROS represents a final event of the PS activation by light leading to PDT cytotoxicity. Ru-based TLD-1433 is able to generate hydroxyl radical \*OH under red light (625 nm, 119 mWcm<sup>−</sup><sup>2</sup> ), although singlet oxygen 1 O2 production is not detected. Importantly, ROS is generated despite very low absorbance of TLD-1433 in red light. This, however, requires certain molecular and ionic environment because ROS is generated only in incomplete DMEM cell culture medium (not complemented with FBS and antibiotics) but not in DI water despite almost identically low absorbance.

Exposure to NIR light (808 nm, 720 mWcm<sup>−</sup><sup>2</sup> ) produces some amount of ROS (\*OH), but it at least an order of magnitude less than under red light. This occurs despite a similar number of absorbed photons and absorbance at 808 nm only 18% less than at 625 nm and greater delivered energy. This may suggest that not only the total delivered energy and number of the absorbed photons but also the photon energy is important for the efficacy in ROS production.

#### **3. Association of the PSs with transferrin**

#### **3.1 Delivery platforms**

To address the challenge of selective uptake of the PSs by tumors, it would be attractive to utilize serum proteins and natural transmembrane transporters as delivery vehicles. Despite numerous approaches for targeted delivery of the PSs including receptors-assisted uptake (as mentioned in the chapter introduction), neither is related to the use of Tf as a vehicle for transition metal-based complexes. The notable exceptions are the works on the interaction between Tf and Cr(III) complexes [38, 39]. It is also known that Ru(II) complexes can associate with albumin and iron transporter transferrin (Tf) [40, 41]. In addition, overexpression of Tf receptors is a common feature of malignant cells that tend to have an increased Fe3+ uptake [42]. The effect of the association of Ru(II)-based PSs with Tf on their photophysical and photobiological properties needs however more elucidation.

#### **3.2 Association signatures and effect of Tf on absorbance spectra**

Upon subtraction of the spectra of the complex and Tf from the spectrum of their premix, a characteristic signature of association between the Ru-based complex and Tf can be detected, with two peaks in UV and visible range. The UV peak indicates conformational changes in aromatic rings (the complex itself or transferrin molecule), and the visible range peak is interpreted as an indicator of LMCT (ligand to metal charge transfer) that represents the interaction between the metal of the complex and transferrin [43].

Premixing of Ru-based complexes with apo-Tf (the Tf not saturated with Fe3+) at 4:1 molar ratio demonstrates the signature with UV and visible range peaks (**Figure 2A**). The absorbance increase in UV range could be due to conformation changes either in the Tf molecule or the complex (as both have UV maxima at similar wavelengths). The peak in visible range indicates a new spectral component distinct (redshifted) from the comparable absorbance peak for the PS alone. This indicates the complex-Tf association and is related to the interaction between the metal in the complex and the Tf molecule.

There is also an increase in absorbance between the signature peaks and, importantly, in the long wavelength tail of the spectrum in the visible range and further into the NIR, which is clinically relevant for PDT. Notably, the absorbance of TLD-1433 alone is very low in red to NIR. The increase in absorbance upon the association with Tf is 16.2-fold (MEC = 3125 vs. 193 M<sup>−</sup><sup>1</sup> cm<sup>−</sup><sup>1</sup> ) in red (635 nm) and 5.7-fold (MEC = 1676 vs. 294 M<sup>−</sup><sup>1</sup> cm<sup>−</sup><sup>1</sup> ) in NIR (800 nm), compared to 5.0-fold (MEC = 8027 vs. 1600 M<sup>−</sup><sup>1</sup> cm<sup>−</sup><sup>1</sup> ) increase in the green (535 nm). Notably, the ability of the PS to associate with Tf depends on the number of thiophene rings in the complex. One

**245**

lesser than for apo-Tf [43].

tude is much lesser.

**Figure 2.**

*media (panel C).*

different pH values.

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes…*

thiophene ring is not enough for this as evident for TLD-1011. Hence, not only metal

*Spectral signatures of association of the Ru(II)-based (panel A) and Os(II)-based (panels B and C) PSs with apo-Tf. The incubation of Ru-based PSs was performed in 10 mM phosphate buffer +100 mM NaCl (pH = 7.4) and of Os(II)-based PSs in the phosphate buffer (panel B) or incomplete RPMI1640 cell culture* 

The association signatures seem to be insensitive to the source of apo-Tf and are very similar for bovine and human Tf. The signatures resemble the signature of Fe3+−Tf binding but are not identical to it. Notably, TLD-1433 can also be associated with Fe3+ saturated holo-Tf although the magnitude of the association signature is

Os-based PSs with biq ligands are also able to associate with apo-Tf, but their signatures (**Figure 2C**) are distinct from those of Ru-based PSs. The visible range peak (observed for TLD-OsH2B and TLD-OsH2dppn but not for TLD-OsH2IP) is however more redshifted (in the range of 500–600 nm), and the signature magni-

Physiologically, when Tf bound with Fe3+ is taken up into a cell, it releases iron in endosomes when pH is decreased to ≈5.5 [44]. TLD-1433-Tf conjugate, in contrast, remains stable during the gradual acidification emulating this process [43]. This is evident by the stability of absorbance at the two peaks of the signature across

Notably, an association of TLD-1433 with holo-Tf also survives the acid environment. The magnitude of the signature peaks is 31–33% lower than for TLD-1433 & apo-Tf at pH = 7.4, but by pH = 5 it increases so the UV peak magnitude catches up with that of TLD-1433 & apo-Tf, and the visible peak magnitude even becomes about 20% greater. Hence, TLD-1433 may remain associated with Tf in the acidic endosome environment. Acidification resistance does not hold however for the

but also organic ligands play a role in the association of the complex with Tf.

**3.3 Stability of the TLD-1433 + Tf association at low pH**

*DOI: http://dx.doi.org/10.5772/intechopen.88519*

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes… DOI: http://dx.doi.org/10.5772/intechopen.88519*

**Figure 2.**

*Tumor Progression and Metastasis*

**3.1 Delivery platforms**

water despite almost identically low absorbance. Exposure to NIR light (808 nm, 720 mWcm<sup>−</sup><sup>2</sup>

energy is important for the efficacy in ROS production.

**3. Association of the PSs with transferrin**

metal of the complex and transferrin [43].

metal in the complex and the Tf molecule.

with Tf is 16.2-fold (MEC = 3125 vs. 193 M<sup>−</sup><sup>1</sup>

cm<sup>−</sup><sup>1</sup>

(MEC = 1676 vs. 294 M<sup>−</sup><sup>1</sup>

cm<sup>−</sup><sup>1</sup>

absorbance of TLD-1433 in red light. This, however, requires certain molecular and ionic environment because ROS is generated only in incomplete DMEM cell culture medium (not complemented with FBS and antibiotics) but not in DI

(\*OH), but it at least an order of magnitude less than under red light. This occurs despite a similar number of absorbed photons and absorbance at 808 nm only 18% less than at 625 nm and greater delivered energy. This may suggest that not only the total delivered energy and number of the absorbed photons but also the photon

To address the challenge of selective uptake of the PSs by tumors, it would be attractive to utilize serum proteins and natural transmembrane transporters as delivery vehicles. Despite numerous approaches for targeted delivery of the PSs including receptors-assisted uptake (as mentioned in the chapter introduction), neither is related to the use of Tf as a vehicle for transition metal-based complexes. The notable exceptions are the works on the interaction between Tf and Cr(III) complexes [38, 39]. It is also known that Ru(II) complexes can associate with albumin and iron transporter transferrin (Tf) [40, 41]. In addition, overexpression of Tf receptors is a common feature of malignant cells that tend to have an increased Fe3+ uptake [42]. The effect of the association of Ru(II)-based PSs with Tf on their photophysical and photobiological properties needs however more elucidation.

**3.2 Association signatures and effect of Tf on absorbance spectra**

Upon subtraction of the spectra of the complex and Tf from the spectrum of their premix, a characteristic signature of association between the Ru-based complex and Tf can be detected, with two peaks in UV and visible range. The UV peak indicates conformational changes in aromatic rings (the complex itself or transferrin molecule), and the visible range peak is interpreted as an indicator of LMCT (ligand to metal charge transfer) that represents the interaction between the

Premixing of Ru-based complexes with apo-Tf (the Tf not saturated with Fe3+)

There is also an increase in absorbance between the signature peaks and, importantly, in the long wavelength tail of the spectrum in the visible range and further into the NIR, which is clinically relevant for PDT. Notably, the absorbance of TLD-1433 alone is very low in red to NIR. The increase in absorbance upon the association

associate with Tf depends on the number of thiophene rings in the complex. One

cm<sup>−</sup><sup>1</sup>

) increase in the green (535 nm). Notably, the ability of the PS to

) in red (635 nm) and 5.7-fold

) in NIR (800 nm), compared to 5.0-fold (MEC = 8027

at 4:1 molar ratio demonstrates the signature with UV and visible range peaks (**Figure 2A**). The absorbance increase in UV range could be due to conformation changes either in the Tf molecule or the complex (as both have UV maxima at similar wavelengths). The peak in visible range indicates a new spectral component distinct (redshifted) from the comparable absorbance peak for the PS alone. This indicates the complex-Tf association and is related to the interaction between the

) produces some amount of ROS

**244**

vs. 1600 M<sup>−</sup><sup>1</sup>

*Spectral signatures of association of the Ru(II)-based (panel A) and Os(II)-based (panels B and C) PSs with apo-Tf. The incubation of Ru-based PSs was performed in 10 mM phosphate buffer +100 mM NaCl (pH = 7.4) and of Os(II)-based PSs in the phosphate buffer (panel B) or incomplete RPMI1640 cell culture media (panel C).*

thiophene ring is not enough for this as evident for TLD-1011. Hence, not only metal but also organic ligands play a role in the association of the complex with Tf.

The association signatures seem to be insensitive to the source of apo-Tf and are very similar for bovine and human Tf. The signatures resemble the signature of Fe3+−Tf binding but are not identical to it. Notably, TLD-1433 can also be associated with Fe3+ saturated holo-Tf although the magnitude of the association signature is lesser than for apo-Tf [43].

Os-based PSs with biq ligands are also able to associate with apo-Tf, but their signatures (**Figure 2C**) are distinct from those of Ru-based PSs. The visible range peak (observed for TLD-OsH2B and TLD-OsH2dppn but not for TLD-OsH2IP) is however more redshifted (in the range of 500–600 nm), and the signature magnitude is much lesser.

#### **3.3 Stability of the TLD-1433 + Tf association at low pH**

Physiologically, when Tf bound with Fe3+ is taken up into a cell, it releases iron in endosomes when pH is decreased to ≈5.5 [44]. TLD-1433-Tf conjugate, in contrast, remains stable during the gradual acidification emulating this process [43]. This is evident by the stability of absorbance at the two peaks of the signature across different pH values.

Notably, an association of TLD-1433 with holo-Tf also survives the acid environment. The magnitude of the signature peaks is 31–33% lower than for TLD-1433 & apo-Tf at pH = 7.4, but by pH = 5 it increases so the UV peak magnitude catches up with that of TLD-1433 & apo-Tf, and the visible peak magnitude even becomes about 20% greater. Hence, TLD-1433 may remain associated with Tf in the acidic endosome environment. Acidification resistance does not hold however for the

increased absorbance in green to NIR range. The increase in red-NIR range due to the association of TLD-1433 with Tf deteriorates completely at low pH, and only in the green range, it shows some resistance: 16% remaining for TLD-1433 & apo-Tf and 66% for TLD-1433 & holo-Tf.

#### **3.4 Effect of Tf on photobleaching**

Association with Tf markedly decreases the extent and rate of photobleaching of TLD-1433 under green light (525 nm, 130 mWcm<sup>−</sup><sup>2</sup> ). At 0.93\*1020 absorbed photons per cm3 , more than 59% of TLD-1433 remains intact in 1:1 TLD-1433 & Tf premix. At the comparable absorbed light (0.23\*1020 absorbed photons per cm3 ), 74% of TLD-1433 in the premix persists compared to 45% of TLD-1433 alone [43]. As mentioned above, a decreased bleaching allows for more efficient ROS production with less PS expended, so the advantage of TLD-1433 & Tf conjugate is evident.

#### **3.5 Effect of Tf on ROS production**

Association of TLD-1433 with Tf dramatically increases ROS production upon irradiation with red light (625 nm, 119 mWcm<sup>−</sup><sup>2</sup> ). In this case, <sup>1</sup> O2 is generated, which does not happen with TLD-1433 alone. The production of \*OH is increased twofold at 1.9\*1022 absorbed photons per cm3 [43]. The association with Tf is therefore advantageous for ROS production considering that \*OH is not only an extremely cytotoxic ROS but can also be produced from 1 O2 [45]. The association with Tf is, however, unable to improve ROS production by TLD-1433 under NIR (808 nm, 720 mWcm<sup>−</sup><sup>2</sup> ) despite the increase in absorbance in this range.

#### **4. In vitro PDT**

Transition metal-based PSs hence are able to absorb light at clinically relevant wavelengths and produce cytotoxic ROS, and the association with Tf is beneficial in that. This warrants assessment of this capacity in biological systems. In vitro, the PDT effects are tested using clinically relevant human cancer cell lines (human glioblastoma U87 cells, human bladder cancer HT1376 cells) or nonhuman cells relevant for preclinical models (rat bladder cancer AY27 cells).

#### **4.1 Ru-based PSs**

The comparative efficacy of the Ru-based PSs on U87 cells is shown in **Table 4**. PDT efficacy of the Ru-based PSs can be very efficient in green light (LD50 in sub-nanomolar range) and moderately efficient in red light (LD50 in micromolar range in red light), but they are not active in NIR light. Notably, the efficacy of the PSs in red light is observed despite negligible absorbance (measured in water). In complete cell culture medium (and potentially intracellularly), absorbance in red is increased due to associaiton of the PSs with proteins but is still low compared to that at the shorter wavelengths.

Depending on the PS, the maximal PDT effect did not reach 100% cell kill. The data at 45 Jcm<sup>−</sup><sup>2</sup> PDT are not shown, but the increase in the light radiant exposure from 45 to 90 Jcm<sup>−</sup><sup>2</sup> significantly (P < 0.05) decreased LD50 for the PDT effect in green light. In red light, the PDT efficacy also could be increased with the increase in the radiant exposure from 45 to 90 and then to 180 Jcm<sup>−</sup><sup>2</sup> . This can be explained by the insufficient number of incident photons per a given concentration of the PS at lower radiant exposure but not by a difference in quantum efficacy of the PDT that

**247**

**Table 4.**

HT1376 cells, LD50 in red light (90 Jcm<sup>−</sup><sup>2</sup>

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes…*

**U87 cells Dark Green Red NIR**

LD50 (μM):0.00595 (CI95 = 0.0050–0.0074)

Maximal kill (%): 49.41 (CI95 = 46.9–52.0)

N = 28 N = 9 N = 5 MEC (water) 2520 151 86

> LD50 (μM): 0.00702 (CI95 = 0.00261– 0.01891)

Maximal kill (%): 65.9 (CI95 = 59.1–72.8)

N = 118 N = 32 N = 32 MEC (water) 3094 158 294

> LD50 (μM): 0.002 (CI95 = 0.00117–0.0040)

Maximal kill (%):74.8 (CI95 = 65.7–83.9)

LD50 (μM): 0.000574 (CI95 = 2.403e-006– 0.005691)

Maximal kill (%): 100.8 (52.2–171.0)

> LD50 (μM): 0.20 (CI95 = 0.16–0.25)

Maximal kill (%): 79.7 (CI95 = 72.5–87.0)

N = 45 N = 18 N = 26 MEC (water) 6947 3046 209 *The cells were incubated with the PS for 4 h, and the PS was removed before PDT. The dose–response provides LD50 (μM) and maximal cell kill (%) for a green and red light and a cell kill for a fixed PS concentration for NIR light.* 

MEC 24,263 4635 1167

MEC (water) 7468 741 0

N = 31 N = 9

N = 14 N = 6

*The data are presented as means and their 95% confidence intervals (SEM for NIR PDT).*

**530 nm 625 nm 808 nm 90 Jcm<sup>−</sup><sup>2</sup> 90Jcm<sup>−</sup><sup>2</sup> 400-600 Jcm<sup>−</sup><sup>2</sup>**

**108 mWcm<sup>−</sup><sup>2</sup> 125 mWcm<sup>−</sup><sup>2</sup> 150 mWcm<sup>−</sup><sup>2</sup>**

LD50 (μM): 0.909 (CI95 = n/d-12.36)

Maximal kill (%): 71.17 (CI95 = 33.7–124.1)

LD50 (μM):3.57 (CI95 = 2.99–4.40)

Maximal kill (%): 76.2(CI95 = 66.7– 85.8)

Inconsistent and low cell kill

Inconsistent and low cell kill

LD50 (μM): 0.23 (CI95 = 0.17–0.31)

Maximal kill (%): 91.8 (CI95 = 83.2–100.4) Insufficient cell kill

Inconsistent and low cell kill

No cell kill

No cell kill

No cell kill

depends only on the photon energy but not on the radiant exposure. The increase in the number of thiophenes in the PS complexes decreases LD50 for the green light and hence increases PDT efficacy. The dark toxicity is however also increasing. In

*In vitro PDT efficacy of Ru(II)-based PSs on U87 cells, in comparison to the FDA-approved Photofrin®.*

) is 15.0 μM (CI95 = 9.1–24.9 μM, N = 30).

*DOI: http://dx.doi.org/10.5772/intechopen.88519*

TLD-1411 LD50 (μM): 101.5

TLD-1433 LD50 (μM): 192.9

TLD-1611 LD50 (μM): 62.9

TLD-1633 LD50 (μM): 31.13

Photofrin® LD50 (μM): 2974

(CI95 = 87.8–117.4)

(CI95 = 146.8–253.3)

(CI95 = 44.9–92.5)

(CI95 = 14.85 to 63.68)

(CI95 = 245.5– 36,027)


*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes… DOI: http://dx.doi.org/10.5772/intechopen.88519*

*The cells were incubated with the PS for 4 h, and the PS was removed before PDT. The dose–response provides LD50 (μM) and maximal cell kill (%) for a green and red light and a cell kill for a fixed PS concentration for NIR light. The data are presented as means and their 95% confidence intervals (SEM for NIR PDT).*

#### **Table 4.**

*Tumor Progression and Metastasis*

and 66% for TLD-1433 & holo-Tf.

**3.4 Effect of Tf on photobleaching**

**3.5 Effect of Tf on ROS production**

(808 nm, 720 mWcm<sup>−</sup><sup>2</sup>

**4. In vitro PDT**

**4.1 Ru-based PSs**

data at 45 Jcm<sup>−</sup><sup>2</sup>

from 45 to 90 Jcm<sup>−</sup><sup>2</sup>

that at the shorter wavelengths.

per cm3

TLD-1433 under green light (525 nm, 130 mWcm<sup>−</sup><sup>2</sup>

irradiation with red light (625 nm, 119 mWcm<sup>−</sup><sup>2</sup>

extremely cytotoxic ROS but can also be produced from 1

relevant for preclinical models (rat bladder cancer AY27 cells).

the radiant exposure from 45 to 90 and then to 180 Jcm<sup>−</sup><sup>2</sup>

twofold at 1.9\*1022 absorbed photons per cm3

increased absorbance in green to NIR range. The increase in red-NIR range due to the association of TLD-1433 with Tf deteriorates completely at low pH, and only in the green range, it shows some resistance: 16% remaining for TLD-1433 & apo-Tf

Association with Tf markedly decreases the extent and rate of photobleaching of

At the comparable absorbed light (0.23\*1020 absorbed photons per cm3

TLD-1433 in the premix persists compared to 45% of TLD-1433 alone [43]. As mentioned above, a decreased bleaching allows for more efficient ROS production with less PS expended, so the advantage of TLD-1433 & Tf conjugate is evident.

Association of TLD-1433 with Tf dramatically increases ROS production upon

which does not happen with TLD-1433 alone. The production of \*OH is increased

therefore advantageous for ROS production considering that \*OH is not only an

with Tf is, however, unable to improve ROS production by TLD-1433 under NIR

Transition metal-based PSs hence are able to absorb light at clinically relevant wavelengths and produce cytotoxic ROS, and the association with Tf is beneficial in that. This warrants assessment of this capacity in biological systems. In vitro, the PDT effects are tested using clinically relevant human cancer cell lines (human glioblastoma U87 cells, human bladder cancer HT1376 cells) or nonhuman cells

The comparative efficacy of the Ru-based PSs on U87 cells is shown in **Table 4**.

Depending on the PS, the maximal PDT effect did not reach 100% cell kill. The

green light. In red light, the PDT efficacy also could be increased with the increase in

the insufficient number of incident photons per a given concentration of the PS at lower radiant exposure but not by a difference in quantum efficacy of the PDT that

PDT are not shown, but the increase in the light radiant exposure

significantly (P < 0.05) decreased LD50 for the PDT effect in

PDT efficacy of the Ru-based PSs can be very efficient in green light (LD50 in sub-nanomolar range) and moderately efficient in red light (LD50 in micromolar range in red light), but they are not active in NIR light. Notably, the efficacy of the PSs in red light is observed despite negligible absorbance (measured in water). In complete cell culture medium (and potentially intracellularly), absorbance in red is increased due to associaiton of the PSs with proteins but is still low compared to

) despite the increase in absorbance in this range.

). In this case, <sup>1</sup>

[43]. The association with Tf is

, more than 59% of TLD-1433 remains intact in 1:1 TLD-1433 & Tf premix.

). At 0.93\*1020 absorbed photons

), 74% of

O2 is generated,

O2 [45]. The association

. This can be explained by

**246**

*In vitro PDT efficacy of Ru(II)-based PSs on U87 cells, in comparison to the FDA-approved Photofrin®.*

depends only on the photon energy but not on the radiant exposure. The increase in the number of thiophenes in the PS complexes decreases LD50 for the green light and hence increases PDT efficacy. The dark toxicity is however also increasing. In HT1376 cells, LD50 in red light (90 Jcm<sup>−</sup><sup>2</sup> ) is 15.0 μM (CI95 = 9.1–24.9 μM, N = 30).

This is a greater value than for U87 cells and suggests lesser PDT sensitivity. The total PDT cell kill is however high, 98.5% (CI95 = 85.6–111.4%). Dark toxicity is, in contrast, low, with LD50 exceeding 200 μM. Importantly, the efficacy of the Ru-based PSs exceeds the efficacy of FDA-approved Photofrin® in green light, although not in red light (**Table 4**). Judging by LD50, the Ru-based PSs have higher dark toxicity than Photofrin®, but this is of less importance because, in addition to their solubility in water, they are effective at much lesser, nontoxic concentrations.

Pure PDT effect elucidates the PS efficacy for PDT neglecting its dark toxicity, which is justifiable scientifically to reveal mechanisms of the PS action. Clinically, however, in the case of selective uptake of the PS into cancer cells vs. normal cells, cancer cell kill can be achieved both by PDT-mediated and cytotoxic mechanisms, and the total PDT-induced cell kill becomes relevant. Considering this, total cell kill close to 100% can be achieved in green light in sub-micromolar (20 nM for TLD-1633, 30 nM for TLD-1433, and 200 nM for TLD-1411) or even sub-nanomolar range (0.5 nM for TLD-1611). For comparison, Photofrin® achieved 100% total cell kill in U87 cells only at concentrations above 300 nM.

Clinically, the balance between the efficacy and safety of the PS is characterized by the therapeutic ratio that indicates how far a dose for a desired therapeutic effect is from the dose that causes undesired toxicity. Dividing PDT effect LD50 to dark toxicity LD50 provides small numbers that are not convenient to operate with. It is easier therefore to use inverted therapeutic ratio, ITR = Dark LD50/PDT effect LD50. In U87 cells, ITR = 17,061 for TLD-1411, 27,486 for TLD-1433, 31,460 for TLD-1611, and 54,252 for TLD-1633 under green light PDT. This exceeds the ITR = 14,870 for Photofrin® and shows thus a clear clinical advantage of Ru-based PSs over an established porphyrin-based PS.

#### **4.2 Os-based PSs**

The comparative efficacy of the Os-based PSs on U87 cells is shown in **Table 5**. Additionally, in HT1376 cells, TLD-OsH2IP has a dark LD50 > 200 μM, N = 43, red light PDT LD50 = 15.0 μM (CI95 = 9.1–24.9, N = 30), and a NIR light PDT LD50 = 39.0 μM (CI95 = 30.6–49.6, N = 5). TLD-OsH2dppn has dark LD50 = 203.2 μM (CI95 = 190.2–217.1, N = 61), red light PDT LD50 = 4.1 μM (CI95 = 2.9–5.7, N = 26) and NIR light PDT LD50 = 27.4 μM (CI95 = 7.2–100.4, N = 9).

The presence of imidazo[4,5-f][1,10]phenanthroline and adding dppn to the complex increase PDT efficacy of the Os-based PSs, although it does not exceed the efficacy of Ru-based PSs. Similarly to the PDT LD50, ITR of the Os-based PSs in red light is also not better than that of Photofrin®; in U87 cells, ITR = 4.9 for TLD-OsH2B, 24.8 for OsH2IP, and 14.7 for TLD-OsH2dppn. In HT1376 cells, ITR > 13.3 for TLD-OsH2IP and equals to 49.6 for TLD-OsH2dppn. The advantage of the Os-based PSs, however, is their PDT activity in NIR light, which both Ru-based PSs and Photofrin® are lacking. ITR for NIR PDT is greater than 5.1 for TLD-OsH2IP and equal to 7.4 for TLD-OsH2dppn.

Another set of experiments focused at three Os-based PSs with bis ligands [31] supplements the data on red light PDT (625 nm, 90Jcm<sup>−</sup><sup>2</sup> , 450 mWcm<sup>−</sup><sup>2</sup> ). In U87 cells, TLD-OsH2IP is the most efficient PS (LD50 = 57 ± 4 μM) exceeding both TLD-OsH2dppn (LD50 = 87 ± 12 μM) and TLD-OsH2B (125 ± 12 μM). In HT1376 cells, TLD-OsH2dppn is the most efficient (LD50 = 83 ± 4 μM); the remaining two PSs have similar LD50 (121 ± 10 μM for TLD-OsH2B and 141 ± 14 μM for TLD-OsH2IP). The inferiority of TLD-OsH2B in red light over the two other PSs is best reproduced across the presented datasets although comparative efficacy of TLD-OsH2IP and TLD-OsH2dppn is less consistent.

**249**

600 Jcm<sup>−</sup><sup>2</sup>

**Table 5.**

TLD-OsH2dppn

, 900 mWcm<sup>−</sup><sup>2</sup>

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes…*

**U87 cells Dark Green Red NIR**

LD50 (μM):36.0(CI95 = 19.4–365.4)

Maximal kill (%): 70.7 (CI95 = 19.9–121.6)

MEC (water) 12,328 3632 2269

LD50 (μM): 3.1 (CI95 = 2.1–13.2)

Maximal kill (%): (CI95 = 30.67–107.9)

MEC (water) 10,761 3119 1957

LD50 (μM): 0.16 (CI95 = 0.08–0.34)

Maximal kill (%): 84.2 (CI95 = 70.5–97.8)

N = 20 N = 10 N = 4 MEC (water) 10,486 4828 2273

> LD50 (μM): 2.1 (CI95 = 1.6–3.4)

Maximal kill (%): 59.4 (CI95 = 46.6–72.3)

MEC (water) 11,716 2914 1376 *The cells were incubated with the PS for 4 h, and the PS was removed before PDT. The dose–response provides LD50 (μM) and maximal cell kill (%) for a green and red light and a cell kill for a fixed PS concentration for NIR light.* 

N = 43 N = 7 N = 12 N = 4

N = 20 N = 10 N = 4 N = 4

**530 nm 625 nm 808 nm 90 Jcm<sup>−</sup><sup>2</sup> 90 Jcm<sup>−</sup><sup>2</sup> 400-600 Jcm<sup>−</sup><sup>2</sup>**

**108 mWcm<sup>−</sup><sup>2</sup> 125 mWcm<sup>−</sup><sup>2</sup> 150 mWcm<sup>−</sup><sup>2</sup>**

LD50 (μM):81.5(CI95 = 16.9–393.3)

Maximal kill (%): 114.3 (CI95 = 7.6–220.9)

LD50 (μM): 12.2 (CI95 = 9.2–15.8)

Maximal kill (%): 54.0 (CI95 = 51.5–56.6)

LD50 (μM): 12.2 (CI95 = 0.7–577.6)

Maximal kill (%): 79.1 (CI95 = -2.0–160.3)

> LD50 (μM): 2.4 (CI95 = 1.8–3.3)

Maximal kill (%): 78.2 (CI95 = 69.0–87.5)

Kill (%):32.1 (SEM = 14.3)

Kill (%): 63.8 (SEM = 13.5)

Inconsistent and low cell kill

Kill (%): 24.2 (SEM = 4.7)

Importantly, the dataset presented in [31] provides LD50 for NIR PDT (808 nm,

Concentration-wise, the PDT efficiency is almost always similar in red and NIR light. The exception is greater efficacy of TLD-OsH2dppn in red vs. NIR in HT1376 cells (P < 0.001). In U87 cells, ITR is 3.3–9.6 for red PDT and 4.2–12.0 for NIR

presented in **Table 5**. TLD-OsH2IP proves to be most effective among the three in U87 cells (LD50 = 45 ± 5 μM), whereas TLD-OsH2B was the most effective PS for HT1376 cells (LD50 = 121 ± 8 μM). For this wavelength, therefore, the efficacy of

TLD-OsH2dppn was the lowest, in contrast to the red light PDT.

*The data are presented as means and their 95% confidence intervals (SEM for NIR PDT).*

), in contrast to the cell kill at a single concentration

*).*

N = 54 N = 33 N = 53 N = 10

*DOI: http://dx.doi.org/10.5772/intechopen.88519*

(CI95 = 323.4– 484.1)

(CI95 = 67.6–314.6)

LD50 (μM): 179.1 (CI95 = 112.6– 284.8)

(CI95 = 107.8– 185.0)

*In vitro PDT efficacy of Os(II)-based PSs on U87 cells (90 Jcm<sup>−</sup><sup>2</sup>*

TLD-OsH2B LD50 (μM):395.7

TLD-OsH2IP LD50 (μM):145.8

TLD-Os14H LD50 (μM): 141.2


*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes… DOI: http://dx.doi.org/10.5772/intechopen.88519*

*The cells were incubated with the PS for 4 h, and the PS was removed before PDT. The dose–response provides LD50 (μM) and maximal cell kill (%) for a green and red light and a cell kill for a fixed PS concentration for NIR light. The data are presented as means and their 95% confidence intervals (SEM for NIR PDT).*

#### **Table 5.**

*Tumor Progression and Metastasis*

This is a greater value than for U87 cells and suggests lesser PDT sensitivity. The total PDT cell kill is however high, 98.5% (CI95 = 85.6–111.4%). Dark toxicity is, in contrast, low, with LD50 exceeding 200 μM. Importantly, the efficacy of the Ru-based PSs exceeds the efficacy of FDA-approved Photofrin® in green light, although not in red light (**Table 4**). Judging by LD50, the Ru-based PSs have higher dark toxicity than Photofrin®, but this is of less importance because, in addition to their solubil-

Pure PDT effect elucidates the PS efficacy for PDT neglecting its dark toxicity, which is justifiable scientifically to reveal mechanisms of the PS action. Clinically, however, in the case of selective uptake of the PS into cancer cells vs. normal cells, cancer cell kill can be achieved both by PDT-mediated and cytotoxic mechanisms, and the total PDT-induced cell kill becomes relevant. Considering this, total cell kill close to 100% can be achieved in green light in sub-micromolar (20 nM for TLD-1633, 30 nM for TLD-1433, and 200 nM for TLD-1411) or even sub-nanomolar range (0.5 nM for TLD-1611). For comparison, Photofrin® achieved 100% total cell

Clinically, the balance between the efficacy and safety of the PS is characterized by the therapeutic ratio that indicates how far a dose for a desired therapeutic effect is from the dose that causes undesired toxicity. Dividing PDT effect LD50 to dark toxicity LD50 provides small numbers that are not convenient to operate with. It is easier therefore to use inverted therapeutic ratio, ITR = Dark LD50/PDT effect LD50. In U87 cells, ITR = 17,061 for TLD-1411, 27,486 for TLD-1433, 31,460 for TLD-1611, and 54,252 for TLD-1633 under green light PDT. This exceeds the ITR = 14,870 for Photofrin® and shows thus a clear clinical advantage of Ru-based

The comparative efficacy of the Os-based PSs on U87 cells is shown in **Table 5**.

(CI95 = 2.9–5.7, N = 26) and NIR light PDT LD50 = 27.4 μM (CI95 = 7.2–100.4, N = 9). The presence of imidazo[4,5-f][1,10]phenanthroline and adding dppn to the complex increase PDT efficacy of the Os-based PSs, although it does not exceed the efficacy of Ru-based PSs. Similarly to the PDT LD50, ITR of the Os-based PSs in red light is also not better than that of Photofrin®; in U87 cells, ITR = 4.9 for TLD-OsH2B, 24.8 for OsH2IP, and 14.7 for TLD-OsH2dppn. In HT1376 cells, ITR > 13.3 for TLD-OsH2IP and equals to 49.6 for TLD-OsH2dppn. The advantage of the Os-based PSs, however, is their PDT activity in NIR light, which both Ru-based PSs and Photofrin® are lacking. ITR for NIR PDT is greater than 5.1 for TLD-OsH2IP and equal to 7.4 for TLD-OsH2dppn. Another set of experiments focused at three Os-based PSs with bis ligands [31]

cells, TLD-OsH2IP is the most efficient PS (LD50 = 57 ± 4 μM) exceeding both TLD-OsH2dppn (LD50 = 87 ± 12 μM) and TLD-OsH2B (125 ± 12 μM). In HT1376 cells, TLD-OsH2dppn is the most efficient (LD50 = 83 ± 4 μM); the remaining two PSs have similar LD50 (121 ± 10 μM for TLD-OsH2B and 141 ± 14 μM for TLD-OsH2IP). The inferiority of TLD-OsH2B in red light over the two other PSs is best reproduced across the presented datasets although comparative efficacy of TLD-OsH2IP and

, 450 mWcm<sup>−</sup><sup>2</sup>

). In U87

Additionally, in HT1376 cells, TLD-OsH2IP has a dark LD50 > 200 μM, N = 43, red light PDT LD50 = 15.0 μM (CI95 = 9.1–24.9, N = 30), and a NIR light PDT LD50 = 39.0 μM (CI95 = 30.6–49.6, N = 5). TLD-OsH2dppn has dark LD50 = 203.2 μM (CI95 = 190.2–217.1, N = 61), red light PDT LD50 = 4.1 μM

ity in water, they are effective at much lesser, nontoxic concentrations.

kill in U87 cells only at concentrations above 300 nM.

PSs over an established porphyrin-based PS.

supplements the data on red light PDT (625 nm, 90Jcm<sup>−</sup><sup>2</sup>

TLD-OsH2dppn is less consistent.

**4.2 Os-based PSs**

**248**

*In vitro PDT efficacy of Os(II)-based PSs on U87 cells (90 Jcm<sup>−</sup><sup>2</sup> ).*

Importantly, the dataset presented in [31] provides LD50 for NIR PDT (808 nm, 600 Jcm<sup>−</sup><sup>2</sup> , 900 mWcm<sup>−</sup><sup>2</sup> ), in contrast to the cell kill at a single concentration presented in **Table 5**. TLD-OsH2IP proves to be most effective among the three in U87 cells (LD50 = 45 ± 5 μM), whereas TLD-OsH2B was the most effective PS for HT1376 cells (LD50 = 121 ± 8 μM). For this wavelength, therefore, the efficacy of TLD-OsH2dppn was the lowest, in contrast to the red light PDT.

Concentration-wise, the PDT efficiency is almost always similar in red and NIR light. The exception is greater efficacy of TLD-OsH2dppn in red vs. NIR in HT1376 cells (P < 0.001). In U87 cells, ITR is 3.3–9.6 for red PDT and 4.2–12.0 for NIR

PDT. In HT1376 cells, it is, respectively, 4.6–6.1 and 2.6–6.1. As in the dataset shown in **Table 5**, this is far behind the ITR value for Photofrin®, but considerable PDT activity in NIR is a decisive asset. This advantage is reinforced by the similar LD50 for red and NIR PDT, which means that (at certain light exposure conditions) NIR PDT can be at least not worse than red PDT.

One should remember however that NIR PDT needs much more energy to be delivered, NIR range photons carry less energy, and absorbance is lesser than for the red range. Red light PDT is still more efficient per absorbed photon than NIR PDT because similar LD50 in μM is achieved at a much lesser number of absorbed photons (P < 0.001). Hence, the NIR PDT advantage of the Os-based PSs must be realized by increasing the delivered energy of light. This does not pose a problem because no thermal effects are observed for 808 nm at 600 Jcm<sup>−</sup><sup>2</sup> .

#### **4.3 Effect of Tf on in vitro PDT efficacy**

Additional apo-Tf increases red light PDT efficacy of the Ru-based TLD-1433 in AY27 cells, together with a decrease in dark toxicity [43]. The PDT improvement effect is however significant (PDT effect LD50 = 11.6–11.9 μM vs. 17.0 μM with no additional Tf, P < 0.05) only after a relatively short (30 minutes) TLD-1433 incubation before PDT. If the incubation time is increased to 90 minutes, the beneficial effect of the additional Tf is not anymore evident, masked by the increased TLD-1433 PDT efficacy.

#### **4.4 PDT efficacy in hypoxia**

Hypoxia in tumors is one of the major challenges for anticancer therapy because both conventional radiotherapy and PDT rely upon oxygen, a mediator of damage to cancer cells. It is known at the same time that the tumors with hypoxic cores are clinically more aggressive [15].

This means that any modality effective under hypoxic conditions is extremely valuable. Among the four Ru-based and six Os-based PSs, Ru-based TLD-1633 and Os-based TLD-OsH2B proved to be active in hypoxic conditions (at 0.1–0.5% O2) after red light PDT (625 nm, 90 Jcm<sup>−</sup><sup>2</sup> , 125 mWcm<sup>−</sup><sup>2</sup> ). Incubation with ALA (having its metabolite PPIX as photosensitizer) is used as a negative control (an oxygendependent PS). For TLD-1633, hypoxia resistance is observed at a concentration as low as 4 μM, with significantly non-zero PDT effect = 67.3% cell kill in normoxia (P = 0.022) and 46.2% in hypoxia (P = 0.036), at moderate (25% cell kill) dark toxicity. For TLD-OsH2B, PDT effect is evident only at 320 μM. PDT effect reaches significantly non-zero effect: 59.8% in hypoxia (P = 0.006) vs. 42.2% in normoxia (P = 0.0006), and at considerable (53% kill) dark toxicity. For both PSs, hypoxia resistance occurs at concentrations above the PDT LD50.

It is noteworthy that TLD-1633 is active at low oxygen concentration corresponding to pO2 = 0.76 mmHg. It is very encouraging because it is known that anticancer efficacy of conventional treatment progressively decreases at pO2 below a critical threshold of 15–35 mmHg [46, 47].

High dark toxicity of the OsH2B hypoxia-effective concentration is a clear limitation, but this demonstrates anyways a possibility of hypoxia-effective Os-based PSs that, as it was shown, have also PDT activity in NIR. NIR light has greater penetration depth into tissues than visible light, and this, together with the PS activity under hypoxia, will pose a double benefit for PDT of bulk tumors.

**251**

**Figure 3.**

*implying its accumulation in the lesions.*

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes…*

translates to the selectivity of TLD-1433 uptake into tumors in vivo.

Intracellular accumulation of TLD-1433 was detected earlier, and association with Tf facilitated this process [43]. Hence, one needs to explore whether this

TLD-1433 is able to accumulate selectively in tumor tissue vs. normal ones even

TLD-1433 accumulation in the tumors is at least one order of magnitude greater than in the adjacent apparently normal tissue: 77 ± 18 mg/kg, N = 6 vs. 0.4 ± 0.09,

The concentration in a tumor, therefore, reaches estimated 76 μM, which is far exceeding in vitro PDT effect LD50 for U87 cells in green light (**Table 4**). Moreover, the foci of coloration are visible outside of a major tumor. This suggests a possibility of detection of very small malignant lesions not readily visible macroscopically

Association of TLD-1433 with apo-Tf is able to increase selectiveness of the PS accumulation in subcutaneous CT26.WT (murine colon adenocarcinoma) tumors in BalbC mice (**Figure 4**). Four hours after systemic injection of 10 mg/kg TLD-1433 premixed with apo-Tf (molar ratio = 1:1), significantly more TLD-1433 is found in a tumor vs. adjacent muscle tissue (P = 0.038); the selectivity ratio is about 1.8. With TLD-1433 injected, the uptake into a tumor is not significantly different from the

Averaging of the individual tumor/muscle uptake ratios for each animal confirms the results shown above. The ratio is significantly above 1 upon injection of the TLD-1433-Tf premix (1.81 ± 0.14, N = 5, P = 0.005) indicating the uptake selectivity. With TLD-1433 alone injected, the uptake into the tumors is not selective (0.74 ± 0.18, N = 4, P = 0.247). This firmly suggests that the association of TLD-1433 with apo-Tf increases selectivity of TLD-1433 uptake by a tumor. Apo-Tf per se cannot be taken up because it has to bind Fe3+ to be recognized by the cell surface TfR. Since selective improvement of the uptake of TLD-1433 & apo-Tf premix by the tumors is

*Accumulation of TLD-1433 in AY27 orthotopic urinary bladder tumors in fisher rats. The bladder was examined 1 h after instillation of 50 μg/mL TLD-1433. The arrows denote the areas of coloration by TLD-1433* 

without premixing with Tf. In AY27 rat urinary bladder tumors, characteristic staining can be seen co-localized with tumors (**Figure 3**) 1 h after instillation of

*DOI: http://dx.doi.org/10.5772/intechopen.88519*

**5.1 Selective uptake by tumors**

**5. In vivo PDT**

50 μg/mL TLD-1433.

without staining by the PS.

adjacent muscle tissue.

N = 6, P = 0.007.

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes… DOI: http://dx.doi.org/10.5772/intechopen.88519*

#### **5. In vivo PDT**

*Tumor Progression and Metastasis*

808 nm at 600 Jcm<sup>−</sup><sup>2</sup>

1433 PDT efficacy.

**4.4 PDT efficacy in hypoxia**

clinically more aggressive [15].

after red light PDT (625 nm, 90 Jcm<sup>−</sup><sup>2</sup>

resistance occurs at concentrations above the PDT LD50.

under hypoxia, will pose a double benefit for PDT of bulk tumors.

a critical threshold of 15–35 mmHg [46, 47].

PDT can be at least not worse than red PDT.

.

**4.3 Effect of Tf on in vitro PDT efficacy**

PDT. In HT1376 cells, it is, respectively, 4.6–6.1 and 2.6–6.1. As in the dataset shown in **Table 5**, this is far behind the ITR value for Photofrin®, but considerable PDT activity in NIR is a decisive asset. This advantage is reinforced by the similar LD50 for red and NIR PDT, which means that (at certain light exposure conditions) NIR

One should remember however that NIR PDT needs much more energy to be delivered, NIR range photons carry less energy, and absorbance is lesser than for the red range. Red light PDT is still more efficient per absorbed photon than NIR PDT because similar LD50 in μM is achieved at a much lesser number of absorbed photons (P < 0.001). Hence, the NIR PDT advantage of the Os-based PSs must be realized by increasing the delivered energy of light. This does not pose a problem because no thermal effects are observed for

Additional apo-Tf increases red light PDT efficacy of the Ru-based TLD-1433 in AY27 cells, together with a decrease in dark toxicity [43]. The PDT improvement effect is however significant (PDT effect LD50 = 11.6–11.9 μM vs. 17.0 μM with no additional Tf, P < 0.05) only after a relatively short (30 minutes) TLD-1433 incubation before PDT. If the incubation time is increased to 90 minutes, the beneficial effect of the additional Tf is not anymore evident, masked by the increased TLD-

Hypoxia in tumors is one of the major challenges for anticancer therapy because both conventional radiotherapy and PDT rely upon oxygen, a mediator of damage to cancer cells. It is known at the same time that the tumors with hypoxic cores are

This means that any modality effective under hypoxic conditions is extremely valuable. Among the four Ru-based and six Os-based PSs, Ru-based TLD-1633 and Os-based TLD-OsH2B proved to be active in hypoxic conditions (at 0.1–0.5% O2)

its metabolite PPIX as photosensitizer) is used as a negative control (an oxygendependent PS). For TLD-1633, hypoxia resistance is observed at a concentration as low as 4 μM, with significantly non-zero PDT effect = 67.3% cell kill in normoxia (P = 0.022) and 46.2% in hypoxia (P = 0.036), at moderate (25% cell kill) dark toxicity. For TLD-OsH2B, PDT effect is evident only at 320 μM. PDT effect reaches significantly non-zero effect: 59.8% in hypoxia (P = 0.006) vs. 42.2% in normoxia (P = 0.0006), and at considerable (53% kill) dark toxicity. For both PSs, hypoxia

It is noteworthy that TLD-1633 is active at low oxygen concentration corresponding to pO2 = 0.76 mmHg. It is very encouraging because it is known that anticancer efficacy of conventional treatment progressively decreases at pO2 below

High dark toxicity of the OsH2B hypoxia-effective concentration is a clear limitation, but this demonstrates anyways a possibility of hypoxia-effective Os-based PSs that, as it was shown, have also PDT activity in NIR. NIR light has greater penetration depth into tissues than visible light, and this, together with the PS activity

, 125 mWcm<sup>−</sup><sup>2</sup>

). Incubation with ALA (having

**250**

#### **5.1 Selective uptake by tumors**

Intracellular accumulation of TLD-1433 was detected earlier, and association with Tf facilitated this process [43]. Hence, one needs to explore whether this translates to the selectivity of TLD-1433 uptake into tumors in vivo.

TLD-1433 is able to accumulate selectively in tumor tissue vs. normal ones even without premixing with Tf. In AY27 rat urinary bladder tumors, characteristic staining can be seen co-localized with tumors (**Figure 3**) 1 h after instillation of 50 μg/mL TLD-1433.

TLD-1433 accumulation in the tumors is at least one order of magnitude greater than in the adjacent apparently normal tissue: 77 ± 18 mg/kg, N = 6 vs. 0.4 ± 0.09, N = 6, P = 0.007.

The concentration in a tumor, therefore, reaches estimated 76 μM, which is far exceeding in vitro PDT effect LD50 for U87 cells in green light (**Table 4**). Moreover, the foci of coloration are visible outside of a major tumor. This suggests a possibility of detection of very small malignant lesions not readily visible macroscopically without staining by the PS.

Association of TLD-1433 with apo-Tf is able to increase selectiveness of the PS accumulation in subcutaneous CT26.WT (murine colon adenocarcinoma) tumors in BalbC mice (**Figure 4**). Four hours after systemic injection of 10 mg/kg TLD-1433 premixed with apo-Tf (molar ratio = 1:1), significantly more TLD-1433 is found in a tumor vs. adjacent muscle tissue (P = 0.038); the selectivity ratio is about 1.8. With TLD-1433 injected, the uptake into a tumor is not significantly different from the adjacent muscle tissue.

Averaging of the individual tumor/muscle uptake ratios for each animal confirms the results shown above. The ratio is significantly above 1 upon injection of the TLD-1433-Tf premix (1.81 ± 0.14, N = 5, P = 0.005) indicating the uptake selectivity. With TLD-1433 alone injected, the uptake into the tumors is not selective (0.74 ± 0.18, N = 4, P = 0.247). This firmly suggests that the association of TLD-1433 with apo-Tf increases selectivity of TLD-1433 uptake by a tumor. Apo-Tf per se cannot be taken up because it has to bind Fe3+ to be recognized by the cell surface TfR. Since selective improvement of the uptake of TLD-1433 & apo-Tf premix by the tumors is

#### **Figure 3.**

*Accumulation of TLD-1433 in AY27 orthotopic urinary bladder tumors in fisher rats. The bladder was examined 1 h after instillation of 50 μg/mL TLD-1433. The arrows denote the areas of coloration by TLD-1433 implying its accumulation in the lesions.*

#### **Figure 4.**

*Accumulation of TLD-1433 without or with apo-Tf at different molar ratios in CT26.WT tumors in BalbC mice 4 h after systemic (IV) injection (10 mg/kg). N = 5 for TLD-1433 & apo-Tf group; N = 4 for TLD-1433 group.*

demonstrated, one can anticipate two possible scenarios: (1) TLD-1433 & apo-Tf still manages to bind Fe3+, and (2) TLD-1433 & apo-Tf conjugate can be recognized by TfR and taken up by the cell without the need to bind Fe3+.

#### **5.2 In vivo PDT efficacy**

#### *5.2.1 Light penetration*

Assessing PDT efficacy in vivo is a necessary step on the way to potential clinical applications. It has however its own challenges to be addressed. Light exposure regime is one of them.

The penetration depth of light at different PDT conditions is crucial for the PDT success. For example, a small penetration depth of green light is because of a strong attenuation by intrinsic chromophores, such as hemoglobins and cytochromes. The calculations estimate the energy attenuation up to 1/8 cm<sup>−</sup><sup>1</sup> in


#### **Table 6.**

*Light attenuation in a phantom tumor (proportion of energy penetrating to the bottom of 1-cm-thick phantom vs. surface) in green (525 nm), red (635 nm), and NIR (808 nm) light.*

**253**

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes…*

more absorbing chromophores [48]. High absorbance of light by the PS is a very desirable property contributing to its efficacy. This could be a double-edged sword however because high absorbance of the PS close to the tumor surface can shield the deeper tissue from the light exposure and hence result in undertreatment of a tumor. The measurements using a tissue-emulating phantom (a piece of meat having a size of an experimental tumor with an overlaying piece of shaved mouse skin) show indeed that the Os-based PSs (TLD-OsH2B, TLD-Os2IP, and TLD-Os14H) affect the penetration of light into a tumor at different wavelengths. Without PSs, 85–90% of energy is lost across the tumor thickness (about 1 cm)

It is noteworthy that the increase in light attenuation across the wavelengths

The limitations of light penetration can be also illustrated by the distribution of PDT-induced damage in tumors. The damage inflicted by red light (660 nm,

after systemic administration of 10 mg/kg of the 1:1 TLD-1433 & apo-Tf premix clearly diminishes as it goes deeper into a tumor (**Figure 5C,D**). The damage area is not necessarily decreased, but the magnitude of the damage has a definite gradient with coagulative necrosis near the surface and the "general damage" that cannot be defined as coagulative necrosis. The damage is incomplete even when TLD-1433 is associated with apo-Tf (which is expected to facilitate PDT effect as evident from in vitro experiments). Notably, the skin on the way of the light beam is not damaged, which can suggest selectivity of the PS uptake into a tumor. Considering that red light is still delivering 60% of the incident energy at 10 mm depth (**Table 6**), much more shallow damage (up to about 3 mm) suggests a steep gradient of PDT efficacy as the delivered energy falls below a certain threshold. The observed damage should be clearly attributed to PDT but not dark toxicity of the PS in a tumor because, without light, there is no visible damage (**Figure 5B**). **Figure 6** shows representative examples of coagulative necrosis as a result of damage and a pattern of gradual transition of the damaged zone from an intact

Thermal effect is another consideration because it can potentially occur in a tumor upon light irradiation. For green light, this is possible due to absorption by intrinsic hemoglobin. Hyperthermia is known and used as an anti-tumor modality [49], but in PDT studies, the thermal effect may mask PDT-specific mechanisms of

In the subcutaneous tumor model (CT26.WT tumor in BalbC mice),

The temperature does not exceed 31–35°C at the end of irradiation even with

) light does not show any signs of overheating at the tumor surface.

continuous-wave irradiation with red (635 nm, 150 mWcm<sup>−</sup><sup>2</sup>

is PS-specific. At the minimal used dose for each PS, TLD-OsH2B does not attenuate green light penetration, TLD-OsH2IP does not attenuate in red and NIR, while TLD-Os14H does this at all three wavelengths. Also, the increase in the PS concentration results in a progressive and disproportional increase in light attenuation. Notably, the absorbance of the PS measured in water (see **Figure 1**) is not translated directly into the PS-dependent light attenuation in the

in a tumor that has a higher density of vasculature and hence

), 40% for red (635 nm, 150 mWcm<sup>−</sup><sup>2</sup>

) photons. The PSs injected into a tumor further

) PDT to CT26.WT subcutaneous tumors in BalbC mice

) and 45%

) or green (525 nm,

*DOI: http://dx.doi.org/10.5772/intechopen.88519*

for green (525 nm, 40 mWcm<sup>−</sup><sup>2</sup>

for NIR (808 nm, 300 mWcm<sup>−</sup><sup>2</sup>

, 125 mWcm<sup>−</sup><sup>2</sup>

tumor to the necrotic area.

TLD-Os14H injected intratumorally.

*5.2.2 Thermal effect*

tumor damage.

40 mWcm<sup>−</sup><sup>2</sup>

diminishes the light penetration (**Table 6**).

skin and 1/20 cm<sup>−</sup><sup>1</sup>

tumor phantom.

90 J/cm<sup>−</sup><sup>2</sup>

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes… DOI: http://dx.doi.org/10.5772/intechopen.88519*

skin and 1/20 cm<sup>−</sup><sup>1</sup> in a tumor that has a higher density of vasculature and hence more absorbing chromophores [48]. High absorbance of light by the PS is a very desirable property contributing to its efficacy. This could be a double-edged sword however because high absorbance of the PS close to the tumor surface can shield the deeper tissue from the light exposure and hence result in undertreatment of a tumor. The measurements using a tissue-emulating phantom (a piece of meat having a size of an experimental tumor with an overlaying piece of shaved mouse skin) show indeed that the Os-based PSs (TLD-OsH2B, TLD-Os2IP, and TLD-Os14H) affect the penetration of light into a tumor at different wavelengths. Without PSs, 85–90% of energy is lost across the tumor thickness (about 1 cm) for green (525 nm, 40 mWcm<sup>−</sup><sup>2</sup> ), 40% for red (635 nm, 150 mWcm<sup>−</sup><sup>2</sup> ) and 45% for NIR (808 nm, 300 mWcm<sup>−</sup><sup>2</sup> ) photons. The PSs injected into a tumor further diminishes the light penetration (**Table 6**).

It is noteworthy that the increase in light attenuation across the wavelengths is PS-specific. At the minimal used dose for each PS, TLD-OsH2B does not attenuate green light penetration, TLD-OsH2IP does not attenuate in red and NIR, while TLD-Os14H does this at all three wavelengths. Also, the increase in the PS concentration results in a progressive and disproportional increase in light attenuation. Notably, the absorbance of the PS measured in water (see **Figure 1**) is not translated directly into the PS-dependent light attenuation in the tumor phantom.

The limitations of light penetration can be also illustrated by the distribution of PDT-induced damage in tumors. The damage inflicted by red light (660 nm, 90 J/cm<sup>−</sup><sup>2</sup> , 125 mWcm<sup>−</sup><sup>2</sup> ) PDT to CT26.WT subcutaneous tumors in BalbC mice after systemic administration of 10 mg/kg of the 1:1 TLD-1433 & apo-Tf premix clearly diminishes as it goes deeper into a tumor (**Figure 5C,D**). The damage area is not necessarily decreased, but the magnitude of the damage has a definite gradient with coagulative necrosis near the surface and the "general damage" that cannot be defined as coagulative necrosis. The damage is incomplete even when TLD-1433 is associated with apo-Tf (which is expected to facilitate PDT effect as evident from in vitro experiments). Notably, the skin on the way of the light beam is not damaged, which can suggest selectivity of the PS uptake into a tumor. Considering that red light is still delivering 60% of the incident energy at 10 mm depth (**Table 6**), much more shallow damage (up to about 3 mm) suggests a steep gradient of PDT efficacy as the delivered energy falls below a certain threshold.

The observed damage should be clearly attributed to PDT but not dark toxicity of the PS in a tumor because, without light, there is no visible damage (**Figure 5B**).

**Figure 6** shows representative examples of coagulative necrosis as a result of damage and a pattern of gradual transition of the damaged zone from an intact tumor to the necrotic area.

#### *5.2.2 Thermal effect*

Thermal effect is another consideration because it can potentially occur in a tumor upon light irradiation. For green light, this is possible due to absorption by intrinsic hemoglobin. Hyperthermia is known and used as an anti-tumor modality [49], but in PDT studies, the thermal effect may mask PDT-specific mechanisms of tumor damage.

In the subcutaneous tumor model (CT26.WT tumor in BalbC mice), continuous-wave irradiation with red (635 nm, 150 mWcm<sup>−</sup><sup>2</sup> ) or green (525 nm, 40 mWcm<sup>−</sup><sup>2</sup> ) light does not show any signs of overheating at the tumor surface. The temperature does not exceed 31–35°C at the end of irradiation even with TLD-Os14H injected intratumorally.

*Tumor Progression and Metastasis*

**5.2 In vivo PDT efficacy**

*5.2.1 Light penetration*

**Figure 4.**

*group.*

regime is one of them.

demonstrated, one can anticipate two possible scenarios: (1) TLD-1433 & apo-Tf still manages to bind Fe3+, and (2) TLD-1433 & apo-Tf conjugate can be recognized

*Accumulation of TLD-1433 without or with apo-Tf at different molar ratios in CT26.WT tumors in BalbC mice 4 h after systemic (IV) injection (10 mg/kg). N = 5 for TLD-1433 & apo-Tf group; N = 4 for TLD-1433* 

Assessing PDT efficacy in vivo is a necessary step on the way to potential clinical

**TLD-OsH2IP MEC (in water) No PS 2.25 mg/kg 3 mg/kg 9 mg/kg** Green 10,761 0.10–15 0.07↓ 0.005↓ 0.06↓ Red 3119 0.60 0.*70* 0.29↓ 0.05↓ NIR 1957 0.55 0.*79* 0.37↓ 0.08↓ **TLD-Os14H MEC (in water) No PS 0.9 mg/kg 1.8 mg/kg 9 mg/kg** Green 11,716 0.10–15 0.04↓ 0.06↓ 0.06↓ Red 2914 0.60 0.22↓ 0.07↓ 0.05↓ NIR 1376 0.55 0.11↓ 0.08↓ 0.08↓

*Light attenuation in a phantom tumor (proportion of energy penetrating to the bottom of 1-cm-thick phantom* 

*vs. surface) in green (525 nm), red (635 nm), and NIR (808 nm) light.*

in

applications. It has however its own challenges to be addressed. Light exposure

**TLD-OsH2B MEC (in water) No PS 4.5 mg/kg 9 mg/kg** Green 12,328 0.10–15 0.10 0.01↓ Red 3632 0.60 0.30↓ 0.08↓ NIR 2269 0.55 0.22↓ 0.15↓

The penetration depth of light at different PDT conditions is crucial for the PDT success. For example, a small penetration depth of green light is because of a strong attenuation by intrinsic chromophores, such as hemoglobins and cytochromes. The calculations estimate the energy attenuation up to 1/8 cm<sup>−</sup><sup>1</sup>

by TfR and taken up by the cell without the need to bind Fe3+.

**252**

**Table 6.**

#### **Figure 5.**

*Tumor damage (H&E staining) after red light (660 nm, 90Jcm<sup>−</sup><sup>2</sup> , 125 mWcm<sup>−</sup><sup>2</sup> ) PDT to CT26.WT subcutaneous tumors in BalbC mice after systemic administration of 10 mg/kg TLD-1433 as 1:1 TLD-1433 & apo-Tf premix. The PDT was performed 4 h after the administration, and the tumors harvested 2 days post-PDT. The Panel a shows untreated tumor, the Panel b shows PS-injected tumor with no irradiation, and the Panels c-d show PDT-treated tumors.*

#### **Figure 6.**

*Coagulative necrosis and "general damage" in a PDT-treated tumor. The Panel a shows an example of coagulative necrosis area; the Panel b shows a gradient transition from non-damaged tumor area to the necrotic one through the area of "general damage".*

**255**

**Figure 7.**

*light (525 nm, 192 Jcm<sup>−</sup><sup>2</sup>*

*, 200 mWcm<sup>−</sup><sup>2</sup>*

*).*

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes…*

Within a tumor, a combination of deeper-penetrating light and less absorbance by the PS also does not result in considerable thermal effect. Under NIR light alone

26.9 to 31.7°C) during 30 minutes of irradiation. In the presence of Ru-based TLD-1433 (50 mg/kg intratumorally in 100 μL per 20 g BW), the temperature rapidly

ing no PDT-dependent thermal effect. TLD-1433 is responsible only for 3.3°C (41%) of the total PDT-induced increase. Notably, in euthanized animals, the total increase in temperature is similar to that in live animals (although with more linear increase dynamics). This may mean that the active removal of heat by circulating blood is not

In CT26.CL25 subcutaneous tumor model in BalbC mice, intratumoral injection of TLD-1411 or TLD-1433 at 1/20 MTD (1.8 and 5.2 mg/kg, respectively)

The dose of both PSs has to be increased to 1/2 MTD (18 and 52 mg/kg, respectively) to obtain significant (P < 0.01–0.05) PDT effect of greater magnitude, with only continuous-wave PDT effective. About 50% of the animals survived beyond 60 days for TLD-1411 and about 75% beyond 90 days for TLD-1433. **Figure 7** shows

*An example of successful tumor destruction by 53 mg/kg TD1433-mediated PDT under continuous-wave green* 

or complete regression of the tumors and a temporary (8–9 days) delay in their growth [50]. This effect was statistically significant (P < 0.05) only for TLD-1433 translating to an increased survival (about 15% of the animals surviving beyond the

an example of the PDT-induced tumor damage and subsequent regression.

critical in maintaining the temperature within the safe range during PDT.

, 200 mWcm<sup>−</sup><sup>2</sup>

), the temperature increases only by 4.8°C (from

). The temperature reaches no more than 36.5°C show-

) light PDT resulted in a fast reduction

delivered to a tumor and only

*DOI: http://dx.doi.org/10.5772/intechopen.88519*

, 300 mWcm<sup>−</sup><sup>2</sup>

increases from 28.4°C to 33.6°C (by 5.2°C) at 50 Jcm<sup>−</sup><sup>2</sup>

(808 nm, 600 Jcm<sup>−</sup><sup>2</sup>

*5.2.3 PDT effect*

green (525 nm, 192 Jcm<sup>−</sup><sup>2</sup>

90 days follow-up period).

by 8.1°C at the end (600 Jcm<sup>−</sup><sup>2</sup>

#### *Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes… DOI: http://dx.doi.org/10.5772/intechopen.88519*

Within a tumor, a combination of deeper-penetrating light and less absorbance by the PS also does not result in considerable thermal effect. Under NIR light alone (808 nm, 600 Jcm<sup>−</sup><sup>2</sup> , 300 mWcm<sup>−</sup><sup>2</sup> ), the temperature increases only by 4.8°C (from 26.9 to 31.7°C) during 30 minutes of irradiation. In the presence of Ru-based TLD-1433 (50 mg/kg intratumorally in 100 μL per 20 g BW), the temperature rapidly increases from 28.4°C to 33.6°C (by 5.2°C) at 50 Jcm<sup>−</sup><sup>2</sup> delivered to a tumor and only by 8.1°C at the end (600 Jcm<sup>−</sup><sup>2</sup> ). The temperature reaches no more than 36.5°C showing no PDT-dependent thermal effect. TLD-1433 is responsible only for 3.3°C (41%) of the total PDT-induced increase. Notably, in euthanized animals, the total increase in temperature is similar to that in live animals (although with more linear increase dynamics). This may mean that the active removal of heat by circulating blood is not critical in maintaining the temperature within the safe range during PDT.

#### *5.2.3 PDT effect*

*Tumor Progression and Metastasis*

**254**

**Figure 6.**

**Figure 5.**

*one through the area of "general damage".*

*the Panels c-d show PDT-treated tumors.*

*Tumor damage (H&E staining) after red light (660 nm, 90Jcm<sup>−</sup><sup>2</sup>*

*Coagulative necrosis and "general damage" in a PDT-treated tumor. The Panel a shows an example of coagulative necrosis area; the Panel b shows a gradient transition from non-damaged tumor area to the necrotic* 

*subcutaneous tumors in BalbC mice after systemic administration of 10 mg/kg TLD-1433 as 1:1 TLD-1433 & apo-Tf premix. The PDT was performed 4 h after the administration, and the tumors harvested 2 days post-PDT. The Panel a shows untreated tumor, the Panel b shows PS-injected tumor with no irradiation, and* 

*, 125 mWcm<sup>−</sup><sup>2</sup>*

*) PDT to CT26.WT* 

In CT26.CL25 subcutaneous tumor model in BalbC mice, intratumoral injection of TLD-1411 or TLD-1433 at 1/20 MTD (1.8 and 5.2 mg/kg, respectively) green (525 nm, 192 Jcm<sup>−</sup><sup>2</sup> , 200 mWcm<sup>−</sup><sup>2</sup> ) light PDT resulted in a fast reduction or complete regression of the tumors and a temporary (8–9 days) delay in their growth [50]. This effect was statistically significant (P < 0.05) only for TLD-1433 translating to an increased survival (about 15% of the animals surviving beyond the 90 days follow-up period).

The dose of both PSs has to be increased to 1/2 MTD (18 and 52 mg/kg, respectively) to obtain significant (P < 0.01–0.05) PDT effect of greater magnitude, with only continuous-wave PDT effective. About 50% of the animals survived beyond 60 days for TLD-1411 and about 75% beyond 90 days for TLD-1433. **Figure 7** shows an example of the PDT-induced tumor damage and subsequent regression.

#### **Figure 7.**

*An example of successful tumor destruction by 53 mg/kg TD1433-mediated PDT under continuous-wave green light (525 nm, 192 Jcm<sup>−</sup><sup>2</sup> , 200 mWcm<sup>−</sup><sup>2</sup> ).*

These results are obtained with a green light that has only a superficial light penetration. TLD-1433-mediated (50 mg/kg =47% MTD) PDT using deeper-penetrating NIR light (808 nm, 600 Jcm<sup>−</sup><sup>2</sup> , 400 mWcm<sup>−</sup><sup>2</sup> ) does not reach however the efficacy of green light PDT despite 6.7 times greater radiant exposure [43]. Only a trend to improvement in survival (P = 0.164–0.179 vs. dark toxicity and light only) could be observed. This is not surprising by itself considering that TLD-1433 has extremely low absorbance in NIR. Nevertheless, the P values allow hypothesizing that a significant effect could be achieved with more powerful experimental design or greater delivered light energy.

More encouraging is a beneficial effect of combining TLD-1433 with Tf. A highly significant PDT effect in the animals survival can be observed when 4:1 TLD-1433 & apo-Tf premix (50 mg/kg TLD-1433) is injected instead of TLD-1433 only (P = 0.0182–0.0032 vs. dark toxicity and light only). No dark toxicity for tumors (effect of the premix with no light on tumor growth) is detected. Although the difference vs. TLD-1433-induced PDT (P = 0.0633) still does not reach statistical significance threshold, the P value, again, is small enough to talk about a trend toward the improvement. The result reinforces the valuable finding of the benefit of TLD-1433-Tf premix in PDT efficacy improvement under NIR light. This is especially noteworthy because the absorbance of TLD-1433-Tf in NIR range is still very low compared to the absorbance in green light despite the facilitating effect of Tf.

Anyways, 600 Jcm<sup>−</sup><sup>2</sup> NIR PDT is able to maintain about 70% of the animals surviving beyond 90 days follow-up (vs. only about 30% after PDT mediated by TLD-1433 that was not mixed with Tf), which is not less than survival after 192 Jcm<sup>−</sup><sup>2</sup> green light PDT. This is especially encouraging considering that NIR PDT is not effective in vitro, either with or without Tf. The failure to detect in vitro PDT effect in NIR is possibly because the short-term viability assay (reflecting metabolic suppression rather than actual cell death) could be not sufficient to detect the effect of NIR that has less energy per photon. The effect *in vivo*, in contrast, is assessed by the long-term follow-up of tumor growth. The activity of the Ru-based complexes under NIR is known from literature [51] but involves multiphoton excitation. In contrast, the results presented above demonstrate the ability of the PSs to be activated by NIR in a continuous-wave regime via singlephoton excitation. Moreover, TLD-14333-Tf premix has an additional benefit of decreased systemic toxicity, with MTD more than twofold greater than that for injection of TLD-1433 only [50].

This double benefit of using apo-Tf as a delivery vehicle for TLD-1433 resembles the already mentioned effect in vitro for red light PDT using AY27 cells where TLD-1433-Tf decreased dark toxicity and increased PDT efficacy. Note however that in vitro experiments using cancer cell line determined dark toxicity in cancer cells and hence can be rather an estimate for dark toxicity of the PS in tumors. In contrast, in vivo model considered the benefit for systemic toxicity.

NIR PDT efficacy in vivo can be also demonstrated by direct quantitation of the tumor damage. Even suboptimal PDT (200 instead of 600 Jcm<sup>−</sup><sup>2</sup> ) shows a trend (P = 0.104, df = 8, one-tailed) to an increase in the relative area of damage in a tumor as compared to the tumors not subjected to PDT (dark and tumor alone data pooled). The damage area is increased to 33.4 ± 10.2% (N = 4) vs. 17.1 ± 2.5% (N = 6). The effect is only moderate and does not reach statistical significance, but this could be because of suboptimal (200 Jcm<sup>−</sup><sup>2</sup> ) radiant exposure.

Among the Os-based PSs (TLD-OsH2B, TLD-OsH2IP, TLD-OsH2dppn), the MTD values vary. TLD-OsH2B is the most toxic (MTD = 1.25 mg/kg) and TLD-OsH2dppn the least toxic (MTD = 47 mg/kg), which is more than one magnitude of difference [31]. For comparison, in vitro dark LD50 for three PSs were much closer

**257**

**Figure 8.**

*TLD-1433-mediated green light (535 nm, 90 Jcm<sup>−</sup><sup>2</sup>*

*image and H&E images are shown.*

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes…*

to one another (416–617 μM for U87 cells and 476–744 μM for HT1376 cells). As it was mentioned already, however, in vitro dark toxicity for cancer cells and in vivo

TLD-OsH2IP-mediated (3 mg/kg = 1/2 MTD) continuous-wave red light PDT

survival vs. light only group (P < 0.01). The effect is however temporary (like TLD-

better result, with the cases of tumor regression and survival significantly increased vs. both dark and light only groups (P < 0.01) and about 80% of the animals surviving beyond the 50 days follow-up. Considering high photostability of TLD-OsH2IP, further increase in power and energy density for red light PDT is possible. This could potentially allow achieving complete tumor-suppressing success, at least in the

We have discussed previously that NIR effect is potentially possible even at suboptimal settings with Ru-based TLD-1433-Tf formulation. This formulation has an absorbance in NIR higher than TLD-1433 but still lower than Os-based TLD-

expect NIR PDT effect for TLD-OsH2dppn because this PS absorbs much better in NIR than TLD-1433. The PDT effect is indeed observed at 3 mg/kg of the PS and

follow-up vs. dark and light only groups (P < 0.01 and 0.0001, respectively). This result further demonstrates the potential of NIR PDT application using transition metal-based PSs. The NIR PDT still requires delivery of at least 3 times more

*Damage to muscle noninvasive AY27 tumor induced orthotopically in fisher rats' urinary bladder 2 days after* 

*and PDT performed after 1 h of incubation and TLD-1433 washing out of the bladder cavity. The macroscopic* 

*) PDT. TLD-1433 at 6 mg/mL was instilled into bladders,* 

cm<sup>−</sup><sup>1</sup>

, with about 60% of the animals surviving beyond 50-day

1433, as discussed above). Increasing the radiant exposure to 266 Jcm<sup>−</sup><sup>2</sup>

) significantly slows down the tumor growth and increases

, respectively). Hence, we could

allows for a

*DOI: http://dx.doi.org/10.5772/intechopen.88519*

(635 nm, 192 or 266 Jcm<sup>−</sup><sup>2</sup>

framework of this in vivo model.

800 nm and 600 Jcm<sup>−</sup><sup>2</sup>

OsH2dppn (MEC = 777–1459 vs. 2273 M<sup>−</sup><sup>1</sup>

MTD as systemic toxicity is not directly comparable.

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes… DOI: http://dx.doi.org/10.5772/intechopen.88519*

to one another (416–617 μM for U87 cells and 476–744 μM for HT1376 cells). As it was mentioned already, however, in vitro dark toxicity for cancer cells and in vivo MTD as systemic toxicity is not directly comparable.

TLD-OsH2IP-mediated (3 mg/kg = 1/2 MTD) continuous-wave red light PDT (635 nm, 192 or 266 Jcm<sup>−</sup><sup>2</sup> ) significantly slows down the tumor growth and increases survival vs. light only group (P < 0.01). The effect is however temporary (like TLD-1433, as discussed above). Increasing the radiant exposure to 266 Jcm<sup>−</sup><sup>2</sup> allows for a better result, with the cases of tumor regression and survival significantly increased vs. both dark and light only groups (P < 0.01) and about 80% of the animals surviving beyond the 50 days follow-up. Considering high photostability of TLD-OsH2IP, further increase in power and energy density for red light PDT is possible. This could potentially allow achieving complete tumor-suppressing success, at least in the framework of this in vivo model.

We have discussed previously that NIR effect is potentially possible even at suboptimal settings with Ru-based TLD-1433-Tf formulation. This formulation has an absorbance in NIR higher than TLD-1433 but still lower than Os-based TLD-OsH2dppn (MEC = 777–1459 vs. 2273 M<sup>−</sup><sup>1</sup> cm<sup>−</sup><sup>1</sup> , respectively). Hence, we could expect NIR PDT effect for TLD-OsH2dppn because this PS absorbs much better in NIR than TLD-1433. The PDT effect is indeed observed at 3 mg/kg of the PS and 800 nm and 600 Jcm<sup>−</sup><sup>2</sup> , with about 60% of the animals surviving beyond 50-day follow-up vs. dark and light only groups (P < 0.01 and 0.0001, respectively). This result further demonstrates the potential of NIR PDT application using transition metal-based PSs. The NIR PDT still requires delivery of at least 3 times more

#### **Figure 8.**

*Tumor Progression and Metastasis*

etrating NIR light (808 nm, 600 Jcm<sup>−</sup><sup>2</sup>

or greater delivered light energy.

facilitating effect of Tf. Anyways, 600 Jcm<sup>−</sup><sup>2</sup>

injection of TLD-1433 only [50].

Jcm<sup>−</sup><sup>2</sup>

These results are obtained with a green light that has only a superficial light penetration. TLD-1433-mediated (50 mg/kg =47% MTD) PDT using deeper-pen-

efficacy of green light PDT despite 6.7 times greater radiant exposure [43]. Only a trend to improvement in survival (P = 0.164–0.179 vs. dark toxicity and light only) could be observed. This is not surprising by itself considering that TLD-1433 has extremely low absorbance in NIR. Nevertheless, the P values allow hypothesizing that a significant effect could be achieved with more powerful experimental design

More encouraging is a beneficial effect of combining TLD-1433 with Tf. A highly significant PDT effect in the animals survival can be observed when 4:1 TLD-1433 & apo-Tf premix (50 mg/kg TLD-1433) is injected instead of TLD-1433 only (P = 0.0182–0.0032 vs. dark toxicity and light only). No dark toxicity for tumors (effect of the premix with no light on tumor growth) is detected. Although the difference vs. TLD-1433-induced PDT (P = 0.0633) still does not reach statistical significance threshold, the P value, again, is small enough to talk about a trend toward the improvement. The result reinforces the valuable finding of the benefit of TLD-1433-Tf premix in PDT efficacy improvement under NIR light. This is especially noteworthy because the absorbance of TLD-1433-Tf in NIR range is still very low compared to the absorbance in green light despite the

surviving beyond 90 days follow-up (vs. only about 30% after PDT mediated by TLD-1433 that was not mixed with Tf), which is not less than survival after 192

 green light PDT. This is especially encouraging considering that NIR PDT is not effective in vitro, either with or without Tf. The failure to detect in vitro PDT effect in NIR is possibly because the short-term viability assay (reflecting metabolic suppression rather than actual cell death) could be not sufficient to detect the effect of NIR that has less energy per photon. The effect *in vivo*, in contrast, is assessed by the long-term follow-up of tumor growth. The activity of the Ru-based complexes under NIR is known from literature [51] but involves multiphoton excitation. In contrast, the results presented above demonstrate the ability of the PSs to be activated by NIR in a continuous-wave regime via singlephoton excitation. Moreover, TLD-14333-Tf premix has an additional benefit of decreased systemic toxicity, with MTD more than twofold greater than that for

This double benefit of using apo-Tf as a delivery vehicle for TLD-1433 resembles

the already mentioned effect in vitro for red light PDT using AY27 cells where TLD-1433-Tf decreased dark toxicity and increased PDT efficacy. Note however that in vitro experiments using cancer cell line determined dark toxicity in cancer cells and hence can be rather an estimate for dark toxicity of the PS in tumors. In

NIR PDT efficacy in vivo can be also demonstrated by direct quantitation of

Among the Os-based PSs (TLD-OsH2B, TLD-OsH2IP, TLD-OsH2dppn), the MTD values vary. TLD-OsH2B is the most toxic (MTD = 1.25 mg/kg) and TLD-OsH2dppn the least toxic (MTD = 47 mg/kg), which is more than one magnitude of difference [31]. For comparison, in vitro dark LD50 for three PSs were much closer

) radiant exposure.

trend (P = 0.104, df = 8, one-tailed) to an increase in the relative area of damage in a tumor as compared to the tumors not subjected to PDT (dark and tumor alone data pooled). The damage area is increased to 33.4 ± 10.2% (N = 4) vs. 17.1 ± 2.5% (N = 6). The effect is only moderate and does not reach statistical significance, but

contrast, in vivo model considered the benefit for systemic toxicity.

the tumor damage. Even suboptimal PDT (200 instead of 600 Jcm<sup>−</sup><sup>2</sup>

this could be because of suboptimal (200 Jcm<sup>−</sup><sup>2</sup>

, 400 mWcm<sup>−</sup><sup>2</sup>

NIR PDT is able to maintain about 70% of the animals

) does not reach however the

) shows a

*Damage to muscle noninvasive AY27 tumor induced orthotopically in fisher rats' urinary bladder 2 days after TLD-1433-mediated green light (535 nm, 90 Jcm<sup>−</sup><sup>2</sup> ) PDT. TLD-1433 at 6 mg/mL was instilled into bladders, and PDT performed after 1 h of incubation and TLD-1433 washing out of the bladder cavity. The macroscopic image and H&E images are shown.*

photons than for red light PDT to match it in efficacy (considering the difference in absorbance and quantum energy), but this does not pose a serious problem because of thermal safety of the light exposure as it was discussed above.

Another anticancer application of PDT using transition metal-based PSs is urothelial non-muscle invasive bladder cancer [52]. As it was mentioned above, Ru-based TLD-1433 accumulated selectively in the orthotopic urinary bladder tumors (instillation with 0.05 mg/mL). At higher concentration of TLD-1433 (6 mg/mL) that is more relevant for the future clinical applications, green light (535 nm, 90 Jcm<sup>−</sup><sup>2</sup> ) PDT causes full depth (2–3 mm) necrosis in a tumor that showed a deep red coloration (**Figure 8**). Importantly, PDT spared the muscle and urothelial tissue adjacent to the tumors, with only a transient local inflammation of the adjacent urothelium. This is a decisive advantage because the collateral muscle damage impairing the bladder function was a reason for the failure of the prior clinical trials on bladder cancer PDT.

#### **6. Clinical PDT efficacy**

The results of the preclinical research allowed planning and initiation of a clinical trial for non-muscle invasive bladder cancer (NMIBC) at the Princess Margaret Cancer Center in Toronto, Canada. It is noteworthy that although several other Ru-based complexes (NAMI-A, KP1019, and KP1339) have currently entered clinical trials as antineoplastic drugs, TLD-1433 is meanwhile the only transition metal complex tested in a trial as a PS for PDT [5, 53].

TLD-1433-mediated PDT (525 nm, 3 W, target dose = 90 Jcm<sup>−</sup><sup>2</sup> ) with intravesical irradiation demonstrated safety and efficacy of the PS in patients with non-muscle invasive urinary bladder cancer (NMIBC) who were previously unresponsive to contemporary anticancer therapy, including the intravesical therapy with Bacillus Calmette-Guérin (BCG) [54]. At therapeutic dose (0.70 mg per cm<sup>2</sup> of bladder surface), 2 of 3 patients were tumor-free at the 180-day posttreatment, with no essential adverse effects and minimal systemic absorption of the PS (complete clearance from the plasma within 72 hrs) and no photosensitivity reactions. This outcome is successful enough to warrant further advance to a phase II trial.

#### **7. PS activation by ionizing radiation**

It is worth noting that at least one of the PSs under discussion, TLD-1433, can be activated not only by nonionizing electromagnetic radiation but also by ionizing one (X-ray). Transition metal complexes are theoretically prone to this because the atoms of transitional metals can attenuate X-rays. For example, Ru attenuates X-ray photons at 75 keV to an extent comparable to iodine, an established X-ray imaging agent [55]. Activation of the PS by X-ray is very advantageous because it allows treatment of the tumors located considerably deeper than reachable by NIR. TLD-1433 retains its functionality after 75 keV irradiation at doses up to 20Gy and retains its ability to generate postirradiation \*OH signal under subsequent red light exposure. In cultured human glioblastoma U87 cells, 20 μM TLD-1433 exerted non-zero radio-enhancement effect after 75 keV X-ray exposure (5 Gy) at the magnitude of 37% cell kill (P = 0.020, df = 3), at dark toxicity of 20% cell kill (P = 0.009, df = 3). Moreover, the effect can be detected in vivo in CT26.WT tumors induced in BalbC mice. At 1 Gy, X-ray resulted in a 2.9-fold increase in coagulative necrosis area in the tumors on day 2 postexposure vs. TL1433 alone and X-ray alone groups pooled

**259**

*Anticancer Photodynamic Therapy Using Ruthenium(II) and Os(II)-Based Complexes…*

(P = 0.007, df = 15) [56]. It is noteworthy that thermal effects at these conditions are highly unlikely because 1 Gy deposits only 0.001 J per gr tissue, which, at the

In vitro and in vivo data suggest that transition metal-based complexes are versatile as PSs with diverse photophysical, photochemical, and biological properties. This includes activation over a wide range of wavelengths and high singlet oxygen yield and photobleaching resistance. The Ru(II)-based PSs may have very high cytotoxic efficacy far exceeding the established porphyrin-based PSs. The Os(II)-based PSs are notable in their PDT activity at deeper-penetrating NIR light PDT. Moreover, even Ru(II)-based PSs could be effective in vivo under NIR light. Transition metal-based PSs demonstrate both Type I and Type II photoreactions and can be active in hypoxic conditions, presenting the potential for the treatment of bulky hypoxic tumors. These properties are further facilitated by their ability to associate with endogenous metal transporter molecules, like human apo-Tf, which enables their targeted endocytosis. Furthermore, the association with Tf increases absorptivity at longer wavelengths (far red to NIR range), ROS generation, and finally tumor destroying potential. The observed capacities of the PSs may allow overcoming notorious challenges of PDT: the necessity for deeper light penetration, the selectivity of accumulation in tumors, and activity under hypoxic conditions. Finally, the research has led to the first clinical trial for this class of PSs, with a successful outcome and potential to further clinical advance. This raises justified hopes that with the ongoing technological improvements, such as the development of transition metal complexes (including the advanced Theralase PSs discussed above), and personalized dosimetry with a treatment planning approach, PDT has the potential to become

K<sup>−</sup><sup>1</sup>

[57], provides a very small

*DOI: http://dx.doi.org/10.5772/intechopen.88519*

(0.0003°C) increase in temperature.

**8. Conclusions**

estimated average specific heat capacity C ≈ 3.7 J g<sup>−</sup><sup>1</sup>

integrated into the mainstream of cancer treatment.

The authors are grateful to the employees of Theralase Inc. for ensuring a very helpful and benevolent working atmosphere and particularly appreciate the contribution of the members of its research team, both past (Jamie Fong, Kamola Kasimova, Yaxal Arenas, and Savo Lazic) and present (Manjunatha Ankathatti Munegowda) for their experimental work and publications used in this chapter. The next acknowledgement is of the invaluable help and crucial support by Prof. Lothar Lilge at Princess Margaret Cancer Centre, Toronto, Ontario, Canada, and his group (including Sarah Forward and Carl Fisher) and of the staff of the other departments, facilities, and services at the center for doing their best to make our work going smoothly and effectively. We greatly appreciate the collaboration with Prof. Sherri McFarland at Acadia University, Wolfville, Nova Scotia, Canada, and her group.

**Acknowledgements**

**Conflict of interest**

No conflict of interest has been declared.

(P = 0.007, df = 15) [56]. It is noteworthy that thermal effects at these conditions are highly unlikely because 1 Gy deposits only 0.001 J per gr tissue, which, at the estimated average specific heat capacity C ≈ 3.7 J g<sup>−</sup><sup>1</sup> K<sup>−</sup><sup>1</sup> [57], provides a very small (0.0003°C) increase in temperature.

#### **8. Conclusions**

*Tumor Progression and Metastasis*

(535 nm, 90 Jcm<sup>−</sup><sup>2</sup>

clinical trials on bladder cancer PDT.

complex tested in a trial as a PS for PDT [5, 53].

**7. PS activation by ionizing radiation**

TLD-1433-mediated PDT (525 nm, 3 W, target dose = 90 Jcm<sup>−</sup><sup>2</sup>

Calmette-Guérin (BCG) [54]. At therapeutic dose (0.70 mg per cm<sup>2</sup>

**6. Clinical PDT efficacy**

photons than for red light PDT to match it in efficacy (considering the difference in absorbance and quantum energy), but this does not pose a serious problem because

Another anticancer application of PDT using transition metal-based PSs is urothelial non-muscle invasive bladder cancer [52]. As it was mentioned above, Ru-based TLD-1433 accumulated selectively in the orthotopic urinary bladder tumors (instillation with 0.05 mg/mL). At higher concentration of TLD-1433 (6 mg/mL) that is more relevant for the future clinical applications, green light

showed a deep red coloration (**Figure 8**). Importantly, PDT spared the muscle and urothelial tissue adjacent to the tumors, with only a transient local inflammation of the adjacent urothelium. This is a decisive advantage because the collateral muscle damage impairing the bladder function was a reason for the failure of the prior

The results of the preclinical research allowed planning and initiation of a clinical trial for non-muscle invasive bladder cancer (NMIBC) at the Princess Margaret Cancer Center in Toronto, Canada. It is noteworthy that although several other Ru-based complexes (NAMI-A, KP1019, and KP1339) have currently entered clinical trials as antineoplastic drugs, TLD-1433 is meanwhile the only transition metal

cal irradiation demonstrated safety and efficacy of the PS in patients with non-muscle invasive urinary bladder cancer (NMIBC) who were previously unresponsive to contemporary anticancer therapy, including the intravesical therapy with Bacillus

surface), 2 of 3 patients were tumor-free at the 180-day posttreatment, with no essential adverse effects and minimal systemic absorption of the PS (complete clearance from the plasma within 72 hrs) and no photosensitivity reactions. This outcome is successful enough to warrant further advance to a phase II trial.

It is worth noting that at least one of the PSs under discussion, TLD-1433, can be activated not only by nonionizing electromagnetic radiation but also by ionizing one (X-ray). Transition metal complexes are theoretically prone to this because the atoms of transitional metals can attenuate X-rays. For example, Ru attenuates X-ray photons at 75 keV to an extent comparable to iodine, an established X-ray imaging agent [55]. Activation of the PS by X-ray is very advantageous because it allows treatment of the tumors located considerably deeper than reachable by NIR. TLD-1433 retains its functionality after 75 keV irradiation at doses up to 20Gy and retains its ability to generate postirradiation \*OH signal under subsequent red light exposure. In cultured human glioblastoma U87 cells, 20 μM TLD-1433 exerted non-zero radio-enhancement effect after 75 keV X-ray exposure (5 Gy) at the magnitude of 37% cell kill (P = 0.020, df = 3), at dark toxicity of 20% cell kill (P = 0.009, df = 3). Moreover, the effect can be detected in vivo in CT26.WT tumors induced in BalbC mice. At 1 Gy, X-ray resulted in a 2.9-fold increase in coagulative necrosis area in the tumors on day 2 postexposure vs. TL1433 alone and X-ray alone groups pooled

) with intravesi-

of bladder

) PDT causes full depth (2–3 mm) necrosis in a tumor that

of thermal safety of the light exposure as it was discussed above.

**258**

In vitro and in vivo data suggest that transition metal-based complexes are versatile as PSs with diverse photophysical, photochemical, and biological properties. This includes activation over a wide range of wavelengths and high singlet oxygen yield and photobleaching resistance. The Ru(II)-based PSs may have very high cytotoxic efficacy far exceeding the established porphyrin-based PSs. The Os(II)-based PSs are notable in their PDT activity at deeper-penetrating NIR light PDT. Moreover, even Ru(II)-based PSs could be effective in vivo under NIR light. Transition metal-based PSs demonstrate both Type I and Type II photoreactions and can be active in hypoxic conditions, presenting the potential for the treatment of bulky hypoxic tumors. These properties are further facilitated by their ability to associate with endogenous metal transporter molecules, like human apo-Tf, which enables their targeted endocytosis. Furthermore, the association with Tf increases absorptivity at longer wavelengths (far red to NIR range), ROS generation, and finally tumor destroying potential. The observed capacities of the PSs may allow overcoming notorious challenges of PDT: the necessity for deeper light penetration, the selectivity of accumulation in tumors, and activity under hypoxic conditions. Finally, the research has led to the first clinical trial for this class of PSs, with a successful outcome and potential to further clinical advance. This raises justified hopes that with the ongoing technological improvements, such as the development of transition metal complexes (including the advanced Theralase PSs discussed above), and personalized dosimetry with a treatment planning approach, PDT has the potential to become integrated into the mainstream of cancer treatment.

#### **Acknowledgements**

The authors are grateful to the employees of Theralase Inc. for ensuring a very helpful and benevolent working atmosphere and particularly appreciate the contribution of the members of its research team, both past (Jamie Fong, Kamola Kasimova, Yaxal Arenas, and Savo Lazic) and present (Manjunatha Ankathatti Munegowda) for their experimental work and publications used in this chapter. The next acknowledgement is of the invaluable help and crucial support by Prof. Lothar Lilge at Princess Margaret Cancer Centre, Toronto, Ontario, Canada, and his group (including Sarah Forward and Carl Fisher) and of the staff of the other departments, facilities, and services at the center for doing their best to make our work going smoothly and effectively. We greatly appreciate the collaboration with Prof. Sherri McFarland at Acadia University, Wolfville, Nova Scotia, Canada, and her group.

#### **Conflict of interest**

No conflict of interest has been declared.

*Tumor Progression and Metastasis*

#### **Author details**

Pavel Kaspler\*, Arkady Mandel, Roger Dumoulin-White and Mark Roufaiel Theralase Inc., Toronto, Canada

\*Address all correspondence to: pkaspler@theralase.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Theralase Inc., Toronto, Canada

provided the original work is properly cited.

Pavel Kaspler\*, Arkady Mandel, Roger Dumoulin-White and Mark Roufaiel

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: pkaspler@theralase.com

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[48] Shi G, Monro S, Hennigar R, Colpitts J, Fong J, Kasimova K, et al. Ru(II) dyads derived from α-oligothiophenes: A new class of potent and versatile photosensitizers for PDT. Coordination Chemistry Reviews. 2015;**282**:127-138. DOI: 10.1016/j.

[49] Raaphorst GP, Heller DP, Bussey A, Ng CE. Thermal radiosensitization by 41″C

hyperthermia during low dose-rate irradiation in human normal and tumour cell lines. International Journal of Hyperthermia. 1994;**10**:263-270. DOI: 10.3109/02656739409009347

[50] Fong J, Kasimova K, Arenas Y, Kaspler P, Lazic S, Mandel A, et al. A novel class of ruthenium-based photosensitizers effectively kills in vitro cancer cells and in vivo tumors. Photochemical & Photobiological Sciences. 2015;**14**:2014-2023. DOI:

[51] Yu B, Ouyang C, Qiu K, Zhao J, Ji L, Chao H. Lipophilic tetranuclear ruthenium(II) complexes as twophoton luminescent tracking non-viral gene vectors. Chemistry - A European Journal. 2015;**21**:3691-3700. DOI:

[52] Lazic S, Kaspler P, Mandel A, Jewett MAS, Kulkarni G, Lilge L. MP61-06. Photodynamic therapy for non-muscle invasive bladder cancer mediated by instilled photosensitizer TLD-1433 and green light activation. The Journal of Urology. 2016;**195**:E805.

DOI: 10.1016/j.juro.2016.02.880

10.1039/c4pp00438h

10.1002/chem.201405151

[41] Śpiewak K, Brindell M. Impact of low- and high-molecular-mass components of human serum on NAMI-A binding to transferrin. JBIC, Journal of Biological Inorganic Chemistry. 2015;**20**:695-703. DOI: 10.1007/s00775-015-1255-5

[42] Shen Y, Li X, Dong D, Zhang B, Xue Y, Shang P. Transferrin receptor 1 in cancer: A new sight for cancer therapy. American Journal of Cancer Research. 2018;**8**(6):916-931 eCollection 2018

[43] Kaspler P, Lazic S, Forward S, Arenas Y, Mandel A, Lilge L. A ruthenium(II) based photosensitizer and transferrin complexes enhance photo-physical properties, cell uptake, and photodynamic therapy safety and efficacy. Photochemical & Photobiological Sciences. 2016. [Epub ahead of print]. DOI: 10.1039/

[44] Quarles CD Jr, Randunu KM, Brumaghim JL, Marcus RK. Metal retention in human transferrin:

Consequences of solvent composition in analytical sample preparation methods. Metallomics. 2011;**3**:1027-1034. DOI:

[45] Ueda J, Takeshita K, Matsumoto S, Yazaki K, Kawaguchi M, Ozawa T. Singlet oxygen-mediated hydroxyl radical production in the presence of phenols: Whether DMPO-\*OH formation really indicates production

of \*OH? Photochemistry and

Photobiology. 2007;**77**:165-170. DOI: 10.1562/0031-8655(2003)0770165SOM

[46] Höckel M, Vaupel P. Tumour hypoxia: Definitions and current

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clinical, biologic, and molecular aspects. Journal of the National Cancer Institute.

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[54] Kulkarni GS, Lilge L, Mandel A, Perlis N, Nesbitt M, Dumoulin-White R, et al. TLD-1433 photodynamic therapy for BCG-unresponsive NMIBC - a phase IB clinical study. In: 19th Annual Meeting of the Society of Urologic Oncology (SUO). Phoenix, AZ, USA; 2018

[55] NIST: X-Ray Mass Attenuation Coefficients – Ruthenium. 2018. Available from https://physics.nist.gov/ PhysRefData/XrayMassCoef/ElemTab/ z44.html [Accessed: 26 December 2018]

[56] Mandel A, Kaspler P, Roufaiel M, Munegowda MA, Lilge L. X-ray and photon mediated in vitro and in vivo activity of ruthenium(II) compounds. In: 16th International Photodynamic Association (IPA) World Congress. Coimbra, Portugal; 2017

[57] Giering K, Lamprecht I, Minet O, Handke A. Determination of the specific heat capacity of healthy and tumorous human tissue. Thermochimica Acta. 1995;**251**:199-205. DOI: 10.1016/0040-6031(94)02047-R

**267**

**Chapter 11**

**Abstract**

**1. Introduction**

hybrid orbitals (e.g., sp, sp2

Treatment

*and Nanda Gopal Sahoo*

Theranostics Application of

Graphene-Based Materials in

Cancer Imaging, Targeting and

*Neha Karki, Anita Rana, Himani Tiwari, Pushpa Negi* 

**Keywords:** graphene, nanocarrier, cancer imaging, drug delivery, DDSs

, and sp3

As a promising interdisciplinary field, nanotechnology acts as a bridge for various disciplines, such as material sciences, engineering, physics and chemistry, and is dedicated to the production of different materials in the nanometer scale (<100 nm), with assorted physical, chemical and mechanical properties. Although nanoscience and nanotechnology are new research interests, the application of nanomaterials for humankind was well-known since ancient times. Among the periodic elements, carbon has the intense ability of catenation and great tendency to form various

smart compounds having different physical and chemical properties according to their structure [1, 2]. Due to the properties mentioned above, carbon have tendency of forming different allotropes of different dimensions, like quantum dots (0D), carbon nanotubes (1D), fullerenes (0D), graphene (2D), graphite (3D), among which, graphene got lot of attention in the past decade. Graphene has hexagonally packed honeycomb like geometry in which a unit layer of carbon atoms are arranged

) which results in the formation of various

Recent advancements in graphene-based nanomaterials provide the opportunity that compliments the limitations of conventional drug delivery systems (DDSs) through simultaneous targeting of the anticancer drug to the cancer cell by reducing the side effects of other administration routes. Graphene with its extraordinary electronic properties like larger surface area, possibilities of surface modification, can efficiently target the tumor cell. At the same time, nanocarriers have the advantages of immune clearance adulteration of physicochemical properties of anticancer drug. The DDSs can be made by biodegradable nanocarriers such as proteins, peptides, biocompatible polymers, antibodies, polymer-drug conjugates, etc. Graphenesupported DDSs in cancer therapy also supports the co-delivery of therapeutic agents, antioxidants, SiRNA, shRNA, etc. as the co-delivery approach, which provide additive or synergistic therapeutic efficacy and can reduce toxic effects.

#### **Chapter 11**

## Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting and Treatment

*Neha Karki, Anita Rana, Himani Tiwari, Pushpa Negi and Nanda Gopal Sahoo*

#### **Abstract**

Recent advancements in graphene-based nanomaterials provide the opportunity that compliments the limitations of conventional drug delivery systems (DDSs) through simultaneous targeting of the anticancer drug to the cancer cell by reducing the side effects of other administration routes. Graphene with its extraordinary electronic properties like larger surface area, possibilities of surface modification, can efficiently target the tumor cell. At the same time, nanocarriers have the advantages of immune clearance adulteration of physicochemical properties of anticancer drug. The DDSs can be made by biodegradable nanocarriers such as proteins, peptides, biocompatible polymers, antibodies, polymer-drug conjugates, etc. Graphenesupported DDSs in cancer therapy also supports the co-delivery of therapeutic agents, antioxidants, SiRNA, shRNA, etc. as the co-delivery approach, which provide additive or synergistic therapeutic efficacy and can reduce toxic effects.

**Keywords:** graphene, nanocarrier, cancer imaging, drug delivery, DDSs

#### **1. Introduction**

As a promising interdisciplinary field, nanotechnology acts as a bridge for various disciplines, such as material sciences, engineering, physics and chemistry, and is dedicated to the production of different materials in the nanometer scale (<100 nm), with assorted physical, chemical and mechanical properties. Although nanoscience and nanotechnology are new research interests, the application of nanomaterials for humankind was well-known since ancient times. Among the periodic elements, carbon has the intense ability of catenation and great tendency to form various hybrid orbitals (e.g., sp, sp2 , and sp3 ) which results in the formation of various smart compounds having different physical and chemical properties according to their structure [1, 2]. Due to the properties mentioned above, carbon have tendency of forming different allotropes of different dimensions, like quantum dots (0D), carbon nanotubes (1D), fullerenes (0D), graphene (2D), graphite (3D), among which, graphene got lot of attention in the past decade. Graphene has hexagonally packed honeycomb like geometry in which a unit layer of carbon atoms are arranged

in two-dimensional (2D) lattice [3–6]. The hybrid orbital of carbon-carbon atoms are in sp2 hybridized form, in which the in-plane σ (C▬C) bonds are much stronger than the out-of-plane π (C▬C) bonds, which is highly accountable for the delocalized array of electrons and come up with the weak polar interaction between graphitic layers of the graphene sheets as well as with graphene and other molecules.

In scheming a potent drug delivery carrier, besides its physicochemical constancy in the biological surroundings, reactivity and toxic issues, diffusivity, immunogenicity, interactions with biological systems, drug loading and release characteristics, blood circulation half-life, drug transportation ability of the biological medium to aim the cells within tissues, etc. are the significant issues (**Figure 1**). Due to the electrostatic interaction and presence of different alkali and alkaline earth metal ions *viz.* Na, Mg, etc. in the physiological medium, the graphene sheets tends to agglomerate which results in reduction of their surface area, decreasing their solubility and increasing their toxicity. Therefore, surface modification of graphene nanosheets is required to overcome such physical and biological effects. Covalent functionalization and noncovalent physisorption are the two well-known strategies universally applied for surface modification to construct desired modified graphene nanosheets [7–9].

Number of research groups have been investigating the surface modification of GO with different kinds of biocompatible polymers as a nanodrug carrier for development of targeted drug delivery systems. The polymers are selectively preferred according to their functional groups, bioavailability and compatibility in the cell medium [10]. Therefore responding to specific stimulus, surface fabrication of GO with various polymers for this particular drug delivery application is limited [11]. For well-organized diagnosis, expressive intracellular drug release is elected over the contemporary arrival of the drug in the system.

With the innovation of the smart material graphene, the curiosity of researchers remarkably moved toward graphene and its oxygenated derivatives from the previously invented other nanomaterials of carbon family, and various scientists are working to organize the surface modification of graphene through the spacious understanding of different functionalization methods [12–14]. From the chemical

**269**

applications [29].

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting…*

GO sheets, or we can say that the reactivity of GO is highly desirable.

point of view, production of graphene oxide that possesses many reactive oxygencontaining groups is appropriate for advance modification and relative aspects of

With reference to different physicochemical properties of nanomaterials of graphene family, it can be projected that they will demonstrate numerous mutual connections with biological moieties such as cells and tissues depending on chemical modification, thickness and dimensions of graphene sheets, etc. [30, 31]. With more supplementary data of graphene application in biomedical field, research on its cellular activity and other intracellular processes is rising. Introduction of polar and reactive oxygenated functionalities give rise to oxidative force in objective cells is supposed to be the effective mechanism for potency of graphene oxide [32, 33]. Due to their dose dependent cytotoxicity and bactericidal activity, graphene-based materials are being explored for applications in antimicrobial products. A number of studies have been performed reporting the antibacterial activities of CNTs, graphene, GO and rGO against *Escherichia coli* and *Staphylococcus aureus* bacteria with rGO having the strongest antibacterial effectiveness [34–38]. With reference to the various studies discussed above, it is apparent that shape and size, of graphene-based materials importantly take part in determining their interactions with cell membrane and intracellular uptake. Moreover *in vitro* studies in various cell lines along with broad perspective of various mechanisms dependent on graphene-based materials are being increasingly explored for various applications in antimicrobial products. In the end, we will briefly discuss the prospects and future challenges regarding graphene-based materials as cancer imaging, targeting and treatment applications.

**2. Synthesis of graphene oxide and its surface modification**

Different methods are used to set up the preparation of graphene sheets according to its structural and chemical behavior with various biocompatible molecules.

Beside this, graphene is sparingly soluble in the water, polar solvents and in the cell environment due to aromatic character [15]. Graphene oxide on the other hand has water contact angle of 30.7° [16] and proficient of composing weak hydrogen bonds and metal incorporated complex ion due to the polar oxygenated groups present on the basal plane and negatively charged carboxylic groups present on the edge site [17–20]. The distinctive arrangement of graphene oxide and its strong carbon-carbon covalent bonding provides outstanding thermal and electrical conductivity with very low thermal expansion quotient. These properties of graphene are also significantly affected by alteration such as edge scattering defect [21] and isotopic doping [22] due to diffusion or localization of phonons at the defect sites. Light absorption and optical imaging are highly dependent on the total number of layers present in GO sheets, as they increase accordingly with the number of layers present in GO [23]. Optoelectronic devices based on GO derivatives are developed as tunable IR detectors, modulators and emitters by electrophysiology and charge multiplexers [24]. This capability to organize the rearrangement and partition of surface electrons can be oppressed in emergent bio-imaging applications [25, 26]. The GO sheets are highly influenced by the divalent ions and some specific polymers which play a crucial role in mechanical properties of GO sheets by inter connecting both the molecules [27, 28]. Due to the superior mechanical properties of GO, it has been reported that by incorporating the different polymers with it, the tensile strength of the respective polymer increases. Graphene supported polymethyl methacrylate (PMMA) and poly-l-lactic acid (PLLA) drastically amplifies the young's modulus and hardness of these polymer nanocomposites for mechanical

*DOI: http://dx.doi.org/10.5772/intechopen.91331*

**Figure 1.** *Properties and applications of graphene oxide.*

#### *Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting… DOI: http://dx.doi.org/10.5772/intechopen.91331*

point of view, production of graphene oxide that possesses many reactive oxygencontaining groups is appropriate for advance modification and relative aspects of GO sheets, or we can say that the reactivity of GO is highly desirable.

Beside this, graphene is sparingly soluble in the water, polar solvents and in the cell environment due to aromatic character [15]. Graphene oxide on the other hand has water contact angle of 30.7° [16] and proficient of composing weak hydrogen bonds and metal incorporated complex ion due to the polar oxygenated groups present on the basal plane and negatively charged carboxylic groups present on the edge site [17–20]. The distinctive arrangement of graphene oxide and its strong carbon-carbon covalent bonding provides outstanding thermal and electrical conductivity with very low thermal expansion quotient. These properties of graphene are also significantly affected by alteration such as edge scattering defect [21] and isotopic doping [22] due to diffusion or localization of phonons at the defect sites. Light absorption and optical imaging are highly dependent on the total number of layers present in GO sheets, as they increase accordingly with the number of layers present in GO [23]. Optoelectronic devices based on GO derivatives are developed as tunable IR detectors, modulators and emitters by electrophysiology and charge multiplexers [24]. This capability to organize the rearrangement and partition of surface electrons can be oppressed in emergent bio-imaging applications [25, 26].

The GO sheets are highly influenced by the divalent ions and some specific polymers which play a crucial role in mechanical properties of GO sheets by inter connecting both the molecules [27, 28]. Due to the superior mechanical properties of GO, it has been reported that by incorporating the different polymers with it, the tensile strength of the respective polymer increases. Graphene supported polymethyl methacrylate (PMMA) and poly-l-lactic acid (PLLA) drastically amplifies the young's modulus and hardness of these polymer nanocomposites for mechanical applications [29].

With reference to different physicochemical properties of nanomaterials of graphene family, it can be projected that they will demonstrate numerous mutual connections with biological moieties such as cells and tissues depending on chemical modification, thickness and dimensions of graphene sheets, etc. [30, 31]. With more supplementary data of graphene application in biomedical field, research on its cellular activity and other intracellular processes is rising. Introduction of polar and reactive oxygenated functionalities give rise to oxidative force in objective cells is supposed to be the effective mechanism for potency of graphene oxide [32, 33]. Due to their dose dependent cytotoxicity and bactericidal activity, graphene-based materials are being explored for applications in antimicrobial products. A number of studies have been performed reporting the antibacterial activities of CNTs, graphene, GO and rGO against *Escherichia coli* and *Staphylococcus aureus* bacteria with rGO having the strongest antibacterial effectiveness [34–38]. With reference to the various studies discussed above, it is apparent that shape and size, of graphene-based materials importantly take part in determining their interactions with cell membrane and intracellular uptake. Moreover *in vitro* studies in various cell lines along with broad perspective of various mechanisms dependent on graphene-based materials are being increasingly explored for various applications in antimicrobial products. In the end, we will briefly discuss the prospects and future challenges regarding graphene-based materials as cancer imaging, targeting and treatment applications.

#### **2. Synthesis of graphene oxide and its surface modification**

Different methods are used to set up the preparation of graphene sheets according to its structural and chemical behavior with various biocompatible molecules.

*Tumor Progression and Metastasis*

graphene nanosheets [7–9].

the contemporary arrival of the drug in the system.

are in sp2

in two-dimensional (2D) lattice [3–6]. The hybrid orbital of carbon-carbon atoms

In scheming a potent drug delivery carrier, besides its physicochemical constancy in the biological surroundings, reactivity and toxic issues, diffusivity, immunogenicity, interactions with biological systems, drug loading and release characteristics, blood circulation half-life, drug transportation ability of the biological medium to aim the cells within tissues, etc. are the significant issues (**Figure 1**). Due to the electrostatic interaction and presence of different alkali and alkaline earth metal ions *viz.* Na, Mg, etc. in the physiological medium, the graphene sheets tends to agglomerate which results in reduction of their surface area, decreasing their solubility and increasing their toxicity. Therefore, surface modification of graphene nanosheets is required to overcome such physical and biological effects. Covalent functionalization and noncovalent physisorption are the two well-known strategies universally applied for surface modification to construct desired modified

Number of research groups have been investigating the surface modification of GO with different kinds of biocompatible polymers as a nanodrug carrier for development of targeted drug delivery systems. The polymers are selectively preferred according to their functional groups, bioavailability and compatibility in the cell medium [10]. Therefore responding to specific stimulus, surface fabrication of GO with various polymers for this particular drug delivery application is limited [11]. For well-organized diagnosis, expressive intracellular drug release is elected over

With the innovation of the smart material graphene, the curiosity of researchers remarkably moved toward graphene and its oxygenated derivatives from the previously invented other nanomaterials of carbon family, and various scientists are working to organize the surface modification of graphene through the spacious understanding of different functionalization methods [12–14]. From the chemical

 hybridized form, in which the in-plane σ (C▬C) bonds are much stronger than the out-of-plane π (C▬C) bonds, which is highly accountable for the delocalized array of electrons and come up with the weak polar interaction between graphitic layers of the graphene sheets as well as with graphene and other molecules.

**268**

**Figure 1.**

*Properties and applications of graphene oxide.*

The methods are widely categorized as colloidal suspension (size specific), arc discharge (electric charge specific), and chemical or mechanical exfoliation. For the broad and extensive production of graphene sheets, mechanical exfoliation method were not used as it is expensive but for the fabrication of electronic devices it is widely applied. Graphene oxide (GO) the oxygenated derivative and replacement of graphene is synthesized by the chemical exfoliation method, in which the sp2 hybridized C▬C hybrid orbitals breaks and the different oxygenated groups such as hydroxyl, carboxyl and epoxy are introduced [39]. The surface modifications of the graphene sheets are site specific as the bulky group carboxyl attached toward the edges of the sheets while on the other hand hydroxyl and epoxy groups tends to form bond with the basal plane of the graphene sheets [40, 41]. These oxygenated and highly reactive functional groups offers reactive handles for a range of surfacefunctionalization reactions covalently and non-covalently, which can be used to build up surface modified GO, its biocompatible composites. For the large-scale synthesis of graphene, the most common methods required exfoliation of graphene. The only variances among graphene made by different methodologies are the defect content and yield of their products [42]. Various methods are available for graphene synthesis, but for the large production of graphene oxide (GO) oxidative-exfoliation methods give excellent results. There are some additional treatments required to reduce typically defective graphene-like nanosheet into reduced graphene oxide (RGO) [43]. During the oxidation process of graphene, functional groups containing oxygen attached to the surface increase the distance among graphitic layers and responsible for enhancing the exfoliation by weakening the van der Waals forces [44]. After the oxidation process, several washing steps are required so that oxidizing agents and some other impurities removed from graphite oxide to enhance the exfoliation. For large-scale washing of graphite oxide, different conventional approaches such as filtration process [45], centrifugation process [46], and dialysis process [47] are mostly used. Among all these processes infiltration processes, after some time, particles of graphite oxide choke the filter pores and make it timeconsuming process.

#### **2.1 Synthesis of graphene oxide (GO)**

For the synthesis of graphene oxide, the most used source of graphite is flake graphite, which occurs as a natural mineral which further use to purify to remove heteroatomic contamination [48]. GO prepared by the use of flake graphite have the property to easily dispersed in water hence used on a large scale [49]. This expanded form of graphite powder has been used for the synthesis of GO sheets by the following method.

#### *2.1.1 Modified Hummer's method*

Graphene oxide is synthesized by modified Hummer's method using graphite powder [50]. In this method, in a round bottom flask, a mixture of 1 g of NaNO3 and calculated graphite powder are mixed. In this mixture drop by drop, 46 mL of H2SO4 was added in an ice water bath with continuous stirring. After 4 h stirring without any pause at 32°C temperature, 6 g of KMnO4 was poured into a slurry mixture of NaNO3 and graphite. Later 2 h continuous stirring 92 mL of DD water was combined in it at 95°C. Again after 2 h, 200 mL of DD water was poured in it and leave for 1 h for constant stirring. Lastly, at room temperature, 20 ml of H2O2 was mixed and mixed it repeatedly for 1 h. The obtained supreme oxidized product washes by 10% HCl solution for purification by abundant quantity by ion free water. Finally, it is filtered by 0.2 μm Nylon membranes until neutralizing the final product.

**271**

**Figure 2.**

*Surface modification of graphene oxide.*

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting…*

From the last decades, the interest has increased in the scientific community for the use of graphene oxide (GO) in biological and biomedical field applications [51]. GO is a two dimensional material with a large surface area containing single-layer

tion in which hydrophilic functional groups with oxygen are present [52]. Thus, GO has many possibilities for surface functionalization due to outstanding solubility in water [53]. There are different methods for the functionalization of the surface of

The nanocarrier thus synthesized was characterized by some advanced spectroscopic techniques, like RAMAN (**Figure 3**), Fourier Transform infrared (FT-IR) (**Figure 4**), Transmittance Electron Microscope (TEM) (**Figure 5**). Thermal stability and quantitative analysis were characterized by Thermo gravimetric Analyzer TGA under a nitrogen atmosphere at a heating rate of 10°C/min from 30 to 600°C

During the processes of oxidation and exfoliation of graphite, there is a large extent of carboxylic group forms on the graphene surface. These groups better modified by different methods; one of them is covalent functionalization. In covalent functionalization, graphene is coupled with reagents, such as 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) [54], or can also be converted to acyl chlorides using thionyl chloride (SOCl) [55]. Covalent functionalization is a multipurpose methodology for modification

graphene oxide; some of them are discussed below (**Figure 2**).

hybridization and carbon sites with sp3

hybridiza-

*DOI: http://dx.doi.org/10.5772/intechopen.91331*

**2.2 Surface functionalization of GO**

*2.2.1 Covalent functionalization of GO*

sheets of carbon atoms with sp2

(**Figure 6**).

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting… DOI: http://dx.doi.org/10.5772/intechopen.91331*

#### **2.2 Surface functionalization of GO**

*Tumor Progression and Metastasis*

consuming process.

ing method.

**2.1 Synthesis of graphene oxide (GO)**

*2.1.1 Modified Hummer's method*

The methods are widely categorized as colloidal suspension (size specific), arc discharge (electric charge specific), and chemical or mechanical exfoliation. For the broad and extensive production of graphene sheets, mechanical exfoliation method were not used as it is expensive but for the fabrication of electronic devices it is widely applied. Graphene oxide (GO) the oxygenated derivative and replacement of graphene is synthesized by the chemical exfoliation method, in which the sp2 hybridized C▬C hybrid orbitals breaks and the different oxygenated groups such as hydroxyl, carboxyl and epoxy are introduced [39]. The surface modifications of the graphene sheets are site specific as the bulky group carboxyl attached toward the edges of the sheets while on the other hand hydroxyl and epoxy groups tends to form bond with the basal plane of the graphene sheets [40, 41]. These oxygenated and highly reactive functional groups offers reactive handles for a range of surfacefunctionalization reactions covalently and non-covalently, which can be used to build up surface modified GO, its biocompatible composites. For the large-scale synthesis of graphene, the most common methods required exfoliation of graphene. The only variances among graphene made by different methodologies are the defect content and yield of their products [42]. Various methods are available for graphene synthesis, but for the large production of graphene oxide (GO) oxidative-exfoliation methods give excellent results. There are some additional treatments required to reduce typically defective graphene-like nanosheet into reduced graphene oxide (RGO) [43]. During the oxidation process of graphene, functional groups containing oxygen attached to the surface increase the distance among graphitic layers and responsible for enhancing the exfoliation by weakening the van der Waals forces [44]. After the oxidation process, several washing steps are required so that oxidizing agents and some other impurities removed from graphite oxide to enhance the exfoliation. For large-scale washing of graphite oxide, different conventional approaches such as filtration process [45], centrifugation process [46], and dialysis process [47] are mostly used. Among all these processes infiltration processes, after some time, particles of graphite oxide choke the filter pores and make it time-

For the synthesis of graphene oxide, the most used source of graphite is flake graphite, which occurs as a natural mineral which further use to purify to remove heteroatomic contamination [48]. GO prepared by the use of flake graphite have the property to easily dispersed in water hence used on a large scale [49]. This expanded form of graphite powder has been used for the synthesis of GO sheets by the follow-

Graphene oxide is synthesized by modified Hummer's method using graphite powder [50]. In this method, in a round bottom flask, a mixture of 1 g of NaNO3 and calculated graphite powder are mixed. In this mixture drop by drop, 46 mL of H2SO4 was added in an ice water bath with continuous stirring. After 4 h stirring without any pause at 32°C temperature, 6 g of KMnO4 was poured into a slurry mixture of NaNO3 and graphite. Later 2 h continuous stirring 92 mL of DD water was combined in it at 95°C. Again after 2 h, 200 mL of DD water was poured in it and leave for 1 h for constant stirring. Lastly, at room temperature, 20 ml of H2O2 was mixed and mixed it repeatedly for 1 h. The obtained supreme oxidized product washes by 10% HCl solution for purification by abundant quantity by ion free water. Finally, it

is filtered by 0.2 μm Nylon membranes until neutralizing the final product.

**270**

From the last decades, the interest has increased in the scientific community for the use of graphene oxide (GO) in biological and biomedical field applications [51]. GO is a two dimensional material with a large surface area containing single-layer sheets of carbon atoms with sp2 hybridization and carbon sites with sp3 hybridization in which hydrophilic functional groups with oxygen are present [52]. Thus, GO has many possibilities for surface functionalization due to outstanding solubility in water [53]. There are different methods for the functionalization of the surface of graphene oxide; some of them are discussed below (**Figure 2**).

The nanocarrier thus synthesized was characterized by some advanced spectroscopic techniques, like RAMAN (**Figure 3**), Fourier Transform infrared (FT-IR) (**Figure 4**), Transmittance Electron Microscope (TEM) (**Figure 5**). Thermal stability and quantitative analysis were characterized by Thermo gravimetric Analyzer TGA under a nitrogen atmosphere at a heating rate of 10°C/min from 30 to 600°C (**Figure 6**).

#### *2.2.1 Covalent functionalization of GO*

During the processes of oxidation and exfoliation of graphite, there is a large extent of carboxylic group forms on the graphene surface. These groups better modified by different methods; one of them is covalent functionalization. In covalent functionalization, graphene is coupled with reagents, such as 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) [54], or can also be converted to acyl chlorides using thionyl chloride (SOCl) [55]. Covalent functionalization is a multipurpose methodology for modification

**Figure 2.** *Surface modification of graphene oxide.*

**Figure 3.** *Raman spectra for GO, GO-PVP, and GO-β-CD.*

**Figure 4.** *Fourier transform infrared (FT-IR) spectra of GO, GO-PVP and GO-βCD.*

**273**

**Figure 6.**

*TGA analysis of GO, PVP, and GO-PVP.*

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting…*

of graphene surface which tailoring the chemical properties as well as electronic properties of graphene [56]. The methodology of GO functionalization by covalent functionalization depends on the environments of the reaction, nature of solvent, temperature condition, different functional groups of the incoming molecules, and other factors like reaction time. When polymer attach to the GO nanosheets it creates stress on GO surface, the covalent mode of functionalization is helpful in controlling the chemical properties of GO and reduces the stress caused by polymer [57]. GO surfaces have the ability for excellent covalent functionalization which makes it a unique nanomaterial which is helpful in developing studies of biological system. According to previous studies functionalization of GO shows excellent results when perform its use in targeted drug delivery applications [58]. There are chances of this because GO surface has the affinity for the adsorption of huge amount of hydrophobic drugs easily and due to specificity of covalent functionalization it releases the drug to particular regions of organisms. This functionalization of GO is also applicable in other biological activities like anti-bacterial activity, bioimaging [59], and photo-dynamic therapy [60]. Even though some procedures of graphene surface modification by covalent functionalization have validated efficient results but some methods generate some supplemental defects on the surface

of graphene which are responsible for the changes in graphene structure.

Non-covalent functionalization is a more effective method in order to make maximum use of the inherent structure and mechanical properties of graphene oxide or graphene. Non-covalent functionalization is largely preferred in place of covalent functionalization as it does not alter the structure and electronic properties of graphene and it simultaneously introduces new chemical groups on the surface. The most common examples of non-covalent functionalization on graphene surface include polymer wrapping, π-π interactions, electron donor-acceptor complexes, hydrogen bonding, and van der Waals forces. Non-covalent functionalization of graphene results in the enhancement of dispersibility, biocompatibility, and reactivity, binding capacity, or sensing properties. Non-covalent interactions also known as supramolecular interactions are found in all types of materials that experience

*2.2.2 Non-covalent functionalization of GO*

*DOI: http://dx.doi.org/10.5772/intechopen.91331*

**Figure 5.** *TEM images of (a) GO (b) GO-PVP, and (c) GO-β-CD.*

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting… DOI: http://dx.doi.org/10.5772/intechopen.91331*

**Figure 6.** *TGA analysis of GO, PVP, and GO-PVP.*

*Tumor Progression and Metastasis*

**272**

**Figure 5.**

**Figure 4.**

**Figure 3.**

*Raman spectra for GO, GO-PVP, and GO-β-CD.*

*TEM images of (a) GO (b) GO-PVP, and (c) GO-β-CD.*

*Fourier transform infrared (FT-IR) spectra of GO, GO-PVP and GO-βCD.*

of graphene surface which tailoring the chemical properties as well as electronic properties of graphene [56]. The methodology of GO functionalization by covalent functionalization depends on the environments of the reaction, nature of solvent, temperature condition, different functional groups of the incoming molecules, and other factors like reaction time. When polymer attach to the GO nanosheets it creates stress on GO surface, the covalent mode of functionalization is helpful in controlling the chemical properties of GO and reduces the stress caused by polymer [57]. GO surfaces have the ability for excellent covalent functionalization which makes it a unique nanomaterial which is helpful in developing studies of biological system. According to previous studies functionalization of GO shows excellent results when perform its use in targeted drug delivery applications [58]. There are chances of this because GO surface has the affinity for the adsorption of huge amount of hydrophobic drugs easily and due to specificity of covalent functionalization it releases the drug to particular regions of organisms. This functionalization of GO is also applicable in other biological activities like anti-bacterial activity, bioimaging [59], and photo-dynamic therapy [60]. Even though some procedures of graphene surface modification by covalent functionalization have validated efficient results but some methods generate some supplemental defects on the surface of graphene which are responsible for the changes in graphene structure.

#### *2.2.2 Non-covalent functionalization of GO*

Non-covalent functionalization is a more effective method in order to make maximum use of the inherent structure and mechanical properties of graphene oxide or graphene. Non-covalent functionalization is largely preferred in place of covalent functionalization as it does not alter the structure and electronic properties of graphene and it simultaneously introduces new chemical groups on the surface. The most common examples of non-covalent functionalization on graphene surface include polymer wrapping, π-π interactions, electron donor-acceptor complexes, hydrogen bonding, and van der Waals forces. Non-covalent functionalization of graphene results in the enhancement of dispersibility, biocompatibility, and reactivity, binding capacity, or sensing properties. Non-covalent interactions also known as supramolecular interactions are found in all types of materials that experience

attractive as well as repulsive forces between them. These type of interactions are found in many natural and synthetic systems [61, 62]. In comparison to covalent bonds the energies of individual non-covalent interactions are normally lower [63]. In graphene, two types of π-π interactions occur between the electron-rich and electron-poor regions, which influence its interaction with other molecules or nanomaterials. This is commonly seen in the face-to-face and edge-to-face arrangement [64]. Graphene materials, with the π-π interactions have dissociation energies less than 50 kJ mol<sup>−</sup><sup>1</sup> . The weakest forces, that is, London-dispersion forces or van der Waals interactions are responsible for the non-covalent interaction affect all atoms in close proximity. The hydrophobic effects caused by different types of interactions are influence not only dispersibility of GO but recognition interactions [65, 66].

#### **3. Challenges in nanotheranostics designing**

In forthcoming nanotheranostics will be accepted as an efficient nanomedicine due to their unique properties like imaging, target selectivity and ability to load the drug in nanocarrier. In the process of nanotheranostics evolution as a potential nanoplatform various challenges encountered for detection of clinical complications. An appropriate technology is required for the treatment and selection of effective therapeutic agents for respective diseases like metallic nanocrystals, image-contrasting agents and choosing an efficient therapeutic agents for corresponding diseases like metallic nanocrystal and concatinate them as nanomaterial. The advanced nanomaterial high selectivity to the target site is required for advance nanotheranostic for excellent delivery of drug targeted nanotheranostic should contain delivery and loading capacity. The biocompatible material should be used in the preparation of nanotheranostics the normal tissue should not damage and easily excreted by human system. Whole designing of nanotheranostics will cheap with no side effects to body.

#### **3.1 Pharmacokinetic and toxicological aspects**

The introduction of a new drug to the site is not only expensive, but also time consuming. It includes discovery, clinical testing, development, and approval. Improving safety/efficacy ratio of marketed drugs is more cost-effective. All this can be done by controlling the time, rate, and place of drug release in the body through a drug-delivery system. Hence, a drug-delivery system could be seen as an interface between the patient and the drug [67]. Since past decades, a growing number of drugs were discovered and were optimized for an enhanced efficiency. However, about 40% of the new drugs, especially those based on biomolecules, like peptides, nucleotides, or proteins, often present a low bioavailability and are rejected by the pharmaceutical industry [68]. For controlled release the ideal materials must control certain important issues like easy reach to the target site in the body, ability to transport the necessary volume of active compounds, and a certain level of release with a certain speed, apart from the properties needed to ensure a better and safe interaction with the human body [69].

#### **4. Graphene-based composites in various biomedical application**

Graphene is considered as the finest and most durable monolayer capable of free existence. The specific 2D geometry and presence of pi electrons in graphene basal plane further applied for valuable drug loading via hydrophobic interactions and

**275**

**Figure 7.**

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting…*

applications in the field of drug delivery, MRI and bioimaging.

*Graphene-based composites for various biomedical applications.*

π-π stacking. Furthermore, large surface area of graphene allows for high density surface fabrication via different surface modification. A number of research on the in vivo behavior and bioactivity of graphene (**Figure 7**) has been investigated

Graphene is the finest and most durable monolayer material which is capable of free existence. In graphene, its 2D structure and presence of delocalized π electrons on its surface can be used for effective drug loading via hydrophobic interactions and π-π stacking. In addition to this, large surface area of graphene allows it for high density bio-functionalization via both covalent and non-covalent surface modification methods. Various studies based on the in vivo behaviour and bioactivity of graphene shows that the nanocarriers interact with the cell membranes and enter into the cells by endocytosis. For targeted drug delivery to the cell nucleus, it is essential that the drug carrier escapes endosomal compartment and release loaded drug into the cytosolic compartments [70, 71]. This process proposed a strategy to reverse cancer drug resistance in DOX resistant MCF-7/ADR cells by loading DOX on graphene oxide surface via physical mixing [72]. High pH dependent release for drug loading with of DOX was observed *in vitro*. GO enhanced accumulations of DOX in MCF-7/ADR cells causing higher cytotoxicity in comparison to free DOX. It is well known that pH is acidic in the cancer micro environment, intracellular lysosomes and endosomes. This fact has been exploited to achieve active drug release in the tumor tissue/cells using chemical modification of graphene [73–76]. For chemotherapeutic efficacy use of graphene-based materials has also been explored for co-delivery of multiple drugs. Zang et al. [77] Loading of DOX and CPT in controlled way inside the same drug delivery system resulted in remarkably higher toxicity in MCF-7 cells compared with GO-loaded only with DOX or CPT. Thus, graphene and GO-modified magnetic nanoparticles results in various biomedical

*DOI: http://dx.doi.org/10.5772/intechopen.91331*

previously.

**4.1 Drug delivery**

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting… DOI: http://dx.doi.org/10.5772/intechopen.91331*

π-π stacking. Furthermore, large surface area of graphene allows for high density surface fabrication via different surface modification. A number of research on the in vivo behavior and bioactivity of graphene (**Figure 7**) has been investigated previously.

#### **4.1 Drug delivery**

*Tumor Progression and Metastasis*

than 50 kJ mol<sup>−</sup><sup>1</sup>

no side effects to body.

attractive as well as repulsive forces between them. These type of interactions are found in many natural and synthetic systems [61, 62]. In comparison to covalent bonds the energies of individual non-covalent interactions are normally lower [63]. In graphene, two types of π-π interactions occur between the electron-rich and electron-poor regions, which influence its interaction with other molecules or nanomaterials. This is commonly seen in the face-to-face and edge-to-face arrangement [64]. Graphene materials, with the π-π interactions have dissociation energies less

Waals interactions are responsible for the non-covalent interaction affect all atoms in close proximity. The hydrophobic effects caused by different types of interactions are influence not only dispersibility of GO but recognition interactions [65, 66].

In forthcoming nanotheranostics will be accepted as an efficient nanomedicine due to their unique properties like imaging, target selectivity and ability to load the drug in nanocarrier. In the process of nanotheranostics evolution as a potential nanoplatform various challenges encountered for detection of clinical complications. An appropriate technology is required for the treatment and selection of effective therapeutic agents for respective diseases like metallic nanocrystals, image-contrasting agents and choosing an efficient therapeutic agents for corresponding diseases like metallic nanocrystal and concatinate them as nanomaterial. The advanced nanomaterial high selectivity to the target site is required for advance nanotheranostic for excellent delivery of drug targeted nanotheranostic should contain delivery and loading capacity. The biocompatible material should be used in the preparation of nanotheranostics the normal tissue should not damage and easily excreted by human system. Whole designing of nanotheranostics will cheap with

The introduction of a new drug to the site is not only expensive, but also time consuming. It includes discovery, clinical testing, development, and approval. Improving safety/efficacy ratio of marketed drugs is more cost-effective. All this can be done by controlling the time, rate, and place of drug release in the body through a drug-delivery system. Hence, a drug-delivery system could be seen as an interface between the patient and the drug [67]. Since past decades, a growing number of drugs were discovered and were optimized for an enhanced efficiency. However, about 40% of the new drugs, especially those based on biomolecules, like peptides, nucleotides, or proteins, often present a low bioavailability and are rejected by the pharmaceutical industry [68]. For controlled release the ideal materials must control certain important issues like easy reach to the target site in the body, ability to transport the necessary volume of active compounds, and a certain level of release with a certain speed, apart from the properties needed to ensure a

**3. Challenges in nanotheranostics designing**

**3.1 Pharmacokinetic and toxicological aspects**

better and safe interaction with the human body [69].

**4. Graphene-based composites in various biomedical application**

Graphene is considered as the finest and most durable monolayer capable of free existence. The specific 2D geometry and presence of pi electrons in graphene basal plane further applied for valuable drug loading via hydrophobic interactions and

. The weakest forces, that is, London-dispersion forces or van der

**274**

Graphene is the finest and most durable monolayer material which is capable of free existence. In graphene, its 2D structure and presence of delocalized π electrons on its surface can be used for effective drug loading via hydrophobic interactions and π-π stacking. In addition to this, large surface area of graphene allows it for high density bio-functionalization via both covalent and non-covalent surface modification methods. Various studies based on the in vivo behaviour and bioactivity of graphene shows that the nanocarriers interact with the cell membranes and enter into the cells by endocytosis. For targeted drug delivery to the cell nucleus, it is essential that the drug carrier escapes endosomal compartment and release loaded drug into the cytosolic compartments [70, 71]. This process proposed a strategy to reverse cancer drug resistance in DOX resistant MCF-7/ADR cells by loading DOX on graphene oxide surface via physical mixing [72]. High pH dependent release for drug loading with of DOX was observed *in vitro*. GO enhanced accumulations of DOX in MCF-7/ADR cells causing higher cytotoxicity in comparison to free DOX. It is well known that pH is acidic in the cancer micro environment, intracellular lysosomes and endosomes. This fact has been exploited to achieve active drug release in the tumor tissue/cells using chemical modification of graphene [73–76]. For chemotherapeutic efficacy use of graphene-based materials has also been explored for co-delivery of multiple drugs. Zang et al. [77] Loading of DOX and CPT in controlled way inside the same drug delivery system resulted in remarkably higher toxicity in MCF-7 cells compared with GO-loaded only with DOX or CPT. Thus, graphene and GO-modified magnetic nanoparticles results in various biomedical applications in the field of drug delivery, MRI and bioimaging.

**Figure 7.** *Graphene-based composites for various biomedical applications.*

Attachment of nanoparticles such as iron oxide with graphene-based nanomaterial makes them super paramagnetic in property and can be useful in drug delivery applications [78]. The resulting magnetic hybrids dispersed uniformly in aqueous solution before and after loading of DOX. Magnetic hybrids show agglomeration behavior in acidic medium and redispersion behavior observed in basic medium. This pH triggered magnetic behavior of GO-Fe3O4 nanoparticle hybrids can be help in controlled drug delivery. Similar pH-dependent drug release system was reported for 5-FU-loaded nanohybrid system composed of graphene nanosheets (GN), carbon nanotube (CNT) and Fe3O4 [79].

#### **4.2 Gene delivery**

Gene therapy is used in many expanding area to treat genetic disorders like Parkinson's disease, cystic fibrosis and cancer. An effective gene therapy needs efficient and safe gene vectors that also protect DNA from nuclease degradation as well as facilitate DNA uptake with high transfection efficiency [80, 81]. According to review of literature, graphene has been reported for wide applications in the field of gene delivery, gene-drug co-delivery and protein delivery with. PEI has been extensively investigated as nonviral gene vector having strong electrostatic interactions with negatively charged phosphates of RNA and DNA. Chemical modification is very easy in PEI which offers increased transfection efficiency, cell selectivity and reduced cytotoxicity however low biocompatibility and high toxicity of (Polyethyleneimine) PEI limit its use for biomedical applications [82]. Chitosan-GO complex are also used for simultaneous drug and gene delivery [83]. Chitosan-GO converts pDNA into stable nano-sized complexes. Amineterminated PEGylated GO was effectively used to deliver high protein payloads due to non-covalent interactions with surface of PEG-GO [84]. Bone morphogenic protein-2 (BMP-2) was loaded onto Ti substrate coated with alternate layers of positively (GO-NH3+) and negatively (GO-COO-) charged GO nanosheets with high loading efficacy and conserved bioactivity. Osteogenic differentiation of MSCs was enriched on Ti coated GO surfaces carrying BMP-2 than only Ti surface with BMP-2. In vivo studies in mouse also exhibited vigorous new bone formation with Ti-GO-BMP2 implants compared to Ti or Ti-GO or Ti-BMP2 implants and making the new composite a very effective carrier for therapeutic drug delivery [85]. All above studies have highlighted potential of graphene-based materials as drug and gene delivery vehicles *in vitro* studies though there is a necessity to validate their potential in vivo with particular focus on safety, biodistribution and efficacy.

#### **4.3 Tissue engineering**

Tissue engineering is an interdisciplinary field that endeavors to develop biological substitutes to resolve, retain or enhance functionality of a tissue or whole organ [86]. Recently, graphene-based materials have been used to treat wound healing, stem cell engineering, regenerative medicine and tissue engineering. Hydrogels have viscoelastic and transport properties to mimicking natural tissues [87], but their weak mechanical properties can limit their use in many tissue engineering applications. Graphene has a platform for tailoring various functionalities on flat surfaces with outstanding mechanical properties like high elasticity, strength, flexibility. Potentially, graphene has a wide range of applications in the field of hydrogels, biodegradable films, electrospun fibers and other tissue engineering scaffolds. When GO incorporated into PVA-based hydrogels it potentially increases tensile stability (132%) and

**277**

**Figure 8.**

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting…*

to inflammatory responses, biocompatibility and regenerative potential.

Raman imaging, and multimodal imaging are highlighted (**Figure 8**).

Graphene-based nanomaterials have been widely explored in biomedical fields such as bioimaging, drug delivery, theranostics, and so on. The recent advances in bioimaging of graphene-based nanomaterials, including graphene, graphene oxide, reduced graphene oxide, graphene quantum dots, and their derivatives, the synthetic methods of graphene-based nanomaterials are included in situ synthesis and binding method. The bioimaging modalities, including optical imaging (fluorescence [FL], two-photon FL), positron emission tomography/single-photon emission computed tomography, magnetic resonance imaging, photoacoustic imaging,

**5. Application of graphene in bioimaging**

*Different types of bioimaging graphene-based bioimaging.*

compressive strength (36%) of composite hydrogel without altering their cytoaffinity [88]. According to Lu et al. graphene-based composite materials are applicable for wound healing by formulating graphene containing chitosan-PVA nanofibrous scaffolds. These three groups, chitosan-PVA-graphene electrospun fibers, chitosan-PVA fibers were also studied without graphene and control (no scaffold), to check wound healing affinity in mice and rabbit [89]. Graphene containing samples healed the wound completely in faster rate in comparison to without graphene-based samples in both mice and rabbit. Graphene-based materials also have applications in the area of musculoskeletal tissue engineering using mouse myoblast C2C12 cell lines [90]. Cellular behavior on graphene derivatives are enhanced by the Surface roughness and surface oxygen content and adsorption of serum proteins. Thus, graphene materials can be useful in reinforcing tissue engineering scaffolds due to its mechanical and electrical properties. Graphene materials have properties like large surface area which adsorb proteins/DNA and can be useful in many therapeutic applications. For instance, Mahmoudi et al. [91] recently reported protective role of GO and proteincoated GO surfaces in amyloid beta fibrillation process, which is implicated in various neurodegenerative disorders. However, along with detailed *in vitro* characterization of scaffolds, more emphasis should be placed on their evaluation in vivo with respect

*DOI: http://dx.doi.org/10.5772/intechopen.91331*

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting… DOI: http://dx.doi.org/10.5772/intechopen.91331*

compressive strength (36%) of composite hydrogel without altering their cytoaffinity [88]. According to Lu et al. graphene-based composite materials are applicable for wound healing by formulating graphene containing chitosan-PVA nanofibrous scaffolds. These three groups, chitosan-PVA-graphene electrospun fibers, chitosan-PVA fibers were also studied without graphene and control (no scaffold), to check wound healing affinity in mice and rabbit [89]. Graphene containing samples healed the wound completely in faster rate in comparison to without graphene-based samples in both mice and rabbit. Graphene-based materials also have applications in the area of musculoskeletal tissue engineering using mouse myoblast C2C12 cell lines [90]. Cellular behavior on graphene derivatives are enhanced by the Surface roughness and surface oxygen content and adsorption of serum proteins. Thus, graphene materials can be useful in reinforcing tissue engineering scaffolds due to its mechanical and electrical properties. Graphene materials have properties like large surface area which adsorb proteins/DNA and can be useful in many therapeutic applications. For instance, Mahmoudi et al. [91] recently reported protective role of GO and proteincoated GO surfaces in amyloid beta fibrillation process, which is implicated in various neurodegenerative disorders. However, along with detailed *in vitro* characterization of scaffolds, more emphasis should be placed on their evaluation in vivo with respect to inflammatory responses, biocompatibility and regenerative potential.

#### **5. Application of graphene in bioimaging**

Graphene-based nanomaterials have been widely explored in biomedical fields such as bioimaging, drug delivery, theranostics, and so on. The recent advances in bioimaging of graphene-based nanomaterials, including graphene, graphene oxide, reduced graphene oxide, graphene quantum dots, and their derivatives, the synthetic methods of graphene-based nanomaterials are included in situ synthesis and binding method. The bioimaging modalities, including optical imaging (fluorescence [FL], two-photon FL), positron emission tomography/single-photon emission computed tomography, magnetic resonance imaging, photoacoustic imaging, Raman imaging, and multimodal imaging are highlighted (**Figure 8**).

**Figure 8.** *Different types of bioimaging graphene-based bioimaging.*

*Tumor Progression and Metastasis*

carbon nanotube (CNT) and Fe3O4 [79].

**4.2 Gene delivery**

Attachment of nanoparticles such as iron oxide with graphene-based nanomaterial makes them super paramagnetic in property and can be useful in drug delivery applications [78]. The resulting magnetic hybrids dispersed uniformly in aqueous solution before and after loading of DOX. Magnetic hybrids show agglomeration behavior in acidic medium and redispersion behavior observed in basic medium. This pH triggered magnetic behavior of GO-Fe3O4 nanoparticle hybrids can be help in controlled drug delivery. Similar pH-dependent drug release system was reported for 5-FU-loaded nanohybrid system composed of graphene nanosheets (GN),

Gene therapy is used in many expanding area to treat genetic disorders like Parkinson's disease, cystic fibrosis and cancer. An effective gene therapy needs efficient and safe gene vectors that also protect DNA from nuclease degradation as well as facilitate DNA uptake with high transfection efficiency [80, 81]. According to review of literature, graphene has been reported for wide applications in the field of gene delivery, gene-drug co-delivery and protein delivery with. PEI has been extensively investigated as nonviral gene vector having strong electrostatic interactions with negatively charged phosphates of RNA and DNA. Chemical modification is very easy in PEI which offers increased transfection efficiency, cell selectivity and reduced cytotoxicity however low biocompatibility and high toxicity of (Polyethyleneimine) PEI limit its use for biomedical applications [82]. Chitosan-GO complex are also used for simultaneous drug and gene delivery [83]. Chitosan-GO converts pDNA into stable nano-sized complexes. Amineterminated PEGylated GO was effectively used to deliver high protein payloads due to non-covalent interactions with surface of PEG-GO [84]. Bone morphogenic protein-2 (BMP-2) was loaded onto Ti substrate coated with alternate layers of positively (GO-NH3+) and negatively (GO-COO-) charged GO nanosheets with high loading efficacy and conserved bioactivity. Osteogenic differentiation of MSCs was enriched on Ti coated GO surfaces carrying BMP-2 than only Ti surface with BMP-2. In vivo studies in mouse also exhibited vigorous new bone formation with Ti-GO-BMP2 implants compared to Ti or Ti-GO or Ti-BMP2 implants and making the new composite a very effective carrier for therapeutic drug delivery [85]. All above studies have highlighted potential of graphene-based materials as drug and gene delivery vehicles *in vitro* studies though there is a necessity to validate their potential in vivo with particular focus on safety, biodistribution and

Tissue engineering is an interdisciplinary field that endeavors to develop biological substitutes to resolve, retain or enhance functionality of a tissue or whole organ [86]. Recently, graphene-based materials have been used to treat wound healing, stem cell engineering, regenerative medicine and tissue engineering. Hydrogels have viscoelastic and transport properties to mimicking natural tissues [87], but their weak mechanical properties can limit their use in many tissue engineering applications. Graphene has a platform for tailoring various functionalities on flat surfaces with outstanding mechanical properties like high elasticity, strength, flexibility. Potentially, graphene has a wide range of applications in the field of hydrogels, biodegradable films, electrospun fibers and other tissue engineering scaffolds. When GO incorporated into PVA-based hydrogels it potentially increases tensile stability (132%) and

**276**

efficacy.

**4.3 Tissue engineering**

#### **5.1 Optical imaging**

GO- and rGO-based composites are extensively used in the bioimaging field as the arrangement of tissues were comprehend with the help of optical imaging and with the application of unique characteristics of photon-based visible light [92]. As compared to the other progression GO derivatives has immense superiority including high-sensitivity non-ionizing energy, relative and economical advantage, real-time imaging, multiplexing capability, short range free space optical communication [93]. Although, this advancement is affected by low tissue penetration (0–2 cm), high tissue spreading of photons in the visible spectra region and considerable conditions because of tissue auto fluorescence and light absorption by macronutrients, oxygen binding groups and even by water molecules [94]. GO-based composites were dynamically developed for future aspects of different optical imaging techniques, such as (Fluorescence Imaging) FL imaging and (Two-Photon Fluorescence Imaging) TPFI.

#### *5.1.1 Fluorescence imaging*

Fluorescence imaging is a nonpersistent technique depends on the intensity of photons emitted from the probe used for fluorescent imaging [95]. A number of research groups focused on organic fluorescent dyes to fabricate GO and rGO *in vitro* and in vivo FL optical imaging. Liu and coworkers subjugated firstly a visible near infrared fluorescent dye, Cyanin 7 (Cy7), complexed with nGO-PEG and form a imaging system (nGO-PEG-Cy7) for in vivo FL imaging of tumor xenografted mice. The nGO-PEG-Cy7 supposed to be enriched in the tumor over time after intravenous vaccination. Prominent uptake of nGO was observed in the infected area compared with other healthy parts of the mouse after 24 h postinjection for different types of tumor modalities. The result shows high tumor accumulation of nGO-PEG-Cy7 based on the improved permeability and retention effect of malignant tumors [96]. Apart from compiling with organic fluorescent dyes, the other non-fluorescence dyes or porphyrin rings are also directly coated on the graphene surface for this particular imaging. Chen and coworkers reported GO conjugated multifunctional system composed of VEGF-loaded (vascular endothelial growth factor) IR-Dye-800 (e.g., GO-IR-Dye-800-VEGF) for fluorescence imaging of ischemic limb cells in the aquatic posterior limb ischemia mold [97]. Further IR-800 dye was firstly compile with the six headed amino groups of poly ethylene glycol (PEG), then later VEGF was loaded onto the two available basal planes of GO via physisorption. The IR-800 shows the same account of FL emission spectra variation even after combing it with various other components at the particular specific maximum wavelength of 800 nm. This specific imaging property of the fluorescence active compounds is used extensively in the imaging application of the GO-based compounds. When we compared the ischemic limbs with the nonischemic limbs the fluorescence intensity ischemic limbs are stronger than that of nonischemic limbs at after intravenous vaccination, explains that GO-IR800- VEGF could exclusively be used to target ischemic limb sowing the enlarged permeability of blood cells. At 24 h time point p.i., the mice were sacrificed and organs of GO-IR800 and GO-IR800-VEGF-injected mice were harvested for *ex vivo* imaging. Interestingly, both of GO-IR800 and GO-IR800-VEGF were mainly distributed throughout the entire ischemic tissue below the ligation site. When we functionalized a biocompatible polymer on the GO surface it acts as a linkage between the GO and these fluorescence active dye [98]. Recently graphene quantum dots are also came into the scenario as they show some photoluminescent properties when incorporated with GO. The lattices inside the GQDs play an important role in

**279**

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting…*

functionalization process as well as show some extraordinary characteristics such as zig zag geometry, electron hole mobile electron carriers, high photostability and lower toxic index [99]. Nahain and its research group demonstrate the particular sized GQDs with the fluid known as hyaluronic acid for proficient CD44-targeted delivery to tumor-effective BALB/c mice, representing the intense fluorescence image of the tumor cell line [100]. However, the QY of GQDs still needs to be improved for further bioimaging applications. Meanwhile, further surface modification is also necessary to improve the optical belongings of GQDs and improve the

Due to some background noise disturb the fluorescence imaging due to single photon fluorescence imaging Two-Photon Fluorescence Imaging came into the picture. Two-Photon Fluorescence Imaging has fascinated much concentration because of its potential applications in fundamental study and medical diagnostics [101]. With the help of TPFI more detailed examination of various in vivo activities of deep located tumorous cells. Compared with one photon excitation using simple continuous-wave lasers, two-photon nonlinear excitation usually uses a nonlinear femtosecond laser to obtain a high reflux of excitation photons. Recently, graphenebased nanomaterials aroused considerable interest in the field of TPFI. Wang and Gu et al. first reported transferrin functionalized GO-PEG with strong two-photon luminescence as a nonbleaching optical probe for three-dimensional TPFI and laser-based cancer microsurgery, using an ultrafast pulsed laser as the excitation source [102]. Gong group employed nitrogen doping GQDs (N-GQDs) with an average size of ∼3 nm as efficient two photon fluorescent probes for cellular and deep-tissue imaging [103]. Taking dimethylformamide as solvent and nitrogen sources, the nitrogen was successfully doping to GQDs by a facile one-pot solvothermal method. Obviously, the TPFI using N-GQDs as fluorescent probe is particularly suitable for in vivo investigation of biostructures in the 800–1500 μm region.

Optical imaging cannot provide quantitative results and sometimes may be interfered by FL quenching of fluorescent dyes, light absorption and scattering of tissues, and autofluorescence background. Radiolabeling method would be able to accurately track the labeled substances in vivo in a quantitative manner with excellent sensitivity (∼10–11–10–12 mol/L) and nearly no penetration depth limit. The radionuclide-based imaging mainly contains PET and SPECT. PET and SPECT images are acquired over a nominally low background signal and require little signal amplification [104]. Graphene-based nanomaterials as promising nanoplatforms are playing an important role in PET/SPECT imaging. In 2011, Liu et al. reported a method to label nGO-PEG with 125I by anchoring iodine atoms on the defects and edges of GO [105]. After that a number of studies have been developed based on this method. Cai group explored in vivo active tumor targeting using 64Cu-labeled nGO-PEG [106]. In comparison to PET, SPECT is ∼10 times less sensitive (∼10–10–10–11 mol/L); however, SPECT enables concurrent imaging of multiple radionuclides of different energies [107]. Cornelissen et al. explored the use of anti-HER2 antibody (trastuzumab)-conjugated nGO, radiolabeled with 111In-benzyl-diethylenetriaminepentaacetic acid (BnDTPA) via π-π stacking, for in vivo targeting and SPECT imaging. The radiolabeled nGO-trastuzumab conjugates demonstrated better pharmacokinetics compared with radiolabeled trastuzumab without NGO, with more rapid clearance from the circulation [108].

*DOI: http://dx.doi.org/10.5772/intechopen.91331*

*5.1.2 Two-photon fluorescence imaging*

**5.2 Radionuclide-based imaging**

tumor accrual rate.

#### *Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting… DOI: http://dx.doi.org/10.5772/intechopen.91331*

functionalization process as well as show some extraordinary characteristics such as zig zag geometry, electron hole mobile electron carriers, high photostability and lower toxic index [99]. Nahain and its research group demonstrate the particular sized GQDs with the fluid known as hyaluronic acid for proficient CD44-targeted delivery to tumor-effective BALB/c mice, representing the intense fluorescence image of the tumor cell line [100]. However, the QY of GQDs still needs to be improved for further bioimaging applications. Meanwhile, further surface modification is also necessary to improve the optical belongings of GQDs and improve the tumor accrual rate.

#### *5.1.2 Two-photon fluorescence imaging*

*Tumor Progression and Metastasis*

Photon Fluorescence Imaging) TPFI.

*5.1.1 Fluorescence imaging*

GO- and rGO-based composites are extensively used in the bioimaging field as the arrangement of tissues were comprehend with the help of optical imaging and with the application of unique characteristics of photon-based visible light [92]. As compared to the other progression GO derivatives has immense superiority including high-sensitivity non-ionizing energy, relative and economical advantage, real-time imaging, multiplexing capability, short range free space optical communication [93]. Although, this advancement is affected by low tissue penetration (0–2 cm), high tissue spreading of photons in the visible spectra region and considerable conditions because of tissue auto fluorescence and light absorption by macronutrients, oxygen binding groups and even by water molecules [94]. GO-based composites were dynamically developed for future aspects of different optical imaging techniques, such as (Fluorescence Imaging) FL imaging and (Two-

Fluorescence imaging is a nonpersistent technique depends on the intensity of photons emitted from the probe used for fluorescent imaging [95]. A number of research groups focused on organic fluorescent dyes to fabricate GO and rGO *in vitro* and in vivo FL optical imaging. Liu and coworkers subjugated firstly a visible near infrared fluorescent dye, Cyanin 7 (Cy7), complexed with nGO-PEG and form a imaging system (nGO-PEG-Cy7) for in vivo FL imaging of tumor xenografted mice. The nGO-PEG-Cy7 supposed to be enriched in the tumor over time after intravenous vaccination. Prominent uptake of nGO was observed in the infected area compared with other healthy parts of the mouse after 24 h postinjection for different types of tumor modalities. The result shows high tumor accumulation of nGO-PEG-Cy7 based on the improved permeability and retention effect of malignant tumors [96]. Apart from compiling with organic fluorescent dyes, the other non-fluorescence dyes or porphyrin rings are also directly coated on the graphene surface for this particular imaging. Chen and coworkers reported GO conjugated multifunctional system composed of VEGF-loaded (vascular endothelial growth factor) IR-Dye-800 (e.g., GO-IR-Dye-800-VEGF) for fluorescence imaging of ischemic limb cells in the aquatic posterior limb ischemia mold [97]. Further IR-800 dye was firstly compile with the six headed amino groups of poly ethylene glycol (PEG), then later VEGF was loaded onto the two available basal planes of GO via physisorption. The IR-800 shows the same account of FL emission spectra variation even after combing it with various other components at the particular specific maximum wavelength of 800 nm. This specific imaging property of the fluorescence active compounds is used extensively in the imaging application of the GO-based compounds. When we compared the ischemic limbs with the nonischemic limbs the fluorescence intensity ischemic limbs are stronger than that of nonischemic limbs at after intravenous vaccination, explains that GO-IR800- VEGF could exclusively be used to target ischemic limb sowing the enlarged permeability of blood cells. At 24 h time point p.i., the mice were sacrificed and organs of GO-IR800 and GO-IR800-VEGF-injected mice were harvested for *ex vivo* imaging. Interestingly, both of GO-IR800 and GO-IR800-VEGF were mainly distributed throughout the entire ischemic tissue below the ligation site. When we functionalized a biocompatible polymer on the GO surface it acts as a linkage between the GO and these fluorescence active dye [98]. Recently graphene quantum dots are also came into the scenario as they show some photoluminescent properties when incorporated with GO. The lattices inside the GQDs play an important role in

**5.1 Optical imaging**

**278**

Due to some background noise disturb the fluorescence imaging due to single photon fluorescence imaging Two-Photon Fluorescence Imaging came into the picture. Two-Photon Fluorescence Imaging has fascinated much concentration because of its potential applications in fundamental study and medical diagnostics [101]. With the help of TPFI more detailed examination of various in vivo activities of deep located tumorous cells. Compared with one photon excitation using simple continuous-wave lasers, two-photon nonlinear excitation usually uses a nonlinear femtosecond laser to obtain a high reflux of excitation photons. Recently, graphenebased nanomaterials aroused considerable interest in the field of TPFI. Wang and Gu et al. first reported transferrin functionalized GO-PEG with strong two-photon luminescence as a nonbleaching optical probe for three-dimensional TPFI and laser-based cancer microsurgery, using an ultrafast pulsed laser as the excitation source [102]. Gong group employed nitrogen doping GQDs (N-GQDs) with an average size of ∼3 nm as efficient two photon fluorescent probes for cellular and deep-tissue imaging [103]. Taking dimethylformamide as solvent and nitrogen sources, the nitrogen was successfully doping to GQDs by a facile one-pot solvothermal method. Obviously, the TPFI using N-GQDs as fluorescent probe is particularly suitable for in vivo investigation of biostructures in the 800–1500 μm region.

#### **5.2 Radionuclide-based imaging**

Optical imaging cannot provide quantitative results and sometimes may be interfered by FL quenching of fluorescent dyes, light absorption and scattering of tissues, and autofluorescence background. Radiolabeling method would be able to accurately track the labeled substances in vivo in a quantitative manner with excellent sensitivity (∼10–11–10–12 mol/L) and nearly no penetration depth limit. The radionuclide-based imaging mainly contains PET and SPECT. PET and SPECT images are acquired over a nominally low background signal and require little signal amplification [104]. Graphene-based nanomaterials as promising nanoplatforms are playing an important role in PET/SPECT imaging. In 2011, Liu et al. reported a method to label nGO-PEG with 125I by anchoring iodine atoms on the defects and edges of GO [105]. After that a number of studies have been developed based on this method. Cai group explored in vivo active tumor targeting using 64Cu-labeled nGO-PEG [106]. In comparison to PET, SPECT is ∼10 times less sensitive (∼10–10–10–11 mol/L); however, SPECT enables concurrent imaging of multiple radionuclides of different energies [107]. Cornelissen et al. explored the use of anti-HER2 antibody (trastuzumab)-conjugated nGO, radiolabeled with 111In-benzyl-diethylenetriaminepentaacetic acid (BnDTPA) via π-π stacking, for in vivo targeting and SPECT imaging. The radiolabeled nGO-trastuzumab conjugates demonstrated better pharmacokinetics compared with radiolabeled trastuzumab without NGO, with more rapid clearance from the circulation [108].

#### **5.3 Magnetic resonance imaging**

Noninvasive technique MRI have been extensive used for the detection of morphological feature of tissue related bioscience in comparison to other optical imaging. But somehow the lower sensitivity for the detection of different concentration and inappropriate signaling time has been assigned as the huge drawbacks of MRI [109]. While the biomolecules and ions with paramagnetic nature of metal ions having manganese (Mn) and gadolinium (Gd) as major contributions are reported as the toxic in most of the cases [110]. Whereas such metallic ions can be incorporated with GO utilizing chelation procedure in between metals and different graphene layers [111]. The Gd (gadolinium) incorporated graphene oxide and amidoamine polymer dendrimer-based composite for the delivery of anticancerous drugs on the targeting sites have been reported by Wei et al. [112]. The composite of nitrogen doped graphene oxide have been studied for the detection of tumor containing sites on the defective cells. Starting from GO andiron hydrate the reduced graphene oxide-based composites were synthesized by Liu et al. following autoclave-based thermal treatment methodology. The hydrophobic interactionbased functionalization of polyetheneglycol and maleic anhydride-alt-1-octadecene molecule with iron-based nanoparticles were reported by the same to restore the magnetic properties along with the enhancement of thermal stability of developed solutions [113]. Graphene oxide/iron oxide nanoparticle-based system was fabricated to study and diagnosis of pancreatic cancer by Fu et al. [114]. The graphene and iron nanoparticle-based composite was reported as the magnificent composite to help the surgeons into the preparations of cancer cells. The dual mapping is main device that can easily radiate the difference in between normal and RLN tissues, thus further the lymph nodes can be treated with laser. The penetration effects of lower energy waves are much larger and deeper, while the radio waves worked as the low scatter for biomedical systems, i.e., tissues, cells or organs, etc. the PAI cells photographic technique, which take advantage of the absorption of longer wavelength containing waves into thermal energy for thermal expansion [115].

#### **5.4 Photoacoustic imaging**

The reduced graphene nanomaterials are irreplaceable candidates to absorb near infra-red light in comparison to graphene oxide that reflects sp2 hybridization of carbon [116]. The reduced nature of reduced GO reflects hydrophobic nature of the graphene oxide thus finally result its poor water solubility. In order to find out a unique solution, the microwave heating-based-reduced graphene sheets having lower oxygen containing functional groups were synthesized by Patel et al. [117]. The Hummers method has been utilized to reduce the major oxygen content present in the graphitic powder, the methodology of Hummers method includes the acidic oxidations. The GO sheets can also be reduced to rGO by single step thermal reduction methodology and reported rGO possess excellent stability and lesser cell toxicity [118].

#### **5.5 Raman imaging**

The advanced characterizations technique, i.e., the RAMAN technique is excellently advanced tool for the analytical and experimental extension for the detection of related various biochemical problems. The RAMAN spectrum including D and G bands exactly mentions and elaborates the enhancements of combinations of various nanoparticles [119]. The folic acid hybrid incorporated Ag/GO composite have been developed for specifically targeting of defective cancerous cells [120].

**281**

shape [128].

**6. Conclusion and future prospects**

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting…*

The in situ synthesis of gold- and GO-based composites have been incorporated in HeLa 229 cells which have been found to display excellent peaks and shifts in Raman spectra. The gold nanoparticle incorporated with nitrogen doped graphene oxide was reported by Ma and coworkers. The in situ synthesis of gold nanocomposites were also assumed to have physical forces of attraction between NOPs and gold particles. The further modifications of GO and reduced graphene sheets with 2-mercaptopyridine were reported by the non-covalent linkage [121]. Gold-based GO composites were reported for the development of good substrate than the Au NPs. Similarly, polyethylene glycol functionalized gold/copper nanoparticles along with graphene were incorporated through CVD method by the group of Tan et al. [122]. The unique Raman signals of graphite-based nanoparticles were reported along with further cell labeling and SERS detection. Recently, bio-imaging applications with more modalities have gained excellent popularity in recent decade [123].

The multimodality of such imaging applications has been referred as the better technique over the individual imaging technique for the higher accuracy and for the better diagnosis [124, 125]. While the multiphase analysis of single agent lack the potential problem on the probe, i.e., tissues blood for the further removal of impurities along with several doses [126]. Liu et al. have developed rGO-IONP for triple modulation, i.e., FL, PA along with MR [127]. The rGO-based composites of iron and GO have been synthesized via hydrothermal methodology where the polyethylene glycol was incorporated with poly(maleicanhydride-alt-1-octadecene) (C18PMH-PEG), further NIR was performed for the detection of magnetic absorbance. Similarly, Chen et al. reported the graphene oxide-based composite of PEG having non-covalent interactions, i.e., π-π stacking for the detection of tumorous cells. The recent reports of Wu et al. have synthesized the BaGdF5 nanoparticles which were reported to formulate on the graphene oxide sheets in moderate presence of polyethylene glycol. In the transmission electron microscopy (TEM), the exact morphology of GO/BaGdF5-based PEG composite was shown. This showed excellent separation of layers, along with the accuracy in size of sheets, where the size was reported to exist as smaller than corresponding pure GO sheets. While the SAED (selected area electron diffraction) spectra showed the excellently good crystal nature of BaGdF nanoparticles having cubic

The unique ability of catenation of carbon and tendency to form various hybrid orbitals results in the formation of various smart compounds with different physical and chemical properties. Its 2D hexagonally packed unique structure of in-plane sigma C▬C bonds accounts for certain physical and chemical properties in biological media has led to its varied applications in the field of drug delivery, gene delivery, tissue engineering and various imaging techniques, etc. The electrostatic interaction and presence of metallic ions in biological media tends to agglomerate and reduces the surface area of graphene sheets. Therefore, covalent and non-covalent methods of surface modifications are used to increase the efficacy of graphene sheets. Further surface fabrication of GO with various polymers allows its use in the fields of drug delivery, tissue engineering and different imaging techniques. Surface modification by way of exfoliation is used for large scale synthesis of graphene. Modified Hummer's method is a common procedure of synthesizing GO from the

*DOI: http://dx.doi.org/10.5772/intechopen.91331*

**5.6 Multimodal imaging**

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting… DOI: http://dx.doi.org/10.5772/intechopen.91331*

The in situ synthesis of gold- and GO-based composites have been incorporated in HeLa 229 cells which have been found to display excellent peaks and shifts in Raman spectra. The gold nanoparticle incorporated with nitrogen doped graphene oxide was reported by Ma and coworkers. The in situ synthesis of gold nanocomposites were also assumed to have physical forces of attraction between NOPs and gold particles. The further modifications of GO and reduced graphene sheets with 2-mercaptopyridine were reported by the non-covalent linkage [121]. Gold-based GO composites were reported for the development of good substrate than the Au NPs. Similarly, polyethylene glycol functionalized gold/copper nanoparticles along with graphene were incorporated through CVD method by the group of Tan et al. [122]. The unique Raman signals of graphite-based nanoparticles were reported along with further cell labeling and SERS detection. Recently, bio-imaging applications with more modalities have gained excellent popularity in recent decade [123].

#### **5.6 Multimodal imaging**

*Tumor Progression and Metastasis*

**5.4 Photoacoustic imaging**

**5.3 Magnetic resonance imaging**

Noninvasive technique MRI have been extensive used for the detection of morphological feature of tissue related bioscience in comparison to other optical imaging. But somehow the lower sensitivity for the detection of different concentration and inappropriate signaling time has been assigned as the huge drawbacks of MRI [109]. While the biomolecules and ions with paramagnetic nature of metal ions having manganese (Mn) and gadolinium (Gd) as major contributions are reported as the toxic in most of the cases [110]. Whereas such metallic ions can be incorporated with GO utilizing chelation procedure in between metals and different graphene layers [111]. The Gd (gadolinium) incorporated graphene oxide and amidoamine polymer dendrimer-based composite for the delivery of anticancerous drugs on the targeting sites have been reported by Wei et al. [112]. The composite of nitrogen doped graphene oxide have been studied for the detection of tumor containing sites on the defective cells. Starting from GO andiron hydrate the reduced graphene oxide-based composites were synthesized by Liu et al. following autoclave-based thermal treatment methodology. The hydrophobic interactionbased functionalization of polyetheneglycol and maleic anhydride-alt-1-octadecene molecule with iron-based nanoparticles were reported by the same to restore the magnetic properties along with the enhancement of thermal stability of developed solutions [113]. Graphene oxide/iron oxide nanoparticle-based system was fabricated to study and diagnosis of pancreatic cancer by Fu et al. [114]. The graphene and iron nanoparticle-based composite was reported as the magnificent composite to help the surgeons into the preparations of cancer cells. The dual mapping is main device that can easily radiate the difference in between normal and RLN tissues, thus further the lymph nodes can be treated with laser. The penetration effects of lower energy waves are much larger and deeper, while the radio waves worked as the low scatter for biomedical systems, i.e., tissues, cells or organs, etc. the PAI cells photographic technique, which take advantage of the absorption of longer wavelength containing waves into thermal energy for thermal expansion [115].

The reduced graphene nanomaterials are irreplaceable candidates to absorb

of carbon [116]. The reduced nature of reduced GO reflects hydrophobic nature of the graphene oxide thus finally result its poor water solubility. In order to find out a unique solution, the microwave heating-based-reduced graphene sheets having lower oxygen containing functional groups were synthesized by Patel et al. [117]. The Hummers method has been utilized to reduce the major oxygen content present in the graphitic powder, the methodology of Hummers method includes the acidic oxidations. The GO sheets can also be reduced to rGO by single step thermal reduction methodology and reported rGO possess excellent stability and lesser cell

The advanced characterizations technique, i.e., the RAMAN technique is excellently advanced tool for the analytical and experimental extension for the detection of related various biochemical problems. The RAMAN spectrum including D and G bands exactly mentions and elaborates the enhancements of combinations of various nanoparticles [119]. The folic acid hybrid incorporated Ag/GO composite have been developed for specifically targeting of defective cancerous cells [120].

hybridization

near infra-red light in comparison to graphene oxide that reflects sp2

**280**

toxicity [118].

**5.5 Raman imaging**

The multimodality of such imaging applications has been referred as the better technique over the individual imaging technique for the higher accuracy and for the better diagnosis [124, 125]. While the multiphase analysis of single agent lack the potential problem on the probe, i.e., tissues blood for the further removal of impurities along with several doses [126]. Liu et al. have developed rGO-IONP for triple modulation, i.e., FL, PA along with MR [127]. The rGO-based composites of iron and GO have been synthesized via hydrothermal methodology where the polyethylene glycol was incorporated with poly(maleicanhydride-alt-1-octadecene) (C18PMH-PEG), further NIR was performed for the detection of magnetic absorbance. Similarly, Chen et al. reported the graphene oxide-based composite of PEG having non-covalent interactions, i.e., π-π stacking for the detection of tumorous cells. The recent reports of Wu et al. have synthesized the BaGdF5 nanoparticles which were reported to formulate on the graphene oxide sheets in moderate presence of polyethylene glycol. In the transmission electron microscopy (TEM), the exact morphology of GO/BaGdF5-based PEG composite was shown. This showed excellent separation of layers, along with the accuracy in size of sheets, where the size was reported to exist as smaller than corresponding pure GO sheets. While the SAED (selected area electron diffraction) spectra showed the excellently good crystal nature of BaGdF nanoparticles having cubic shape [128].

#### **6. Conclusion and future prospects**

The unique ability of catenation of carbon and tendency to form various hybrid orbitals results in the formation of various smart compounds with different physical and chemical properties. Its 2D hexagonally packed unique structure of in-plane sigma C▬C bonds accounts for certain physical and chemical properties in biological media has led to its varied applications in the field of drug delivery, gene delivery, tissue engineering and various imaging techniques, etc. The electrostatic interaction and presence of metallic ions in biological media tends to agglomerate and reduces the surface area of graphene sheets. Therefore, covalent and non-covalent methods of surface modifications are used to increase the efficacy of graphene sheets. Further surface fabrication of GO with various polymers allows its use in the fields of drug delivery, tissue engineering and different imaging techniques. Surface modification by way of exfoliation is used for large scale synthesis of graphene. Modified Hummer's method is a common procedure of synthesizing GO from the

natural mineral source, i.e., flake graphite. The carboxylic group is found on the surface of graphite during the process of oxidation and exfoliation is modified by covalent functionalization making its use possible in the studies of biological systems and also found applications in biological activities like anti-bacterial activity, bioimaging and photodynamic therapy. Non-covalent functionalization has added advantage of not altering the structure or electronic properties of graphene while introducing new chemical groups on its surface. This results in enhancement of its dispersibility, biocompatibility, reactivity, binding capacity and sensing properties. Graphene has the properties of high surface region, distinctive geometry and structure, flexibility, extra ordinary physico-chemical properties, counting the high fracture strength, high Young's modulus, great thermal and electrical conductivity, highly mobile charge carriers and biocompatibility. All mentioned properties makes graphene a valuable and important material for applications in biological systems and other biomedical processes.

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting…*

enable commercial scale applications of GO-based nanomaterials.

graphene from a little known nanoparticle to wide spread interest in the field of development of graphene-based nanomaterial for applications in biochemical and biophysical systems and processes. However, much research work is still desired to

Authors acknowledge the financial assistance from NMHS research grant KU/

NMHS/MG/2016/002/8603/007, GBPIHED, Kosi-Katarmal, Almora, India.

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Department of Chemistry, Prof. Rajendra Singh Nanoscience and Nanotechnology

2 Department of Chemistry, Graphic Era Hill University, Uttarakhand, India

, Pushpa Negi<sup>2</sup>

and Nanda Gopal Sahoo1

\*

, Himani Tiwari1

Centre, Kumaun University, Nainital, Uttarakhand, India

\*Address all correspondence to: ngsahoo@yahoo.co.in

*DOI: http://dx.doi.org/10.5772/intechopen.91331*

**Acknowledgements**

**283**

**Author details**

Neha Karki1

provided the original work is properly cited.

, Anita Rana1

In contrast to pristine graphene synthesized GO has high dispersibility in physiological media leading to better contact with biologically important organic molecules. Outstanding thermal and electrical conductivity and very low thermal expansion quotient of GO allows its use in energy conversion storage devices and bio sensors. GO derivative-based optoelectronic devices have been developed as IR detectors and electrophysiological modulators and emitters. The optical properties like intense light transmittance, fluorescence, photoluminescence and high mobility of charge make graphene an important material for application in MRI and biomedical imaging. Superior mechanical properties of GO like high tensile strength and extensive stiffness has enabled its use in the field of biomedical implant and tissue engineering. Cell-graphene and biomolecule-graphene studies have opened a vast area of GOs exploration in the fields of cellular biology, genomics and development of antibiotics, etc. Despite its varied uses certain challenges still remain in the field of nanotheranostic designing in terms of bioavailability, selectivity, biocompatibility and safety. In the field of pharmacology, better targeted and relatively lower dose drug delivery with graphene complex has proved cheaper and efficient than the discovery of newer drug. Graphene-based materials as drug and gene delivery vehicles have used successfully in *in vitro* studies, however much research in in vivo studies is still in early stages. Many researchers have focused on developing graphene-based materials for wound healing, stem cell imaging, regenerative medicine and tissue engineering. Graphene finds its application in bio imaging by way of optical imaging, fluorescence Imaging (FL) and two photon fluorescence imaging (TPFI), etc. High sensitivity non-ionizing energy, real time imaging, multiplexing capability, short range free space optical communication and economic advantages makes GO derivative superior for use in optical imaging. However, low tissue penetration, high tissue spreading of photons in the visible spectra, tissue auto fluorescence and light absorption by oxygen binding groups and water limits the use of GO derivatives in optical imaging. Labeling of fluorescent dyes on GO surface and their detection by photons emission from probe has been enabled the development of fluorescent imaging technique for the study of biological systems. Recently certain desirable properties of GQDs like luminescence, zig zag geometry, electron hole mobile electron carriers, high photostability and lower toxic index have enabled development of better FL imaging system. The limitations of FL imaging leads to development of TPFI using non-linear femto second laser to obtain a high reflux of excitation photons, thereby enabling the development of better and deeper fluorescent imaging probes. Radio labeling of graphene-based nanomaterials has increased the sensitivity of qualitative imaging studies like PET/SPECT further doped GO has found its application in MRI for detailed study of different tissues in humans. Past few years have witnessed the development of

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting… DOI: http://dx.doi.org/10.5772/intechopen.91331*

graphene from a little known nanoparticle to wide spread interest in the field of development of graphene-based nanomaterial for applications in biochemical and biophysical systems and processes. However, much research work is still desired to enable commercial scale applications of GO-based nanomaterials.

#### **Acknowledgements**

*Tumor Progression and Metastasis*

and other biomedical processes.

natural mineral source, i.e., flake graphite. The carboxylic group is found on the surface of graphite during the process of oxidation and exfoliation is modified by covalent functionalization making its use possible in the studies of biological systems and also found applications in biological activities like anti-bacterial activity, bioimaging and photodynamic therapy. Non-covalent functionalization has added advantage of not altering the structure or electronic properties of graphene while introducing new chemical groups on its surface. This results in enhancement of its dispersibility, biocompatibility, reactivity, binding capacity and sensing properties. Graphene has the properties of high surface region, distinctive geometry and structure, flexibility, extra ordinary physico-chemical properties, counting the high fracture strength, high Young's modulus, great thermal and electrical conductivity, highly mobile charge carriers and biocompatibility. All mentioned properties makes graphene a valuable and important material for applications in biological systems

In contrast to pristine graphene synthesized GO has high dispersibility in physiological media leading to better contact with biologically important organic molecules. Outstanding thermal and electrical conductivity and very low thermal expansion quotient of GO allows its use in energy conversion storage devices and bio sensors. GO derivative-based optoelectronic devices have been developed as IR detectors and electrophysiological modulators and emitters. The optical properties like intense light transmittance, fluorescence, photoluminescence and high mobility of charge make graphene an important material for application in MRI and biomedical imaging. Superior mechanical properties of GO like high tensile strength and extensive stiffness has enabled its use in the field of biomedical implant and tissue engineering. Cell-graphene and biomolecule-graphene studies have opened a vast area of GOs exploration in the fields of cellular biology, genomics and development of antibiotics, etc. Despite its varied uses certain challenges still remain in the field of nanotheranostic designing in terms of bioavailability, selectivity, biocompatibility and safety. In the field of pharmacology, better targeted and relatively lower dose drug delivery with graphene complex has proved cheaper and efficient than the discovery of newer drug. Graphene-based materials as drug and gene delivery vehicles have used successfully in *in vitro* studies, however much research in in vivo studies is still in early stages. Many researchers have focused on developing graphene-based materials for wound healing, stem cell imaging, regenerative medicine and tissue engineering. Graphene finds its application in bio imaging by way of optical imaging, fluorescence Imaging (FL) and two photon fluorescence imaging (TPFI), etc. High sensitivity non-ionizing energy, real time imaging, multiplexing capability, short range free space optical communication and economic advantages makes GO derivative superior for use in optical imaging. However, low tissue penetration, high tissue spreading of photons in the visible spectra, tissue auto fluorescence and light absorption by oxygen binding groups and water limits the use of GO derivatives in optical imaging. Labeling of fluorescent dyes on GO surface and their detection by photons emission from probe has been enabled the development of fluorescent imaging technique for the study of biological systems. Recently certain desirable properties of GQDs like luminescence, zig zag geometry, electron hole mobile electron carriers, high photostability and lower toxic index have enabled development of better FL imaging system. The limitations of FL imaging leads to development of TPFI using non-linear femto second laser to obtain a high reflux of excitation photons, thereby enabling the development of better and deeper fluorescent imaging probes. Radio labeling of graphene-based nanomaterials has increased the sensitivity of qualitative imaging studies like PET/SPECT further doped GO has found its application in MRI for detailed study of different tissues in humans. Past few years have witnessed the development of

**282**

Authors acknowledge the financial assistance from NMHS research grant KU/ NMHS/MG/2016/002/8603/007, GBPIHED, Kosi-Katarmal, Almora, India.

#### **Author details**

Neha Karki1 , Anita Rana1 , Himani Tiwari1 , Pushpa Negi<sup>2</sup> and Nanda Gopal Sahoo1 \*

1 Department of Chemistry, Prof. Rajendra Singh Nanoscience and Nanotechnology Centre, Kumaun University, Nainital, Uttarakhand, India

2 Department of Chemistry, Graphic Era Hill University, Uttarakhand, India

\*Address all correspondence to: ngsahoo@yahoo.co.in

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[111] Yoo JM, Kang JH, Hong BH. Graphene-based nanomaterials for versatile imaging studies. Chemical Society Reviews. 2015;**44**(14):4835-4852

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[113] Yang K, Gong H, Shi X, Wan J, Zhang Y, Liu Z. In vivo biodistribution and toxicology of functionalized nano-graphene oxide in mice after oral and intraperitoneal administration. Biomaterials. 2013;**34**(11):2787-2795

[114] Wang YW, Fu YY, Peng Q, Guo SS, Liu G, Li J, et al. Dye-enhanced graphene oxide for photothermal therapy and photoacoustic imaging. Journal of Materials Chemistry B. 2013;**1**(42):5762-5767

[115] Huang J, Zong C, Shen H, Liu M, Chen B, Ren B, et al. Mechanism of cellular uptake of graphene oxide studied by surface-enhanced Raman spectroscopy. Small. 2012;**8**(16):2577-2584

[116] Lalwani G, Cai X, Nie L, Wang LV, Sitharaman B. Graphenebased contrast agents for photoacoustic and thermoacoustic tomography. Photoacoustics. 2013;**1**(3-4):62-67

[117] Patel MA, Yang H, Chiu PL, Mastrogiovanni DD, Flach CR, Savaram K, et al. Direct production of graphene nanosheets for near infrared

**291**

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting…*

therapy. Advanced Materials. 2014;**26**(37):6401-6408

2012;**116**(48):14062-14070

2012;**338**(6109):903-910

[126] Cheng Z, Al Zaki A, Hui JZ, Muzykantov VR, Tsourkas A. Multifunctional nanoparticles: Cost versus benefit of adding targeting and imaging capabilities. Science.

[127] Rong P, Yang K, Srivastan A, Kiesewetter DO, Yue X, Wang F, et al. Photosensitizer loaded nanographene for multimodality imaging guided tumor photodynamic therapy. Theranostics. 2014;**4**(3):229-239

[128] Wu H, Shi H, Wang Y, Jia X, Tang C, Zhang J, et al. Hyaluronic acid conjugated graphene oxide for targeted drug delivery. Carbon.

2014;**69**(12):379-389

[125] Ma J, Huang P, He M, Pan L, Zhou Z, Feng L, et al. Folic acidconjugated Laf3:Yb,Tm@SiO2 nanoprobes for targeting dual-modality imaging of upconversion luminescence and X-ray computed tomography. The Journal of Physical Chemistry. B.

*DOI: http://dx.doi.org/10.5772/intechopen.91331*

photoacoustic imaging. ACS Nano.

[118] Sheng Z, Song L, Zheng J, Hu D, He M, Zheng M, et al. Proteinassisted fabrication of nano-reduced graphene oxide for combined in vivo photoacoustic imaging and photothermal therapy. Biomaterials.

[119] Kim YK, Han SW, Min DH. Graphene oxide sheath on Ag

nanoparticle/graphene hybrid films as an antioxidative coating and enhancer of surface-enhanced Raman scattering. ACS Applied Materials & Interfaces.

[120] Liu Z, Guo Z, Zhong H, Qin X, Wan M, Yang B. Graphene oxide based surface enhanced Raman scattering probes for cancer cell imaging.

Physical Chemistry Chemical Physics.

[121] Ma X, Qu Q, Zhao Y, Luo Z, Zhao Y, Ng KW, et al. Graphene oxide wrapped gold nanoparticles for intracellular Raman imaging and drug delivery. Journal of Materials Chemistry B. 2013;**1**(47):6495-6500

[122] Song ZL, Chen Z, Bian X, Zhou LY, Ding D, Liang H, et al. Alkyne-functionalized superstable graphitic silver nanoparticles for Raman imaging. Journal of the American Chemical Society. 2014;**136**(39):13558-13561

[123] Park K, Lee S, Kang E, Kim K, Choi K, Kwon IC. New generation of multifunctional nanoparticles for cancer imaging and therapy. Advanced Functional Materials.

[124] Huang P, Rong P, Jin A, Yan X, Zhang MG, Lin J, et al. Dye-loaded ferritin nanocages for multimodal imaging and photothermal

2009;**19**(10):1553-1566

2013;**7**(9):8147-8157

2013;**34**(21):5236-5243

2012;**4**(12):6545-6551

2013;**15**(8):2961-2966

*Theranostics Application of Graphene-Based Materials in Cancer Imaging, Targeting… DOI: http://dx.doi.org/10.5772/intechopen.91331*

photoacoustic imaging. ACS Nano. 2013;**7**(9):8147-8157

*Tumor Progression and Metastasis*

Chemie, International Edition.

[103] Liu Q, Guo B, Rao Z, Zhang B, Gong JR. Strong two-photon-induced fluorescence from photostable, biocompatible nitrogen-doped graphene quantum dots for cellular and deep-tissue imaging. Nano Letters.

[104] Park K, Lee S, Kang E, Kim K, Choi K, Kwon IC. New generation of multifunctional nanoparticles for cancer imaging and therapy. Advanced Functional Materials.

[105] Yang K, Zhang S, Zhang G,

Letters. 2010;**10**(9):3318-3323

[106] Hong H, Yang K, Zhang Y, Engle JW, Feng L, Yang Y, et al. In vivo targeting and imaging of tumor vasculature with radiolabeled, antibodyconjugated nanographene. ACS Nano.

[107] Sun Z, Huang P, Tong G,

Lin J, Jin A, Rong P, et al. VEGF-loaded graphene oxide as theranostics for multi-modality imaging-monitored targeting therapeutic angiogenesis of ischemic muscle. Nanoscale.

2012;**6**(3):2361-2370

2013;**5**(1):6857-6866

2013;**34**:1146-1154

[108] Cornelissen B, Able S,

Kersemans V, Waghorn PA, Myhra S, Jurkshat K, et al. Nanographene oxidebased radioimmunoconstructs for in vivo targeting and SPECT imaging of HER2-positive tumors. Biomaterials.

[109] Jiang L, Fan Z. Design of advanced porous graphene materials: From

Sun X, Lee ST, Liu Z. Graphene in mice: Ultrahigh *in vivo* tumor uptake and efficient photothermal therapy. Nano

2012;**51**(8):1830-1834

2013;**13**(6):2436-2441

2009;**19**(10):1553-1566

nanoparticles as nonbleaching optical probe for two-photon luminescence imaging and cell therapy. Angewandte graphene nanomesh to 3D architectures.

Nanoscale. 2014;**6**(4):1922-1945

[110] Caravan P, Ellison JJ, TJ MM, Lauffer RB. Gadolinium(III) chelates as MRI contrast agents: Structure, dynamics, and applications. Chemical Reviews. 1999;**99**(9):2293-2352

[111] Yoo JM, Kang JH, Hong BH. Graphene-based nanomaterials for versatile imaging studies. Chemical Society Reviews. 2015;**44**(14):4835-4852

[112] Yang HW, Huang CY, Lin CW, Liu HL, Huang CW, Liao SS, et al. Gadolinium-functionalized

[113] Yang K, Gong H, Shi X, Wan J, Zhang Y, Liu Z. In vivo biodistribution and toxicology of functionalized nano-graphene oxide in mice after oral and intraperitoneal administration. Biomaterials. 2013;**34**(11):2787-2795

[114] Wang YW, Fu YY, Peng Q,

2013;**1**(42):5762-5767

Guo SS, Liu G, Li J, et al. Dye-enhanced graphene oxide for photothermal therapy and photoacoustic imaging. Journal of Materials Chemistry B.

[115] Huang J, Zong C, Shen H, Liu M, Chen B, Ren B, et al. Mechanism of cellular uptake of graphene oxide studied by surface-enhanced Raman spectroscopy. Small. 2012;**8**(16):2577-2584

[116] Lalwani G, Cai X, Nie L, Wang LV, Sitharaman B. Graphenebased contrast agents for photoacoustic and thermoacoustic tomography. Photoacoustics. 2013;**1**(3-4):62-67

[117] Patel MA, Yang H, Chiu PL, Mastrogiovanni DD, Flach CR, Savaram K, et al. Direct production of graphene nanosheets for near infrared

2014;**35**(24):6534-6542

nanographene oxide for combined drug and microrna delivery and magnetic resonance imaging. Biomaterials.

**290**

[118] Sheng Z, Song L, Zheng J, Hu D, He M, Zheng M, et al. Proteinassisted fabrication of nano-reduced graphene oxide for combined in vivo photoacoustic imaging and photothermal therapy. Biomaterials. 2013;**34**(21):5236-5243

[119] Kim YK, Han SW, Min DH. Graphene oxide sheath on Ag nanoparticle/graphene hybrid films as an antioxidative coating and enhancer of surface-enhanced Raman scattering. ACS Applied Materials & Interfaces. 2012;**4**(12):6545-6551

[120] Liu Z, Guo Z, Zhong H, Qin X, Wan M, Yang B. Graphene oxide based surface enhanced Raman scattering probes for cancer cell imaging. Physical Chemistry Chemical Physics. 2013;**15**(8):2961-2966

[121] Ma X, Qu Q, Zhao Y, Luo Z, Zhao Y, Ng KW, et al. Graphene oxide wrapped gold nanoparticles for intracellular Raman imaging and drug delivery. Journal of Materials Chemistry B. 2013;**1**(47):6495-6500

[122] Song ZL, Chen Z, Bian X, Zhou LY, Ding D, Liang H, et al. Alkyne-functionalized superstable graphitic silver nanoparticles for Raman imaging. Journal of the American Chemical Society. 2014;**136**(39):13558-13561

[123] Park K, Lee S, Kang E, Kim K, Choi K, Kwon IC. New generation of multifunctional nanoparticles for cancer imaging and therapy. Advanced Functional Materials. 2009;**19**(10):1553-1566

[124] Huang P, Rong P, Jin A, Yan X, Zhang MG, Lin J, et al. Dye-loaded ferritin nanocages for multimodal imaging and photothermal

therapy. Advanced Materials. 2014;**26**(37):6401-6408

[125] Ma J, Huang P, He M, Pan L, Zhou Z, Feng L, et al. Folic acidconjugated Laf3:Yb,Tm@SiO2 nanoprobes for targeting dual-modality imaging of upconversion luminescence and X-ray computed tomography. The Journal of Physical Chemistry. B. 2012;**116**(48):14062-14070

[126] Cheng Z, Al Zaki A, Hui JZ, Muzykantov VR, Tsourkas A. Multifunctional nanoparticles: Cost versus benefit of adding targeting and imaging capabilities. Science. 2012;**338**(6109):903-910

[127] Rong P, Yang K, Srivastan A, Kiesewetter DO, Yue X, Wang F, et al. Photosensitizer loaded nanographene for multimodality imaging guided tumor photodynamic therapy. Theranostics. 2014;**4**(3):229-239

[128] Wu H, Shi H, Wang Y, Jia X, Tang C, Zhang J, et al. Hyaluronic acid conjugated graphene oxide for targeted drug delivery. Carbon. 2014;**69**(12):379-389

**Chapter 12**

**Abstract**

**1. Introduction**

**293**

Intelligence

*Nolan D. Rea and José Abella*

Tumor Malignancy

Characterization in Clinical

Using the FYC-Index of

Spiculation and Artificial

*Fernando Yepes-Calderón, Flor M. Medina,*

According to the World Health Organization, cancer is the second leading cause of death in the world. The myriad of variations, paths of development, and mutations make this abnormality challenging to treat. With the advent of medical imaging, complex qualitative information is collected with the aim of characterizing the pathology; however, the uncomfortable and time-consuming histology remains the state of care within hospitals. This manuscript presents a strategy to extract quantifiable features from the images. The method captures shape perturbation as variations in reference to a perfect circle that is used in a standardized dimensional space. A multifeatured scheme is created when the quantification is applied in all slices produced by imaging modalities such as computed tomography, magnetic resonance imaging, and tomosynthesis. Later, the numbers obtained by the introduced algorithm are used in an artificial intelligence pipeline that correlates spicularity with aggressiveness using the histology as supervising factor.

**Keywords:** medical image analysis, tumor grading, cancer, tumor characterization

Classifying cancer lesions in form and intensity from the images is of interest in radiology units [1–3]. Currently, histology is the gold standard to define cancer type, stage, and grade; nevertheless, histology comes with its associated costs and delays and has been reported to increase morbidity [4, 5]. When diagnosing from the images, the desired classification is accurate and repeatable only if the operator includes the quantitative domain to the set of available tools that are mostly from the qualitative domain. The quantification is accomplished by separating the neo-

mass from the anatomical parts in the image employing segmentation.

Environments: An Approach

#### **Chapter 12**

## Tumor Malignancy Characterization in Clinical Environments: An Approach Using the FYC-Index of Spiculation and Artificial Intelligence

*Fernando Yepes-Calderón, Flor M. Medina, Nolan D. Rea and José Abella*

#### **Abstract**

According to the World Health Organization, cancer is the second leading cause of death in the world. The myriad of variations, paths of development, and mutations make this abnormality challenging to treat. With the advent of medical imaging, complex qualitative information is collected with the aim of characterizing the pathology; however, the uncomfortable and time-consuming histology remains the state of care within hospitals. This manuscript presents a strategy to extract quantifiable features from the images. The method captures shape perturbation as variations in reference to a perfect circle that is used in a standardized dimensional space. A multifeatured scheme is created when the quantification is applied in all slices produced by imaging modalities such as computed tomography, magnetic resonance imaging, and tomosynthesis. Later, the numbers obtained by the introduced algorithm are used in an artificial intelligence pipeline that correlates spicularity with aggressiveness using the histology as supervising factor.

**Keywords:** medical image analysis, tumor grading, cancer, tumor characterization

#### **1. Introduction**

Classifying cancer lesions in form and intensity from the images is of interest in radiology units [1–3]. Currently, histology is the gold standard to define cancer type, stage, and grade; nevertheless, histology comes with its associated costs and delays and has been reported to increase morbidity [4, 5]. When diagnosing from the images, the desired classification is accurate and repeatable only if the operator includes the quantitative domain to the set of available tools that are mostly from the qualitative domain. The quantification is accomplished by separating the neomass from the anatomical parts in the image employing segmentation.

Regarding segmentation, authors have proposed assisting techniques that partially or fully accomplish the tasks with different levels of accuracy [6–8].

voxels of 1 mm before selecting the biggest mask through area calculation. Next, the dimensions of the biggest bounding box are used as a dimensional template. Then, the other slices in the study—including those of other tumors in case we are working with a population—are scaled to the dimensions of the biggest bounding box. This process also centers the masks. Distortion in the mask growing process is avoided by using the adaptive supersampling method [23, 24]. After scaling, all the images share the same field of view (FOV) and

**Block II.** Then, the edges of the masks are detected using the Canny edge

**Block III.** As the Canny detector does not create single-pixel edges, the system detects two paths corresponding to the outer and inner edges. The Euclidean distance from the artificially created center of coordinates to each point in the edge is saved in two arrays, one corresponds to the outer edge and the other to the inner edge. The two arrays are averaged in an ordered array of distances (AoD). The run along the edge that creates the AoD is standardized by starting the distance calculation at the top center of the image and taking the edging points in a clockwise fashion until the starting point is located at a distance of

ð Þ<sup>2</sup> <sup>p</sup> mm of the current point or below. Recall that voxels are all set to 1 mm. **Block IV.** The AoD is Gaussian filtered creating the FAoD. This filtering is intended to eliminate the high-frequency components produced by the digital grid. The filter is implemented in the frequency domain, keeping 80% of the original spectral power. According to [26], maintaining the 80% of the signal spectral power assures that the important content of the signals is kept. **Block V.** A five-point differentiation is applied to find the regions of rapid change; next, a second five-point differentiation is executed to recover the inflection points. These operations are generalized to each point in FAoD as

*<sup>n</sup>* ¼ ½ � ð Þþ *dn* � *dn*�<sup>2</sup> ð Þþ *dn* � *dn*�<sup>1</sup> ð Þþ *dn*þ<sup>1</sup> � *dn* ð Þ *dn*þ<sup>2</sup> � *dn =*4 (1)

The second derivative "p"— obtained with the second pass of Eq. (1)—is where peaks are detected. The peak elements on FAoD are exalted, while regions of low dynamics in the same array are diminished when raising FAoD to the fourth power. **Block VI.** A moving window integration selects peaks in the exponential second derivative. Given points *p s*ðÞ¼ ð Þ *s; d* , the area ð Þ *A* under the curve section with a width ð Þ *N* is calculated for step ð Þ*s* , as it is shown in Eq. (2). If *A s*ð Þ>*T* for a

> *sN* 2 � �

**Block VII.** The spiculation quantifying process is executed in axial and sagittal

**Block VIII.** The location of the detected peaks is then crossed; we only kept the

**Block IX.** These points when mapped back in the images uplift the regions where the tumor presents a highly disorganized growing pattern.

**Block X.** Each slice in the study contributes to the histogram signature of the tumor. The FYC-Index defines the span of the histograms by using the maximum and minimum amount of points found in the slices when working on a single mass or among all analyzed tumors when working on populations.

� *<sup>N</sup>* <sup>2</sup> <sup>þ</sup> *<sup>i</sup>*

� � � � (2)

chosen value of *T*, *s* is added to this list of locations of peaks:

*N* 2 *<sup>i</sup>*¼�*<sup>N</sup>* 2 *p*

*A s*ðÞ¼ ∑

points that coincide in both views.

therefore, the same planar coordinates for the center point.

*Tumor Malignancy Characterization in Clinical Environments: An Approach…*

detector [25].

*DOI: http://dx.doi.org/10.5772/intechopen.82145*

ffiffiffiffiffiffi

shown in **Eq. 1**:

*p*0

views.

**295**

After segmentation, the challenge is finding a repeatable and performant method for all kinds of cancer manifestations. Some quantifying approaches target cancer in specific parts of the body [9, 10], while others focus on particular kinds of cancer [11, 12].

Although technology has invaded the medical facilities, currently assisting tools are not of help in diagnosing cancer. The tasks are still performed by human experts employing purely qualitative judgment. There is a need to quantify and thus abandon the uncertainty produced by human variability.

In practice, qualitative features suggested by X-rads [13, 14] such as roughness and stiffness are difficult to conceptualize with mathematical models; therefore, indexes based on these features are complicated to model [15]. However, the shape of the captured objects is a stable feature in the field of view [16, 17] and, conveniently, has the required sensibility across all cancer manifestations because it captures the core manifestations of the disease, the disordered growth pattern [18, 19]. More importantly, tumor shape is quantified in a feature-enriched scheme to favor further machine-learning implementation. In this document, we employ the FYC-Index of spiculation [20] to assert quantification on the edges of breast tumors imaged with tomosynthesis [21, 22]. The numbers yielded by the FYC-Index strategy are fed to an artificial intelligence classifier that initially differentiates between benign and malign neo-masses, showing a high degree of accuracy in supervised experiments. The presented strategy is equally performant in all imaging techniques that generate volumetric representations by slicing, including MRI, CT, and tomosynthesis.

#### **2. Materials and methods**

#### **2.1 Clinical data**

A cohort of 48 breast tomosynthesis images underwent segmentation performed by an expert radiologist. Histology was performed on the 48 masses yielding 29 malignant cases and 19 benign. The resulting masks hold the specifications of the original images regarding the field of view and spatial resolution. Since the algorithm explained in Section 2.2 is immune to resolution changes and the field of view is standardized, records of the images'specifications are not provided in this document.

#### **2.2 The FYC-Index of spiculation**

The reader is invited to refer to **Figure 1**. Recall that the procedure explained below is used on both views, axial and sagittal.

**Block I.** Images of CT, MRI, or tomosynthesis are suitable for this processing due to their slicing nature. The FYC-Index re-sample all masks to isometric

**Figure 1.** *Block diagrams for the FYC-Index pipeline.* *Tumor Malignancy Characterization in Clinical Environments: An Approach… DOI: http://dx.doi.org/10.5772/intechopen.82145*

voxels of 1 mm before selecting the biggest mask through area calculation. Next, the dimensions of the biggest bounding box are used as a dimensional template. Then, the other slices in the study—including those of other tumors in case we are working with a population—are scaled to the dimensions of the biggest bounding box. This process also centers the masks. Distortion in the mask growing process is avoided by using the adaptive supersampling method [23, 24]. After scaling, all the images share the same field of view (FOV) and therefore, the same planar coordinates for the center point.


$$p'\_n = [(d\_n - d\_{n-2}) + (d\_n - d\_{n-1}) + (d\_{n+1} - d\_n) + (d\_{n+2} - d\_n)]/4 \tag{1}$$

The second derivative "p"— obtained with the second pass of Eq. (1)—is where peaks are detected. The peak elements on FAoD are exalted, while regions of low dynamics in the same array are diminished when raising FAoD to the fourth power.

**Block VI.** A moving window integration selects peaks in the exponential second derivative. Given points *p s*ðÞ¼ ð Þ *s; d* , the area ð Þ *A* under the curve section with a width ð Þ *N* is calculated for step ð Þ*s* , as it is shown in Eq. (2). If *A s*ð Þ>*T* for a chosen value of *T*, *s* is added to this list of locations of peaks:

$$A(s) = \sum\_{i=-\frac{N}{2}}^{\frac{N}{2}} p\left[\left(\frac{sN}{2}\right) - \left(\frac{N}{2} + i\right)\right] \tag{2}$$

**Block VII.** The spiculation quantifying process is executed in axial and sagittal views.

**Block VIII.** The location of the detected peaks is then crossed; we only kept the points that coincide in both views.


Regarding segmentation, authors have proposed assisting techniques that par-

Although technology has invaded the medical facilities, currently assisting tools are not of help in diagnosing cancer. The tasks are still performed by human experts employing purely qualitative judgment. There is a need to quantify and thus aban-

In practice, qualitative features suggested by X-rads [13, 14] such as roughness and stiffness are difficult to conceptualize with mathematical models; therefore, indexes based on these features are complicated to model [15]. However, the shape of the captured objects is a stable feature in the field of view [16, 17] and, conveniently, has the required sensibility across all cancer manifestations because it captures the core manifestations of the disease, the disordered growth pattern [18, 19]. More importantly, tumor shape is quantified in a feature-enriched scheme to favor further machine-learning implementation. In this document, we employ the FYC-Index of spiculation [20] to assert quantification on the edges of breast tumors imaged with tomosynthesis [21, 22]. The numbers yielded by the FYC-Index strategy are fed to an artificial intelligence classifier that initially differentiates between benign and malign neo-masses, showing a high degree of accuracy in supervised experiments. The presented strategy is equally performant in all imaging techniques that generate volumetric representations by slicing, including MRI, CT,

A cohort of 48 breast tomosynthesis images underwent segmentation performed

The reader is invited to refer to **Figure 1**. Recall that the procedure explained

**Block I.** Images of CT, MRI, or tomosynthesis are suitable for this processing due to their slicing nature. The FYC-Index re-sample all masks to isometric

by an expert radiologist. Histology was performed on the 48 masses yielding 29 malignant cases and 19 benign. The resulting masks hold the specifications of the original images regarding the field of view and spatial resolution. Since the algorithm explained in Section 2.2 is immune to resolution changes and the field of view is standardized, records of the images'specifications are not provided in this document.

tially or fully accomplish the tasks with different levels of accuracy [6–8]. After segmentation, the challenge is finding a repeatable and performant method for all kinds of cancer manifestations. Some quantifying approaches target cancer in specific parts of the body [9, 10], while others focus on particular kinds of

don the uncertainty produced by human variability.

cancer [11, 12].

*Tumor Progression and Metastasis*

and tomosynthesis.

**2.1 Clinical data**

**Figure 1.**

**294**

**2. Materials and methods**

**2.2 The FYC-Index of spiculation**

*Block diagrams for the FYC-Index pipeline.*

below is used on both views, axial and sagittal.

Under the FYC-Index domain, while more spiculation, the histogram profiles are more populated in the right side.

ndata = est . transform (mdata) estsvm = svm . LinearSVC ()

*DOI: http://dx.doi.org/10.5772/intechopen.82145*

**3. Results**

**Figure 2.**

**297**

**3.1 FYC-Index extraction**

gs = grid\_search . GridSearchCV (estsvm, {'C' : np . logspace (–4,3)})

*Tumor Malignancy Characterization in Clinical Environments: An Approach…*

experiment that yielded the highest accuracy values per folding.

tscore = np . mean (cross\_validation . cross\_val\_score (gs, ndata, lbls, n\_jobs=5)) As it is shown in Listing 1, the SVM classification is done after progressively adding features which are grabbed from the *mdata* matrix using the indexes saved in the features' array. The accuracy records presented in Section 4 correspond to the

**Figure 2** shows how the algorithm yields two different outcomes based on the tortuosity of the two analyzed shapes. The small shape refers to a mostly rounded region of interest (ROI), therefore, does not present abrupt changes in the distances from the edging points to the center of FOV. In contrast, the same measure yields rapidly changing distances in the big ROI. Those rapidly changing distances are captured by the first derivate and framed in their inflection points by the second derivate. Later, those points are amplified and made all positive by the fourth power function, while the same fourth power function diminishes changes in which the derivate yielded values in the range (1, 1). As the moving window adds up all values encountered in its domain, the regions of rapid change represented by large values compute to higher numbers within the domain of the moving window, and that is where the enhanced points appear in the plot. As all the points are mapped with their original coordinates, a crossing of 3D positions among the selected points

*FYC-Index extraction. The inner loop is the detailed block diagram similar to the one shown in Figure 1 but*

*specific for tomosynthesis images. The outer loop shows sampling images on each block.*

#### **2.3 Proof of concept on synthetic data**

Testing on extremes is a common practice in engineering. Unfortunately, finding extremes on clinical data is cumbersome. The difficulty relies on the nature of the information; in the clinics, where the patients are imaged on the presumption that some abnormality is present, the images often yield moderately spiculated masses, posing a problem overall for the lower extreme reference. Regarding the highly spiculated reference, one can use the histogram signature to pinpoint the slice yielding the most right-filling pattern. However, a sounded proof of concept should comply with the common complexity found in the clinics, where two masses can have similar volumes and have a different nature regarding malignancy; thus, conventional methods are unable to detect differences. To overcome this problem, we have created a synthetic framework where lower references are created by stacking the less spiculated slice among all the data analyzed. A mildly spiculated mass is created by stacking a mildly spiculated slice among the study, and, analogously, the extreme spiculated sample is created by stacking the most spiculated slice found in the study. For the three samples, the stacking is driven in a manner that the masses end by having a similar volume.

#### **2.4 Artificial intelligence (AI) implementation**

Every column in the histogram signature created by employing the procedure in Section 2.2 is seen as a feature in classification postulate that aims to distinguish between malign and benign samples. This is possible due to the independence of the peaks counting in a slice by slice fashion. In general, the perturbations on the slice *n* do not have any correlation with the perturbations on slice *m*; therefore, orthogonality is granted. In addition to the bins counting, the number of bins fulfilled some bins may end empty—those filled from the middle bin to the right and those filled to the middle bin to the left, is also used in the featuring space.

Every tumor population has a different span in the histogram signature; however, the amount of peak-counting-derived features have been set constant by forcing seven equally spaced bins regardless of the peak-counting range. Thus, the experiments always create an analyzing matrix containing 11 columns, 10 columns for the features, and 1 column to register the supervising factor provided by the histology. The current exercise presents a boolean support vector machine (SVM) classifier, where the machine is trained to provide a benign or malign verdict.

The data matrix is scaled and normalized using Python-Pandas [27, 28]. The classifier estimators are proved by cross correlation where the train and test samples are gathered from the original dataset using (0*:*7 : 0*:*3) (train:test) in a fivefold experimental scheme. For the classification experiments, Scikit-learn [29] is employed.

Listing 1: Python code use to run the SVM classifier while progressively adding features.

**def** run Features Testing Classification (features, mdata, lbls): atregs = [] ascores = [] **for** i **in** length(features): est = feature\_selection.SelectKBest (k=i) est . fit (mdata, lbls) tregs = est . get\_support (indices = True)

*Tumor Malignancy Characterization in Clinical Environments: An Approach… DOI: http://dx.doi.org/10.5772/intechopen.82145*

ndata = est . transform (mdata) estsvm = svm . LinearSVC () gs = grid\_search . GridSearchCV (estsvm, {'C' : np . logspace (–4,3)}) tscore = np . mean (cross\_validation . cross\_val\_score (gs, ndata, lbls, n\_jobs=5)) As it is shown in Listing 1, the SVM classification is done after progressively

adding features which are grabbed from the *mdata* matrix using the indexes saved in the features' array. The accuracy records presented in Section 4 correspond to the experiment that yielded the highest accuracy values per folding.

#### **3. Results**

Under the FYC-Index domain, while more spiculation, the histogram profiles

Testing on extremes is a common practice in engineering. Unfortunately, finding extremes on clinical data is cumbersome. The difficulty relies on the nature of the information; in the clinics, where the patients are imaged on the presumption that some abnormality is present, the images often yield moderately spiculated masses, posing a problem overall for the lower extreme reference. Regarding the highly spiculated reference, one can use the histogram signature to pinpoint the slice yielding the most right-filling pattern. However, a sounded proof of concept should comply with the common complexity found in the clinics, where two masses can have similar volumes and have a different nature regarding malignancy; thus, conventional methods are unable to detect differences. To overcome this problem, we have created a synthetic framework where lower references are created by stacking the less spiculated slice among all the data analyzed. A mildly spiculated mass is created by stacking a mildly spiculated slice among the study, and, analogously, the extreme spiculated sample is created by stacking the most spiculated slice found in the study. For the three samples, the stacking is driven in a manner

Every column in the histogram signature created by employing the procedure in Section 2.2 is seen as a feature in classification postulate that aims to distinguish between malign and benign samples. This is possible due to the independence of the peaks counting in a slice by slice fashion. In general, the perturbations on the slice *n* do not have any correlation with the perturbations on slice *m*; therefore, orthogonality is granted. In addition to the bins counting, the number of bins fulfilled some bins may end empty—those filled from the middle bin to the right and those

Every tumor population has a different span in the histogram signature; however, the amount of peak-counting-derived features have been set constant by forcing seven equally spaced bins regardless of the peak-counting range. Thus, the experiments always create an analyzing matrix containing 11 columns, 10 columns for the features, and 1 column to register the supervising factor provided by the histology. The current exercise presents a boolean support vector machine (SVM) classifier, where the machine is trained to provide a benign or malign verdict. The data matrix is scaled and normalized using Python-Pandas [27, 28]. The classifier estimators are proved by cross correlation where the train and test samples are gathered from the original dataset using (0*:*7 : 0*:*3) (train:test) in a fivefold experimental scheme. For the classification experiments, Scikit-learn [29] is

Listing 1: Python code use to run the SVM classifier while progressively adding

filled to the middle bin to the left, is also used in the featuring space.

**def** run Features Testing Classification (features, mdata, lbls):

are more populated in the right side.

*Tumor Progression and Metastasis*

**2.3 Proof of concept on synthetic data**

that the masses end by having a similar volume.

**2.4 Artificial intelligence (AI) implementation**

employed.

features.

**296**

atregs = [] ascores = []

**for** i **in** length(features):

est . fit (mdata, lbls)

est = feature\_selection.SelectKBest (k=i)

tregs = est . get\_support (indices = True)

#### **3.1 FYC-Index extraction**

**Figure 2** shows how the algorithm yields two different outcomes based on the tortuosity of the two analyzed shapes. The small shape refers to a mostly rounded region of interest (ROI), therefore, does not present abrupt changes in the distances from the edging points to the center of FOV. In contrast, the same measure yields rapidly changing distances in the big ROI. Those rapidly changing distances are captured by the first derivate and framed in their inflection points by the second derivate. Later, those points are amplified and made all positive by the fourth power function, while the same fourth power function diminishes changes in which the derivate yielded values in the range (1, 1). As the moving window adds up all values encountered in its domain, the regions of rapid change represented by large values compute to higher numbers within the domain of the moving window, and that is where the enhanced points appear in the plot. As all the points are mapped with their original coordinates, a crossing of 3D positions among the selected points

#### **Figure 2.**

*FYC-Index extraction. The inner loop is the detailed block diagram similar to the one shown in Figure 1 but specific for tomosynthesis images. The outer loop shows sampling images on each block.*

**Figure 3.**

*Histogram signature of the FYC-Index on two tumors and intermediate steps (b, c, d, g) of processing. The circles in frames in (c) and (g) correspond to perfectly rounded regions where the area is equal to the one of the mask.*

in two image views filters out positions erroneously selected. Finally, the presented procedure allocates an item of frequency in a histogram where the bins contain ranges of point counting. Naturally, highly spiculated slices contribute mostly to the right bins of the histogram. When all slices in a tumor have been analyzed, the operator could be sure that the histogram is descriptive of the degree of homogeneity of the mass which is also associated with aggressiveness (see **Figure 3**).

A sample of the process where the 3D reconstruction of the masses together with the respective normalized FYC-Index histogram is presented in **Figure 4**.

#### **3.2 Analysis of synthetic data**

As explained in Section 2.3, extreme references are created to demonstrate the span of the method and the capacity to deliver a representation of easy interpretation. The synthetic creations are shown in **Figure 5**.

The results obtained on synthetic data corroborates that the FYC-Index is sensible to the changes in the edges that distinguish between malign and benign masses. In contrast, commonly used geometrical indexes are not sensitive to changes. In this exercise, we have isolated the spiculation by equalizing the volumes of the studied software objects. A complete set of 3D geometrical functions are applied on the clinical data in use, with the aim of comparing the performance of standard of care tools in the clinics, and the FYC-Index is shown in **Figure 6**.

**5. Discussion**

*A sample of the processed tumors and their FYC-Index signatures.*

*Tumor Malignancy Characterization in Clinical Environments: An Approach…*

*DOI: http://dx.doi.org/10.5772/intechopen.82145*

**Figure 4.**

**299**

The proposed method is sensitive to slight changes in the edges of the masses that are characteristically malignant. The same method includes a stage of quantification that has proven to be descriptive at a simple glance even for nonspecialized operators. Since the procedure has been automated, it is compliant with the confidentiality regulations and, therefore, can be easily implemented in hospitals and clinics. The FYC-Index is a flexible method equally performant when analyzing masses in individuals and populations. The method presents a signature which results in a measure of lobularity. This strategy works regardless of factors such as size and spatial resolution. Moreover, the results are direct and easy to interpret.

#### **4. Verdicts dictated by (AI) implementation**

The fivefolding SVM exercise proposed in Section 2.4 was executed using a Python-Pandas dataframe and Scikit-Learn SVM. The results are registered in **Table 1**.

The strong-force algorithm presented in Section 2.4 executed the supervised classification with a high degree of accuracy. The design of the experiments turns the classification into the capacity to differentiate whether a mass is benign or malign.

*Tumor Malignancy Characterization in Clinical Environments: An Approach… DOI: http://dx.doi.org/10.5772/intechopen.82145*

**Figure 4.** *A sample of the processed tumors and their FYC-Index signatures.*

#### **5. Discussion**

in two image views filters out positions erroneously selected. Finally, the presented procedure allocates an item of frequency in a histogram where the bins contain ranges of point counting. Naturally, highly spiculated slices contribute mostly to the right bins of the histogram. When all slices in a tumor have been analyzed, the operator could be sure that the histogram is descriptive of the degree of homogene-

*Histogram signature of the FYC-Index on two tumors and intermediate steps (b, c, d, g) of processing. The circles in frames in (c) and (g) correspond to perfectly rounded regions where the area is equal to the one of the*

A sample of the process where the 3D reconstruction of the masses together with

As explained in Section 2.3, extreme references are created to demonstrate the span of the method and the capacity to deliver a representation of easy interpreta-

The results obtained on synthetic data corroborates that the FYC-Index is sensible to the changes in the edges that distinguish between malign and benign masses. In contrast, commonly used geometrical indexes are not sensitive to changes. In this exercise, we have isolated the spiculation by equalizing the volumes of the studied software objects. A complete set of 3D geometrical functions are applied on the clinical data in use, with the aim of comparing the performance of standard of care

The fivefolding SVM exercise proposed in Section 2.4 was executed using a Python-Pandas dataframe and Scikit-Learn SVM. The results are registered in

The strong-force algorithm presented in Section 2.4 executed the supervised classification with a high degree of accuracy. The design of the experiments turns the classification into the capacity to differentiate whether a mass is benign or malign.

ity of the mass which is also associated with aggressiveness (see **Figure 3**).

the respective normalized FYC-Index histogram is presented in **Figure 4**.

**3.2 Analysis of synthetic data**

*Tumor Progression and Metastasis*

**Table 1**.

**298**

**Figure 3.**

*mask.*

tion. The synthetic creations are shown in **Figure 5**.

**4. Verdicts dictated by (AI) implementation**

tools in the clinics, and the FYC-Index is shown in **Figure 6**.

The proposed method is sensitive to slight changes in the edges of the masses that are characteristically malignant. The same method includes a stage of quantification that has proven to be descriptive at a simple glance even for nonspecialized operators. Since the procedure has been automated, it is compliant with the confidentiality regulations and, therefore, can be easily implemented in hospitals and clinics. The FYC-Index is a flexible method equally performant when analyzing masses in individuals and populations. The method presents a signature which results in a measure of lobularity. This strategy works regardless of factors such as size and spatial resolution. Moreover, the results are direct and easy to interpret.


with sufficient samples in all grading range. This multilevel classification should be designed to follow the classification directives presented in the X-RADS standards; thus, the existing automatic tools can also provide insights for selecting more accurate treatments. To the best of our knowledge, no other authors are integrating the tools as we have proposed. The use of the features we have proposed is a novel view of the solution; therefore, we do not include in this report a comparison with

*Results for the fivefolding experiments on histograms acquired with the FYC-Index of spiculation.*

*Tumor Malignancy Characterization in Clinical Environments: An Approach…*

*DOI: http://dx.doi.org/10.5772/intechopen.82145*

**Folding Accuracy (%) Sensibility (%) Specificity (%)** 93.4 92.1 89.1 91.3 92.4 90.4 90.3 90.2 89.6 92.7 90.1 89.3 89.9 90.0 89.6

Cancer is the second most threating disease which humanity has not been able to

neutralize. Other diseases that were considered pandemics in the past, costing millions of human lives, have been eradicated through vaccination. Rapidly mutating diseases such as AIDS have been downgraded from mortal to chronic. Maladies like high blood pressure, stroke, or cirrhosis among several other chronic afflictions have been associated with race, genetics, habits, or exposition factors, providing a way to reduce the probability of acquiring them or a path of development where scientists still have space to explore. Cancer instead affects all humans regardless of any factor. The only aspect that increases the surviving expectations, without a doubt, is early detection, and it is here where the method presented in this manuscript gains relevance. Detection from the images is possible, and automatic diagnosis not only avoids the painful and uncomfortable biopsy, but it also contributes

other methods.

**Table 1.**

*SVM-supervised classification.*

**6. Conclusion**

**301**

to faster and more accurate verdicts.

#### **Figure 5.**

*Performance of the FYC-Index in software-created references. On the right, a table with records of often used 3D geometrical indexes. Note that these indexes are not sensible within the characteristics that require to be quantified.*

#### **Figure 6.**

*The two boxes per colored column correspond to the clinical data detailed in Section 2.1. Normality was discarded by Kolmogorov test [30] done in the two groups separately. As normality was not met, the nonparametric Kruskal-Wallis test [31] was employed. The p-values are mapped back and forward in the chisquare distribution.*

The specifications of the FYC-Index make it suitable to analyze all sort of cancer manifestations, regardless of localization or pathogenic roots. The presented strategy uses a machine-learning classifier to rapidly characterize the malignancy of a mass. However, the real challenge consists of defining malignancy together with aggressiveness. Such an approach requires more rounds of training/testing sessions


*Tumor Malignancy Characterization in Clinical Environments: An Approach… DOI: http://dx.doi.org/10.5772/intechopen.82145*

#### **Table 1.**

*SVM-supervised classification.*

with sufficient samples in all grading range. This multilevel classification should be designed to follow the classification directives presented in the X-RADS standards; thus, the existing automatic tools can also provide insights for selecting more accurate treatments. To the best of our knowledge, no other authors are integrating the tools as we have proposed. The use of the features we have proposed is a novel view of the solution; therefore, we do not include in this report a comparison with other methods.

#### **6. Conclusion**

Cancer is the second most threating disease which humanity has not been able to neutralize. Other diseases that were considered pandemics in the past, costing millions of human lives, have been eradicated through vaccination. Rapidly mutating diseases such as AIDS have been downgraded from mortal to chronic. Maladies like high blood pressure, stroke, or cirrhosis among several other chronic afflictions have been associated with race, genetics, habits, or exposition factors, providing a way to reduce the probability of acquiring them or a path of development where scientists still have space to explore. Cancer instead affects all humans regardless of any factor. The only aspect that increases the surviving expectations, without a doubt, is early detection, and it is here where the method presented in this manuscript gains relevance. Detection from the images is possible, and automatic diagnosis not only avoids the painful and uncomfortable biopsy, but it also contributes to faster and more accurate verdicts.

The specifications of the FYC-Index make it suitable to analyze all sort of cancer manifestations, regardless of localization or pathogenic roots. The presented strategy uses a machine-learning classifier to rapidly characterize the malignancy of a mass. However, the real challenge consists of defining malignancy together with aggressiveness. Such an approach requires more rounds of training/testing sessions

*nonparametric Kruskal-Wallis test [31] was employed. The p-values are mapped back and forward in the chi-*

*The two boxes per colored column correspond to the clinical data detailed in Section 2.1. Normality was discarded by Kolmogorov test [30] done in the two groups separately. As normality was not met, the*

*Performance of the FYC-Index in software-created references. On the right, a table with records of often used 3D geometrical indexes. Note that these indexes are not sensible within the characteristics that require to be*

**Figure 5.**

*Tumor Progression and Metastasis*

*quantified.*

**Figure 6.**

**300**

*square distribution.*

*Tumor Progression and Metastasis*

### **Author details**

Fernando Yepes-Calderón<sup>1</sup> \*, Flor M. Medina<sup>2</sup> , Nolan D. Rea<sup>1</sup> and José Abella<sup>2</sup> **References**

rsnarights

06.066

17512669

1356178

**303**

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*DOI: http://dx.doi.org/10.5772/intechopen.82145*

*Tumor Malignancy Characterization in Clinical Environments: An Approach…*

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1 Children's Hospital Los Angeles, Los Angeles, CA, USA

2 Fundación Valle del Lili, Cali, Colombia

\*Address all correspondence to: fernandoyepesc@gmail.com

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Tumor Malignancy Characterization in Clinical Environments: An Approach… DOI: http://dx.doi.org/10.5772/intechopen.82145*

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**Author details**

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Fernando Yepes-Calderón<sup>1</sup>

*Tumor Progression and Metastasis*

\*, Flor M. Medina<sup>2</sup>

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Children's Hospital Los Angeles, Los Angeles, CA, USA

\*Address all correspondence to: fernandoyepesc@gmail.com

2 Fundación Valle del Lili, Cali, Colombia

provided the original work is properly cited.

, Nolan D. Rea<sup>1</sup> and José Abella<sup>2</sup>

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### *Edited by Ahmed Lasfar and Karine Cohen-Solal*

This book offers significant coverage on different aspects of cancer from risk factors to the mechanisms leading to tumor progression and metastasis. Although tremendous progress has been made in cancer research and treatment, cancer metastasis remains a major unmet clinical need. The life and death of many cancer patients hangs on the degree of metastasis. This book provides new perspectives for diagnosis and cancer therapy. It includes new technologies and a new basis for current cancer therapies. To guarantee the high quality of this book, important topics are included and rigorously discussed in a simple and authentic way. The book addresses important challenges governing tumor progression and metastasis and brings new responses to both diagnosis and therapy. This book is a great source of knowledge and will be useful for researchers, medical doctors, oncologists, graduate and medical students, continued medical educators, health care providers, and all individuals interested in understanding cancer and its challenges.

Published in London, UK © 2020 IntechOpen © dzika\_mrowka / iStock

Tumor Progression and Metastasis

IntechOpen Book Series

Physiology, Volume 8

Tumor Progression

and Metastasis

*Edited by Ahmed Lasfar and Karine Cohen-Solal*