**3.4 Combination of gene and drug delivery**

The combination of gene and drug delivery is another factor to enhance the chance of success in oncotherapy. For example, Both paclitaxel (PTX) and focal adhesion kinase (FAK) siRNA loading HA-labeled poly(d,l-lactide-co-glycolide) NPs (HA-PLGA-NP-PTX+FAK siRNA) were used for ovarian oncotherapy. Tumor cells due to the presence of CD44 obtain more HA-PLGA-NP-PTX+FAK siRNA which decreases cell viability by inducing apoptosis in both SKOV3-TR and HeyA8-MDR (multidrug resistance) cells. Knockdown of AKT pathway that has a role in metastasis and drug resistance, have occurred by FAK siRNA [58].

In another experiment, a novel combination of chemotherapy and gene therapy for A2780DDP cells and xenograft nude mice model was developed. Platinum(IV)− azide complexes (Pt(IV) prodrugs) and the siRNA of c-fos (si(c-fos)) embedded in a photoactivatable polymeric NP. Pt(IV) prodrugs are nontoxic in dark but after mild light (blue light) irradiation, it released the Pt(II) drug that has cytotoxic activity. This nano vehicle has high drug loading properties and extraordinary stability that lead to cytotoxicity and antitumor characterization (**Figure 7**) [59].

**185**

*Nano Technology and Gas Plasma as Novel Therapeutic Strategies for Ovarian Cancer…*

*Anti-Pin1 and cyclodextrins loaded to liposome as a new therapy for OC. This figure was obtained with* 

*permission from [56] under the terms of creative commons CC BY license.*

Furthermore, A2780R cells treated by two separate NPs were developed for the delivery of drug and gene. CIS resistance leads from overexpression of miRNA-21 in OC. So, anti-miRNA-21 by PEGylated poly(lactic-co-glycolic acid) NPs which decorated with AS1411 antinucleolin aptamer for developing target delivery (Ap anti-miR-21-NPs) and NPs contain CIS (Ap–CIS–NPs) deliver to A2780R cells. It is caused to the reduction in drug resistance by inhibiting miRNA-21 and increased

*Photoactivatable polymeric nanoparticles as a gene and drug delivery for platinum-resistant OC. This figure* 

Gas plasma is a novel technology with potential and actual applications ranging from energy and water to food sciences [61]. Management of gas plasma effects on biological objects is related to different factors including charged particles, electric fields, UV radiation, and RONS. These chemical and physical factors are involved in combination or multimodal forms, provide a solution for a variety of medical applications [62] (**Figure 8**). Cancer therapy, wound healing, virus inactivation,

**4. Gas plasma based therapy for ovarian cancer oncotherapy**

*was obtained with permission from [63] under the terms of creative commons CC BY license.*

*DOI: http://dx.doi.org/10.5772/intechopen.96387*

**Figure 6.**

**Figure 7.**

mortality via induction apoptosis [60].

**4.1 Gas plasma: key features and applications**

*Nano Technology and Gas Plasma as Novel Therapeutic Strategies for Ovarian Cancer… DOI: http://dx.doi.org/10.5772/intechopen.96387*

### **Figure 6.**

*Ovarian Cancer - Updates in Tumour Biology and Therapeutics*

induces cell death by apoptosis and necrosis [54].

inhibition effect on tumor growth and volume [57].

**3.4 Combination of gene and drug delivery**

*3.3.2 NPs for shRNA delivery*

*3.3.3 NPs for miRs delivery*

implement on the PTX-resistant and CIS-resistant, SKOV3-TR and A2780-CP20 cells respectively. The observations indicated an increment in cellular uptake that

shRNA is a stem-loop RNA that in comparison with siRNA cause prolong gene silencing and highly effective. In the following, 2 examples of this procedure have been brought. PEG NPs with a peptide of FSH β 33-53 for specific target delivery encapsulate shRNA for silencing growth-regulated oncogene α (gro-α) (FSH33- G-NP). Internalization in FSHR positive cells like HEY cells is more. FSH33-G-NP decrement cell proliferation, invasion and migration and also in vivo experiments showed antitumor activity [55]. Also, overexpression of pin1 is related to cancer malignancy by regulating oncogenes and tumor suppressor genes, so silencing of pin1 can inhibit the tumor growth in a syngeneic mouse model and induce apoptosis in OC cells. Proteasome-dependent degradation of Pin1 happened via liposomebased NPs that were modified by cyclodextrins for shRNA delivery (**Figure 6**) [56].

Micro RNAs (miRs) are short and non-coding RNAs that modulate gene expression at the level of post-transcriptional. The existence of them is necessary for the regulation of cell metabolism, differentiation, proliferation, and apoptosis. But sometimes, dysregulation and improper expression of miRs (oncomiRs) are related to the early and advanced stages of cancers, so, anti-miR delivery for downregulating oncomiR is an anticancer strategy. A high level of miR-21 is related to the incidence of many cancers including OC. In order to improve cancer therapy porous silicon NPs that were modified by MAL-PEG-SVA enclosed anti-miR-21 (LAN) to target OAW42 ovarian cells. In this study, CREK peptide as a control peptide for no targeting activity in cell culture and CGKRK peptide for displaying tumorhoming and tumor penetrating properties were analyzed for comparison. Findings illustrated a decrease in cell viability due to apoptosis by evaluating caspase-3, and also, COV-318 xenograft tumors subcutaneously transplanted into nude mice after treatment represented the higher accumulation of NPs in tumor tissue that lead to

The combination of gene and drug delivery is another factor to enhance the chance of success in oncotherapy. For example, Both paclitaxel (PTX) and focal adhesion kinase (FAK) siRNA loading HA-labeled poly(d,l-lactide-co-glycolide) NPs (HA-PLGA-NP-PTX+FAK siRNA) were used for ovarian oncotherapy. Tumor cells due to the presence of CD44 obtain more HA-PLGA-NP-PTX+FAK siRNA which decreases cell viability by inducing apoptosis in both SKOV3-TR and HeyA8-MDR (multidrug resistance) cells. Knockdown of AKT pathway that has a

In another experiment, a novel combination of chemotherapy and gene therapy for A2780DDP cells and xenograft nude mice model was developed. Platinum(IV)− azide complexes (Pt(IV) prodrugs) and the siRNA of c-fos (si(c-fos)) embedded in a photoactivatable polymeric NP. Pt(IV) prodrugs are nontoxic in dark but after mild light (blue light) irradiation, it released the Pt(II) drug that has cytotoxic activity. This nano vehicle has high drug loading properties and extraordinary stability that lead to cytotoxicity and antitumor characterization (**Figure 7**) [59].

role in metastasis and drug resistance, have occurred by FAK siRNA [58].

**184**

*Anti-Pin1 and cyclodextrins loaded to liposome as a new therapy for OC. This figure was obtained with permission from [56] under the terms of creative commons CC BY license.*

### **Figure 7.**

*Photoactivatable polymeric nanoparticles as a gene and drug delivery for platinum-resistant OC. This figure was obtained with permission from [63] under the terms of creative commons CC BY license.*

Furthermore, A2780R cells treated by two separate NPs were developed for the delivery of drug and gene. CIS resistance leads from overexpression of miRNA-21 in OC. So, anti-miRNA-21 by PEGylated poly(lactic-co-glycolic acid) NPs which decorated with AS1411 antinucleolin aptamer for developing target delivery (Ap anti-miR-21-NPs) and NPs contain CIS (Ap–CIS–NPs) deliver to A2780R cells. It is caused to the reduction in drug resistance by inhibiting miRNA-21 and increased mortality via induction apoptosis [60].

## **4. Gas plasma based therapy for ovarian cancer oncotherapy**

### **4.1 Gas plasma: key features and applications**

Gas plasma is a novel technology with potential and actual applications ranging from energy and water to food sciences [61]. Management of gas plasma effects on biological objects is related to different factors including charged particles, electric fields, UV radiation, and RONS. These chemical and physical factors are involved in combination or multimodal forms, provide a solution for a variety of medical applications [62] (**Figure 8**). Cancer therapy, wound healing, virus inactivation,

**Figure 8.** *The key role of reactive agents from generation in the plasma to interaction with the biological objects.*

biofilm removal, dentistry, and ophthalmology, as well as cosmetic uses, are some of the applications of plasma in medicine. It is now clear that plasma is a promising therapeutic candidate for the multivariate condition of cancer [63, 64].

Recent studies revealed, gas plasma oncotherapy provides insights into the wide context challenging of cancer treatment through physical and chemical effects. Until now, the underlying mechanisms of plasma action were ascribed to RONS, but more recently, the role of physical factors (UV and EM) is also emphasized [62, 65]. These cocktails inducing dose-dependent effects, redox flux increase to cells, flexibility in use, multimodality nature, and the mild effect that are primary features of gas plasma [8]. Also, these unique physicals, chemical and biological properties have a high potential to act synergistically and will be crucial to the achievement of selectivity for cancer cells, enhancing cancer chemosensitivity, stimulation of the immune system, elimination of cancer stem cells, halting cancer metastasis as medical features of gas plasma oncotherapy [6, 63]. Thus, plasma as an alternative effective technology eliminates some of the most important undesirable consequences and side effects of common treatments. The great antitumor impact of plasma for all types of cancer have been reported [66].

### **4.2 Direct and indirect plasma treatment: role of the device and liquid**

Plasma treatment is divided into two general direct and indirect modalities in order to offer new solutions to its increasingly diverse range of applications, as well as to cover the requirement related to them. In addition to exposing biological objects to plasma radiation, another method was developed. In the indirect treatment modality that has known as plasma activated liquid, the solution is exposed to plasma irradiation and then is added to the biological target [15, 67, 68]. It seems like the direct method is suitable for superficial tumors, but for peritoneal tumors,

**187**

1

(HOCl) and ●

(**Figure 9**).

activities.

*Nano Technology and Gas Plasma as Novel Therapeutic Strategies for Ovarian Cancer…*

the indirect method or plasma-activated liquid is a good option and can be used as innovative technology. By ignoring the unknown complexities of plasma liquid

Atmospheric Pressure Plasma Jets (APPJ) and volume and surface Dielectric Barrier Discharge (DBD) are three configuration types of common plasma devices used for biomedical application. Regarding the feeding gas, noble gases, air, nitrogen, or a combination of that, are utilized for the generation of plasma depending on the configuration used [69, 70]. Toward the APPJ, the DBD seems to be appropriate for the production of plasma-activated liquid due to the larger volume of

Culture mediums (DMEM, RPMI, alpha-MEM), Phosphate-buffered saline (PBS), and Ringer's solution have been reported as an exposed solution for PAL generation. Currently, all three types of solutions are used to produce PAL. As it was previously mentioned, aside from plasma device and process parameters, the compositions of the liquid have a pivotal role in the plasma action [15, 71]. It is appropriate to use solutions that have less interaction with plasma and do not change their function. However, it is well established that the sensitivity of cells to culture conditions is another limitation of this method and many cells are destroyed by changing the culture medium. Taken together, further research in this regard is

**4.3 Ovarian cancer oncotherapy through gas plasma: selectivity, restores** 

OC, colorectal cancer, pancreatic/appendiceal cancer, stomach cancer, peritoneal mesothelioma, and primary peritoneal cancer are the most common cancers that cause peritoneal carcinomatosis. In recent years, treatment strategies for these cancers, improved by combining several existing methods. [72]. Nevertheless, peritoneal carcinomatosis treatments are ineffective and require new multiple strategies

OC the most important type of cancer in the cancer cells response discovering process to the plasma. Albeit the number of relevant studies examining OC with gas plasma is limited compared to other cancers. Most studies have been examined the selectivity of gas plasma oncotherapy on cancer or healthy cells. Regarding chemotherapy resistance, the impact of plasma on acquired and intrinsic resistance cells also have been investigated. Besides, various evidence suggests that gas plasma plays a crucial role in OC by mediating several genes involved in proliferation, apoptosis,

The selectivity mechanism of gas plasma oncotherapy has been demonstrated

O2, thereby inactivating some of the catalase of cancer cells. Then, Due to the differences between healthy and cancerous cells, cell-based secondary <sup>1</sup>

tion is high, and therefore more catalase is inactivated. So, H2O2 with penetrating the cells through aquaporin causes depletes GSH or activities Hypochlorous acid

Here, we outline the existing studies about the application of gas plasma and the mechanisms responsible for their expression strictly in OC. PAL has great potential to act as an innovative approach and overcome multiple biological barriers and treatment challenges in peritoneal cancers. Thus, PAL is a commonly used therapeutic option in this chapter. Also, it seems Ringer Lactate solution will be a proper liquid for future plasma activated liquid and has direct anti-cancer

−

signaling that leads to caspase-mediated cell death [74]

produce primary

O2 genera-

in our previous work [73], briefly, plasma-derived H2O2 and NO2

**chemotherapy sensitivity, and metastasis inhibition**

that provide targeted drug delivery on a large scale.

migration, and metastasis.

NO/ONOO−

*DOI: http://dx.doi.org/10.5772/intechopen.96387*

exposed solution [69].

very vital.

interaction, the RONS play a dominant role in PAL.

*Nano Technology and Gas Plasma as Novel Therapeutic Strategies for Ovarian Cancer… DOI: http://dx.doi.org/10.5772/intechopen.96387*

the indirect method or plasma-activated liquid is a good option and can be used as innovative technology. By ignoring the unknown complexities of plasma liquid interaction, the RONS play a dominant role in PAL.

Atmospheric Pressure Plasma Jets (APPJ) and volume and surface Dielectric Barrier Discharge (DBD) are three configuration types of common plasma devices used for biomedical application. Regarding the feeding gas, noble gases, air, nitrogen, or a combination of that, are utilized for the generation of plasma depending on the configuration used [69, 70]. Toward the APPJ, the DBD seems to be appropriate for the production of plasma-activated liquid due to the larger volume of exposed solution [69].

Culture mediums (DMEM, RPMI, alpha-MEM), Phosphate-buffered saline (PBS), and Ringer's solution have been reported as an exposed solution for PAL generation. Currently, all three types of solutions are used to produce PAL. As it was previously mentioned, aside from plasma device and process parameters, the compositions of the liquid have a pivotal role in the plasma action [15, 71]. It is appropriate to use solutions that have less interaction with plasma and do not change their function. However, it is well established that the sensitivity of cells to culture conditions is another limitation of this method and many cells are destroyed by changing the culture medium. Taken together, further research in this regard is very vital.

### **4.3 Ovarian cancer oncotherapy through gas plasma: selectivity, restores chemotherapy sensitivity, and metastasis inhibition**

OC, colorectal cancer, pancreatic/appendiceal cancer, stomach cancer, peritoneal mesothelioma, and primary peritoneal cancer are the most common cancers that cause peritoneal carcinomatosis. In recent years, treatment strategies for these cancers, improved by combining several existing methods. [72]. Nevertheless, peritoneal carcinomatosis treatments are ineffective and require new multiple strategies that provide targeted drug delivery on a large scale.

OC the most important type of cancer in the cancer cells response discovering process to the plasma. Albeit the number of relevant studies examining OC with gas plasma is limited compared to other cancers. Most studies have been examined the selectivity of gas plasma oncotherapy on cancer or healthy cells. Regarding chemotherapy resistance, the impact of plasma on acquired and intrinsic resistance cells also have been investigated. Besides, various evidence suggests that gas plasma plays a crucial role in OC by mediating several genes involved in proliferation, apoptosis, migration, and metastasis.

The selectivity mechanism of gas plasma oncotherapy has been demonstrated in our previous work [73], briefly, plasma-derived H2O2 and NO2 − produce primary 1 O2, thereby inactivating some of the catalase of cancer cells. Then, Due to the differences between healthy and cancerous cells, cell-based secondary <sup>1</sup> O2 generation is high, and therefore more catalase is inactivated. So, H2O2 with penetrating the cells through aquaporin causes depletes GSH or activities Hypochlorous acid (HOCl) and ● NO/ONOO− signaling that leads to caspase-mediated cell death [74] (**Figure 9**).

Here, we outline the existing studies about the application of gas plasma and the mechanisms responsible for their expression strictly in OC. PAL has great potential to act as an innovative approach and overcome multiple biological barriers and treatment challenges in peritoneal cancers. Thus, PAL is a commonly used therapeutic option in this chapter. Also, it seems Ringer Lactate solution will be a proper liquid for future plasma activated liquid and has direct anti-cancer activities.

*Ovarian Cancer - Updates in Tumour Biology and Therapeutics*

biofilm removal, dentistry, and ophthalmology, as well as cosmetic uses, are some of the applications of plasma in medicine. It is now clear that plasma is a promising

*The key role of reactive agents from generation in the plasma to interaction with the biological objects.*

Recent studies revealed, gas plasma oncotherapy provides insights into the wide context challenging of cancer treatment through physical and chemical effects. Until now, the underlying mechanisms of plasma action were ascribed to RONS, but more recently, the role of physical factors (UV and EM) is also emphasized [62, 65]. These cocktails inducing dose-dependent effects, redox flux increase to cells, flexibility in use, multimodality nature, and the mild effect that are primary features of gas plasma [8]. Also, these unique physicals, chemical and biological properties have a high potential to act synergistically and will be crucial to the achievement of selectivity for cancer cells, enhancing cancer chemosensitivity, stimulation of the immune system, elimination of cancer stem cells, halting cancer metastasis as medical features of gas plasma oncotherapy [6, 63]. Thus, plasma as an alternative effective technology eliminates some of the most important undesirable consequences and side effects of common treatments. The great antitumor impact of plasma for all types of cancer have been

therapeutic candidate for the multivariate condition of cancer [63, 64].

**4.2 Direct and indirect plasma treatment: role of the device and liquid**

Plasma treatment is divided into two general direct and indirect modalities in order to offer new solutions to its increasingly diverse range of applications, as well as to cover the requirement related to them. In addition to exposing biological objects to plasma radiation, another method was developed. In the indirect treatment modality that has known as plasma activated liquid, the solution is exposed to plasma irradiation and then is added to the biological target [15, 67, 68]. It seems like the direct method is suitable for superficial tumors, but for peritoneal tumors,

**186**

reported [66].

**Figure 8.**

### **Figure 9.**

*Flow chart of major steps in CAP leading to selective apoptosis of tumor cells. Step 1: CAP generates NO2 − and H2O2 in cell containing medium for 1 minute. Alternatively, CAP is used to treat medium, creating PAM (step 1′). Defined concentrations of NO2 − and H2O2 containing medium are used in reconstitution experiments (step 1"). Step 2: NO2 − - and H2O2 create primary 1 O2 near cells following O2NOOH pathway, as described in reference. Step 3: Few catalase molecules on a few cells are inactivated due to primary 1 O2 near cells. Step 4: At the site of inactivated catalase, H2O2 and ONOO− (generated through NOX1 and NOS) are no longer decomposed. Step 5: The reaction between H2O2 and ONOO− is leading ultimately to secondary 1 O2. Step 6: This additional <sup>1</sup> O2 leads to further catalase inactivation and the process cycles back to step 4. Step 7: Increased H2O2 resulting from catalase loss from secondary 1 O2 leads to H2O2 entering cells via aquaporins, leading to antioxidant glutathione depletion. Step 8: In parallel with step 7, increased H2O2 resulting from catalase loss from secondary <sup>1</sup> O2 also leads to HOCl generation by peroxidase, in the presence of Cl<sup>−</sup> . The interaction between NOX1 derived O2 ●− leads to ● OH formation near the cell membrane and lipid oxidation. Step 8′: If HOCl signaling is suppressed, an alternative ● NO/ONOO− signaling can also lead to lipid peroxidation. Step 9: If both lipid peroxidation and glutathione depletion occur, then caspase-associated apoptosis can take place, finally leading to cell death. Steps 1–3 correspond to CAP triggering or activation of a few cells, thereby initiating propagating bystander signaling in steps 4–6. Steps 7–9 are the steps that lead to the final cell apoptosis. These steps are activated only if the repeated performance of steps 4–6 has caused a sufficiently high degree of catalase inactivation for reactivation of HOCl or ● NO/ONOO− - mediated apoptosis-inducing signaling. This figure was obtained with permission from [74] under the terms of creative commons CC BY license.*

Selectivity towards cancer cells, chemotherapy-resistance elimination, restore sensitivity to chemotherapy, inhibition of metastasis, and more recently the possible mechanism of plasma action has been achieved in these studies.

Gas plasma effects on OC were first examined on two human epithelial ovarian carcinoma cell lines, SKOV3 and HRA and normal human fetal lung fibroblast cell lines, WI-38 and MRC-5. Nonequilibrium atmospheric pressure plasma (NEAPP) was utilized to assess toxicity and proliferation inhibition. Cell proliferation, flow cytometry, western blot analysis along with pH, temperature, and volume of the medium before and after plasma treatments were evaluated. NEAPP selectively targets two cancer cells and induces apoptosis in them, while normal cells were not damaged. Although the authors do not address the mechanism of action, they assume a pivotal role in the process of plasma application for UV radiation, charged particles, and free radicals such as reactive oxygen species (ROS). Also, pH, temperature, and volume of culture medium did not affect by plasma irradiation [75].

Given that compositions of culture medium act as key mediators of biological responses triggered by gas plasma and can affect results. In a study by Boehm et al. hypothesized that instead of a culture medium, PBS to be used. The solution

**189**

*Nano Technology and Gas Plasma as Novel Therapeutic Strategies for Ovarian Cancer…*

compounds used can play an important role in investigating the cytotoxic effect of plasma on HeLa and CHO-K1 cell lines. They found that the surrounding milieu and the presence of anti-oxidants such as pyruvate in PBS can change and influence the

In addition, cell proliferation and cell motility of SKOV-3, OVCAR-3, TOV-21G, and TOV-112G cells as OC cells investigated by direct and indirect exposure to gas plasma. In accordance with other studies, CAP and PAM have similar cytotoxicity effects on the mentioned cell lines. Also, dose-response effects depending on cell

Bekeschus and colleagues attempt to insight the interaction of gas plasma with tumor microenvironment and immunomodulatory properties. Accordingly, human OC cell lines OVCAR-3 and SKOV-3 as well as human THP-1 monocytes have been used to examined gas plasma effect. The results indicate that plasma can trigger cell death in a caspase 3/7 independent and dependent manner for OVCAR-3 and SKOV-3 OC cell lines, respectively. Also, tumor cell-induced monocyte/macrophage

Owing to clinical facts and desirability of Ringer's Lactate solution in comparison to the culture medium, Bisag et al. investigated the efficacy of plasma-activated Ringer's Lactate solution (PA-RL) on OC cell lines (SKOV-3 and OV-90) and noncancer cells (HOSE cell line and two lines of immortalized fibroblasts (F1 and F2)). It was the first time that a multiwire plasma source without needing technical gas was used to activate a solution with a volume of 20 mL. Chemical characterization and measurement of long-lived RONS concentration in different PA-RL dilutions were performed. Results confirm that PA-RL showed selective cytotoxicity towards cancer cells, whereas normal cells remained unaffected. These observations are

in the PA-RL [79].

Improving the performance of conventional treatments is a significant part of oncotherapy. The cancer treatment new strategy requires advantages over conventional treatment methods. This is achieved by exploring new approaches to restore sensitivity to chemotherapy. Thus, gas plasma oncotherapy to introducing as an innovative oncotherapeutics agent should have been effective than conventional drugs. Besides, able to re-sensitize chemotherapy resistance cells to chemotherapeu-

Combination effects of CAP and PAL with conventional therapy like chemotherapy, radiation therapy, pulsed electric fields, nanoparticles, and plant origin have been discussed in recent years to improve the effectiveness of these methods. Here we also report the last work about the combination of chemotherapy drugs with gas

In a most recent research, to develop an innovative strategy for OC treatment, Rasouli et al. focused on the selective effect of gas plasma oncotherapy and eliminating chemotherapy resistance. For this purpose, hypodiploid human cell line, A2780 CP, SKOV-3 as OC cell lines, and Granulosa cells (GCs) as normal primary cells were used. As shown in **Figure 10**, we further utilized several treatment modalities including chemotherapeutic agents (carboplatin (CAR), PTX, a combination of CAR and PTX), gas plasma (direct exposure (CAP), plasma activated medium (PAM)), and combination of PAM whit chemotherapy drugs. IC50 of mentioned cells and selectivity index of cancer cell lines were obtained. Our results demonstrated the calculated selectivity indices of the CAR and PAM for A2780 CP, SKOV-3 smaller than the three that specified for the interesting selectivity index. Among all plasma treatment methods, PAM 10% FBS induced high selectivity

−

*4.3.1 Gas plasma restores chemotherapy sensitivity in chemoresistance OC cells*

tic agents while maintaining selective effect toward normal and cancer cells.

plasma that has been conducted for OC treatment.

*DOI: http://dx.doi.org/10.5772/intechopen.96387*

generation of H2O2 and related results [76].

phenotype reverted by plasma therapy [78].

related to the pH and H2O2 and NO2

type and exposure time [77].

*Nano Technology and Gas Plasma as Novel Therapeutic Strategies for Ovarian Cancer… DOI: http://dx.doi.org/10.5772/intechopen.96387*

compounds used can play an important role in investigating the cytotoxic effect of plasma on HeLa and CHO-K1 cell lines. They found that the surrounding milieu and the presence of anti-oxidants such as pyruvate in PBS can change and influence the generation of H2O2 and related results [76].

In addition, cell proliferation and cell motility of SKOV-3, OVCAR-3, TOV-21G, and TOV-112G cells as OC cells investigated by direct and indirect exposure to gas plasma. In accordance with other studies, CAP and PAM have similar cytotoxicity effects on the mentioned cell lines. Also, dose-response effects depending on cell type and exposure time [77].

Bekeschus and colleagues attempt to insight the interaction of gas plasma with tumor microenvironment and immunomodulatory properties. Accordingly, human OC cell lines OVCAR-3 and SKOV-3 as well as human THP-1 monocytes have been used to examined gas plasma effect. The results indicate that plasma can trigger cell death in a caspase 3/7 independent and dependent manner for OVCAR-3 and SKOV-3 OC cell lines, respectively. Also, tumor cell-induced monocyte/macrophage phenotype reverted by plasma therapy [78].

Owing to clinical facts and desirability of Ringer's Lactate solution in comparison to the culture medium, Bisag et al. investigated the efficacy of plasma-activated Ringer's Lactate solution (PA-RL) on OC cell lines (SKOV-3 and OV-90) and noncancer cells (HOSE cell line and two lines of immortalized fibroblasts (F1 and F2)). It was the first time that a multiwire plasma source without needing technical gas was used to activate a solution with a volume of 20 mL. Chemical characterization and measurement of long-lived RONS concentration in different PA-RL dilutions were performed. Results confirm that PA-RL showed selective cytotoxicity towards cancer cells, whereas normal cells remained unaffected. These observations are related to the pH and H2O2 and NO2 − in the PA-RL [79].

### *4.3.1 Gas plasma restores chemotherapy sensitivity in chemoresistance OC cells*

Improving the performance of conventional treatments is a significant part of oncotherapy. The cancer treatment new strategy requires advantages over conventional treatment methods. This is achieved by exploring new approaches to restore sensitivity to chemotherapy. Thus, gas plasma oncotherapy to introducing as an innovative oncotherapeutics agent should have been effective than conventional drugs. Besides, able to re-sensitize chemotherapy resistance cells to chemotherapeutic agents while maintaining selective effect toward normal and cancer cells.

Combination effects of CAP and PAL with conventional therapy like chemotherapy, radiation therapy, pulsed electric fields, nanoparticles, and plant origin have been discussed in recent years to improve the effectiveness of these methods. Here we also report the last work about the combination of chemotherapy drugs with gas plasma that has been conducted for OC treatment.

In a most recent research, to develop an innovative strategy for OC treatment, Rasouli et al. focused on the selective effect of gas plasma oncotherapy and eliminating chemotherapy resistance. For this purpose, hypodiploid human cell line, A2780 CP, SKOV-3 as OC cell lines, and Granulosa cells (GCs) as normal primary cells were used. As shown in **Figure 10**, we further utilized several treatment modalities including chemotherapeutic agents (carboplatin (CAR), PTX, a combination of CAR and PTX), gas plasma (direct exposure (CAP), plasma activated medium (PAM)), and combination of PAM whit chemotherapy drugs. IC50 of mentioned cells and selectivity index of cancer cell lines were obtained. Our results demonstrated the calculated selectivity indices of the CAR and PAM for A2780 CP, SKOV-3 smaller than the three that specified for the interesting selectivity index. Among all plasma treatment methods, PAM 10% FBS induced high selectivity

*Ovarian Cancer - Updates in Tumour Biology and Therapeutics*

Selectivity towards cancer cells, chemotherapy-resistance elimination, restore

Gas plasma effects on OC were first examined on two human epithelial ovarian carcinoma cell lines, SKOV3 and HRA and normal human fetal lung fibroblast cell lines, WI-38 and MRC-5. Nonequilibrium atmospheric pressure plasma (NEAPP) was utilized to assess toxicity and proliferation inhibition. Cell proliferation, flow cytometry, western blot analysis along with pH, temperature, and volume of the medium before and after plasma treatments were evaluated. NEAPP selectively targets two cancer cells and induces apoptosis in them, while normal cells were not damaged. Although the authors do not address the mechanism of action, they assume a pivotal role in the process of plasma application for UV radiation, charged particles, and free radicals such as reactive oxygen species (ROS). Also, pH, temperature, and volume of culture medium did not affect by plasma irradiation [75]. Given that compositions of culture medium act as key mediators of biological responses triggered by gas plasma and can affect results. In a study by Boehm et al. hypothesized that instead of a culture medium, PBS to be used. The solution

sensitivity to chemotherapy, inhibition of metastasis, and more recently the possible mechanism of plasma action has been achieved in these studies.

*Flow chart of major steps in CAP leading to selective apoptosis of tumor cells. Step 1: CAP generates NO2*

*H2O2 in cell containing medium for 1 minute. Alternatively, CAP is used to treat medium, creating PAM* 

*to antioxidant glutathione depletion. Step 8: In parallel with step 7, increased H2O2 resulting from catalase* 

*O2 also leads to HOCl generation by peroxidase, in the presence of Cl−*

*NO/ONOO−*

*Step 9: If both lipid peroxidation and glutathione depletion occur, then caspase-associated apoptosis can take place, finally leading to cell death. Steps 1–3 correspond to CAP triggering or activation of a few cells, thereby initiating propagating bystander signaling in steps 4–6. Steps 7–9 are the steps that lead to the final cell apoptosis. These steps are activated only if the repeated performance of steps 4–6 has caused a sufficiently* 

*signaling. This figure was obtained with permission from [74] under the terms of creative commons CC BY* 

 *and H2O2 containing medium are used in reconstitution experiments* 

*O2 leads to further catalase inactivation and the process cycles back to step 4. Step 7: Increased* 

*O2 near cells following O2NOOH pathway, as described* 

 *is leading ultimately to secondary <sup>1</sup>*

*O2 leads to H2O2 entering cells via aquaporins, leading* 

*OH formation near the cell membrane and lipid oxidation. Step 8′:* 

*NO/ONOO−*

 *(generated through NOX1 and NOS) are no longer* 

 *signaling can also lead to lipid peroxidation.* 

*−*

*in reference. Step 3: Few catalase molecules on a few cells are inactivated due to primary 1*

*- and H2O2 create primary 1*

*− and* 

*O2 near cells. Step* 

*. The interaction* 

 *- mediated apoptosis-inducing* 

*O2. Step 6:* 

**188**

**Figure 9.**

*(step 1"). Step 2: NO2*

*This additional <sup>1</sup>*

*license.*

*loss from secondary <sup>1</sup>*

*between NOX1 derived O2*

*(step 1′). Defined concentrations of NO2*

*−*

*4: At the site of inactivated catalase, H2O2 and ONOO−*

*●− leads to ●*

*H2O2 resulting from catalase loss from secondary 1*

*If HOCl signaling is suppressed, an alternative ●*

*decomposed. Step 5: The reaction between H2O2 and ONOO−*

*high degree of catalase inactivation for reactivation of HOCl or ●*

### **Figure 10.**

*Diagram of treatment methods in this study. CAP (cold atmospheric plasma), PAM (plasma activated medium), PTX (paclitaxel), CAR (carboplatin). All treatment methods were performed on three A2780 CP, SKOV-3, and GCs cells.*

towards OC cells. Also, selectivity performance of other plasma therapies such as CAP 1% FBS, CAP 10% FBS, and PAM 1% FBS compared with chemotherapy drugs were desirable. According to the carboplatin resistance of cancer cells, it was a very interesting result [19].

In another part of this study, to improve the performance of chemotherapy drugs, co-treatment of these agents with PAM was investigated. Although PAM improves efficacy and selectivity indices of CAR and PTX but induces high selectivity in conjunction with CAR. In general, we concluded that PAM alone and simultaneous with CAR selectively induced apoptosis in chemotherapy-resistant OC cells accompanied with high expression of P53, BAX, and CASP-3. The novelty of PAM and combination treatment led to developing a new trend in OC oncotherapy associated with produced RONS (H2O2, NO2 − , NO3 − ), reduced pH in plasma activated medium and physical factors such as UV and electric field [19].

Assuming gas plasma oncotherapy is closer to the therapeutic facts, Utsumi et al. used NEAPP-activated medium (NEAPP-AM) as an intraperitoneal (IP) treatment modality. To this end, for the first time, NOS2 and NOS3 as chronic paclitaxel/cisplatin-resistant OC cells and xenografted tumors in a mouse model were investigated by NEAPP-AM. Also, they assessed the role of ROS or their scavengers in NOS2 and NOS3 OC cells. Given fact that NOS2 and NOS3 are acquired resistance to paclitaxel, the study was a very crucial role in plasma oncotherapy research. The results revealed PAM has an interesting cytotoxicity effect on chemo-resistant OC cells. Besides, PAM can induce an anti-tumor effect on the xenograft model. There is no difference between direct and indirect treatment, but due to the benefits that PAM creates the authors suggested it as future intraperitoneal administration [80].

Clear cell carcinoma (CCC) of the ovary is a rare histological subtype of epithelial OC (EOC), has the worst prognosis and exhibits high rates of recurrence

**191**

**Figure 11.**

*Nano Technology and Gas Plasma as Novel Therapeutic Strategies for Ovarian Cancer…*

and low chemosensitivity. Therefore, developed a novel approach to combat CCC is critical. Hence, Utsumi et al investigated the influence of gas plasma on TOV21G as a CCC cell line by NEAPP-AM. The ES-2, SKOV3, and NOS2 as other EOC cell lines and omentum derived human fibroblastic cells (OHFC) and human peritoneal mesothelial cells (HPMC) as normal cells were examined. The study demonstrated that PAM with high selectivity induces apoptosis in CCC cells which is resistant to chemotherapy. Also, ROS produced by PAM in cancer cells were considered as a

E-cadherin has pivotal roles in epithelial cell behavior, tissue formation, and suppression of cancer and is a critical part of epithelial cell adhesion and epithelialto-mesenchymal transition (EMT). Furthermore, transforming growth factor-β1 (TGF-β1) is a multifunctional growth factor that plays a crucial role in chronic inflammation in various tissues and regulates several cellular processes, including cell cycle arrest, differentiation, morphogenesis, and apoptosis. From this viewpoint, Wang et al. focused on various factors such as cell numbers and the morphological characteristics of cells, that are thought to be effective in the interaction of plasma and cells. Four human OC cell lines, OVCAR-3, TOV21G, NOS2, and ES-2 used to examine differences in responses to gas plasma oncotherapy through direct and indirect irradiation. The point to consider was the different sensitivities of the used cancer cells to conventional chemotherapy drugs. They concluded compared with the other two cell lines, TOV21G and ES-2 cells were drastically sensitive to PAM treatment, as well as, sensitivity to PAM therapy in OC cells is related to their number and morphology. Having a negative impact of cell density on cell proliferation inhibition rate (PIR) is more evident in OVCAR-3 and NOS2 cells. Regarding cell morphology and PAM sensitivity, low E-cadherin expression was suggested as a factor for more PAM sensitivity. Also, TGF-β1 with inducing mesenchymal mor-

OC is one of the gynecological malignancies that penetrates the peritoneum. That means cancer developed a spread of largest volume and treatment of it is challenging. Intraperitoneal therapy is a concept utilized in these cases to focused

*Mechanisms of the anti-metastatic effect of PAM. ROS in PAM diffuses into ES2 cells and down-regulates MMP-9 expression via inhibiting of MAPK pathway, suppressing cancer cell adhesion, migration and invasion onto mesothelial cells lining the peritoneal cavity. Finally, PAM prevents intraperitoneal metastasis. This figure* 

*was obtained with permission from [83] under the terms of creative commons CC BY license.*

*DOI: http://dx.doi.org/10.5772/intechopen.96387*

phologic change can sensitize cancer cells to PAM [82].

selectivity factor [81].

### *Nano Technology and Gas Plasma as Novel Therapeutic Strategies for Ovarian Cancer… DOI: http://dx.doi.org/10.5772/intechopen.96387*

and low chemosensitivity. Therefore, developed a novel approach to combat CCC is critical. Hence, Utsumi et al investigated the influence of gas plasma on TOV21G as a CCC cell line by NEAPP-AM. The ES-2, SKOV3, and NOS2 as other EOC cell lines and omentum derived human fibroblastic cells (OHFC) and human peritoneal mesothelial cells (HPMC) as normal cells were examined. The study demonstrated that PAM with high selectivity induces apoptosis in CCC cells which is resistant to chemotherapy. Also, ROS produced by PAM in cancer cells were considered as a selectivity factor [81].

E-cadherin has pivotal roles in epithelial cell behavior, tissue formation, and suppression of cancer and is a critical part of epithelial cell adhesion and epithelialto-mesenchymal transition (EMT). Furthermore, transforming growth factor-β1 (TGF-β1) is a multifunctional growth factor that plays a crucial role in chronic inflammation in various tissues and regulates several cellular processes, including cell cycle arrest, differentiation, morphogenesis, and apoptosis. From this viewpoint, Wang et al. focused on various factors such as cell numbers and the morphological characteristics of cells, that are thought to be effective in the interaction of plasma and cells. Four human OC cell lines, OVCAR-3, TOV21G, NOS2, and ES-2 used to examine differences in responses to gas plasma oncotherapy through direct and indirect irradiation. The point to consider was the different sensitivities of the used cancer cells to conventional chemotherapy drugs. They concluded compared with the other two cell lines, TOV21G and ES-2 cells were drastically sensitive to PAM treatment, as well as, sensitivity to PAM therapy in OC cells is related to their number and morphology. Having a negative impact of cell density on cell proliferation inhibition rate (PIR) is more evident in OVCAR-3 and NOS2 cells. Regarding cell morphology and PAM sensitivity, low E-cadherin expression was suggested as a factor for more PAM sensitivity. Also, TGF-β1 with inducing mesenchymal morphologic change can sensitize cancer cells to PAM [82].

OC is one of the gynecological malignancies that penetrates the peritoneum. That means cancer developed a spread of largest volume and treatment of it is challenging. Intraperitoneal therapy is a concept utilized in these cases to focused

### **Figure 11.**

*Ovarian Cancer - Updates in Tumour Biology and Therapeutics*

towards OC cells. Also, selectivity performance of other plasma therapies such as CAP 1% FBS, CAP 10% FBS, and PAM 1% FBS compared with chemotherapy drugs were desirable. According to the carboplatin resistance of cancer cells, it was a very

*Diagram of treatment methods in this study. CAP (cold atmospheric plasma), PAM (plasma activated medium), PTX (paclitaxel), CAR (carboplatin). All treatment methods were performed on three A2780 CP,* 

In another part of this study, to improve the performance of chemotherapy drugs, co-treatment of these agents with PAM was investigated. Although PAM improves efficacy and selectivity indices of CAR and PTX but induces high selectivity in conjunction with CAR. In general, we concluded that PAM alone and simultaneous with CAR selectively induced apoptosis in chemotherapy-resistant OC cells accompanied with high expression of P53, BAX, and CASP-3. The novelty of PAM and combination treatment led to developing a new trend in OC oncotherapy asso-

> − , NO3 −

Assuming gas plasma oncotherapy is closer to the therapeutic facts, Utsumi et al. used NEAPP-activated medium (NEAPP-AM) as an intraperitoneal (IP) treatment modality. To this end, for the first time, NOS2 and NOS3 as chronic paclitaxel/cisplatin-resistant OC cells and xenografted tumors in a mouse model were investigated by NEAPP-AM. Also, they assessed the role of ROS or their scavengers in NOS2 and NOS3 OC cells. Given fact that NOS2 and NOS3 are acquired resistance to paclitaxel, the study was a very crucial role in plasma oncotherapy research. The results revealed PAM has an interesting cytotoxicity effect on chemo-resistant OC cells. Besides, PAM can induce an anti-tumor effect on the xenograft model. There is no difference between direct and indirect treatment, but due to the benefits that PAM creates the authors suggested it as future intraperito-

Clear cell carcinoma (CCC) of the ovary is a rare histological subtype of epithelial OC (EOC), has the worst prognosis and exhibits high rates of recurrence

medium and physical factors such as UV and electric field [19].

), reduced pH in plasma activated

**190**

interesting result [19].

*SKOV-3, and GCs cells.*

**Figure 10.**

neal administration [80].

ciated with produced RONS (H2O2, NO2

*Mechanisms of the anti-metastatic effect of PAM. ROS in PAM diffuses into ES2 cells and down-regulates MMP-9 expression via inhibiting of MAPK pathway, suppressing cancer cell adhesion, migration and invasion onto mesothelial cells lining the peritoneal cavity. Finally, PAM prevents intraperitoneal metastasis. This figure was obtained with permission from [83] under the terms of creative commons CC BY license.*

on local delivery. For this purpose, Nakamura et al. introducing PAM intraperitoneal therapy as an innovative option for OC oncotherapy. The experiments were designed to assess the inhibit metastasis effectiveness of PAM on OC ES2, SKOV3, and WI-38 cell lines in vitro and ES2 in in-vivo levels. They mentioned that PAM treatment suppressed ES2 cell migration, invasion, and adhesion while cell viability changes were negligible [83].

Most importantly, PAM inhibited peritoneal dissemination of ES2 cells, resulting in prolonged survival in an in-vivo mouse model of intraperitoneal metastasis. Furthermore, the evaluated underlying mechanism revealed PAM inhibited the phosphorylation of JNK1/2 and p38 MAPK and prevented the MAPK pathway activation. Besides, PAM was decreased MMP-9 expression [83] (**Figure 11**).

### **5. Conclusion and perspective**

Despite rapid advancements for OC oncotherapy, our understanding of the cause and management of OC is limited. Cancer cells become resistant to conventional chemotherapy and increasing the concentration of drugs just enhances the side effects, and does not cause any improvement in recovery. Besides, approved oncotherapy drugs for clinical and preclinical administration, faces several obstacles to treatment. Introducing combined therapeutic strategies such as nanoparticle and gas plasma that used the synergizing advantage of these approaches holds great potential for future combination or multimodal OC treatment.

The bioavailability property of NPs enhances their efficacy in drug loading and protects them from physiological barriers. To provide a suitable platform for clinical trials, it is very crucial to analyze the NPs safety at the level of in vitro and in vivo. Therefore, the reviewed NPs need more experiments in the level of in vivo for entrance into the clinical arena. Furthermore, gas plasma is not considered as the therapeutic strategy for modern medicine unless focused studies are performed on the design and manufacturing of simple, accurate, standard, and low-cost plasma devices.

While the identification of the underlying mechanism of each gas plasma and nanocarriers technology is under debate, promising observations open up interesting avenues for them as an emerging candidate in future oncotherapy. Independently from action mechanisms of gas plasma and nanoparticles, these therapies rely on the selective ability of them to discriminate between healthy cells and cancerous ones.

Indeed, gas plasma and nanoparticles as novel biomedical fields need funding from a wide range of government agencies and international research centers to be specifically targeted towards research at the intersection of these disciplines and resolve modern challenges such as cancer. Finally, we hope that this chapter will enhance collaboration between researchers in interdisciplinary research fields including physics, chemistry, biology, oncology, and medicine, and provide the needed interplay to address current challenges in OC management. Aside from providing new knowledge on molecular mechanisms in the mentioned modalities, to overcome the failure of oncological ovarian treatment, synergizing of innovative therapeutic approaches can be useful.

Collectively, our strategy potentially opens a new and accessible approach and led to addresses several cancer challenges. As a future direction, we hope to combine new approaches with conventional treatments to obtain finer modalities, improve the efficiency of each of them, and resolve oncotherapy challenges.

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*Nano Technology and Gas Plasma as Novel Therapeutic Strategies for Ovarian Cancer…*

*DOI: http://dx.doi.org/10.5772/intechopen.96387*

**Acronyms and abbreviations**

DDS Drug delivery system GDS Gene delivery system CTX Chemotherapy ECT Electrochemotherapy

PDT Photodynamic Therapy

RT Radiotherapy

HT Hyperthermia UV Ultraviolet NPs Nanoparticles miRNA microRNA

Tf Transferrin R8 Octaarginine DOX Doxorubicin HA Hyaluronic acid

shRNA Short Hairpin RNA

FRα Folate Receptor α FA Folic Acid NE Nanoemulsion DTX Docetaxel TQR Tariquidar

MNPs Magnetic NPs ETB Erlotinib

CB Cubosomes

Wtmn Wortmannin CIS Cisplatin

GEM Gemcitabine

p70S6K p70 S6 kinase

pHSL pH-Sensitive Liposome CHEMS Cholesteryl Hemisuccinate

MRI Magnetic Resonance Image

EGFR Growth Factor Receptor

NDCs NP-Drug Conjugates ADCs Antibody-Drug Conjugates MMAE Monomethyl Auristatin E

EOC Epithelial Ovarian Cancer

KSP Kinesin Spindle Protein

PROC Platinum-Resistant Ovarian Cancer

AONS Antisense Oligonucleotides siRNA Small interfering RNA

CIS-pARG-HA NPs Cisplatin-loaded polyarginine-HA NPs

PIPAC Pressurized Intraperitoneal Aerosol Chemotherapy

DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

PEI-g-PCL-b-PEG-FA Polyethylenimine-graft-polycaprolactone-block-

poly(ethyleneglycol) modified FA

GO-PVP-NPs Polyvinylpyrrolidone functionalized Graphene Oxide NPs

The authors have no conflict of interest to declare.

RONS Reactive Oxygen and Nitrogen Species

**Conflict of interest**

*Nano Technology and Gas Plasma as Novel Therapeutic Strategies for Ovarian Cancer… DOI: http://dx.doi.org/10.5772/intechopen.96387*
