**3.1 The concept of targeting**

Paul Ehrlich first pioneered the theory of targeting [69]. Targeted delivery is a process of binding a drug into a specific site of the body, to minimize the toxicity

*Porphyrinoid Photosensitizers for Targeted and Precise Photodynamic Therapy: Progress… DOI: http://dx.doi.org/10.5772/intechopen.109071*

**Figure 7.**

*The concept of treatment of PDT with targeted PS into tumor tissue [70].*

by avoiding the localization of the drug into other body parts (**Figure 7**) [71]. By efficient targeting, the concentration of active ingredients should be more at the site of diseased tissue, this helps in reducing the dose and side effects due to decreased body distribution and accumulation of the drug. Drug targeting has many advantages over conventional delivery of drugs; such as better therapeutic effect, reduced expense by decreasing the dose, increase concentration of drug at the specific target site, minimizing the adverse effect, and preventing the accumulation of drugs in the non-targeting site.

.In the case of PDT, the PS has disadvantages such as it can accumulate in the normal vegetative tissues and can cause unfavorable conditions, for example, phototoxicity, photophobia, etc. The only solution to avoid such dangerous situations associated with PDT is targeted photodynamic therapy. Targeted PDT helps in preventing such types of adverse reactions [72, 73]. As per reports PS, they self-have an inherent tumor-targeting ability, but in many cases, it fails to achieve efficient targeting. Better targeting is achieved by the encapsulation of the PS into a proper nanocarrier to improve its accumulation in a specific target site. S. Nair et al. developed a unique nanomedicine to inhibit the migration of metastatic breast cancer cells. The team fabricated a core shell with PLGA nanocore encapsulated with temoporphin (mTHPC) as PS [74]. Manzoor et al. developed a photosensitizer (Ce6 or m-THPC) conjugated nanoparticle with an optical and magnetic contrast agent. Interestingly the system showed better targeting ability with higher singlet oxygen-producing efficiency and fluorescence quantum yield [75]. The process of targeting can be divided into active targeting and passive targeting based on their mechanisms as shown in **Figure 8**.

In passive targeting, the nanocarriers (in the case of tumor targeting) move across and target the cell based on the properties of the tumor microenvironment (TME), which is distinct from the normal tissue. So, passive targeting happens based on the biodistribution (i.e., physicochemical factors and pathophysiological factors) of the target tissue [76]. The limitations of passive targeting are ineffective transfection at the target site and quick clearance of some smaller drug particles due to variable blood flow in the target site.

Active targeting can be used to overcome such limitations to improve the binding of PS to the specific targeted site [77]. It is the process of drug delivery to a specific targeting site, based on the interaction between the ligands, receptors, etc. [78]. **Figure 8** depicts passive and active PDT schematically.

#### **3.2 .Passive targeting**

In passive accumulation, the drugs or drugs in nanocarriers will naturally penetrate the targeted tissue due to the change in the milieu of the target site concerning its healthy surroundings. Here the driving force to reach the target site include various physicochemical factors of drug or nanocarrier, material composition, size, and

**Figure 8.** *Presentation of the passive and active PDT schematically [75].*

surface characters (e.g., cationic or anionic nature) and pathophysiological factors of the target site, such as TME as well as EPR effect. For example, the lower diameter of nanovesicles promotes passive targeting of rapidly growing carcinoma due to the enhanced permeability and retention effect (EPR) [79]. Maeda and Matsumura first narrated the EPR effect, the macromolecules or smaller nanoparticles can progressively accumulate into the tumor, due to the leaky vasculature and poor lymphatic drainage in the tumor tissue [80, 81].

To maximize the effectiveness of PDT, the nanocarrier has the potential to enhance the selectivity and sensitivity of PS by altering the distribution pattern. In the tumor area, the permeability is high to promote the rapid multiplication of the tumor, and at the same time, the lymphatic drainage is poor in the area. As a result of that, the macromolecules and nanoparticles are not cleared and get accumulated in the tumor site. The biophysical and morphological characteristics of the nanocarriers play a significant role in the targeted distribution of PS-loaded nanocarriers. Due to its large size, it will not quickly revert to the bloodstream and thus results in its accumulation in the specific tumor site.

To facilitate longer circulation periods and enhance accumulations at the desired region, hydrophilic polymers, surfactants, or biodegradable copolymers are typically used to coat nanoparticles. Because of poor biodistribution of hydrophobic PS, low cellular uptake, and low efficiency in treating bulky tumors, nanoparticles are developed [82]. Liposomes, polymer micelles, polymeric nanoparticles, gold nanoparticles, and carbon-based nanoparticles are used as drug carriers to enhance the drug loading capacity, improve drug delivery, increase bioavailability, etc. [83].

Liposomes are spherical structured vesicles with hydrophobic cavities and hydrophilic cavities. Such vesicular structures will help to increase drug loading efficiency and therapeutic efficiency. It will enter the cancer cell by endocytosis and maintain the constant release of PS at a specific site and it is achieved by passive targeting. The liposome can be activated by modifying its surface with ligands.

Passive PSs are more readily available, less expensive, and simpler to administer. For example, amphiphilic cationic porphyrin-based photosensitizers. The efficacy of *Porphyrinoid Photosensitizers for Targeted and Precise Photodynamic Therapy: Progress… DOI: http://dx.doi.org/10.5772/intechopen.109071*

a PS in PDT depends on its amphiphilicity; hydrophilicity will promote the distribution and improved clearance rate, while lipophilicity will aid in cellular uptake. PS can accumulate in the targeted area by the passive target [78].

#### **3.3 Active targeting**

In active targeting, the pharmacophore directs to the specific receptor site by peripherally decorated targeting moieties in the delivery vesicle [84]. The process of active targeting is a promising methodology. It helps to overcome the limitations of the passive targeting method by improving the binding of PS to a specific tumor site. So, this method reduces the major adverse effects of PDT by avoiding the accumulation of PS in the non-targeting area. Various research in nanotechnology significantly contributed to the area of active targeting. Well-developed selective tumor-targeting ligands, such as monoclonal antibodies (mAb), antibody fragments, peptides, aptamers (singlestranded oligonucleotides), and nucleic acid are coupled with nanoparticles [72]. Such targeting ligand-incorporated nanoparticles or vesicles shows active binding of PS into tumor receptors. Usually in PDT to enhance the tumor cell uptake of PS, these modified nanoparticles will target overexpressed tumor receptors like folate receptor, transferrin receptor, CD44, and growth factor receptor (EGFR) [85]. For example, targeted photodynamic therapy (TPDT) was attained by encapsulating PS into polymeric micelles for breast cancer treatment and diagnosis [86]. Breast cancer is the most common cancer in women around the world, and it is currently treated with traditional methods such as chemotherapy, radiotherapy, and surgery. The challenges in using PDT in breast cancer were poor water solubility and the non-specificity of PSs. These problems frequently reduced the overall efficacy of this novel cancer treatment. But PDT driven with monoclonal antibody targeted drug delivery systems based on gold NP showed improved specificity of PSs in breast cancer tumors. In approximately 25% of breast cancer cases, HER2 monoclonal antibodies are conjugated with gold nanoparticles, and this antibody binds to the HER2 receptors that are overexpressed on the cellular surface [87]. In recent years, monoclonal antibody-based targeted drug delivery gains much attention because of its high accuracy in targeting and better therapeutic activity. For example, porphyrin-trastuzumab complexes are effectively used to deliver the PS to a specific target site. Trastuzumab is an FDA-approved Mab against HER2 (Epidermal growth factor 2 receptor)-positive breast cancer [88, 89].

Another major overexpressed receptor in tumors is the transferrin receptor (TfR), it can be targeted using antibodies against TfR and transferrin itself. HpD with albumin and transferrin conjugates showed better-prolonged phototoxicity. Nowadays transferrin-conjugated liposome is one of the important targeting aids, but M. O Senge reported that temoporfin-loaded transferrin-conjugated liposomes do not improve the efficiency of invitro PDT [90]. On the other hand, L. E. Xodo and coworkers developed a successful RAS-driven active targeting system with cationic porphyrins and studied it in pancreatic adenocarcinoma cells. The confocal microscopic results showed the photoactivated PS binds and induces apoptosis even in nanomolar concentration by RAS gene suppression system [91].

#### **3.4 Peptide-mediated targeting**

Peptide-mediated targeting in PDT is also known as peptide-based supramolecular photodynamic therapy. As the name represents here the PS and the peptide molecules are allowed to self-assemble either covalently or non-covalently, thus directing the PS

into the cancer lesion site effectively [92]. As discussed above to achieve the targeted photodynamic therapy, one should localize the PS into the intracellular space where ever required. But such targeting is hard to achieve in many cases due to various structural and morphological characteristics of the PS, such as molecular size, shape, penetrability, and solubility. Here comes the importance of peptide targeting. It's reported that the number of receptors in the tumor periphery is more, so the small peptides can bind with it. Thus, a PS-bound peptide can effectively deliver them to the target site.

Small peptides are having various advantages, such as ease in synthesis, good penetrability even into the BBB, small size, rapid tumor access, ease in linking to a spacer through amide bonds, and high clearance rate [93]. Some of the major examples of peptides used widely for PDT-mediated anti-cancer therapy include:


H. Lin *et al* formulated a pH-responsive peptide targeted PDT. Here protoporphyrin IX (PpIX) coupled with the N terminal of the P12 peptide to form a PpIX-P12 conjugate. This peptide conjugate targets the acidic microenvironment of the tumor-as

#### **Figure 9.**

*pH-responsive targeted PDT; protoporphyrin IX (PpIX) coupled with P12 peptide (PpIX-P12 conjugate) binds the cancer cells in the acidic microenvironment [99].*

*Porphyrinoid Photosensitizers for Targeted and Precise Photodynamic Therapy: Progress… DOI: http://dx.doi.org/10.5772/intechopen.109071*

shown in **Figure 9**. It could localize specifically into the tumor cell and a greater phototoxic effect was shown within the acidic medium. The team first explained the activity through in vitro studies and found that PpIX-P12 is having the drift to get attached to cancerous cells. Thus, they proved that this will be an asset in treating solid tumors [99].

.Another example is the use of epidermal growth factor (EGF) peptide-labeled formulations showed better killing efficiency than unlabeled formulations. The peptide is combined with gold nanoparticles (EGF pep-Au NP) encapsulated with silicone phthalocyanine (PS-4). The nanoformulation efficiently targeted the early endosomes, and *in vitro* studies showed that this combination was twofold effective in destroying tumor cells as compared to conventional systems. As the silicone phthalocyanines have the potential to generate fluorescence, they also conducted *in vivo* investigations to support their claim and were able to perform fluorescence imaging to ensure targeting [100].

Formation of a dual targeting PS-peptide amphiphile conjugate containing encapsulated doxorubicin for increased chemo-photodynamic cancer therapy. When the Doxorubicin-PS-Peptide nano micelle enters the tumor via R8 peptide. The doxorubicin and PpIX were released as a result of cathepsin B-triggered hydrolysis [77]. X. Z. Zhang et al. created a cell membrane targeting PpIX-PS by loading a charge-reversible selfdelivery chimeric peptide. This self-assembled peptide can achieve a long-term photodynamic effect for about 14 hours, by delivering the PS through enhanced permeability and retention effect. Under an acidic environment, the peptide-PS complex changes the charge and in the presence of light, ROS is generated which leads to direct necrosis [101].

A chip made of a chimeric peptide and PpIX was developed by C. Hong et al. This chip has a hydrophilic PEG chain, and a bioactive peptide series with a double targeting ability. This double-targeted chip can bind with the mitochondria and plasma membrane, hence creating a synergistic effect. During the introduction of light, generated ROS can cause tumor necrosis [102]. Another example of a synergistic effect is the development of a nanorod with dual targeting capacity (plasma membrane and Nucleus). The PS loaded inside the self-assembling chimeric peptide upon light irradiation, generated ROS can induce necrosis and thereby cell death [103].

The disadvantages of ALA targeting can be mitigated by incorporating an ALA prodrug and a zwitterionic stealth peptide into a nanoparticle. The ALA-conjugated prodrug nanoparticles were created through conjugation with a thiolated stealth peptide sequence called CPPPPEKEKEKEKEKEDGR. The release of ALA prodrugs is dependent on lysosomal/endosomal pH 5.5 [104].

The development of a protease activatable-cell-penetrating peptide-PS conjugate can induce proteolysis using peptide activity at the tumor site which will result in the release of the PS into the tumor site. The fluorescence produced by the conjugate after getting accumulated into the tumor will aid in tumor diagnosis and image-driven PDT [105].

Tumor hypoxia during PDT is a significant issue that affects efficacy; to address this, an arginine-peptide complex that is compatible with a *m*THPP to generate a stable nanoparticle has been designed. This combination will both release nitric oxide and can deliver PS directly into the tumor microenvironment. Nitric oxide will be released, which will help to enhance cellular oxygen levels and prevent cells from respiring through their mitochondria. Thereby resolving the difficulty [106].

## **4. Diagnostics and theranostics**

The detection of illness is broadly known as diagnosis, and biomedical imaging is one of the commonly used diagnostic tools. Biomedical imaging is a fast-growing area of research and various advanced imaging techniques include Near-Infrared (NIR) fluorescence Imaging, shortwave infrared (SWIR) fluorescence imaging, positron emission tomography (PET), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), *invivo* nuclear magnetic resonance (NMR) imaging and photoacoustic imaging [107]. Many of the imaging techniques are still at the research level, in 2020 first PET Imaging drug Gallium (68Ga) gozetotide accepted for prostate cancer in men [108]. In 1960 itself Lipson proved that the hematoporphyrin derivative discovered by T. Doughtery has the fluorescence property to diagnose tumors (**Figure 4**) [8]. He observed hematoporphyrin derivatives produce fluorescence selectively from cancer cells. Currently, porphyrinoids and their derivatives are utilized in a variety of diagnostic imaging areas, including MRI, PET/SPECT, and NIR imaging [109].

During the course of treatment for several disorders, including cancer, rheumatoid arthritis, and neurodegenerative diseases, there may be some variation (tissue alteration, physiological change, and change in medication disposition). Therefore, during therapy, frequent monitoring is necessary which theranostics can fulfill [110]. Thus, theranostics is a fast-growing research area, that combines diagnosis with narrowly focused therapy to provide patients with individualized care [111]. The use of theranostic agents can improve disease destruction with high localized cytotoxicity and minimal collateral damage. The fluorescence that is released can be used to diagnose diseases as photo diagnosis and molecular imaging, known as photosensitizer fluorescence detection (PFD). The combination of PFD and PDT will diagnose and treat the diseases. But PFD has some limitations such as autofluorescence and tissue absorption leading to decreased tissue penetration (**Figure 3**). So, such limitations can be reduced by using NIR (from 700 nm to 1000 nm) and SWIR (from 1000 nm to 2500 nm) radiations, because of their high image resolution they can deeply penetrate the tissues [112]. For example, Lanthanide (Ln) porphyrinoids show near-infrared emission and can be used for fluorescence imaging [113].

Gadolinium-porphyrin-based polymer nanotheranostics showed excellent fluorescence imaging properties, good singlet oxygen-producing efficiency, and excellent long-term colloidal stability. As shown in **Figure 10**, the nanotheranostics acted like a therapeutic tool, fluorophore, and MRI contrasting agent [114]. The coupling of porphyrinoids with nanoparticles has greater importance in cancer targeting and theranostics. Different theranostic formulations of porphyrin nanomaterials based on silica nanoparticles, fullerene, virus capsids, protein, steroids, carbohydrates, iron oxide nanoparticles, polymers, graphene oxide, and liposomes have a greater future in cancer treatment [115].

.Instead of this nano theranostics, various single molecular theranostics also gained research interest. In 2014 Zijian Guo reported a single molecule cancer theranostics *i.e.,* platinum (II)–gadolinium (III) complexes with better biocompatibility [116]. Texaphyrin, an expanded porphyrinoid, which is developed' by Sessler *et al.* showed applications in PDT. Now finding its uses as MRI contrast agents and anticancer agents [117]. Recently the same group reported Indium-111radiolabelleddmetallatedd luteum texaphyrin for prostate-specific membrane antigen (PSMA) targeting. This novel texaphyrin system showed high-resolution SPECT imaging and better PDT action toward prostate carcinoma [118]. The challenges in porphyrin-based compounds toward their theranostic or biological applications are their poor water solubility, this can be addressed by the use of cationic and anionic porphyrinoids. In the case of the lipophilic porphyrinic systems, the problem can be addressed by designing the proper nanocarrier drug delivery system such as liposomes, micelles, and silica nanoparticles [119].

*Porphyrinoid Photosensitizers for Targeted and Precise Photodynamic Therapy: Progress… DOI: http://dx.doi.org/10.5772/intechopen.109071*

**Figure 10.**

*Schematic representation of (a) components and (b) formulation of gadolinium-porphyrin-based polymer nanotheranostics; (c) its invivo administration, (d) PDT action, and (e) fluorescence (FL) and MRI imaging [114].*

Recent years of research in the metal-organic framework (MOF) gained considerable attention in catalysis and hydrogen storage cells [120]. Various nano-MOF (NMOFs) showed interesting biomedical applications in drug delivery, imaging, chemotherapy, photodynamic and photothermal therapy [120–122]. For example NMOF (Zr-NMOFs) is used for bioimaging and targeted photodynamic therapy [123]. Nowadays, covalent organic frameworks (COFs) have a promising future in the field of PDT. Integrating a dye-labeled oligonucleotide onto porphyrin-based COF nanoparticles are incredibly effective in cancer treatment and diagnosis. This COF-based nanoplatform operates well in vitro, and in vivo and is superior to theranostic probes based on other nanomaterials, such as metal-organic frameworks, in terms of stability, biocompatibility, and high integration [124].

The principles of PDT are not limited to cancer therapy, it can be used in many diseases like rheumatoid arthritis (RA), skin diseases and microbial infections, etc. Tetra sulphonatophenyl porphyrin has many applications in the field of cancer and other inflammatory diseases. The combination of tetra sulphonatophenyl porphyrin and nanoparticle is used as a diagnostic and theranostic agent for many cancer and other diseases [125].
