**2.2 Nanomaterials used for PDT enhancement**

Nanostructured delivery systems for photosensitizers can provide some major advantages in PDT. The first one is regarding the increased quantity of dyes that can be delivered to the target cell due to the large surface-to-volume ratio, while the second one refers to the prevention of the premature release of the dyes before reaching the target, enhancing the specific accumulation in the target tissue and diminishing the side effects. The third is somehow related to the second, since the loaded dyes find few obstacles in the blood stream and acquire an amphiphilic character once conjugated with nanostructures, enhancing the tumor accumulation as well. Another advantage is the privileged accumulation of nanosized materials in tumor tissues due to the enhanced permeability and retention (EPR) effect. Finally, their surface can be functionalized with a plethora of groups, so that their biodistribution, pharmacokinetics, cell uptake and surface chemistry can be tuned according to the desired application [8]. **Figure 1** summarizes the main advantages of nanotechnology combined with PDT.

Both biodegradable and non-biodegradable nanoparticles can be used to potentiate photodynamic therapy (PDT). In the case of biodegradable nanoparticles

(generally polymers and lipid-based structures), the photosensitizers are trapped inside them and are released in a controlled manner, so that singlet oxygen can be generated due to the exposition to light. On the other hand, when non-biodegradable nanoparticles are used, usually the photosensitizers are adsorbed on their surface (either external or internal, in case of porous structures), and they do not need to be released completely to generate singlet oxygen [1].

Regarding biodegradable nanoparticles, many photosensitizers have been encapsulated with water-soluble polymers, such as meso-tetra(hydroxyphenyl) porphyrin, bacteriochlorophyll, verteporfin, various phthalocyanines, methylene blue, and hypericin. Their singlet oxygen efficiency depends much on the polymer. Poly lactic glycolic acid (PLGA), for instance, has demonstrated good results compared to other polymers, along with poly lactic acid (PLA) and poly ethylene glycol (PEG). The pharmacodynamics of different polymer nanoparticles may differ from one another, and so do their bioavailability, thus the PDT efficiency may be different according to the nature of the polymer [1].

Non-biodegradable nanoparticles are mostly metallic or ceramic-based (especially silica), but polyacrylamide was also reported as a photosensitizer nanocarrier. Nevertheless, solid silica nanoparticles present higher singlet oxygen yield than polyacrylamide nanoparticles, i.e. 2 to 3-fold more singlet oxygen production by silica nanoparticles loaded with methylene blue compared to their polyacrylamide counterparts [1].

Plasmonic materials, a very important class of non-biodegradable nanomaterials, have proven to act as photosensitizers in the right conditions, and if they have photosensitizers attached to their surface, they can enhance the photodynamic efficiency of the dye. It was observed that semiconductor nanoparticles that present the suitable energy gap can be used as photosensitizers and can also be conjugated with other organic dyes. In these conjugated materials, energy can be transferred from the excited nanoparticles to the photosensitizers through a FRET mechanism [5].

Gold and silver nanoparticles are more stable and present higher extinction coefficients than organic dyes, but if one desires to use them to generate singlet oxygen, O2 molecules must be adsorbed on their surface in order to provide a rapid energy transfer between the two. Furthermore, the energy transfer from the nanoparticles to the adsorbed oxygen molecules is more efficient in low-energy surface states of metal nanoparticles rather than the high-energy states. When these conditions are fulfilled, it is believed that the PDT antitumor efficiency can be up to 10 times that of chemotherapeutics like doxorubicin [5].

Gold nanorods have been developed by different authors in order to carry phthalocyanines via adsorption onto the nanoparticle surface, either chemically with a thiol group or via electrostatic interaction. The formation of a phthalocyanine layer covering the nanoparticle prevents the aggregation of the hydrophobic photosensitizer and enhances the photodynamic activity [2, 9].

Camerin and collaborators compared the efficacy of a phthalocyanine in its free form and conjugated with gold nanoparticles in ablating B78H1 amelanotic melanoma tumors in mice. The results showed that the accumulation of photosensitizer in the tumor is enhanced when they are bound to the nanoparticles, and as a consequence, the damage was significantly more intense and the tumor growth was significantly slower than the tumors treated with the free phthalocyanines [2].

Several cases of enhancement of the photosensitizer efficiency by plasmonic nanoparticles (due to a strong energy transfer and the prevention of photobleaching) have been reported in the literature, with various photosensitizers and nanoparticle morphologies and materials [9–13]. One interesting example was demonstrated by [14], because the PDT efficiency of the photosensitizer conjugated with gold

#### *Nanomaterials for Enhanced Photodynamic Therapy DOI: http://dx.doi.org/10.5772/intechopen.94255*

nanoparticles was comparable to the free photosensitizer, but the hyperthermal effect contributed to a more intense cytotoxicity against the tumor cells.

The material and the morphology of the plasmonic nanoparticles influence on the extinction coefficients and, consequently, on the energy transfer efficiency. Gold nanourchins, for instance, present an intense extinction coefficient at 940 nm, which is within the therapeutic window, and the singlet oxygen production was intense and sufficed to eliminate cancer HeLa cells while preserving normal NIH-3 T3 fibroblasts. Gold bipyramids can be efficient singlet oxygen generators if the wavelength used overlaps with the surface plasmon resonance peak, even more efficient than methylene blue. Silver and gold nanocubes are unable to generate singlet oxygen, while in the form of nanoprisms the opposite occurs [5].

This dependence on the morphology can be explained by theoretical calculations showing that O2 can be adsorbed on Au(111), Ag(111), Au(110), Ag(110), Au(100), and Ag(100) surfaces, but on Au(111), Au(100), and Ag(111) surfaces oxygen can remain in molecular form and be excited to its singlet state, whereas on the other surfaces it dissociates into its atomic form. This can only be altered when some defects are present in the crystalline structure [5].

Quantum dots are other promising materials for photodynamic therapy. Graphene quantum dots, for example, reduced tumor cell viability to 20% in a 1.8 μM concentration, compared to 35% cell viability when the same concentration of PpIX was used. Similar results were obtained for ZnO quantum dots irradiated with blue light [5].

Silica is also widely used as a nanomaterial because it is non-toxic and optically transparent, and their surface chemical functionalization is easily achieved due to the presence of several hydroxyl groups on its surface. When it comes to PDT, silica can act as a carrier of photosensitizers, protecting them from enzymatic degradation and enhancing their permeation in tumors [5]. **Figure 2** shows some of the most important nanomaterials used in combination with PDT.

## **2.3 Recent advances in X-PDT**

X-ray driven PDT makes use of scintillating materials and/or radiosensitizers (**Figure 3**). High-Z elements, for instance, have inner shell electrons which are very efficient in capturing X-ray photons and converting them into relaxed electrons and visible light photons. Thus, the most common scintillators are nanoparticles of high-Z elements doped with rare earth elements, and present

**Figure 3.**

*Deeper penetration of X-rays and it use for X-PDT. The red circles symbolize singlet oxygen generated by the interaction of the excited photosensitizer and molecular oxygen.*

useful properties for medical imaging and high-energy physics. The materials can be designed as films, coordination compounds, vitroceramics, metal-organic frameworks (MOFs), and hybrid organic-inorganic materials, and characteristics such as nanometric size, defects, coatings and media interaction influence on their scintillation properties [4].

Nanoscintillators can be basically divided in doped and semi-conductors scintillators. Lantanides are the most explored as doped scintillators due to their high density, high-Z, and significant intensity of luminescence, while semiconductor scintillators are mostly composed of porous Si, Si nanocrystals, ZnO, CdSe, CdS, PbS, and CuBr [4].

The efficiency of X-PDT is largely affected by the intensity of X-Ray luminescence, the singlet oxygen yield of the photosensitizer, and the way the photosensitizer is bound to the nanomaterial (either by covalent bonding, electrostatic interactions, or by pore loading). Furthermore, part of the tumor ablation might be due to the generation of UV photons during the scintillation process, apart from the photodynamic effect. The radiosensitization effect must also be considered, since high-Z materials generate ROS whenever their electrons are excited by X-rays into states above the conduction band edge, consequently producing electron hole pairs that interact with water producing hydroxyl radicals, and the electrons generate superoxide and peroxide radicals when they react with O2. Those ROS increase the cytotoxicity of the materials under X-ray irradiation [4].

Nanoscale metal-organic frameworks (nMOFs) consist of the self-assembly of metal ions or clusters and bridging ligands, usually organic polydentate. These materials are used as a means to put scintillators and photosensitizers closer to each other, enhancing the singlet oxygen generation efficiency [4].

Nanosized MOFs are usually biodegradable, offering a significant advantage over other nanomaterials, depending on the desired application. They encompass a virtually infinite possibility of structures due to the large availability amount of organic linkers and metallic parts; however, it is of utmost importance to select the components in accordance with the desired application in order to optimize the results. In the medical field, the use of MOFs is still in the prelude, since more pharmacokinetic, pharmacodynamics and biological characterization studies must be performed so that these materials reach clinical trials [15].

Regarding the use of lanthanides as scintillators, Dou et al. synthesized UCNs (NaYF4:Yb,Tm) covalently conjugated with chlorin e6 to prevent the premature

*Nanomaterials for Enhanced Photodynamic Therapy DOI: http://dx.doi.org/10.5772/intechopen.94255*

release of the photosensitizer and tested them *in vitro*. The nanoparticles were more efficient than the free photosensitizer even at low concentrations, and the efficacy can be fine-tuned by adjusting the dose of Ce6-UCNs and the laser power [7]. On the other hand, non-lanthanide materials such as SiC/SiOx core/shell nanowires functionalized with azide groups and porphyrin derivatives were tested for X-PDT and demonstrated significant efficacy. This material showed to be non-cytotoxic in the dark, and emits fluorescence at 545 nm when irradiated with X-rays, exciting the porphyrin derivative in the process [4].

Another example of experimental X-PDT was performed by Sivasubramanian et al. using BaFBr:Eu2+ nanoparticles loaded with porphyrins. When irradiated with 3 Gy of X-rays, the nanoparticles generated luminescence that matched the excitation wavelengths of the photosensitizer, leading to photodynamic effect that damaged the DNA, the mitochondria, and generated intense oxidative stress, significantly killing prostate cancer cells *in vitro* [16].

One of the main concerns about X-PDT is the radiation dose that needs to be applied to the patients. In order to diminish the amount of radiation that the patient must be exposed to, some scintillators that present persistent luminescence upon irradiation, rather than fluorescence, are the option. Fluorescence is a phenomenon that lasts for a few nanoseconds, while persistent luminescence can persist for minutes to hours after the excitation, therefore the required dose of radiation for excitation can be significantly decreased. There are evidences that persistent luminescence decreases the rate of oxygen consumption during PDT and may avoid the undesired hypoxia that hinders the photodynamic efficacy [4].

## **2.4 Recent advances in CR-PDT**

Cherenkov radiation-driven PDT, symbolized in **Figure 4**, takes advantage of the fact that most radiopharmaceuticals accumulate in tumors in a selective manner, therefore the photodynamic ablation may occur in a more localized way. However, the generation of Cherenkov radiation occurs in low fluence rates, usually not enough to enable a good photodynamic efficiency [4].

There is a significant advantage, though, of CR-PDT over X-PDT, which is the possibility of targeting multiple metastases easier than with external X-rays.

#### **Figure 4.**

*Cherenkov radiation being generated after radionuclide decay, and its ability to excite photosensitizers in order to perform PDT. The red circles symbolize singlet oxygen.*

Furthermore, even if the photons generated by the radionuclides are in much lower number than external irradiation (and possibly insufficient to exert significant phototoxicity), it is likely that the damage induced directly by the radionuclides contribute synergistically for the success of tumor ablation with CR-PDT [17].

An example of experimental CR-PDT was performed by Kamkaew et al. The authors encapsulated the radionuclide 89Zr with chlorin e6 into a mesoporous silica nanoparticle. The zirconium Cherenkov radiation emission is mostly in the UV region, but there is a significant emission in the blue region around 400 nm, corresponding to one of the absorption peaks of chlorin e6. The results *in vitro* showed high levels of DNA damage when the photosensitizer is present compared to the radionuclide alone, while in vivo results showed complete tumor remission after 14 days, even with a sublethal radiation dose of 15 MBq. However, a significant amount of radioactive nanoparticles were found in the liver after 14 days, so strategies to avoid toxicity to health tissues must be applied [18].

Nevertheless, much progress is yet to be made before X-PDT and CR-PDT become official clinic protocols, despite all the successful results that have been obtained so far. The mechanisms of cell death by the combination of radiotherapy and PDT must be fully understood, and the materials used as scintillators must be fully characterized and optimized [4].

#### **2.5 Hypoxia-reverting strategies**

PDT efficacy in tumors is limited by the oxygen supply to the tumors, which tends to be reduced due to deteriorated microcirculation, especially in the tumor center. Since PDT consumes oxygen, it increases even further the local hypoxia, preventing the technique to reach its full potential. Therefore, some strategies to increase the availability of oxygen to the tumors while PDT is occurring have been developed in order to increase the tumor ablation [3]. Cheng and co-workers, for instance, loaded photosensitizers that are activated at 780 nm into perfluorocarbon nanodroplets enriched with oxygen with average size of 200 nm. The use of the nanodroplets also increases the half-life of singlet oxygen, so the PDT efficiency is enhanced both *in vitro* and *in vivo*. With intravenous administration, the tumors were significantly ablated, but with intratumor administration the tumors were eliminated completely [19].

It was observed by Kim et al. that O2 can be efficiently produced via Fenton reaction in cancer tissues due to the abundancy of H2O2 derived from the tumor metabolism, especially when mesoporous silica nanoparticles are conjugated to manganese ferrite nanoparticles, which are classical Fenton catalysts, and loaded with chlorin e6. This system enabled a continuous PDT process by providing the tissue with the necessary amount of O2 via Fenton reaction, and could act as a contrast agent for magnetic resonance imaging, acting as a theranostic material [20].

In this regard, cerium oxide nanoparticles provide a good alternative for converting hydrogen peroxide into molecular oxygen and water, even in the absence of light irradiation. They are, therefore, a smart strategy to provide the hypoxic tissues with oxygen to enhance PDT efficacy, as demonstrated by Jia et al. The authors used a mesoporous core-shell structure consisting of NaGdF4:Yb,Tm@NaGdF4 upconversion nanoparticles coated with CeOx capable of converting NIR light into UV light, which activates cerium oxide to produce ROS. Since the nanoparticles have a hollow interior, they can also be used as a drug carrier for a combined chemotherapy, besides being very efficient in tumor ablation by PDT [21].

Although most of the oxygen-generating strategies make use of the excess of hydrogen peroxide caused by the intense metabolism of tumors, which can react

#### *Nanomaterials for Enhanced Photodynamic Therapy DOI: http://dx.doi.org/10.5772/intechopen.94255*

with iron cations generating O2 and hydroxyl radicals. There is a class of materials, however, that uses water as the source of oxygen, the so-called water-splitting materials, commonly used for solving energy and environmental problems. Since water is the major component of the organism, there is a virtually endless supply of oxygen to be used for PDT enhancement. Metal-free C3N4 decorated with carbon dots (in order to enhance the water-splitting upon irradiation with red light) was used by Zheng et al. as a water-splitting material. The nanocomposite was conjugated with the compound PpIx-PEG-RGD, consisting of the photosensitizer protoporphyrin IX with polyethylene glycol and the peptide sequence RGD (arginine, glycine, and asparagine) for active tumor targeting and photodynamic therapy. Under 630 nm irradiation, there was an increased O2 concentration and singlet oxygen production, enabling a significant cell killing without the occurrence of hypoxia [22].

Red blood cells (RBCs) can be used as photosensitizer and oxygen carriers at the same time in order to increase the efficacy of PDT in hypoxic situations. Wang et al., for example, coupled the photosensitizer Rose Bengal and a hypoxic probe on the surface of RBCs. Upon low levels of oxygen, the hypoxic probe can switch to an active state and undergo an orthogonal near-infrared upconversion, resulting in the release of O2 from the oxygenated hemoglobin when 980 nm light is applied. The photodynamic process is, thus, kept for longer and results in a better tumor ablation [3].

Oh the opposite side of the previous strategies, a protocol has been developed in order not to avoid the hypoxia in the tumors, but to use it to potentiate chemotherapy after PDT has been performed. This is possible with the use of hypoxiaactivated prodrugs such as triapazamine or apaziquone. He and collaborators used nanoscale metal-organic frameworks (NMOFs) as porous nanocarriers of photosensitizers and hypoxia-activated chemotherapeutics. Both *in vitro* and in vivo results indicate an on-demand release behavior of the nanoparticles and an intense tumor ablation, therefore it consists on a promising antitumor strategy [23].
