**2.6 Tumor-targeting and specific delivery strategies for PDT using nanomaterials**

One of the most common strategies of actively targeting specific organs or tissues is by the use of antibodies. Stuchinskaya and collaborators combined the versatility of gold nanoparticles with a hydrophobic photosensitizer (zinc phthalocyanine derivative), preventing its aggregation before reaching the target, and decorated the nanoparticle with tumor-specific antibodies (anti-HER2 for breast cancer) by covalent bonds formed with the coating layer of polyethylene glycol. There was a high efficiency in singlet oxygen generation in cancer cells after a selective targeting [24].

Active targeting can also make use of membrane proteins that are overexpressed in tumor cells, i.e. some integrins and neuropilin-1. By coupling ligands like RGD (a tripeptide composed of arginine, glycine, and aspartate), biotin, and folic acid to nanocarriers, the tumor accumulation is significantly enhanced [3]. Organelle targeting is also an option, especially when it comes to mitochondria. Several lines of evidence show that targeting the mitochondria for PDT avoids drug-resistance by tumor cells via a decreased level of intracellular ATP (the drug resistance phenotype in tumor cells is often associated with overexpressed ATP-driven transmembrane efflux pumps), besides the fact that damage to the mitochondria often leads to cell death [25]. Targeting the lysosomes can be additionally useful because the leakage of protons and hydrolases into the cytoplasm can damage inner structures and lead to cell death [3].

Another reason that makes organelle-targeting important is the short action range of singlet oxygen (no more than 20 nm), so a localized photosensitizer excitation is required. Hou et al. developed a Fe3O4@Dex-TPP nanoparticles that enhance the oxygen concentration in tumor cells via Fenton reaction, target the mitochondria (via the triphenylphosphine group, TPP), and are able to be imaged by magnetic resonance imaging due to the magnetic behavior of Fe3O4. This system was loaded with the photosensitizer protoporphyrin IX and grafted with a reduced glutathione-responsive moiety. Upon internalization, Fe2+ and Fe3+ ions are liberated from the Fe3O4 core and diffuse into the cytoplasm, then oxygen is produced by Fenton reaction (Fe2+ reacting with the excess of H2O2 producing O2 and hydroxyl radical (•OH). This allows the PDT process to keep occurring, enhancing the therapeutic efficacy [25].

One of the strategies for specific delivery is the development of pH-sensitive materials that make use of the mild acidity environment found in tumors (around 6.5 to 7.2). Ai and co-workers developed upconversion nanoparticles with a low-pH insertion peptide that in acidic environments allow the insertion of the nanoparticles into the plasma membrane. They observed a large accumulation in the tumor tissue compared to healthy tissues [26].

Calcium phosphate is a biocompatible and biodegradable material, as it is the main component of hard tissues such as bones and teeth. It is sensitive to pH, maintaining its stable structure in physiological pH and dissolves in acidic environments, therefore it can be useful for controlled delivery to tumors. Another advantage relies on the fact that, once inside the cells, calcium phosphate nanoparticles dissolve and liberate calcium ions across lysosomal membranes, impairing the osmotic pressure of the cell and leading it to necrosis [27].

Liu et al. fabricated calcium phosphate-encapsulated core-shell structured nanoparticles (UCNPs-Ce6@SiO2@Calcium phosphate-Doxorubicin), characteristic for being biodegradable, biocompatible, pH-sensitive (which enables the liberation of the chemotherapeutic in the tissue), and provides therapeutic efficiency by PDT upon irradiation with 808 nm due to the presence of Chlorin e6 in its structure. Finally, due to the presence of rare earth elements, it can be used as an imaging tool for diagnostic purposes [27].

Another strategy is the development of nanomaterials that can be degraded by enzymes that are overexpressed in tumors, such as matrix metalloproteinases (MMPs) and hyaluronidase. One good example is the nanomaterial developed by Li et al. [28], which consisted of hyaluronic acid nanoparticles conjugated with chlorin e6 that disassemble in the presence of hyalurinodase and liberate the photosensitizer. This way, they can act as theranostic materials, meaning they can use as diagnostic tools and therapeutic agents. Another example was the MMP2-responsive chimeric peptide nanoparticles coupled with protoporphyrin-IX, which turn from a sphere into large fibers when MMP-2 is present, and this sphere-to-fiber transition contributes to the augmented tumor retention of the nanoparticles [29].

Dai et al. developed a peptide nanoparticle coupled with protoporphyrin-IX (PpIX-Ahx-K8(DMA)-PLGVR-PEG8) responsive to both pH and enzyme. This nanoparticle assumes a spherical shape while in circulation and avoids nonspecific uptake, and when in tumor environments they undergo a charge reversal and cleavage of the PLGVR sequence by MMP-2. Simultaneously, the DMA group is detached because of the low pH. This logic worked to enhance even more the specific uptake by tumor tissues [23].

A very intricate nanosystem combining tumor-targeted PDT with antiangiogenesis therapy and reduced glutathione (GSH) was developed by Min et al.. It consisted on a porphyrinic zirconium-metal-organic framework nanoparticle that

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

can act simultaneously as a photosensitizer and a carrier of the vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor apatinib. MnO2 covers the nanoparticle core in order to consume the intratumoral GSH, and the whole system is decorated with a camouflage made of a tumor cell membrane. The tumor specificity was much enhanced, and so was the ablative efficiency of the combined treatment provided by this nanomaterial [30].

In addition to the previous strategies, some researchers have developed nanoparticles that are activated by near-infrared light for selective photodynamic therapy, protecting healthy tissues like the skin. The mechanism of action of these systems is based on the blockage of photodynamic action from the photosensitizer by a co-loaded NIR dye via a fluorescence resonance energy transfer (FRET) effect. The photosensitizer action is recovered once the NIR dye is photobleached by NIR light irradiation in the specific site. Dong et al. developed CaCO3-PDA-PEG hollow and porous nanoparticles loaded with chlorin e6 for this purpose, and observed that they are degraded in acidic environments such as tumors, liberating the photosensitizer in a selective manner. The generation of singlet oxygen was enhanced in the acidic environment, and the photosensitizer was taken up more efficiently when administered within the nanoparticles, compared to the free photosensitizer and other formulations. It is worthy to mention that when chlorin e6 is injected in a liposomal formulation, the mice present significant weight loss, probably due to an intrinsic toxicity, and this does not happen in the CaCO3-PDA-PEG formulation [31].

Jeong et al. tested human serum albumin nanoparticles loaded with chlorin e6 in order to develop a more biocompatible system for enhanced PDT efficacy. The nanoparticles, with circa 88 nm in diameter, proved to be non-cytotoxic in the dark, but produced significant amounts of singlet oxygen upon irradiation with the appropriate wavelength. Remarkably, when injected in mice they provided a very specific tumor delivery compared with the free photosensitizer, and simultaneously provided a good imaging property die to the fluorescence of chlorin e6 [32].

PDT can be not only an adjuvant for chemotherapy, but also for immunotherapy, and nanotechnology can potentiate the results and enable the combination of the two therapies in one single approach. That is what was demonstrated by Xu et al. when they developed mesoporous silica nanoparticles made of amorphous silicon dioxide. The nanoparticles were relatively small (around 80 nm in diameter) in order to enhance the cell internalization and avoid side effects, and the pores were large (around 5–10 nm) in order to optimize the drug loading capacity. The nanoparticles were loaded with CpG oligodeoxynucleotide, which is a Toll-like receptor-9 agonist for immunotherapy, and chlorin e6. The authors observed an effective accumulation in tumors in vivo after intravenous injection, and the treatment induced cell damage and the recruitment of dendritic cells. With the immune response elicited, there was a strong cancer vaccination effect, therefore tumors in distant sites can also be affected by the treatment [21].

Finally, a novel phenomenon has been calling the attention of researchers, namely aggregation-induced emission (AIE) of photosensitizers. Some fluorophores are poor light emitters when they are in a single molecule state, but they become strong emitters when aggregated, enabling bioimaging with significant biocompatibility and photostability. Besides, they can generate singlet oxygen in the aggregated state, so they can act as efficient PDT agents. Liu and coworkers synthesized AIEsomes, which are lipid structures conjugated with compounds with AIE property, and tested their efficacy in vivo. Their compounds were biocompatible, provided efficient bioimaging and loading efficiency, ultimately leading to a significant photodynamic effect [33].
