**3. Photobleaching**

Experimental results have shown that the photobleaching process can hinder the photodynamic efficiency of photosensitive compounds [36, 37]. Photodegradation of photosensitizers can reduce the concentration of these photoactive compounds in diseased tissue, decreasing the efficacy of PDT to reduce cell viability, leading to an incomplete treatment. On the other hand, photobleaching can reduce the photosensitivity of healthy tissues after irradiation due to the lower amount of reactive oxygen species generated in the photodynamic process motivated by the destruction of photosensitizer molecules. Considering that phthalocyanines are photosensitizers that tend to suffer photobleaching, [37] as well as to aggregate in aqueous medium, our group evaluated the ability of polymeric nanoparticles to reduce the aggregation of these lipophilic molecules and also the effect of photobleaching [29].

The laser power and the concentration of free phthalocyanine significantly influenced the photobleaching for concentrations in which the molecule is in the monomeric state since the photosensitizer aggregation state tends to decrease the photobleaching process due to the difficulty to produce reactive oxygen species [29].

Works have shown that the encapsulation of photosensitizers decrease the effect of photobleaching in phthalocyanines when compared to free molecules due to the scattering of light caused by the polymeric matrix [5, 7, 8, 29]. More soluble photosensitizers tend to be more susceptible to suffer photobleaching and even encapsulated can be photodegraded according to the laser power and irradiation dose used, limiting its ability to be used as a good photosensitizer in photodynamic therapy [29]. The short storage period at several temperatures did not cause significant influence in the photobleaching behave of the encapsulated phthalocyanines, probably due to the aggregation process of the particles (**Figures 6**, **7**) (results not showed).

### **4. Photooxidation**

Phthalocyanines are a class of compounds used as photosensitizers due to their chemical, electronic and spectroscopic properties, [38, 39] in particular, due to the intense absorption of these compounds in the therapeutic window and their ability to generate singlet oxygen in the presence of a light source.

Researchers have shown that the presence of heavy atom in the phthalocyanine structure favors the generation of singlet oxygen due to the increase in spin-orbital coupling and, consequently, the transition of the photosensitizer from an excited singlet state to a triplet state (intersystem crossing) [40–42]. In addition, the literature suggests that photooxidative mechanisms for singlet oxygen are usually more efficient due to their greater diffusibility and higher reaction rate constants with substrates [43, 44]. However, the metallophthalocyanines present limited solubility in certain solvents due to the symmetry of molecular structure, hamper their application in PDT [29].

The chemical structure of phthalocyanines has been modified by introducing substitutes in the peripheral or non-peripheral positions of the phthalocyanine nucleus to reduce the molecule symmetry and consequently increase the polarity and solubility of the phthalocyanines [41, 42]. Besides, studies show that encapsulation improves the photodynamic efficiency of the photosensitizer, as well as decreases side effects such as photosensitivity of the skin after photodynamic treatment, and reduces molecular aggregation compared to phthalocyanines dissolved in aqueous medium [5, 7, 8, 29, 45].

We have studied the photodynamic efficiency of different porphyrins and phthalocyanines encapsulated in polymeric nanoparticles (**Figures 2**-**4**). As an example, gallium phthalocyanine (GaPc - **Figure 2A**) and 1,4-(tetrakis[4-(benzyloxy) phenoxy] phthalocyaninato) indium(III) chloride (InTBPPc - **Figure 4**) are convenient photosensitizers for PDT. These compounds have high singlet oxygen (0.41 and 0.94, respectively) and triplet (0.69 and 0.97, respectively) quantum yield. However, InTBPPc has more interesting features for use in PDT [29].

The photooxidation of simple molecules (as dimethylanthracene (DMA) and tryptophan (Trp)) was used to evaluate the photodynamic efficiency of each free and encapsulated phthalocyanines. It is notable that the asymmetry caused by (benzyloxy)phenoxy group in phthalocyanine seems to increase the photodegradation of InTBPPc, due to the greater solubility of the photosensitizer which favors the reduction of its aggregation state. The decrease in the aggregation state favors the generation of singlet oxygen and consequently, the efficacy of the free photosensitizer in photooxidizing simple molecules such as DMA and Trp, as well as the phthalocyanine photobleaching [29]. Therefore, free InTBPPc was more efficient than free GaPc in photooxidate DMA and Trp molecules, due to its lower aggregation state and the higher capacity of free InTBPPc to generate singlet oxygen. However, the encapsulated GaPc proved to be more efficient than the encapsulated InTBPPc in photooxidate the Trp molecules, corroborating that the encapsulation can enhance the photosensitizer photocytotoxicity and reduce the aggregation state of the free photosensitizer [29].

We have demonstrated that the photocytotoxicity of encapsulated photosensitizers depends on the incubation time, the photosensitizer concentration and the laser power [5, 7, 8, 16, 29]. However, these observations cannot be considered a fact for all free or encapsulated photosensitizers due to their solubility characteristics, their states of aggregation and the influences of the parameters used in the nanoparticles preparation on the nanoparticulate properties. For example, the aggregation state of free InPc decreases the photodynamic efficiency of this photosensitizer, so that the viability of tumor cells is not altered by increasing the concentration and laser power [7].

We have observed that each nanoparticulate formulation should be treated in a particular way, taking care to do generalizations about certain conclusions as susceptible to be applied to all formations. An example was the result observed with InTBPPc, the encapsulation process did not increase its efficiency in the photooxidation process of Trp due to the photobleaching process suffered by the

*Synergic Influence of Parameters Involved in the Polymeric Nanoparticle Preparation… DOI: http://dx.doi.org/10.5772/intechopen.94176*

photosensitizer [29]. In general, the encapsulation process of photosensitizers has not created barriers for the singlet oxygen generation, and has increased the uptake of photosensitizer into the cancer cells, improving the efficiency of the phthalocyanines and porphyrins to reduce the viability of cancer cells [7, 8, 16].

It was also shown that the photocytotoxic activity of nanoparticles loaded with porphyrins did not depend on the different amphiphilic characteristics of the compounds, probably due to the encapsulation process [16]. Even the similarities in photodynamic efficacy are related to the degree of similarity in the internalization of each encapsulated photosensitizer inside tumor cells [16].
