# Advances in Purpurin 18 Research: On Cancer Therapy
## 4 Photodynamic Therapy
In the case of 4T1 2D cell cultures, significant phototoxicity of pu18 (0.5 uM) was observed after light illumination at 630 nm (35 s, 3.71 J\(\cdot\)cm\({}^{-2}\)). Without photoactivation, pu18 did not exhibit any signs of toxicity up to 4 uM concentration. During the microscopic analysis of PDT-treated cells, a change in the cell shape (size reduction, rounding, vacuolation) and their separation from the surface of the culture dish was detected. Flow cytometry analysis showed that PDT treatment of 4T1 cells using 0.5 uM pu18 (0.724 J\(\cdot\)cm\({}^{-2}\)) successfully induced apoptosis in 50 % of the cells. For 4T1 cells cultured as 3D spheroids, the cells were exposed to 4 uM pu18 for 24 h and were photoactivated at 630 nm (4 min, 25.2 J\(\cdot\)cm\({}^{-2}\)),  
Figure 3: A general principle of photodynamic therapy (PDT). PDT is based on the action of light radiation on a photosensitive drug (PS). After receiving energy, it passes into a higher energy state. Then, it transfers the received energy to surrounding molecules and turns back to its ground state. In the energy transfer mechanism Type I, the energy is transferred to a biomolecule which reacts with \({}^{3}\)O\({}_{2}\) to form reactive oxygen species (ROS). During the Type II transfer, there is a direct reaction of the PS with the \({}^{3}\)O\({}_{2}\) molecule, with the formation of singlet oxygen \({}^{1}\)O\({}_{2}\), which also belongs to the ROS. and further cultured for 6 days. On the second day after PDT, a decrease in the mean of 4T1 spheroids was observed compared to the control group. However, further cultivation showed that in the PDT-treated group, cell growth was not completely inhibited, since the 4T1 spheroids were further increasing their volume. However, it was slower than in the case of PDT-untreated 4T1 spheroids. The PDT effect of pu18 administered intratumoral was verified using experimental Balb/c mice with subcutaneously implanted 4T1 cells. After PDT treatment (pu18, 0.625 mg\(\cdot\)kg\({}^{-1}\), 600 J\(\cdot\)cm\({}^{-2}\)), the tumor size was monitored every second day for two weeks. Six days after PDT, a significant decrease in the tumor volume (60%) was observed in the PDT-treated group compared to control groups, in which a constant growth of the tumors was observed. Although PDT significantly suppressed the size of the tumor, its overall disappearance did not occur here either. However, the survival time of PDT-treated mice was longer compared to the control group. The obtained results indicate a high efficacy of pu18 as a potent PS for the use in PDT. Nevertheless, there are still big weaknesses of high pu18 hydrophobicity (leading to the formation of pu18 aggregates), low bioavailability (difficult delivery of pu18 to a tumor tissue), and rapid removal from the tumor tissue [33]. Researchers have tried to solve these shortcomings by derivatization of the pu18 structure, such as creating imides, or anhydrides [34; 35; 36; 37], conjugating it to a molecule of a carrier or creating various types of pu18-containing nanoparticles. A solution to reduce pu18 hydrophobicity and improve its bioavailability is, for example, conjugation of pu18 with a polyethylene glycol (PEG) spacer, as described in our previous article [38]. It was observed that the newly prepared PEGylated pu18 conjugates efficiently accumulated in human cancer cells of various origins (breast--MCF-7, prostate--PC-3 and LNCaP, cervix--HeLa, and pancreas--MiaPaCa-2) in vitro with preferential localization in PDT of important organelles such as mitochondria and endoplasmic reticulum (ER). Attachment of the PEG\({}_{3}\) arm also led to an increase in phototoxicity after light illumination (4 J\(\cdot\)cm\({}^{-2}\), 13 min) compared to the parental compound. The increase in phototoxicity was probably due to the increase in the conjugate hydrophilicity and, thus, improved solubility of the compound in aqueous media. This further led to a reduction in the formation of pu18 aggregates and better drug bioavailability.