# Advances in Purpurin 18 Research: On Cancer Therapy
## 4 Photodynamic Therapy
To increase the PEGylated conjugate modality, it was further modified with a zinc ion which was incorporated into the pu18 structure. The presence of a zinc ion led to an increase in the absorption in the red region of the visible spectrum, which could enable also imaging of this compound. Besides PEGylation, conjugating of pu18 to biocompatible graphene oxide (GO), has been described. Thanks to its unique surface properties (carboxyl, hydroxyl, and epoxy groups, free \(\pi\) electron, pH-dependent negative surface charge), GO can bind drug molecules and, thus, contribute to their efficient transport into tumor cells. The properties of GO as a PS carrier were used in the work of [PERSON] et al. [39], who prepared GO-Pu18 conjugates composed of GO with non-covalently bound (\(\pi\)-\(\pi\) stacking, hydrophobic interactions) pu18 methyl ester, with the aim to increase water solubility, improve bioavailability, control pH-release of pu18 and possibly use in theranostics (see Chapter 9). A similar approach was used by [PERSON] et al. [40], who prepared two types of nanoparticles GO-PS2 and GO-PS3, composed of GO as a carrier and pu18-\(N\)-ethylamine as a PS. These nanoparticles differed from each other in the type of bonds in the molecule, GO-PS2 contained covalently bound PS, which led to the high stability of the particles under different conditions. In contrast, GO-PS3 contained, like the aforementioned GO-pu18, non-covalently bound PS, which was released at a slightly acidic pH (Figure 4A). Based on the microscopic examination, it was found that all prepared conjugates (GO-Pu18, GO-PS2, GO-PS3) are transported into human tumor cells (HepG2, A549) to a greater extent than the unconjugated pu18. Increased phototoxicity of conjugates GO-Pu18 (HepG2 cell line, xenon lamp, 700 nm, 5 mW\(\cdot\)cm\({}^{-2}\), 10 min) and GO-PS2, GO-PS3 (A549 cell line, laser, 610-710 nm, 2 J\(\cdot\)cm\({}^{-2}\), 15 min) vs. free pu18 after photoactivation was detected. Dark toxicity (without photoactivation) corresponded to a free PS for GO-pu18 and GO-PS2 conjugates. No toxicity was observed for GO-PS3 in the specified concentration range. Although GO-PS3 showed a lower rate of \({}^{1}\)O\({}_{2}\) generation compared to free pu18 (in vitro), higher PDT activity was determined for GO-PS3. Thiswas probably due to the more efficient delivery of GO-PS3 to the cells and the release of non-covalently bound pu18-\(N\)-ethylamine in the acidic environment of the tumor cells. Both research groups have shown that GO can be a suitable carrier for the efficient delivery of a PS to cancer cells. From the point of view of the targeted release of a PS in tumor tissue, it is advantageous to use noncovalent binding for linking the carrier to the PS molecule. Currently, great potential in the development of PSs for PDT have nanoparticles. These can be used, for example, for reducing the PS hydrophobicity and to improve its bioavailability. This approach was chosen by [PERSON] et al. [41], who prepared three types of water-soluble organic nanoparticles (WSONs) containing chlorine derivatives such as PS: methyl pyropheophorbide \(a\) (MPPa), zinc metal-introduced MPPa (ZnMPPa), and pu18 methyl ester (pu18 ME). All prepared WSONs reached a size of 87-112 nm and showed good solubility and stability in water. Fluorescence microscopy analysis showed the entry of all WSONs into human HeLa tumor cells and their localization in mitochondria. High phototoxicity of WSONs after light-emitting diode (LED) illumination was also recorded (640-710 nm, 2 J\(\cdot\)cm\({}^{-2}\), 15 min) which corresponded to the parental PS. The newly prepared WSONs thus preserve or even improve the properties of the original PS, such as absorption in the red region of the visible spectrum, high phototoxicity, and reduced dark toxicity. Besides, the high solubility of WSONs ensures good bioavailability compared to free PS. Additionally, other research groups have been involved in increasing PS bioavailability.