# Advances in Purpurin 18 Research: On Cancer Therapy
## 6 Photoacoustic Imaging and Therapy
A 3-fold increase in heat conversion efficiency was observed for the PA1 and PA3 aggregates compared to their monomeric state. An increase in heat conversion efficiency was also observed for PA7, although not to such an extent. The degree of aggregation of the conjugates in the aqueous medium correlated with heat conversion efficiency, which is proportional to the photoacoustic signal. [PERSON] et al. [25] also defined the relationship between the degree of aggregation and the relative photoacoustic ratio as a useful tool for aggregation research. RGD-Dex/NPBA-pu18 (RDNP) monomers were generated to monitor targeted drug delivery, and the structural changes induced by the acidic environment of the cells. By different molar ratios of biocompatible peptide Cys-Gly-Gly-Arg-Gly-Asp (RGD) with dextran (RGD-Dex) as a carrier and a photoacoustic probe pu18 modified with boric acid (NPBA-pu18), a total of five types of monomers (P1-P5) with different properties were prepared. It was found that when NPBA-pu18 is in a 3-fold and higher molar excess over RGD-Dex, RDNP monomers in an aqueous medium can be self-assembled into nanoparticles [84]. The hydrophobic NPBA-pu18 forms a core of nanoparticles that is surrounded by a hydrophilic RGD-Dex. It serves to stabilize the nanoparticle. In an acidic environment, RDNP may cleave due to a pH-dependent phenyl borate bond. The released NPBA-pu18 can repackage into the structure of highly organized nanofibers due to high hydrophobicity and \(\pi\)-\(\pi\) interactions (Figure 11). After laser irradiation, it is then possible to monitor the PA signal and distinguish the structural changes that are characteristic for the monomeric and aggregated state of NPBA-pu18. P2 (single-stranded) and P5 (nanogranulated) labeled with Cy5 were used for cell entry assays in HeLa cells. Based on the intensity of the fluorescent signal, it was determined that P5 internalized into cells more efficiently than P2 by caveolae-mediated and clathrin-mediated endocytosis. Both conjugates were localized in cell lysosomes/endosomes. 1h after the treatment of HeLa cells with P5, an increasing photoacoustic signal was detected and after 3 h when all nanoparticles were likely to dissociate to form NPBA-pu18 aggregates, a stable PA signal was achieved. The entire process of structural transformation of RDNP was monitored in real-time using PAI, which makes this method an important tool for following many cell processes, such as targeted drug delivery or autophagy, which is often connected to cancer. In general, autophagy is a natural catabolic process, in which proteins, organelles, and other cell components are degraded. It is one of the essential cellular processes for maintaining homeostasis, which occurs in response to stressful conditions such as hypoxia, presence of ROS, or lack of nutrients. Some drugs, such as DOX, have been reported to up-regulate autophagy, which can, however, paradoxically lead also to resistance of the cell to chemotherapy [85; 86]. Therefore, autophagy inhibitors are commonly co-administered with chemotherapeutics to help increase the success of anticancer therapy [87]. However, the combination of these drugs often leads to high systemic toxicity and damage to the organism. Thus, preventive monitoring of autophagy in real time, directly in tumor cells using PAI, could be beneficial. With this goal, [PERSON] et al. [88] developed a novel photoacoustic in vivo self-assembled probe (DDP18, Figure 12). It is composed of hydrophobic pu18 as a signaling molecule, Gly-Lys-Gly-Ser-Phe-Gly-Phe-Thr-Gly (GKGSFGFG) peptide, the cleavage site of autophagy-related cysteine protease, and poly(amidoamine) dendrimer, which serves as a carrier increasing the solubility and biocompatibility of the molecule. Upon entry of the probe into cells undergoing autophagy, the pu18 residue is enzymatically cleaved with a linker to aid in its folding.