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- Comparative analysis of metallated phthalocyanines for photodynamic therapy of solid tumorsPublication . Dias, Lionel Mendes; Heger, Michal; Cavaco, José Eduardo Brites; Ding, BaoyuePhotodynamic therapy (PDT) comprises the administration of a photosensitizer (PS) and its accumulation within the tumor site, followed by irradiation with light of a specific wavelength. Consequently, singlet oxygen and other reactive oxygen/nitrogen species (ROS/RNS) are produced from bioavailable oxygen at the tumor’s microenvironment and are responsible for the tumor’s eradication. PDT works effectively in certain types of cancer, but poorly in tumors that reside in internal organs and organ structures such as the pancreas and biliary tree. Moreover, adverse effect such as skin phototoxicity is a major obstacle to more widespread clinical applicability. To that end, we encapsulated a second-generation of PS into liposomal carriers that are targeted to the tumor interstitium after intravenous administration. Therefore, this doctoral thesis describes essentially the evaluation and comparison between lipophilic metallated-phthalocyanines (ZnPC and AlPC) encapsulated in interstitially-targeted liposomes (ITLs), and their corresponding hydrophilic derivatives (ZnPCS4 and AlPCS4). To do so, we initiated the doctoral research through our first study by performing an attritional assessment in vitro using A431 cells as a template for tumor cells with a dysfunctional P53 tumor suppressor gene and epidermal growth factor receptor (EGFR) overexpression. As a methodology for our investigations, we have first assessed the dark toxicity as a function of PS concentration using the water-soluble tetrazolium salt (WST-1) and sulforhodamine B dyes as an indication of the cell viability. Using the same principle, we then drew the LC50 values for each PS through PDT at 671 nm and a light exposure of 15 J/cm2 following 1 hour of PS exposure. We continued our research looking into a time-dependent uptake and intracellular distribution of the PS, and we finalized our first study with the assessment of the mode of cell death as well as the cell cycle arrest at 24 hours after PDT. Through this research we observed that, in the absence of illumination, AlPC and ZnPC in ITLs were not toxic to cells up to a 1.5 μM PS concentration and exposure for up to 72 h, but for AlPCS4 and ZnPCS4, the dark toxicity was at 5 μM and at 2.5 μM, respectively. However, PDT of cells photosensitized with ZnPC, AlPC, and AlPCS4 yielded LC50 values of 0.13 μM, 0.04 μM, and 0.81 μM, respectively (24 hours post-PDT based on sulforhodamine B assay). The uptake of all PSs was observed as early as 1 min after PS addition to cells and increased in amplitude during a 2-h incubation period. ZnPCS4 did not induce notable phototoxicity, which was echoed in the mode of cell death and cell cycle arrest data. However, AlPCS4 induced considerable necrosis in addition to apoptosis, whereby most of the cell death had already manifested as early as 2 h after PDT. Cell death signaling coincided with a reduction in cells in the G0/G1 phase (ZnPC, AlPC, AlPCS4) and cell cycle arrest in the S-phase (ZnPC, AlPC, AlPCS4) and G2 phase (ZnPC and AlPC). With the intention of validating our previous research, we have moved forward with the investigations through a second study by using our comparative model with the four metallated-phthalocyanines in a human cholangiocarcinoma cell line and tumor-comprising cells (endothelial cells, fibroblasts, and macrophages), as a representation of the tumor’s microenvironment. In addition to all parameters assessed in the previous study, we went one step further and evaluated the systemic toxicity of each PS in zebrafish and in chicken embryos, and while using BALB/c nude mice as our in vivo model, we researched for signs of skin phototoxicity. A pilot study on PDT efficacy was also performed in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts. The key findings were that photodynamically active PSs (all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells. In addition, PSs in study did not induced any notable systemic toxicity in zebrafish and chicken embryos. However, ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum containing PSs did not exert any detectable sign of cutaneous phototoxicity. Last but not least, ITL-delivered ZnPC and AlPC are equally effective in their tumor-killing capacity in human tumor breast cancer xenografts, and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis. In summary, the research on the applications of ZnPCS4 for oncological PDT will be discontinued in our group as it failed the attrition step regarding phototoxicity and it showed alarming signs of cutaneous phototoxicity. It is therefore concluded that AlPC and its derivative AlPCS4 are the least toxic and most effective PSs to employ with respect to ITLs as part of the comprehensive tumor targeting and PS delivery platform.