Comparative analysis of whole cell-derived vesicular delivery systems for photodynamic therapy of extrahepatic cholangiocarcinoma

Abstract

This first-in-its-class proof-of-concept study explored the use of bionanovesicles for the delivery of photosensitizer into cultured cholangiocarcinoma cells and subsequent treatment by photodynamic therapy (PDT). Two types of bionanovesicles were prepared: cellular vesicles (CVs) were fabricated by sonication-mediated nanosizing of cholangiocarcinoma (TFK-1) cells, whereas cell membrane vesicles (CMVs) were produced by TFK-1 cell and organelle membrane isolation and subsequent nanovesicularization by sonication. The bionanovesicles were loaded with zinc phthalocyanine (ZnPC). The CVs and CMVs were characterized (size, polydispersity index, zeta potential, stability, ZnPC encapsulation efficiency, spectral properties) and assayed for tumor (TFK-1) cell association and uptake (flow cytometry, confocal microscopy), intracellular ZnPC distribution (confocal microscopy), dark toxicity (MTS assay), and PDT efficacy (MTS assay). The mean ± SD diameter, polydispersity index, and zeta potential were 134 ± 1 nm, -16.1 ± 0.9, and 0.220 ± 0.013, respectively, for CVs and 172 ± 3 nm, -16.4 ± 1.1, and 0.167 ± 0.022, respectively, for CMVs. Cold storage for 1 wk and incorporation of ZnPC increased bionanovesicular diameter slightly but size remained within the recommended range for in vivo application (136-220 nm). ZnPC was incorporated into CVs and CMVs at an optimal photosensitizer:lipid molar ratio of 0.006 and 0.01, respectively. Both bionanovesicles were avidly taken up by TFK-1 cells, resulting in homogenous intracellular ZnPC dispersion. Photosensitization of TFK-1 cells did not cause dark toxicity, while illumination at 671 nm (35.3 J/cm²) produced LC₅₀ values of 1.11 μM (CVs) and 0.51 μM (CMVs) at 24 h post-PDT, which is superior to most LC₅₀ values generated in tumor cells photosensitized with liposomal ZnPC. In conclusion, CVs and CMVs constitute a potent photosensitizer platform with no inherent cytotoxicity and high PDT efficacy in vitro.

Keywords: Anti-cancer treatment; Biliary tumors; Cell death; Endocytosis; Intracellular distribution; Oxidative stress; Photosensitized; Reactive oxygen species; Targeted photosensitizer delivery; Vesicles.