Evaluation of nano-formulation of Pheophorbide-a-mediated photodynamic therapy against 3D Lung cancer cells

Pheophorbide-a (PPBa) is a potent photosensitizer (PS) with some limitations such as poor water solubility, low bioavailability, etc. [1]. As a strategy, liposomes can provide a platform in co-delivery of drugs in cancer therapy [2]. In this study, we synthesized two nanocomplexes (Lipo@AuNPs@PPBa) from PPBa and gold nanoparticles (AuNPs) for photodynamic therapy (PDT) purposes using a 660 nm laser on A549 lung cancer spheroid cells. The thin-film hydration method was used to synthesize Lipo@AuNPs@ PPBa and characterization was carried out using UV-VIS spectroscopy, FTIR, DLS, EDS, and SEM. Thereafter, the cytotoxic effects of nanocomplexes in PDT on A549 spheroid cells were evaluated by morphological changes, MTT, ATP, amd LDH assays and then the apoptotic rate was determined with immunofluorescence (IF), flow cytometry, and real-time PCR.

UV-VIS spectroscopy, FTIR, EDS, and DLS results showed successful co-loading of PPBa and AuNPs in liposomes at 6 µM and 10 µg/mL concentrations, respectively. SEM and TEM images showed the shape of Lipo@AuNPs@ PPBa was a rod-like and the size was 87 nm. Cellular response indicated that IC₅₀ of Lipo@AuNPs@ PPBa was 60 µg/mL and caused significant cellular death, ATP reduction, LDH release, and spheroid shrinkage post-PDT (15 J/cm²). Additionally, IC₅₀ of Lipo@AuNPs@PPBa post-PDT caused 46.4% and 21.4% of early and late apoptotic rates, respectively. Moreover, Lipo@AuNPs@PPBa nanocomplexes induced activation of apoptotic proteins such as BAX, Cyt c, and CASP9 in A549 spheroids post-PDT. Additionally, IF and real-time PCR showed that the nanoformulation of PPBa caused up-regulation of apoptotic proteins and genes more than in its free form post-PDT. In conclusion, AuNPs demonstrated a synergistic effect on PPBa in PDT. Hence, liposomes can provide a remarkable platform for the co-delivery of PPBa and AuNPs.