Cytochrome c (Cyt c) is a protein that induces apoptosis via activation of the caspase cascade. Our group has previously developed a drug delivery system consisting of Cyt c nanoparticles decorated with the amphiphilic copolymer folate-poly(ethylene glycol)-poly(lactic-co-glycolic acid)-thiol (FA-PEG-PLGA-SH). In this study we optimized the synthesis of a nanoparticle free formulation for Cyt c in which the ligand FA-PEG is stoichiometrically conjugated to the protein (FA-PEG-Cyt c). FA-PEG ligand was conjugated to Cyt c using an amine-to-sulfhydryl crosslinker. The conjugation of FA-PEG to Cyt c was studied using UV-Vis spectroscopy. The integrity of the protein was accessed using circular dichroism (CD) and caspase activity assay. Cell viability was determined using the MTS assay. The levels of FA receptor on LLC cell membrane and internalization of the formulations was studied using confocal microscopy. We were able to conjugate FA-PEG to Cyt c and according to CD spectra and caspase activity assay results the protein integrity was maintained after the conjugation. MTS experiments did not show significant reduction of LLC viability after 24 h of treatment with FA-PEG-Cyt c, while the treatment with FA-PEG-PLGA-Cyt c nanoparticles induced apoptosis in LLC cells after 6h of treatment. The confocal microscopy results showed overexpression of FA receptors in LLC cells as well as internalization of FA-PEG-PLGA-Cyt c nanoparticles. Our results show that FA-PEG-PLGA-Cyt c nanoparticles may be the best formulation for Cyt c probably due to a higher drug payload. We intend to determine the differences between FA-PEG-Cyt c and FA-PEG-PLGA-Cyt c nanoparticles from the stand point of protein integrity and overall outcome of the treatment using Lewis Lung Carcinoma (LLC) as a model. In vivo studies are being made using a lung cancer mouse model to test the specificity and effectiveness in decreasing tumor size of these nanoparticles

Comparison of nanoparticle and nanoparticle-free formulations for the active delivery of cytochrome c by targeting folate receptors

2019 Spring National Meeting

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