Smart Release Nano-formulation of Cytochrome C and Hyaluronic Acid Induces Apoptosis in Cancer Cells

Abstract

Herein we tested a nanosized cancer-cell targeted delivery system based on cytochrome c (Cyt c) and hyaluronic acid. Cyt c was chosen since it is a per se non-toxic protein but causes apoptosis when delivered to the cytoplasm of target cells. Hyaluronic acid was employed to create the nanosized delivery system with passive targeting capability in order to exploit the enhanced permeation and retention (EPR) effect and active targeting capability of hyaluronic acid. In addition, our goal was to incorporate a smart release strategy to only promote protein release upon reaching its target.Nanoparticles were formed by a simple yet precise nanoprecipitation process based on desolvation. They were physically characterized to select precipitation conditions leading to adequate size, shape, protein bioactivity, and protein loading to produce a feasible targeted cancer treatment. We synthesized nanoparticles of around 500 nm diameter with a 60% protein loading and more than 80% of protein bioactivity. In vitro, cumulative release of 92% of Cyt c was observed after 8 h under conditions mimicking the reductive intracellular environment, while under non-denaturing conditions only 20% was released. The nanoparticles displayed a selective cytotoxic effect on cancer cells. After 6 h of incubation with the nanoparticles, hyaluronic acid receptor over expressing A549 human lung adenocarcinoma cells showed a viability of ca. 20% at 0.16 mg/ml of Cyt c concentration. Only a negligible effect was observed on viability of COS-7 African green monkey kidney fibroblast, a normal cell line notoverexpressing the hyaluronic acid receptor. Confocal microscopy confirmed that the drug delivery system indeed delivered Cyt c to the cytoplasm of the target cells. We conclude that we were able to create a smart stimuli-responsive targeted drug delivery system with significant potential in cancer therapy.

Combined Effect of Anticancer Agents and Cytochrome C Decorated Hybrid Nanoparticles for Liver Cancer Therapy

“…Apoptosis induction in A549 cells has previously been reported when exposed to cytochrome C loaded formulations compared to the free protein as imaged under confocal microscope. Here, green fluorescent Annexin-V signals were observed due to early stage apoptosis and red fluorescent signals were observed due to late apoptotic cells [31]. Similarly in our studies, we were able to visualise the presence of early apoptosis after combination treatment, demonstrating the more rapid and effective treatment of the co-therapy approach.…”