Folic acid-modified bovine serum albumin nanoparticles with doxorubicin and chlorin e6 for effective combinational chemo-photodynamic therapy

Abstract

We herein describe a facile method to synthesize stable bovine serum albumin-based nanoparticles (BNPs) loaded with two anticancer therapeutics, doxorubicin (DOX) and a photosensitizer, chlorin e6 (Ce6), in combination with folic acid (FA) as a target cancer cell receptor for the development of an effective combined chemo and photodynamic (FA-Ce6/DOX/BNPs) therapy against cervical cancer. FA-Ce6/DOX/BNPs exhibited excellent monodispersity with an average diameter of 103.5 ± 3.8 nm, a negative zeta potential of approximately -30.44 ± 0.35 mV, and long-term stability. As a result, FA-Ce6/DOX/BNPs exhibited severe toxicity to cervical HeLa cancer cells. Also, a higher drug release rate was observed under acidic pH conditions (pH 5.0). Moreover, FA-Ce6/DOX/BNPs potentiated mitochondrial reactive oxygen species (ROS) production in HeLa cells under 671-nm laser exposure, leading to activation of key regulator proteins of apoptosis such as BH3 interacting-domain death agonist (BID), B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X (BAX), as well as induction of the caspase cascade and mitochondrial ROS-mediated cell death. Confocal microscopy analysis further validated cellular uptake of FA-Ce6/DOX/BNPs by HeLa cells. Furthermore, results of real-time quantitative PCR (RT-qPCR) and western blot analysis further validated the anticancer effect of FA-Ce6/DOX/BNPs, as evidenced by elevated gene/protein expression levels of apoptotic biomarkers p53, BID, caspase-3, cleaved poly(ADP-ribose) polymerase 1 (PARP-1), and BAX, contrary to levels of the anti-apoptotic marker Bcl-2. Moreover, in vivo toxicity results of FA-Ce6/DOX/BNPs using laser irradiation in zebrafish larvae, as a chemo-photodynamic therapy confirmed that it does not affect the larval development without causing any adverse toxic effect in zebrafish larvae. Altogether these findings strongly support the anticancer effect of FA-Ce6/DOX/BNPs combinational chemo-photodynamic therapy, which could be a promising candidate for cervical cancer therapy.

Keywords: Biodegradable BNPs; Drug delivery system; Mitochondrial apoptosis; Photodynamic therapy; ROS.