Synthesis and characterization of nanocarriers for the delivery of siRNA silencing the EZH2 gene

Abstract

In recent years, gene therapy has emerged as a powerful treatment option for cancer therapy using RNA interference, such as small interfering RNA (siRNA), microRNA (miRNA) and short harpin RNA (shRNA), able to suppress pathological gene expression. In particular, siRNAs are double stranded RNA molecules consisting of 21-23 nucleotides that can regulate gene expression and protein silencing by inducing sequence specific cleavage of the complementary mRNA. However, naked siRNAs have important shortcomings, such as poor cellular uptake, rapid degradation in the blood, lack of specific cell targeting and toxicity, that greatly hamper their therapeutic application. In order to overcome these drawbacks, non-toxic, biocompatible and non-immunostimulatory carriers should be found to safely and effectively transport siRNA at the target tissue, protecting it from degradation. In this work, different systems were studied for the delivery of a specifically designed siRNA targeting the EZH2 gene, known for its great potential in the down-regulation of specific genes involved in cancer cells proliferation (sarcoma and prostate cancer therapy). In particular, for the delivery of siRNA-EZH2 we described the use of positively and negatively charged polymethylmethacrylate nanoparticles (PMMANPs), protein-based nanoparticles (keratin and albumin), and gold nanoparticles (AuNPs). PMMANPs, consisting of a hydrophobic inner core surrounded by a cationic or anionic shell made of ammonium quaternary salts or sulphonated groups, respectively, were synthetized and characterized by dynamic light scattering, scanning electron microscopy and UV-Vis spectroscopy. In vitro preliminary studies on PMMANPs@siRNA on a panel of cancerous cell lines accounted for a good cellular uptake, while none of the optimized systems provided the expected silencing of the target gene. Protein-based nanoparticles made of keratin or albumin, have been shown as promising carriers for drug delivery due to their biocompatibility, low toxicity and biodegradability. Keratin nanoparticles were obtained starting from high molecular weight and water-soluble keratin, and subsequently coated with polyethyleneimine (PEI) to create a polyplex able to electrostatically interact with siRNA. Moreover, keratin nanoparticles were tested with an alternative approach based on the combination of tazemetostat, a new epigenetic drug for the treatment of soft tissue sarcoma, with the cytotoxic drug paclitaxel. This innovative multi-modal system displayed promising synergistic/additive in vitro anticancer activity in different cancerous cell lines. Finally, based on their unique properties, including the possibility of fine tune their size, shape and surface coating, we explored the use of gold nanoparticles as potential carriers of siRNA-EZH2. To this end, AuNPs of different sizes and with diverse degrees of surface functionalization were obtained and characterized. Unfortunately, in vitro preliminary studies demonstrated that none of the herein reported AuNPs@siRNA particles were able to induce EZH2 gene silencing under the tested conditions. Despite being a promising approach for treating cancer, siRNA-based therapy is still hampered from clinical application due to significant drawbacks, including the high instability in blood and biological fluids. To address this issue several nanotechnology-based systems have been herein described, but further studies are still needed to optimize the system and secure an effective delivery at the target tissues.