Cellular uptake and trafficking of peptide-based drug delivery systems for miRNA

Today, macromolecular compounds such as microRNAs (miRNAs) are becoming more and more widespread as leading therapeutics. However, their application is limited mostly due to their poor stability, limited cellular uptake, and poor target specificity. Cell-penetrating peptides (CPPs), a group of positively charged peptides, represent a breakthrough as delivery systems for macromolecules. In the present study, we used two types of nanoparticles which differ in the type of CPP used for their manufacturing. The first type is composed of protamine, an arginine rich CPP, which is highly positively charged. The arginine residues are able to form electrostatic interactions with miRNAs, stabilize them, and deliver them to cells. The second type is composed of the N-Ter peptide (also known as MPG), an amphipathic peptide rich in lysine. The positively charged parts of the N-Ter peptide electrostatically stabilize miRNAs, whereas its amphipathic character allows it to successfully traverse cell membranes. We used miRNA-27a, a negative regulator of adipogenesis, to form nanoparticles with the peptides and traced their uptake in 3T3-L1 preadipocytes. Motivated by the lengthy discourse regarding the uptake mechanism of CPPs, the focus of our study was to analyse and understand the internalization of proticles (protamine nanoparticles) and N-Ter complexes. The nanoparticles were characterized regarding size, size distribution, and zeta potential, and their cytotoxicity was tested in 3T3-L1 cells. The uptake studies were performed by varying the experimental conditions such as time, concentration, and temperature, as well as by applying different inhibitors of endocytosis. Furthermore, we assessed the biological effect of miRNA-27a on the pro-adipogenic machinery. The obtained data have shown that protamine and the N-Ter peptide form positively charged nanoparticles through non-covalent complexation. The uptake of proticles and N-Ter complexes was found to be dependent on time, concentration, and temperature, and different uptake pathways were discovered to be involved in the internalization of the different nanoparticles. Furthermore, both types of nanoparticles induced the anti-adipogenic effect of miRNA-27a, demonstrating that this approach can be used as a novel miRNA replacement therapy in the treatment of obesity and obesity-related disorders.