Internalisation and biological activity of nucleic acids delivering cell-penetrating peptide nanoparticles is controlled by the biomolecular corona

Abstract

Nucleic acid molecules can be transferred into cells to alter gene expression and, thus, alleviate certain pathological conditions. Cell-penetrating peptides (CPPs) are vectors that can be used for transfecting nucleic acids as well as many other compounds. CPPs associate nucleic acids non-covalently, forming stable nanoparticles and providing efficient transfection of cells in vitro. However, in vivo, expected efficiency is achieved only in rare cases. One of the reasons for this discrepancy is formation of protein corona around nanoparticles, once they are exposed to a biological environment, e.g. blood stream. In this study, we compared CPP-nucleic acid nanoparticles formed in the presence of bovine, murine and human serum. We used Western blot and mass-spectrometry to identify the major constituents of protein corona forming around nanoparticles, showing that proteins involved in transport, haemostasis and complement system are its major components. We investigated physical features of nanoparticles, and measured their biological efficiency in splice-correction assay. We showed that protein corona constituents might alter the fate of nanoparticles in vivo, e.g. by subjecting them to phagocytosis. We demonstrated that composition of protein corona of nanoparticles is species-specific that leads to dissimilar transfection efficiency and should be taken into account while developing delivery systems for nucleic acids.

Divalent metal ions boost effect of nucleic acids delivered by cell-penetrating peptides

“…To assess if the positive effect of the ions is also present at the mRNA level, we incubated HeLa pLuc 705 cells with differently prepared nanoparticles for 24 h and performed PCR analysis of luciferase gene expression (Fig. 1C) [64].…”

“…RNA was subjected to DNase treatment (TURBO DNase, Thermo Scientific) and cDNA was synthesized from 1.34 µg of RNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). PCR of luciferase gene and GAPDH gene (used as a housekeeper) was performed using Platinum II Hot-Start Green PCR Master Mix (Invitrogen) as described earlier [64]. Agarose gel electrophoresis was performed to analyze the splicing correction, and resulting bands were quantified using ImageJ software.…”