Padari, K. | Search bioRxiv

bioRxiv2021

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

Cell-penetrating peptides (CPPs) are promising tools for transfection of various substances, including nucleic acids, into cells. The aim of current work was to search for novel safe and effective approaches for enhancing transfection efficiency of nanoparticles formed of CPP and splice-correcting oligonucleotide (SCO) without increasing the concentration of peptide. We analyzed an effect of inclusion of calcium and magnesium ions into nanoparticles on CPP-mediated transfection in cell culture. We also studied the mechanism of such transfection as well as its efficiency, applicability in case of different cell lines, nucleic acid types and peptides, and possible limitations. We discovered a strong positive effect of these ions on transfection efficiency of SCO, that translated to enhanced synthesis of functional reporter protein. We observed significant changes in intracellular distribution and trafficking of nanoparticles formed with addition of the ions, without increasing cytotoxicity. We propose a novel strategy of preparing CPP-oligonucleotide nanoparticles with enhanced efficiency and, thus, higher therapeutic potential. Our discovery may be translated to primary cell cultures and, possibly, in vivo studies, in the aim to increase CPP-mediated transfection efficiency and likelihood of using CPPs in clinics.

cell biology↗

bioRxiv2021

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

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.

cell biology↗