Divalent Metal Ions Boost Effect of Nucleic Acids Delivered by Cell-Penetrating Peptides

Abstract

Cell-penetrating peptides (CPPs) are promising tools for the transfection of various substances, including nucleic acids, into cells. The aim of the current work was to search for novel safe and effective approaches for enhancing transfection efficiency of nanoparticles formed from CPP and splice-correcting oligonucleotide (SCO) without increasing the concentration of peptide. We analyzed the 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 by the addition of the ions, without increasing cytotoxicity. We propose a novel strategy for 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, with the aim of increasing CPP-mediated transfection efficiency and the likelihood of using CPPs in clinics.

Choosing an Optimal Solvent Is Crucial for Obtaining Cell-Penetrating Peptide Nanoparticles with Desired Properties and High Activity in Nucleic Acid Delivery

“…The inclusion of biologically relevant divalent cations Ca 2+ and Mg 2+ into PF14 nanoparticles with oligonucleotides strongly increased the biological effect of the latter inside the cells ( Figure 2 ), consistent with earlier reports [ 48 , 51 , 75 , 76 , 77 , 78 , 79 , 80 , 81 ]. Therefore, we next analysed the morphology of metal ion-complemented PF14-SCO nanoparticles by AFM, SEM, and TEM ( Figure 5 , Table S5 ).…”

“…We may conclude that when PF14 was dissolved in an optimal solvent mixture, it assembled into spherical nanoparticles after dilution in an aqueous media, and the aggre- The inclusion of biologically relevant divalent cations Ca 2+ and Mg 2+ into PF14 nanoparticles with oligonucleotides strongly increased the biological effect of the latter inside the cells (Figure 2), consistent with earlier reports [48,51,[75][76][77][78][79][80][81]. Therefore, we next analysed the morphology of metal ion-complemented PF14-SCO nanoparticles by AFM, SEM, and TEM (Figure 5, Table S5).…”

“…As can be seen in Figure 2A, all of the SCO-PF14 nanoparticles prepared with PF14 dissolved in organic solvents showed activity similar to the ones prepared using water stock. The inclusion of biocompatible divalent cations, Ca 2+ and Mg 2+ , into the nanoparticles strongly increased the SCO delivery efficiency of PF14, as we observed earlier [51]. Although the increase was observed in the case of all the PF14 stocks used, it was more prominent and consistent in the case of the solutions that contained TMC, and respective SCO-PF14-Ca 2+ /Mg 2+ particles led to a significantly higher splicing correction than the ones prepared with water-dissolved PF14.…”

“…The advantageous properties of PF14 nanoparticles were not compromised by complexation with SCO or siRNA or by the further supplementation of particles with divalent metal ions, i.e., Ca 2+ and Mg 2+ ( Table S3 ). The metal ions have earlier been shown to increase the efficiency of PF14-NA complexes multiple fold and were, therefore, included in this study [ 51 ]. Particles of PF14 in complex with SCO were of similar size to the particles that consisted of the peptide only (112 and 119 nm, respectively).…”

“…For the splice-correction assay, we prepared CPP-SCO nanoparticles by mixing them at a 5:1 molar ratio (MR) in Milli-Q (MQ) water in 1/10th of the final volume [ 51 ]. After mixing by pipetting, the solutions were incubated at room temperature (RT) for 15 min.…”