Non-viral Delivery of Nucleic Acids: Insight Into Mechanisms of Overcoming Intracellular Barriers

Abstract

Delivery of genes, including plasmid DNAs, short interfering RNAs (siRNAs), and messenger RNAs (mRNAs), using artificial non-viral nanotherapeutics is a promising approach in cancer gene therapy. However, multiple physiological barriers upon systemic administration remain a key challenge in clinical translation of anti-cancer gene therapeutics. Besides extracellular barriers including sequestration of gene delivery nanoparticles from the bloodstream by resident organ-specific macrophages, and their poor extravasation and tissue penetration in tumors, overcoming intracellular barriers is also necessary for successful delivery of nucleic acids. Whereas for RNA delivery the endosomal barrier holds a key importance, transfer of DNA cargo additionally requires translocation into the nucleus. Better understanding of crossing membrane barriers by nucleic acid nanoformulations is essential to the improvement of current non-viral carriers. This review aims to summarize relevant literature on intracellular trafficking of non-viral nanoparticles and determine key factors toward surmounting intracellular barriers. Moreover, recent data allowed us to propose new interpretations of current hypotheses of endosomal escape mechanisms of nucleic acid nanoformulations.

Keywords: endosomal escape; gene delivery; intracellular trafficking; lipoplexes; polyplexes; siRNA delivery.

FIGURE 1

Intracellular trafficking of non-viral nucleic acid nanoformulations. Comments can be found in the text. EE, early endosome; MVB, multivesicular body; LE, late endosome; L, lysosome; ERC, endocytic recycling compartment; TGN, trans-Golgi network; EEA1, early endosome antigen 1; PI3P, phosphatidylinositol 3-phosphate; PI(3,5)P₂, phosphatidylinositol (3,5)-bisphosphate; LBPA, lysobisphosphatidic acid; LAMP1, lysosomal-associated membrane protein 1. “+” and “±” mean high and moderate levels of abundance, respectively.

FIGURE 2

Lipoplex endosomal escape. Escape of lipoplex-formulated genetic cargo from an endosome in HeLa cells takes seconds followed by diffusion of siRNA throughout entire cytosol (A). Efficacy of GFP knockdown in HeLa-GFP cells upon incubation with lipoplexes carrying siGFP in the presence of inhibitors of endosomal acidification bafilomycin A1 (BAF) or chloroquine (CHQ) (B). “Lipid-mixing” mechanism of endosomal escape of lipoplex-formulated nucleic acids in assumption of primary role of BMP/LBPA, concentrated in the luminal leaflet of endosomal membrane in area of ILV formation (see the text for additional comments) (C). (A,B) Figures are reprinted by permission from Nature Publishing Group (Wittrup et al., 2015).

FIGURE 3

Polyplex endosomal escape. Accumulation of chloride in endosomal vesicles upon cellular uptake of polyamidoamine (PAM), polyethylenimine (PEI), and polylysine (POL) reflects higher buffering capacity of PAM and PEI (A). Lack of acidic endosomal pH increase in vesicles containing branched PEI (BPEI), compared to bafilomycin A-treated cells, contradicts the “proton sponge” hypothesis (B). Released polyplex-formulated genetic cargo (FITC-labeled oligodeoxyribonucleotides, ODNs) from an endosome in HeLa cells spreads in a single direction (white arrow) (C). Proposed mechanism of endosomal escape of polyplex-formulated nucleic acids driven by osmotic pressure and local permeabilization of endosomal membrane due to electrostatic interactions between polycation and anionic phospholipids like BMP/LBPA (D). Figure adapted with permission from: (A) (Sonawane et al., 2003), ASBMB; (B) (Benjaminsen et al., 2013), Elsevier; (C) (ur Rehman et al., 2013), copyright (2013) American Chemical Society.