Non-Viral Carriers for Nucleic Acids Delivery: Fundamentals and Current Applications

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TL;DR

This review summarizes current knowledge on non-viral carriers for nucleic acid delivery, including polyplexes, liposomes, peptide-based carriers, exosomes, and nanomaterials. It covers the key barriers to effective gene delivery—cell membrane crossing, endosomal escape, and nuclear transport—and strategies to overcome them through chemical modifications and targeting ligands. The clinical significance lies in the potential of non-viral carriers to provide safer alternatives to viral vectors for gene therapy, though efficacy limitations remain a major hurdle.

Full summary

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**Background:** Gene therapy is a promising approach for treating inherited and acquired diseases by delivering therapeutic DNA or RNA into cells. Viral vectors are the most efficient delivery vehicles but carry risks of immunogenicity and cytotoxicity. Non-viral carriers—including liposomes, polycations, peptides, and inorganic compounds—offer safer alternatives but have limited clinical translation due to insufficient efficacy in overcoming biological barriers. This review summarizes recent developments in non-viral carriers based on essential requirements for gene therapy.

**Methods:** The authors conducted a narrative review of the literature on non-viral nucleic acid delivery systems, focusing on the fundamental barriers to transfection and the strategies used to overcome them. The review covers polyplexes (polymer-based complexes), lipid-based carriers, peptide carriers, polymeric carriers (PEI, PBAE, pDMAEMA, dendrimers, chitosan), DNA nanostructures, exosomes, microvesicles, and nanomaterials (graphene oxide, carbon nanotubes, mesoporous silica nanoparticles).

**Key Results:** The review identifies four major barriers to non-viral gene delivery: (1) cell membrane crossing, (2) endosomal escape, (3) cytoplasmic transport, and (4) nuclear entry (for plasmid DNA). For cell membrane binding, both non-specific charge interactions (with proteoglycans, heparan sulfate proteoglycans) and specific receptor-ligand interactions (e.g., folate receptor, CD44) are described. Endocytic pathways include clathrin-mediated endocytosis, caveolar-type endocytosis, macropinocytosis, and phagocytosis. For endosomal escape, four main mechanisms are discussed: pH-buffering effects (proton sponge effect, e.g., PEI), membrane pore formation (e.g., pHLIP peptides), endosomal membrane fusion (e.g., hemagglutinin HA-2), and photochemical disruption (e.g., TPCS2a). For nuclear delivery, the most commonly used nuclear localization signal (NLS) is the SV40 large T antigen peptide (PKKKRKV). The review notes that on average only 0.7% of injected nanoparticles reach the tumor via the enhanced permeability and retention (EPR) effect. For tumor targeting, ligands discussed include hyaluronic acid (CD44 targeting), phenylboronic acid (sialic acid targeting), monoclonal antibodies (e.g., VEGFab), peptides (e.g., RGD, iRGD), folic acid, and biotin. For CNS delivery, strategies include blood-brain barrier disruption (e.g., hyperosmotic mannitol), adsorptive-mediated transcytosis (e.g., Tat-derived CPP), and receptor-mediated transcytosis (e.g., transferrin receptor-targeted T7 peptide, rabies virus-derived RVG29 peptide). For muscle targeting, the ASSLNIA and A2G80 (VQLRNGFPYFSY) peptides are highlighted. For fibroblasts, linear-branched hybrid poly(β-amino ester) achieved ~93% transfection efficacy. Lipid-based carriers (liposomes, LPD nanoparticles, SNALPs) are noted as the most extensively researched class of mRNA delivery carriers, including their use in SARS-CoV-2 mRNA vaccines (mRNA-1273, BNT162b2/Comirnaty). Peptide carriers include cell-penetrating peptides (e.g., HIV-1 TAT, penetratin), targeted peptides (e.g., RGD, iRGD), NLS-carrying peptides, and fusogenic peptides (e.g., diINF-7, DIV3W). For polymeric carriers, PEI modifications (PEGylation, amino acid conjugation, disulfide cross-linking) aim to reduce cytotoxicity and improve transfection. PAMAM dendrimer modifications include alkylcarboxylation, PEGylation, and targeting peptides. Exosomes and microvesicles are described as naturally occurring carriers capable of delivering CRISPR/Cas9 plasmids and siRNAs in vivo.

**Clinical Implications:** Non-viral carriers represent a safer alternative to viral vectors for gene therapy, with advantages including lower immunogenicity and cytotoxicity. However, their clinical translation remains limited by insufficient transfection efficacy. The development of multifunctional carriers combining targeting ligands, stimuli-responsive release mechanisms, and improved endosomal escape strategies is critical for advancing non-viral gene therapy. The successful use of lipid nanoparticles in COVID-19 mRNA vaccines demonstrates the clinical potential of non-viral carriers. Further research is needed to identify more specific ligands for tissue targeting and to optimize carrier modifications for overcoming biological barriers.