Chemical vectors for gene delivery a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers

Purpose: Non-viral nucleic acid delivery systems—lipoplexes, polyplexes, and lipopolyplexes—are limited mainly by poor endosomal escape. The review examines whether histidine- and imidazole-containing carriers can exploit the acidic endosomal environment to destabilize membranes and improve cytosolic delivery of DNA, mRNA, siRNA, and oligonucleotides.

Hypothesis: The imidazole ring of histidine is a weak base that becomes protonated/cationic when pH drops below ~6. In acidic endosomes, this can induce membrane fusion and/or membrane permeation and, upon accumulation, a proton sponge effect. Therefore, histidylated or imidazolylated polymers, peptides, and lipids should enhance endosomal escape and transfection efficiency, often with lower cytotoxicity than permanently cationic amine-based carriers.

Aims: Review polymers, peptides, and lipids containing histidine or imidazole as nucleic acid carriers. - Describe proposed mechanisms: pH-sensitive membrane fusion/permeation and proton sponge effect. - Summarize in vitro and in vivo applications for pDNA, mRNA, siRNA, and ODN delivery. - Highlight structure–activity relationships, transfection efficiency, and cytotoxicity.

Delivery system: Chemical vectors include: - Polyplexes: histidylated polylysine (HpK), histidine-rich HK copolymers, HHHK8b, His6 reducible polycations, imidazole-modified chitosan (UAC70), imidazolylated bPEI (IP/IPP), cyclodextrin-based CDPim. - Peptides: cRGD-hk, HoKC, LAH4/LAH4-L1, Tat-10H, C-5H-Tat-5H-C, O10H6, FGF-2/HK recombinant fusions. - Lipids: imidazole/imidazolium-headed cationic lipids, histidylated cholesterol, lipophosphoramides, imidazolium bolaamphiphiles, and pH-sensitive helper lipids. - Payloads: plasmid DNA, antisense oligonucleotides, siRNA, mRNA, ribozymes. - Targeting/extra features: RGD, galactose, FGF-2, Sendai virosomes, PEGylation, aerosol or systemic administration.

Approach: Review article, not a primary experimental study. It synthesizes published in vitro cell-culture data (HepG2, HEK293, COS-1, HeLa, CHO, A549, dendritic cells, cancer cell lines) and in vivo rodent models (MDA-MB-435 xenograft, B16 melanoma, K-ras null lung tumorigenesis). No original group structure, n, doses, or controls are reported.

Key methods: Transfection/gene expression assays. - Bafilomycin A1 inhibition to test endosome-acidification dependence. - pH-dependent membrane fusion/permeation and buffering-capacity measurements. - FRET, particle sizing, and zeta-potential characterization. - siRNA target mRNA knockdown and apoptosis assays. - In vivo tumor growth inhibition, survival, aerosol lung delivery, and CTL immune response.

Key results: HpK transfection was 3–4.5 orders of magnitude higher than polylysine in HepG2; optimal with 63–100 histidyl residues (33–50%). - HpK delivered DNA but not 25-mer ODN; highly histidylated HoK delivered ODN but not DNA. - Im-PK luciferase activity approached bPEI 25 kDa with lower cytotoxicity. - UAC70 (~37 mol% imidazole substitution) maintained ~90% viability in HEK293/A549 vs 20–40% for PEI. - IPP viability was 78% vs 37% for bPEI; IP transfection was ~3× bPEI at 39 mol% imidazolyl substitution. - Systemic HHHK8b/Raf-1 siRNA inhibited MDA-MB-435 xenograft growth with reduced normal-tissue toxicity. - MART1 mRNA lipopolyplexes delayed B16 melanoma growth and induced a CTL response; mRNA alone, polyplexes, or lipoplexes did not. - Tat-10H improved gene expression up to 7000-fold over controls, comparable to bPEI. - Imidazole helper lipids improved transfection up to 100-fold vs DOPE. - Aerosol UAC70/PTEN suppressed pulmonary tumorigenesis by ~50% in K-ras null mice.

Interpretation: Histidine/imidazole carriers are promising non-viral vectors because endosomal acidification triggers imidazole protonation and membrane destabilization/proton sponge effects, often with lower cytotoxicity than amine-only carriers. However, the exact escape mechanism remains unidentified, and transfection efficiency is still below viral vectors. Comparative studies are needed to determine the best carrier for each application.

Limitations: Review only; no new primary data. - Endosomal escape mechanism not definitively established. - Transfection efficiency is cell-type dependent and generally below viral vectors. - pDNA nuclear import remains a critical bottleneck. - Direct quantification of endosomal escape at the cellular level is lacking. - Most in vivo evidence is preclinical; long-term safety, immunogenicity, and clinical translation remain unresolved.