pH-Responsive Charge-Conversional Poly(ethylene imine)–Poly(l‑lysine)–Poly(l‑glutamic acid) with Self-Assembly and Endosome Buffering Ability for Gene Delivery Systems

Poly­(ethylene imine)–poly­(l-lysine)–poly­(l-glutamic acid) (PKE) polymers with various glutamic acid portions were synthesized by ring opening polymerization of l-lysine N-carboxyanhydride (NCA) and l-glutamic acid NCA from poly­(ethylene imine) 1.8 kDa (PEI1.8k) as a macroinitiator. It was found that their glutamic acid residues could buffer endosomal pH. PK5E9 polymer could form nanoparticles by self-assembly and nanosized polyplexes, possessing pH-responsive charge-conversion properties. PK5E9 or its polyplex nanoparticles showed polyhedral structures with bumpy surfaces. Its cytotoxicity was marginal at both pH 7.4 and 6.0, and its transfection efficiency was highly increased at pH 6.0. The improved transfection efficiency in acidic conditions was thought to be induced by elevated cellular uptake of the polyplexes via charge-conversion from negative to positive charges. Its transfection was also found to be mediated by endosomal escape through endosome buffering by bafilomycin A1-treated transfection. In conclusion, PK5E9 polymer with self-assembly and endosome buffering ability was found to possess potentials for efficient gene delivery systems in acidic conditions via charge conversion, which may be applied for tumor microenvironment-targeting

Conditional Antisense Oligonucleotides Triggered by miRNA

Antisense oligonucleotides (ASOs) are single-stranded short nucleic acids that silence the expression of target mRNAs and show increasing therapeutic potential. Since ASOs are internalized by many cell types, both normal and diseased cells, gene silencing in unwanted cells is a significant challenge for their therapeutic use. To address this challenge, we created conditional ASOs that become active only upon detecting transcripts unique to the target cell. As a proof-of-concept, we modified an HIF1α ASO (EZN2968) to generate miRNA-specific conditional ASOs, which can inhibit HIF1α in the presence of a hepatocyte-specific miRNA, miR-122, via a toehold exchange reaction. We characterized a library of nucleic acids, testing how the conformation, thermostability, and chemical composition of the conditional ASO impact the specificity and efficacy in response to miR-122 as a trigger signal. Optimally designed conditional ASOs demonstrated knockdown of HIF1α in cells transfected with exogenous miR-122 and in hepatocytes expressing endogenous miR-122. We confirmed that conditional ASO activity was mediated by toehold exchange between miR-122 and the conditional ASO duplex, and the magnitude of the knockdown depended on the toehold length and miR-122 levels. Using the same concept, we further generated another conditional ASO that can be triggered by miR-21. Our results suggest that conditional ASOs can be custom-designed with any miRNA to control ASO activation in targeted cells while reducing unwanted effects in nontargeted cells

Agmatine-Containing Bioreducible Polymer for Gene Delivery Systems and Its Dual Degradation Behavior

Agmatine-containing bioreducible polymer, poly­(cystaminebis­(acrylamide)-agmatine) (poly­(CBA-AG)) was synthesized for gene delivery systems. It could form 200–300 nm sized and positively charged polyplexes with pDNA, which could release pDNA in reducing the environment due to the internal disulfide bonds cleavage. Poly­(CBA-AG) also showed a spontaneous degradation behavior in aqueous condition in contrast to the backbone polymer, poly­(cystaminebis­(acrylamide)-diaminobutane) (poly­(CBA-DAB)) lacking guanidine moieties, probably due to the self-catalyzed hydrolysis of internal amide bonds by guanidine moieties. The cytotoxicity of poly­(CBA-AG) was cell-dependent but minimal. Poly­(CBA-AG) exhibited highly enhanced transfection efficiency in comparison with poly­(CBA-DAB) and even higher transfection efficiency than PEI25k. However, cellular uptake efficiency of the polyplexes did not show positive correlation with the transfection efficiency. Confocal microscopy observation revealed that pDNA delivered by poly­(CBA-AG) was strongly accumulated in cell nuclei. These results suggested that high transfection efficiency of poly­(CBA-AG) may be derived from the efficient pDNA localization in cell nuclei by guanidine moieties and that the polyplexes dissociation via self-catalyzed hydrolysis as well as disulfide bonds cleavage in cytosol also may facilitate the transfection process. Finally, poly­(CBA-AG)/pJDK-apoptin polyplex showed a high anticancer activity induced by apoptosis, demonstrating a potential of poly­(CBA-AG) as a gene carrier for cancer gene therapy

Effects of the Physicochemical, Colloidal, and Biological Characteristics of Different Polymer Structures between α‑Poly(l‑lysine) and ε‑Poly(l‑lysine) on Polymeric Gene Delivery

Though α-poly­(l-lysine) (APL) has been well-studied in gene delivery, ε-poly­(l-lysine) (EPL) with same repeating unit of l-lysine but different structure has been rarely investigated. This study compared various effects of their different structures in gene delivery processes. EPL showed less cytotoxicity and more proton buffering capacity for endosomal release than APL. Also, EPL/pDNA polyplexes represented higher nucleus preference than APL/pDNA polyplexes. However, EPL had weaker affinities with pDNA than APL, leading to formation of larger EPL/pDNA complexes with less compactness and successively faster decomplexation. The resultant difference of their pDNA binding affinity caused lower cellular uptake and lower transfection efficiency of EPL/pDNA complexes than APL/pDNA complexes. Thus, this study confirmed that various effects of gene delivery processes are changed by chemical structure of polymeric gene carriers. Especially, despite the low transfection efficiency of EPL-based polyplexes, the study found potentials of EPL in cytocompatibility, endosomal release, and nuclear import