Reversible PEGylation and Schiff-base linked imidazole modification of polylysine for high-performance gene delivery

Abstract

Gene carriers made from polylysine are of interest in relation to gene therapy but suffer from the lack of transfection efficiency due to limited stability and endosomal escape ability. To address this problem, we designed and developed Schiff-base linked imidazole modified polylysine with a reversible-PEGylation catiomer (SL-ImPEG-SS-PLL) for high efficiency gene delivery. The reversible PEGylation was introduced for in vivo circulation, as well as selective PEG detachment to augment the cellular internalization, while introducing Schiff-base linked imidazole residues into polylysine was expected to accelerate the endosomal escape of the DNA payload, as well as facilitate intracellular DNA unpacking and release, thus significantly enhancing gene delivery efficiency. The size alteration of the SL-I 15 mPEG-SS-PLL/pDNA polyplexes in the presence of 10 mM GSH suggests stimulus-induced PEG detachment under tumor relevant reduction conditions. Acid-base titration assays indicate that imidazole residues confer on polylysine remarkable buffering ability. The agarose gel retardation assay suggests that the Schiff-base linkages provide an increased DNA binding ability to protect DNA against nucleases and timely intracellular DNA unpacking to permit DNA dissociation from polylysine and access to transcriptional machinery. Biological efficacy assessment of this multifunctional carrier, using pEGFP and pGL-3 as reporter genes, indicates comparable to or even higher transfection efficiencies than gold standard PEI and their transfection efficiencies are slightly affected by serum. More importantly, in vivo transfection of pEGFP reveals that GFP expression was found not only in some of the important organs, such as the liver, spleen, kidneys and lungs, but also in the transplanted carcinoma. These experimental results suggest that the reversible PEGylation and Schiff-base linked imidazole modification make SL-ImPEG-SS-PLL a great potential candidate for an effective and biocompatible gene delivery system.

Effective co-delivery of nutlin-3a and p53 genes via core–shell microparticles for disruption of MDM2–p53 interaction and reactivation of p53 in hepatocellular carcinoma

“…Therefore, numerous efforts have been directed towards producing non-viral gene carriers (artificial viruses) for the safe delivery of DNA and RNA interference (RNAi). [25][26][27][28][29][30] For instance, Kumar et al prepared new lipopolymers in which low molecular weight and branched polyethylene amine (LMW PEI) molecules hydrophobically modified with ferrocene-terminated alkyl tails. Their results showed a greater transfection efficiency as compared to high molecular weight PEI (25 kDa) and commercially available transfection agents.…”

“…27 In another study, a new multifunctional gene carrier was prepared via crosslinking LMW PEI with lactitol diacrylate (LDA) which caused higher cellular uptake, cell targeting, and a rapid intracellular release of genes in vitro and in vivo. 28 To date, chitosan (CS), a natural linear cationic polysaccharide with a random distribution of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) units, has gained much attention as a gene delivery vector owing to its biocompatibility, low immunogenicity, ease of modification, and biodegradability. 31,32 Although CS primary amino groups can be readily protonated at low pH, its poor solubility under physiological conditions (pH B 7.0) and rather low transfection efficiency in vitro and in vivo have significantly confined its extensive applications in the biomedical domain.…”

A block copolymer containing PEG and histamine-like segments: well-defined functions for gene delivery

“…19,20 The PEG grafted or copolymerized polymer complex with anionic DNA produced core-shell polymeric micelles, where the polycation-DNA complex made up the core and the hydrophilic PEG segment made up the shell. 26,27 As a moiety of histamine, an imidazole ring has been employed to modify polymeric vectors for enhancing biocompatibility and gene transfection efficiency due to its buffering capacity near endosomal pH, which promotes a fast endosomal escape via the so-called ''proton sponge'' effect. 19,21,22 For instance, compared to PLL/DNA complexes, polyion complex micelles from complexes of PEG-b-PLL with DNA have exhibited high stability in a serum containing medium and prolonged blood circulation in experimental animals.…”

“…It has been confirmed that the hydrophilic PEG-shell can increase colloidal solubility, reduce systematic toxicity and improve circulation time. [26][27][28][29] Moreover, considering the deprotonation as the physiological pH, the imidazole moiety was further quaternized for the improvement of DNA-binding ability. 23 Moreover, Kataoka's group firstly compared the different dynamic states of polyplexes and PEGylated polyplex micelles in the bloodstream, and demonstrated the significance of PEGylation to prevent polyplex agglomeration in vivo.…”

Preparation and Characterization of Chitosan‐Based Nanoparticles as Protein Delivery System

“…Furthermore, PLL has high positive charge density which will cause undesirable cytotoxicity. So, when used as biomaterial, biocompatibility, toxicity, and buffering capacity of PLL should be improved, conjugating with various hydrophilic or amphiphilic polymers can realize desirable modifications …”