Imidazole/poly(Ethylene glycol)-substituted poly(n-(8-aminooctyl)acrylamide) as biocompatible and efficient gene carrier.

A novel cationic polymer was developed by conjugating imidazole and poly(ethylene glycol) (PEG) on poly(N-(8-aminooctyl)acrylamide) (P8Am) for complexing with pDNA to exhibit high gene expression with low cytotoxicity and the resistance against erythrocyte agglutination and serum inhibition. Cytotoxicity results indicated that these P8Am derivatives with varying substitutions were more biocompatible than unmodified P8Am and PEI control. Moreover, the particle size and zeta potential experiment demonstrated that they were capable of complexing pDNA into sub-micrometer (135-625 nm) and positively charged (+10 to +43 mV) particles, while the high degree of substitution might impede their pDNA complexation ability that formed less positive and larger polyplexes. Flow cytometry analysis demonstrated that the cellular uptake efficiency was dependent on the degree of substitution; low degree of substitution would mediate high uptake efficiency. The gene-transfection ability, evaluated by luciferase assay, revealed low substitution in P8Am-IM11 (substituted with 11 mol% imidazole moieties) and P8Am-PG7 (substituted with 7 mol% PEG moieties) in transfected cells more efficient than unmodified P8Am. Therefore, a multi-functional P8Am derivative, P8Am-IM11-PG7, containing both imidazole and PEG, was developed according to the optimized contents. In the presence of serum, P8Am-IM11-PG7 polyplexes significantly enhanced the gene-transfection efficiency relative to unmodified P8Am polyplexes. Moreover, they exhibited minimal cytotoxicity and the erythrocyte aggregation assay showed that P8Am-IM11-PG7 polyplexes had good blood compatibility as compared to P8Am and PEI polyplexes. This indicated that, by chemical modification, P8Am-IM11-PG7 could possess the required abilities to overcome the difficulties encountered in gene transfection and be a promising alternative of a gene carrier.

Design, synthesis and evaluation of cationic poly(N-substituent acrylamide)s for in vitro gene delivery.

A series of poly(N-substituent acrylamide)s (PAms) that differ in alkylamine side-chain was synthesized via free radical polymerization. The PAms were designed to examine the effects of the methylene numbers (from 2 to 12) in the alkylamine side-chain on cytotoxicity, plasmid DNA (pDNA) binding affinity, cellular uptake efficiency and gene expression. The cytotoxicity of PAms evaluated in HEK293 cells using the MTT assay showed a trend of decreasing toxicity with increasing side-chain length and the IC50 values of all PAms were lower than that of polyethylenimine (PEI) control. The primary amine-based polymers were able to efficiently condense pDNA to form complexes with size ranging from 100 to 350 nm. The gene transfection ability of PAms is dominantly determined by the specific side-chain length that P8Am (with an octylamine side-chain) reveals higher gene expression than other PAms containing the same backbone structure. Although the gene transfection efficiency of PEI was better than all of PAms, PAms were found not to be uptake-limited. This was supported by the effect of chloroquine on transfection activity, based on the protease inhibition activity of chloroquine. Especially, complexes formed from P8Am displayed high uptake level relative to PEI, which was attributed to the proper structure of P8Am to compact pDNA to form stable nanoparticles in the heparin replacement assay. The present study offers the understanding to polymer structure that influences the transfection ability and gives useful information when designs efficient polymeric gene carrier.

Multilayered polyplexes with the endosomal buffering polycation in the core and the cell uptake-favorable polycation in the outer layer for enhanced gene delivery.

In the present study, quaternary polyplexes were prepared by sequential addition of polycations (polyethylenimine (PEI) or poly (N-(8-aminooctyl)-acrylamide) (P8Am)) for loading pDNA into the core polyplexes and poly (acrylic acid) (PAA) for reversing charges to deposit additional polycation (PEI or P8Am) layer. It was found the cytotoxicity and cellular uptake expression of PEI core polyplexes could be improved by coating a cell uptake-favorable P8Am layer. Conversely, P8Am could not facilitate endosomal release through the proposed proton sponge effect so the PEI core was required for the P8Am-coated quaternary polyplexes to ensure efficient transfection. Consequently, an efficient and safe non-viral gene vehicle was constructed by layer-by-layer deposition, using alternate polyanion and polycation with required functionalities to overcome the obstacles met in the process of transfection. Maximum transfection activity with minimal toxicity was observed when the quaternary polyplex of pDNA/PEI/PAA/P8Am was prepared at a weight ratio of 1/1.5/3/5. Conversely, the same composition in different position such as the cell-favorable P8Am core was externally deposited with the endosome lytic moiety, PEI showed high toxicity and low efficiency. This indicates the pDNA/PEI/PAA/P8Am sequence for a quaternary polyplex is as important as the functional polymer selection for designing safe and reliable gene delivery vehicles. We demonstrate here that gene delivery efficiency may be improved by increasing the uptake level and the endosomal buffering release through an additional layer of cell uptake-favorable polycations associated with the core polycations possessing endosomal release ability.