Design, synthesis and evaluation of cell uptake-favorable polycation for gene delivery and other biomedical applications
Date Issued
2010
Date
2010
Author(s)
Ke, Jin-He
Abstract
Therapeutic nucleic acid delivery has been considered as a powerful strategy for treating gene-related diseases. Development of safe and efficient gene delivery vector is essential for clinical use in gene therapy. Polycations are the major type of the nonviral gene vectors widely investigated for gene delivery. The purpose of this dissertation attempts to develop an efficient with minimal toxicity polycationic gene vector for gene delivery.
The first part of this dissertation gives a broad discussion of the current comprehension of the biological barriers and common discussed polycations in gene delivery.
In the second part of this dissertation, 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 two to twelve) 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 the 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 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 under the heparin replacement assay. This offers the understanding to polymer structure that influences the transfection ability and gives useful information to develop efficient polymeric gene vector.
In the third part of this dissertation, chemical modification was performed to give P8Am multi-functionalities to overcome the gene delivery barriers encountered during transfection. Hence, a novel cationic polymer was developed by conjugating imidazole and polyethylene glycol (PEG) on poly(N-(8-aminooctyl)acrylamide) (P8Am) to exhibit high gene expression with low cytotoxicity and the resistance against erythrocyte agglutination and serum inhibition. Cytotoxicity results indicated that these P8Am derivatives in varied substitutions were more of biocompatibility than unmodified P8Am and PEI control. Moreover, the particle size and zeta potential experiment demonstrated that they were capable of complexing pDNA into sub-micro (135 ~ 625 nm) and positive charge (+10 ~ +43 mV) particles, while high degree of substitution might impede their pDNA complexation ability that formed less positive and larger polyplexes. Flow cytometry analysis demonstrated the cellular uptake efficiency was depended on the degree of substitution; low degree of substitution would mediate high uptake efficiency. The gene transfection ability was evaluated by luciferase assay that revealed low substitution P8Am-IM11 (substituted with 11 mole % of imidazole moiety) and P8Am-PG7 (substituted with 7 mole % of PEG moiety) transfected cells more efficient than unmodified P8Am, respectively. Therefore, the 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, it exhibited minimal cytotoxicity and the erythrocyte aggregation assay showed that P8Am-IM11-PG7 polyplexes revealed good blood compatibility as compared to P8Am polyplexes and PEI polyplexes. This indicated that by the efforts of chemical modification, P8Am-IM11-PG7 could possess required abilities to overcome the difficulties encountering in gene transfection. However, the chemical strategy seems to impede the cell-uptake favorable property of P8Am.
In the fourth part of this dissertation, 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.
In the last part of the dissertation, achievements of each chapter in this dissertation were concluded, and some suggestions and prospection were provided according to the present findings.
Subjects
gene therapy
polycation
cell uptake
polymer synthesis
Type
thesis
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