Su Young Chae

Preparation of a Hydrophobized Chitosan Oligosaccharide for Application as an Efficient Gene Carrier

To prepare chitosan-based polymeric amphiphiles that can form nanosized core-shell structures (nanoparticles) in aqueous milieu, chitosan oligosaccharides (COSs) were modified chemically with hydrophobic cholesterol groups. The physicochemical properties of the hydrophobized COSs (COSCs) were investigated by using dynamic light scattering and fluorescence spectroscopy. The feasibility of applying the COSCs to biomedical applications was investigated by introducing them into a gene delivery system. The COSCs formed nanosized self-aggregates in aqueous environments. Furthermore, the physicochemical properties of the COSC nanoparticles were closely related to the molecular weights of the COSs and the number of hydrophobic groups per COS chain. The critical raggregation concentration values decreased upon increasing the hydrophobicity of the COSCs. The COSCs efficiently rcondensed plasmid DNA into nanosized ion-complexes, in contrast to the effect of the unmodified COSs. An investigation of gene condensation, performed using a gel retardation assay, revealed that COS6(Mn= 6,040 Da) containing 5% of cholesteryl chloroformate (COS6C5) formed a stable DNA complex at a COS6C5/DNA weight ratio of 2. In contrast, COS6, the unmodified COS, failed to form a stable COS/DNA complex even at an elevated weight ratio of 8. Furthermore, the COS6C5/DNA complex enhanced thein vitro transfection efficiency on Human embryonic kidney 293 cells by over 100 and 3 times those of COS6 and poly(L-lysine), respectively. Therefore, hydrophobized chitosan oligosaccharide can be considered as an efficient gene carrier for gene delivery systems.

Development of polymeric gene delivery carriers: PEGylated copolymers of l-lysine and l-phenylalanine

Block copolymers consisting of poly(ethylene glycol) (PEG) and poly(amino acid)-based random copolymers were successfully synthesized by the ring opening polymerization of the N-carboxy anhydrides (NCA) of l-lysine and l-phenylalanine. The synthesized copolymers had a molecular weight of around 30,000 and contained l-lysine and l-phenylalanine residues with molar ratios of 10/0, 9/1, 8/2, 7/3 and 6/4. The complex formation of the copolymer and pCMV-luc plasmid DNA was confirmed by the gel retardation assay and zeta potential measurement. Complete neutralization was achieved at an N/P ratio of more than 1.0 and the size of the complex was determined to be around 150 nm by dynamic light scattering. The cytotoxicity and transfection efficiency were tested on the HEK 293T cell line. The synthesized copolymers displayed negligible cytotoxicity, resulting in a cell viability of more than 95%, while those of the poly(l-lysine) (PLL) and poly(ethylenimine) (PEI) homopolymer were around 65 and 55%, respectively, under comparable conditions. The introduction of the hydrophilic PEG is believed to reduce the toxicity of the copolymer, due to its enhanced biocompatibility, and to impart improved stability to the complex under physiological conditions. The transfection efficiency at the optimized charge ratio of 7 was dramatically improved as the molar content of the l-phenylalanine residues in the copolymers increased and reached a maximum value at an l-phenylalanine content of 30 mol%. The transfection efficiency of the PEGK7/plasmid DNA complex was around 80 times higher than that of PLL, despite the presence of neutral PEG as a block segment.

Synthesis and Characterization of Poly(L-Lysine-co-L-proline) as a Non-viral Gene Delivery Vector

Gene therapy, the expression of genetic materials with therapeutic activity, has been considered as an encouraging approach to heal life threatening diseases with genetic deficiency. Since direct delivery of the DNA in the absence of a carrier experiences rapid degradation by nuclease and generally exhibits poor cellular uptake, it is one of the main challenges facing today’s gene therapy to establish safe and efficient vectors for delivering therapeutic genes to specific cells. Gene delivery systems investigated so far include viral and non-viral vectors. Viral vectors are intensively used in clinical applications because they efficiently integrate their genetic information into the host chromosomes; however, toxic immunological reactions and recombination events into virulent products still remain as major drawbacks. Non-viral vectors, including liposomes and cationic polymers, display nonimmunogenicity, low acute toxicity, and flexibility to design a carrier with well-defined structures and chemical properties, while they have major problems in transfection efficiency which is limited as only few percentage of that by viral vectors. Among non-viral vectors, poly(L-lysine) (PLL) has been widely used due to the reasonable efficiency and biocompatible nature of peptide bonds in the backbones. However, transfection efficiency of PLL is not so high enough that modification of PLL, such as introduction of targeting ligands or endosomal escape moieties, has been investigated for the use of PLL in vivo. Proline is the only amino acid with a cyclic structure containing a secondary α-amino group and this structural feature gives unique stereochemical and biological properties to the peptides containing proline residue. Water soluble prolinerich peptides have been reported as naturally occurring cellpermeant peptides, which are able to break the cell membrane and deliver the attached carriers into the cells without causing lethal membrane disruption. In this communication, we report the synthesis of lysine and proline based amino acid copolymers. Characterization of the copolymer and the effect of proline content on the formation of copolymer/DNA complex, in vitro cytotoxicity and transfection efficiency were investigated to examine the possibility of the synthesized copolymers as a polymeric gene delivery carrier.