A block copolymer composed of cationic polymer and polyethylene glycol(PEG) was used as a DNA carrier. Poly(2-(dimethylamino)ethyl methacrylate(DMAEMA)-N-vinyl-2-pyrrolidone (NVP)) having a terminal carboxylic group was synthesized by free radical polymerization using an initiator, 4,4'-azobis(4-cyanovaleric acid). The terminal carboxylic acid was activated by N-hydroxysuccinimide (NHS) with dicyclohexylcarbodiimide (DCC) and then conjugated with PEG bisamine. For a specific gene targeting to asialoglycoprotein receptor of hepatocytes, a galactose moiety was incorporated into the PEG terminal end of poly(DMAEMA-NVP)-b-PEG by reductive coupling using lactose and sodium cyanoborohydride. Plasmid RSV luciferase was used as a reporter gene and in vitro gene transfection efficiency was measured by using HepG2 human hepato carcinoma cells. Poly(DMAEMA-NVP)-b-PEG-gal/DNA complexes formed at 0.5-2 polymer/plasmid weight ratio had compacted structures around 200 nm particle size and exhibited slightly negative surface charge. These complexes were coated with positively charged KALA, a cationic, pH sensitive, endosomal disruptive peptide, for generating positively charged poly(DMAEMA-NVP)-b-PEG-gal/DNA/KALA complex particles. In the presence of serum proteins, both the PEG block and the galactose targeting moiety of poly(DMAEMA-NVP)-b-PEG-gal greatly enhanced the gene transfection efficiency, which was very close to that of Lipofectamine plus. Irrespective of the presence of serum proteins, as the KALA/DNA weight ratio increased, poly(DMAEMA-NVP)-b-PEG-gal enhanced the transfection due to the pH dependent endosomal disruptive property of KALA. This study demonstrates that sufficient transfection efficiency as high as a commercial agent could be attained by judicious formulation of molecular engineered poly(DMAEMA-NVP)-b-PEG-gal combined with an endosomal disruptive peptide, KALA.
양이온성 고분자와 Polyethylene glycol로 구성된 블록 공중합체는 유전자 전달체로 사용된다. 말단기에 carboxylic acid를 가진 poly(DMAEMA-NVP)는 개시제인 4,4'-azobis(4-cyanovaleric acid)을 이용해서 라디칼 중합에 의해 합성했다. 고분자 말단의 carboxylic acid은 DCC와 NHS로 차례로 활성화시킨 뒤, PEG bisamine로 결합시켰다. 간세포의 asialoglycoprotein receptor 표적 지향성 유전자 전달을 위해, lactose와 sodium cyanoborohydride를 이용한 reductive coupling을 통해 galactose를 poly(DMAEMA-NVP)-b-PEG 말단기에 도입했다. Plasmid RSV luciferase는 reporter gene으로 사용하고, in vitro 유전자 전달 효율은 HepG2 human hepatocarcinoma cells을 통해 측정했다. 고분자/유전자 무게비가 0.5-2인 범위에서 형성된 poly(DMAEMA-NVP)-b-PEG-galactose/DNA 복합체들은 약 200 nm 크기의 compacted structures 를 보이고, 음이온의 표면 전하를 가졌다. 이 복합체들은 양이온성, pH sensitivity, endosome disruptive property 를 가진 KALA peptide로 ionic interaction을 통해 coating 시켜서, 양이온 표면 전하를 가진 poly(DMAEMA-NVP)-b-PEG-galactose/DNA/KALA 복합체를 만들었다. Gene transfection 할 때 serum proteins이 있는 경우, poly(DMAEMA-NVP)-b-PEG-galactose의 PEG와 galactose 표적 지향체는 모두 유전자 전달 효율을 급격히 증가시켰는데, 그 효율은 상품화된 Lipofectamine plus와 거의 유사한 효율성을 보였다. 특히 serum proteins의 존재 유무와는 상관없이 KALA/DNA 무게비가 증가시켰을 때, KALA의 pH dependent endosomal distruptive property 때문에 유전자 전달 효율이 동시에 증가했다. 이 연구는 상용화된 유전자 전달체 만큼 높은 유전자 전달 효율은 KALA와 결합된 poly(DMAEMA-NVP)-b-PEG-galactose의 적합한 formulation을 통해 얻을 수 있다.
