유전자 전달을 위한 덱사메타손이 접합된 폴리아미도아민 덴드리머의 합성 및 특성

Abstract

국문요지

Polyamidoamine(PAMAM) 덴드리머는 양이온성 고분자로 유전자 전달에 넓게 이용되고 있다. PAMAM 덴드리머는 세대 (generation,G)가 높을수록 표면에 많은 아민을 포함하고 있으며 DNA와 고분자가 핵산과 같은 폴리아민과 함께 전기적 상호작용을 통해서 복합체를 형성한다. 따라서 PAMAM 덴드리머는 잘 정의된 구조, 표면의 기능 제어 용의성과 함께 상대적으로 높은 유전자 전달 효율을 지니고 있는 고분자 유전자 전달체로 개발 되었다. 본 연구에서 PAMAM G1과 PAMAM G2를 소수성 약물인 덱사메타손과 접합하여 PAMAM G1-Dexa와 PAMAM G2-Dexa를 합성하였다. PAMAM G1과 PAMAM G2를 선택하게 된 이유는 낮은 분자량의 PAMAM 덴드리머는 다른 높은 세대의 PAMAM에 비해 낮은 세포독성을 지닐 것으로 예측되기 때문이다. 젤 지연 분석법 (gel retardation)을 통해서 PAMAM G1-Dexa (or PAMAM G2-Dexa)가 낮은 비율에서도 DNA와 복합체를 형성함을 확인 하였다. 헤파린 경쟁 분석법 (heparin competition assay)를 통해 PAMAM G2-Dexa가 PEI25k 보다 DNA와 단단하게 복합체를 형성함을 확인 할 수 있었다. HEK293 세포와 N2A 세포에서 트랜스팩션 분석법 시행 하였으며, 이를 통해 PAMAM G1-Dexad 와 PAMAM G2-Dexa 복합체들이 PAMAM G1, PAMAM G2, PEI25k 보다 높은 유전자 전달 효율을 보임을 확인 하였다. 가장 높은 유전자 전달 효율은 PAMAM G2-Dexa 1:10 (DNA:무게비)에서 보였으며, 유세포 분석기 (flow cytometry)와 형광현미경을 통하여 HEK 293 세포와 N2A 세포에서 pcDNA-EGFP/PAMAM G1-D, pcDNA-EGFP/PAMAM G2-D 복합체의 녹색 형광 단백질의 발현을 분석하였다. 그 결과 PAMAM G1-Dexa 복합체의 형광발현 효율이 가장 높음을 HEK293 cell에서 확인 하였으며 PAMAM G2-Dexa 복합체는 N2A 세포에서 형광발현이 가장 잘 보임을 확인하였다. 세포독성 분석을 통하여 PAMAM G1-Dexa와 PAMAM G2-Dexa의 독성이 lipofectamin에 비해 낮고 PEI25k와는 비슷함을 확인 하였다. 항 염증효과를 보기 위한 TNF-α 효소면역측정법 (ELISA)을 시행하였다. LPS가 유도된 Raw264.7 대식세포에서 PAMAM G1-Dexa, PAMAM G2-Dexa 복합체 모두 TNF-α 사이토카인을 감소시킴을 확인 하였다. 이러한 실험을 통하여 PAMAM G1-Dexa와 PAMAM G2-Dexa 마이셀이 덱사메타손의 항 염증효과를 지닌 유용한 유전자 전달체로 이용될 수 있을 것이라 예상된다. |ABSTRACT

Synthesis and characterization of dexamethasone conjugated polyamidoamine dendrimer for gene delivery

Kim, Jinyoung Dept. of Bioengineering The Graduate School Hanyang University

Polyamidoamine (PAMAM) dendrimers widely used cationic polymer for gene delivery. PAMAM dendrimers contain large number of surface amines that are generation-dependent and are capable of forming DNA/polymer complex through the electrostatic interaction with polyanions such as nucleic acids. Thus, PAMAM dendrimers have been developed as polymeric gene carriers with their well-defined structure, ease of control of surface functionality, and relatively high gene transfection efficiency. In this study, PAMAM G1-Dexa or PAMAM G2-Dexa was synthesized with hydrophobic drug dexamethasone conjugated PAMAM G1 or G2 dendrimer. I contacted PAMAM G1 and PAMAM G2, because low molecular weight PAMAM dendrimer expected low cytotoxcicity than other high generation. In gel retardation assay, PAMAM G1-Dexa (or PAMAM G2-Dexa) impeded pDNA low ratios. Heparin competition assay sighted that PAMAM G2-Dexa complex more mighty binding with DNA and more than safety complex than PEI25k. Transfection assays were performed with pβ-Luc/PAMAM G1-Dexa (or PAMAM G2-Dexa) complexes into HEK293 and N2A cells. Evaluated PAMAM G1-Dexa and PAMAM G2-Dexa complexes have higher than transfection efficiency PAMAM G1, PAMAM G2 and Polyethylenimine (PEI). The most transfection efficiency at 1:10 weight ratio DNA:PAMAM G2-Dexa. Flow cytometry and fluorescence microscopy were analysis with pcDNA-EGFP/PAMAM G1-Dexa (or PAMAM G2-Dexa) complex of EGFP expression into HEK293 and N2A cells. The monitoring results PAMAM G1-Dexa show highest EGFP expression into HEK293 cells and PAMAM G2-Dexa show highest fluorescence expression into N2A cells. Cell viability measured MTT assay. MTT assay evaluated PAMAM G1-Dexa, PAMAM G2-Dexa less cytotoxicity than lipofectamine. Anti- inflammatory effect measured TNF-α ELISA. Evaluated PAMAM G1-Dexa and PAMAM G2-Dexa complexes decreased TNF-α cytokine level more efficiently than dexamethasone in lipopolysaccharides (LPS) induced Raw264.7 cells. I experiment PAMAM G1-Dexa (or PAMAM G2-Dexa) micelles are expected as a useful gene delivery polymeric carriers and anti-inflammatory effect of dexamethasone.

Keywords: Dexamethasone, Polyamidoamin (PAMAM) dendrimer, gene delivery; ABSTRACT

Synthesis and characterization of dexamethasone conjugated polyamidoamine dendrimer for gene delivery

Kim, Jinyoung Dept. of Bioengineering The Graduate School Hanyang University

Polyamidoamine (PAMAM) dendrimers widely used cationic polymer for gene delivery. PAMAM dendrimers contain large number of surface amines that are generation-dependent and are capable of forming DNA/polymer complex through the electrostatic interaction with polyanions such as nucleic acids. Thus, PAMAM dendrimers have been developed as polymeric gene carriers with their well-defined structure, ease of control of surface functionality, and relatively high gene transfection efficiency. In this study, PAMAM G1-Dexa or PAMAM G2-Dexa was synthesized with hydrophobic drug dexamethasone conjugated PAMAM G1 or G2 dendrimer. I contacted PAMAM G1 and PAMAM G2, because low molecular weight PAMAM dendrimer expected low cytotoxcicity than other high generation. In gel retardation assay, PAMAM G1-Dexa (or PAMAM G2-Dexa) impeded pDNA low ratios. Heparin competition assay sighted that PAMAM G2-Dexa complex more mighty binding with DNA and more than safety complex than PEI25k. Transfection assays were performed with pβ-Luc/PAMAM G1-Dexa (or PAMAM G2-Dexa) complexes into HEK293 and N2A cells. Evaluated PAMAM G1-Dexa and PAMAM G2-Dexa complexes have higher than transfection efficiency PAMAM G1, PAMAM G2 and Polyethylenimine (PEI). The most transfection efficiency at 1:10 weight ratio DNA:PAMAM G2-Dexa. Flow cytometry and fluorescence microscopy were analysis with pcDNA-EGFP/PAMAM G1-Dexa (or PAMAM G2-Dexa) complex of EGFP expression into HEK293 and N2A cells. The monitoring results PAMAM G1-Dexa show highest EGFP expression into HEK293 cells and PAMAM G2-Dexa show highest fluorescence expression into N2A cells. Cell viability measured MTT assay. MTT assay evaluated PAMAM G1-Dexa, PAMAM G2-Dexa less cytotoxicity than lipofectamine. Anti- inflammatory effect measured TNF-α ELISA. Evaluated PAMAM G1-Dexa and PAMAM G2-Dexa complexes decreased TNF-α cytokine level more efficiently than dexamethasone in lipopolysaccharides (LPS) induced Raw264.7 cells. I experiment PAMAM G1-Dexa (or PAMAM G2-Dexa) micelles are expected as a useful gene delivery polymeric carriers and anti-inflammatory effect of dexamethasone.

Keywords: Dexamethasone, Polyamidoamin (PAMAM) dendrimer, gene delivery