利用聚乙二烯胺修飾作為基因載體的明膠奈米微粒

Abstract

在基因治療的領域中，建立低毒性及高效率的基因傳送系統是相當重要的目標。本研究利用低分子量的聚乙二烯胺大量連接在明膠奈米微粒的表面，在不影響細胞毒性的情況下，提升奈米微粒的轉染效率。 無生物毒性的明膠奈米微粒為核心材料，以低分子量的聚乙二烯胺(MW 600)交聯於其表面上，增加其介面電位(42.47 mV)及對pH值的緩衝能力，藉以提昇明膠─聚乙二烯胺奈米微粒的穩定性。在確認明膠─聚乙二烯胺奈米微粒表面的氨基（amino group）濃度後，即可利用不同奈米顆粒及不同N/P下測試轉染的效率。實驗結果顯示接上分子量1.8k的聚乙二烯胺奈米微粒在N/P比值為30時，具有最好的轉染效率（2.12×104 RLU/μg protein）。與市售產品ExGen 500（1.17×105 RLU/μg protein）和Lipofectamine 2000（3.12×104 RLU/μg protein）比較發現，明膠─聚乙二烯胺奈米微粒的轉染效率達到ExGen 500的20 %，且與Lipofectamine 2000的效率接近。而在細胞活性方面，明膠─聚乙二烯胺奈米微粒的細胞活性可以達到86.4 %，表現優於ExGen 500(67.7%)及Lipofectamine 2000（33.4%）。 根據實驗結果，經聚乙二烯胺修飾的明膠奈米微粒，其轉染效率大幅提昇高，且細胞毒性低於市售商業產品，為一具有高轉染效率與低生物毒性的基因轉染載體。

In gene therapy, developing non-toxic and efficient gene delivery system is vital. Although there has been numerous studies regarding viral gene vectors, safety concern limits the application. For this reason, recent studies have shown that non-viral gene vectors, especially polymer nanoparticles, play an important role in gene therapy. Polymer nanoparticles offer several significant advantages, including biosafety, easier modification, cost-effectiveness and so on; polyethylenimine is particularly attractive for non-viral gene therapy. The high affinity with DNA of PEI is due to its large amount of positive net charges from nitrogen atoms. High cationic surface charge also gives PEI an excellent buffer capacity in acidic environment. However, high transfection efficiency of PEI (25kDa), along with its cytotoxicity, strongly depends on its molecular weight. To maintain the transfection efficiency and minimize cytotoxicity, many studies performed cross-linked low molecular weight PEI. In this study, gelatin, a biopolymer without cytotoxicity, was made to nanoparticle. Then PEI was cross-linked into the surface of nanoparticles. Because of high positive zeta potential (42.47 mV) and buffering effect, GA-PEI 1.8k can form compact and stable complex with DNA. The results showed that GA-PEI 1.8k NPs with N/P ratio 30 had excellent transfection efficiency （2.12×104 RLU/μg Protein）, which is about 20% of ExGen 500（1.17×105 RLU/μg Protein）and comparable to Lipofectamine 2000 （3.12×104 RLU/μg Protein）. The cell viability of GA-PEI 1.8k (86.4%) is higher than ExGen 500 (67.7%) and Lipofectamine 2000 (33.4%). We successfully cross-linked PEI into the surface of gelatin nanoparticle. As a gene vector, GA-PEI 1.8k NPs had high trnasfection efficiency and low cell toxicity, and it can be a potential gene delivery system used in gene therapy.