Delivery of a granzyme B inhibitor gene using carbamate-mannose modified PEI protects against cytotoxic lymphocyte killing
Delivery of a granzyme B inhibitor gene using carbamate-mannose modified PEI protects against cytotoxic lymphocyte killing
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DOI：
10.1016/j.biomaterials.2013.01.090
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发表时间：
2013-05-01
期刊：
影响因子：
14
通讯作者：
Ashton-Rickardt, Philip G.
中科院分区：
文献类型：
--
作者：
Cheng, Wei;Yang, Chuan;Ashton-Rickardt, Philip G.
细胞毒性T淋巴细胞（CTL）和自然杀伤（NK）细胞通过使用颗粒酶B（GrB）杀死感染或转化的细胞以诱导细胞凋亡来保护脊椎动物。然而，GrB诱导的靶细胞凋亡导致炎性疾病和慢性移植排斥，因此是重要的疾病靶点。本研究的目的是通过使用阳离子聚合物作为非病毒载体递送编码GrB抑制剂蛋白酶抑制剂-9（PI-9）的质粒来防止靶细胞的凋亡。聚乙烯亚胺（PEI，支链，Mn 10 kDa）在许多类型的细胞系中提供高度的基因转染效率，但它具有高度的细胞毒性。为了减少这种细胞毒性，我们通过伯胺和受保护的甘露糖官能化的环状碳酸酯（MTC-ipman）之间的亲核加成来封闭伯胺基团，从而通过环状碳酸酯的开环产生氨基甲酸酯键来修饰PEI。甘露糖的脱保护产生用碳水化合物修饰的PEI聚合物。67个伯胺基团中有7个或20个被碳水化合物取代的PEI与未修饰的PEI相比具有相似的基因结合能力，导致GFP报告质粒在HEK 293 T人胚肾细胞中几乎100%的转染效率。此外，PEI的修饰导致PEI/DNA复合物的细胞毒性降低。然而，所有伯胺基团被封闭的PEI不能与DNA形成复合物，因此报告基因转染可以忽略不计。使用具有最佳伯胺取代度的修饰的PEI将PI-9编码质粒有效地转染到HEK 293 T细胞中，保护高达80%的HEK 293 T细胞免受人天然细胞因子样白血病YT细胞的杀伤。因此，这些氨基甲酸酯-甘露糖修饰的PEI/PI-9编码质粒复合物在预防慢性移植排斥和GrB引起的炎性疾病中具有潜在的临床应用。(C)2013爱思唯尔有限公司保留所有权利。
Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells protect vertebrates by killing infected or transformed cells using granzyme B (GrB) to induce apoptosis. However, GrB-induced apoptosis of target cells causes inflammatory disease and chronic transplant rejection and so is an important disease target. The aim of this study was to prevent apoptosis of the target cells by delivering a plasmid encoding GrB inhibitor proteinase inhibitor-9 (PI-9) using cationic polymers as a non-viral vector. Polyethyleneimine (PEI, branched, Mn 10 kDa) gives a high degree of gene transfection efficiency in many types of cell lines, but it is highly cytotoxic. To reduce this cytotoxicity, we modified PEI by blocking primary amine groups through nucleophilic addition between primary amine and a protected mannose-functionalized cyclic carbonate (MTC-ipman), generating a carbamate linkage through the ring-opening of the cyclic carbonate. Deprotection of the mannose yielded a PEI polymer that is decorated with the carbohydrate. PEI with 7 or 20 of 67 primary amine groups substituted by the carbohydrate had similar gene binding ability compared to unmodified PEI, leading to almost 100% transfection efficiency of a GFP-reporter plasmid in HEK293T human embryonic kidney cells. Furthermore, modification of PEI resulted in a decrease in the cytotoxicity of PEI/DNA complexes. However, PEI with all primary amine groups blocked was unable to form a complex with DNA, and so reporter transfection was negligible. The PI-9 encoding plasmid was transfected into HEK293T cells effectively using the modified PEIs with the optimal degree of primary amine substitution, protecting up to 80% HEK293T cells from killing by human natural killer-like leukemic YT cells. Therefore, these carbamate-mannose modified PEI/PI-9 encoding plasmid complexes have potential clinical utility in the prevention of chronic transplant rejection and inflammatory disease caused by GrB. (C) 2013 Elsevier Ltd. All rights reserved.