Nano-carriers for DNA delivery to the lung based upon a TAT-derived peptide covalently coupled to PEG-PEI
Nano-carriers for DNA delivery to the lung based upon a TAT-derived peptide covalently coupled to PEG-PEI
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DOI：
10.1016/j.jconrel.2005.09.036
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发表时间：
2005-12-05
影响因子：
10.8
通讯作者：
Kissel, T
中科院分区：
文献类型：
--
作者：
Kleemann, E;Neu, M;Kissel, T
针对呼吸上皮的基因治疗具有治疗囊性纤维化和肺癌等疾病的潜力。聚乙烯亚胺（PEI）已被用于基因传递到气道。在这项研究中，我们描述了一种新的PEI修饰，其中与HIV-1 TAT蛋白转导结构域相关的寡肽通过异双功能聚乙二醇（PEG）间隔物与25 kDa PEI （PEI）共价偶联，形成TAT-PEG-PEI偶联物。观察到小（类似于90 nm）多聚体的DNA报告基因络合和保护得到改善，并显著提高了对多阴离子、Alveofact（(R)）、支气管肺泡衬里液和DNA酶的稳定性。为了确定体外多重毒性，进行了MTT试验，在体内试验中，研究了小鼠支气管肺泡灌洗的总细胞计数、中性粒细胞数量、总蛋白和tnf - α浓度。所有参数均表明TAT-PEG-PEI的毒性显著降低。在体外条件下（A549）和气管内灌注小鼠体内，研究了PEI和TAT-PEG-PEI复合物与DNA的转染效率。虽然TAT-PEG-PEI （0.2 ng/mg蛋白）在A549细胞中的荧光素酶表达量远低于PEI （2 ng/mg蛋白），但在小鼠中检测到TAT-PEG-PEI的转染效率显著提高。报告基因表达分布于支气管和肺泡组织。因此，TAT-PEG-PEI代表了一种用于肺部治疗的非病毒基因载体的新方法，包括对质粒DNA的保护，低毒性和在体内条件下显著提高转染效率。(c) 2005 Elsevier B.V.版权所有
Gene therapy aimed at the respiratory epithelium holds therapeutic potential for diseases such as cystic fibrosis and lung cancer. Polyethylenimine (PEI) has been utilized for gene delivery to the airways. In this study, we describe a new modification of PEI, in which an oligopeptide related to the protein transduction domain of HIV-1 TAT was covalently coupled to 25 kDa PEI (PEI) through a heterobifunctional polyethylenglycol (PEG) spacer resulting in a TAT-PEG-PEI conjugate. Improved DNA reporter gene complexation and protection was observed for small (similar to 90 nm) polyplexes as well as significantly improved stability against polyanions, Alveofact((R)), bronchial alveolar lining fluid and DNase. To determine polyplex toxicity in vitro, MTT assays were performed and, for in vivo testing, the mice bronchial alveolar lavage was investigated for total cell counts, quantity of neutrophils, total protein and TNF-alpha concentration. All parameters suggest significantly lower toxicity for TAT-PEG-PEI. Transfection efficiencies of both PEI and TAT-PEG-PEI polyplexes with DNA were studied under in vitro conditions (A549) and in mice after intratracheal instillation. While luciferase expression in A549 cells was much lower for TAT-PEG-PEI (0.2 ng/mg protein) than for PEI (2 ng/mg), significantly higher transfection efficiencies for TAT PEG-PEI were detected in mice. Reporter gene expression was distributed through bronchial and alveolar tissue. Thus, TAT-PEG-PEI represents a new approach to non-viral gene carriers for lung therapy, comprising protection for plasmid DNA, low toxicity and significantly enhanced transfection efficiency under in vivo conditions. (c) 2005 Elsevier B.V. All rights reserved.