Spatial and Temporal Control of Transfection Using Ferrocene-Containing Lipids

描述(由申请人提供)：这项探索性/开发性的提案寻求开发一种方法，使用可通过电化学转化的含有二茂铁的阳离子脂类来实现对细胞转染性的主动控制。该方法建立在初步观察的基础上，即由含二茂铁阳离子脂质制备的脂质/DNA复合体在脂质处于还原氧化还原状态时导致高水平的细胞转染率，而当脂质处于氧化状态时转染率非常低。这一重要的观察结果为一种普遍而简便的方法提供了基础，该方法可用于通过脂簇的原位电化学激活来实现对体外或体内细胞的转基因的主动控制。这项拟议的研究试图验证这样一种假设，即氧化还原活性的、含二茂铁的阳离子脂质可以用来形成对细胞转染具有活性或非活性的脂合物，并且可以通过施加低电位来外部和主动地控制“非活性”配方的激活。以下具体目标旨在评估这一假说，并确定这一方法的可行性。它们是：1)使用由质粒DNA和完全氧化或完全还原的模型二茂铁阳离子脂(BFDMA)形成的脂合物来表征一组细胞系的转染率和细胞毒性。这一目的的第二个目标是评估还原和氧化的BFDMA通过尾静脉注射给药时在体内介导转基因表达的能力的差异；2)测量BFDMA在脂丛中氧化状态的改变的速率，并表征转化后脂丛的重组速率。这个目标的第二个目标是研究在用来转化脂质的电化学势作用下，DNA在脂复合体中的完整性；以及3)证明DNA/BFDMA脂复合体在体内和体外的电化学转化会导致细胞转染度的变化。我们预计这一探索性/发展性研究的潜在结果至少在两个方面具有重大的基础和应用影响：第一，通过引入基础生物和生物医学研究的新工具，第二，通过引入最终可用于在治疗或临床背景下实现对转基因的空间和时间控制的方法。

DESCRIPTION (provided by applicant): This exploratory/developmental proposal seeks to develop methods for achieving active control over the transfection of cells using a ferrocene-containing cationic lipid that can be transformed electrochemically. The approach is founded on preliminary observations that lipid/DNA complexes prepared from a ferrocene- containing cationic lipid lead to high levels of cell transfection when the lipid is in a reduced redox state, but very low levels of transfection when the lipid is in the oxidized state. This critical observation suggests the basis of a general and facile approach that could be used to achieve active control over the transfection of cells in vitro or in vivo though the in situ electrochemical activation of lipoplexes. The proposed research seeks to test the hypothesis that a redox-active, ferrocene-containing cationic lipid can be used to formulate lipoplexes that are either active or inactive toward cell transfection, and that the activation of `inactive' formulations can be can be controlled externally and actively through the application of low electrical potentials. The following Specific Aims are designed to evaluate this hypothesis and determine the feasibility of this approach. They are: 1) To characterize levels of transfection and cytotoxicity in a panel of cell lines using lipoplexes formed from plasmid DNA and either the completely oxidized or completely reduced forms of a model ferrocene-containing cationic lipid (BFDMA). A second goal of this Aim seeks to evaluate differences in the ability of reduced and oxidized BFDMA to mediate transgene expression in vivo when administered to mice by tail vein injection; 2) To measure the rates at which changes in the oxidation state of BFDMA can be effected within lipoplexes and to characterize the rates of reorganization of lipoplexes upon transformation. A second goal of this Aim seeks to characterize the integrity of DNA within lipoplexes upon exposure to the electrochemical potentials used to transform the lipid; and 3) To demonstrate that electrochemical transformation of DNA/BFDMA lipoplexes in situ leads to changes in the extent of transfection of cells in vitro and in vivo. We envisage the potential outcomes of this exploratory/developmental research as having substantial fundamental and applied impacts in at least two ways: first, through the introduction of new tools for basic biological and biomedical research, and, secondly, through the introduction of methods that could ultimately be used to achieve spatial and temporal control of transfection in therapeutic or clinical contexts.