Spatial and Temporal Control of Transfection Using Ferrocene-Containing Lipids

描述（由申请人提供）：本探索性/开发性提案旨在开发使用可电化学转化的含二茂铁阳离子脂质实现对细胞转染的主动控制的方法。该方法建立在初步观察的基础上，即由含二茂铁的阳离子脂质制备的脂质/DNA复合物在脂质处于还原的氧化还原状态时导致高水平的细胞转染，但在脂质处于氧化状态时导致非常低水平的转染。这一关键的观察结果表明，一个通用的和简单的方法，可用于实现在体外或体内通过原位电化学激活的lipoplexes的细胞转染的主动控制的基础。拟议的研究旨在测试的假设，氧化还原活性，含二茂铁的阳离子脂质可用于制定lipoplexes，无论是积极的或消极的细胞转染，和激活的“非活性”的配方可以是可以控制外部和积极通过应用低电位。以下具体目标旨在评估该假设并确定该方法的可行性。它们是：1）使用由质粒DNA和完全氧化或完全还原形式的模型含二茂铁阳离子脂质（BFDMA）形成的脂质复合物来表征一组细胞系中的转染和细胞毒性水平。该目的的第二个目标是评估当通过尾静脉注射施用给小鼠时，还原和氧化的BFDMA在体内介导转基因表达的能力的差异; 2）测量在脂质复合物内可以实现BFDMA氧化态变化的速率，并表征转化后脂质复合物重组的速率。该目的的第二个目标寻求在暴露于用于转化脂质的电化学电势时表征脂质复合物内DNA的完整性;和3）证明DNA/BFDMA脂质复合物的原位电化学转化导致体外和体内细胞转染程度的变化。我们设想这种探索性/发展性研究的潜在结果具有实质性的基本和应用的影响，至少在两个方面：第一，通过引入新的工具，为基础的生物和生物医学研究，第二，通过引入的方法，最终可以用来实现空间和时间控制转染治疗或临床情况。

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.