Spatial and Temporal Control of Transfection Using Ferrocene-Containing Lipids

描述（由申请人提供）：本探索性/发展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，即当脂质处于还原氧化还原状态时，由含二茂铁阳离子脂质制备的脂质/DNA复合物导致高水平的细胞转染，但当脂质处于氧化状态时，转染水平非常低。这一关键的观察结果表明，一种通用的、简单的方法可以通过脂丛的原位电化学激活，在体外或体内实现对细胞转染的主动控制。提出的研究旨在验证一种假设，即氧化还原活性，含二茂铁的阳离子脂质可以用来配制对细胞转染有活性或无活性的脂质体，并且“无活性”配方的激活可以通过低电位的应用从外部和主动地控制。以下具体目标旨在评估这一假设，并确定这种方法的可行性。它们是：1)利用质粒DNA和模型含二茂铁阳离子脂质（BFDMA）的完全氧化或完全还原形式形成的脂质体，表征转染水平和细胞系的细胞毒性。本研究的第二个目标是评估通过尾静脉注射给小鼠后，还原和氧化BFDMA在体内介导转基因表达能力的差异；2)测量在脂丛中影响BFDMA氧化态变化的速率，表征转化后脂丛的重组速率。本Aim的第二个目标是在暴露于用于转化脂质的电化学电位时，表征脂丛内DNA的完整性；3)原位DNA/BFDMA脂质体的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至少在两个方面具有实质性的基础和应用影响：首先，通过引入基础生物学和生物医学研究的新工具，其次，通过引入最终可用于实现治疗或临床环境中转染的时空控制的方法。

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.