Development of Bioresponsive Lipids for Intracellular Delivery

描述(申请人提供)：拟议研究的主要目标是合成可用于控制脂质介导膜融合的新化合物。描述了一个跨学科的项目，该项目将扩大可用材料的范围，以加速这一根本重要的过程。建议的材料将作为掩蔽的、非融合性化合物并入客膜囊泡中，这些化合物在暴露于酸性或氧化环境时将成为融合性化合物--这一触发过程在概念上类似于酸性内体中由pH诱导的基于病毒蛋白的膜融合。初步实验指导了可裂解乙烯基醚-聚乙二醇脂的设计，这种脂类在触发乙烯基醚键降解后促进膜融合。PI实验室开发的合成方法将用于在一系列相分离、可切割的聚乙二醇脂中安装可调节的乙烯基醚键。这些化合物将包含疏水棒段，其一端由可切割的亲水性乙烯基醚-聚乙二醇单元体遮盖，另一端通过相分离的氢键疏水块固定在膜上。平均场单链理论将被用来指导聚乙二醇脂的设计，它在激活前保持分散，但在触发发生后形成热力学稳定的相分离状态。分子动力学模拟将被用来为拟议的平均场计算产生初始输入。然后将测试该FusoGen文库在聚乙二醇裂解和将未屏蔽的疏水杆状结构域插入相对的双层时，在模型膜系统中促进膜融合的能力。高效液相分析和基于荧光的分析将被用来监测在酸性或氧化触发条件下的聚乙二醇脂裂解、膜脂混合和囊泡内容物混合的速率。还将使用31P核磁共振、单层膜天平、C-TEM/C-SEM和DSC对触发前后的膜结构进行物理表征。使用流式细胞术和激光共聚焦显微镜技术将检测最有效的融合子，以确定它们是否能够通过受体介导的内吞作用在细胞中影响质粒DNA货物的胞质释放，从而使这些载体内化。 公共卫生相关性：拟议研究的主要目标是合成可用于控制脂质介导膜融合的新化合物。描述了一个跨学科的项目，该项目将扩大可用材料的范围，以加速这一根本重要的过程。建议的材料将作为掩蔽的、非融合性化合物并入客膜囊泡中，这些化合物在暴露于酸性或氧化环境时将成为融合性化合物--这一触发过程在概念上类似于酸性内体中由pH诱导的基于病毒蛋白的膜融合。使用流式细胞术和激光共聚焦显微镜技术将检测最有效的融合子，以确定它们是否能够通过受体介导的内吞作用在细胞中影响质粒DNA货物的胞质释放，从而使这些载体内化。

DESCRIPTION (provided by applicant): The primary objective of the proposed research is to synthesize new compounds that can be used to control lipid-mediated membrane fusion. An interdisciplinary project is described that will expand the range of materials available for accelerating this fundamentally important process. The proposed materials will be incorporated within guest membrane vesicles as masked, nonfusogenic compounds that will become fusogenic upon exposure to acidic or oxidative environments--a triggering process that is conceptually similar to pH-induced viral protein-based membrane fusion within acidic endosomes. Preliminary experiments have guided the design of cleavable vinyl ether-PEG lipids that promote membrane fusion after triggering vinyl ether bond degradation. Synthetic methodology developed in the PI's laboratory will be used to install vinyl ether linkages of tunable lability within a family of phase-segregating, cleavable PEG lipids. These compounds will contain hydrophobic rod segments that are masked on one end by a cleavable hydrophilic vinyl ether-PEG unit and anchored to the membrane on the other via a phase-segregating, hydrogen-bonded hydrophobic block. Mean- field single chain theory will be used to guide the design of PEG lipids that will remain dispersed prior to activation, but form a thermodynamically stable phase-separated state after triggering has occurred. Molecular dynamics simulations will be used to generate initial inputs for the proposed mean-field calculations. This fusogen library will then be tested for their ability to promote membrane fusion in model membrane systems upon PEG cleavage and insertion of the unmasked hydrophobic rod domains into apposed bilayers. HPLC analysis and fluorescence-based assays will be used to monitor the rates of PEG lipid cleavage, membrane lipid mixing, and vesicle contents mixing under acidic or oxidative triggering conditions. Physical characterization of the membrane structures, before and after triggering, will also be performed using 31P NMR, monolayer film balance, C-TEM/C-SEM, and DSC. The most efficient fusogens will be assayed, using flow cytometry and laser confocal microscopy techniques, for their ability to effect cytoplasmic release of plasmid DNA cargo in cells targeted to internalize these carriers via receptor mediated endocytosis. PUBLIC HEALTH RELEVANCE: The primary objective of the proposed research is to synthesize new compounds that can be used to control lipid-mediated membrane fusion. An interdisciplinary project is described that will expand the range of materials available for accelerating this fundamentally important process. The proposed materials will be incorporated within guest membrane vesicles as masked, nonfusogenic compounds that will become fusogenic upon exposure to acidic or oxidative environments--a triggering process that is conceptually similar to pH-induced viral protein-based membrane fusion within acidic endosomes. The most efficient fusogens will be assayed, using flow cytometry and laser confocal microscopy techniques, for their ability to effect cytoplasmic release of plasmid DNA cargo in cells targeted to internalize these carriers via receptor mediated endocytosis.