Development of Bioresponsive Lipids for Intracellular Delivery

描述（由申请人提供）：拟议研究的主要目的是合成可用于控制脂质介导的膜融合的新化合物。描述了一个跨学科项目，将扩大可用于加速这一基本重要过程的材料范围。提议的材料将作为掩蔽的、非融合性的化合物被纳入客体膜囊泡中，这些化合物在暴露于酸性或氧化环境时将成为融合性的——这是一个在概念上类似于酸性内体中ph诱导的基于病毒蛋白的膜融合的触发过程。初步实验指导了可切割乙烯醚- peg脂类的设计，该脂类在引发乙烯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相分离、可切割的PEG脂类家族中安装可调稳定度的乙烯醚连接。这些化合物将包含疏水棒段，其一端被可切割的亲水性乙烯醚- peg单元掩盖，另一端通过相分离的氢键疏水块固定在膜上。平均场单链理论将用于指导PEG脂类的设计，该脂类在激活前保持分散，但在触发发生后形成热力学稳定的相分离状态。分子动力学模拟将用于生成所提出的平均场计算的初始输入。然后，将测试该融合原库在PEG切割和将暴露的疏水棒结构域插入相对双层时促进模型膜系统中膜融合的能力。HPLC分析和基于荧光的测定将用于监测PEG脂质裂解，膜脂混合和囊泡内容物混合在酸性或氧化触发条件下的速率。触发前后膜结构的物理表征也将使用31P NMR，单层膜平衡，C-TEM/C-SEM和DSC进行。我们将使用流式细胞术和激光共聚焦显微镜技术，检测最有效的融合原在细胞内释放质粒DNA的能力，通过受体介导的内吞作用将这些载体内化。

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.