Development of Bioresponsive Lipids for Intracellular Delivery

用于细胞内递送的生物响应性脂质的开发

• 批准号: 8018991
• 负责人: DAVID H THOMPSON
• 金额: $ 29.03万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8018991
• 关键词: Acids; Adsorption; Behavior; Biological Assay; Biological Availability; Biology; Biophysics; Cell division; Cells; Cellular biology; Complex; Confocal Microscopy; Cytoplasm; Defect; Development; Dimensions; Drug Formulations; Endosomes; Environment; Equilibrium; Exposure to; Family; Fertilization; Film; Flow Cytometry; Fluorescence; Goals; High Pressure Liquid Chromatography; Hydrogen Bonding; Kinetics; Laboratories; Lasers; Libraries; Lipids; Masks; Mediating; Membrane; Membrane Fusion; Membrane Lipids; Methodology; Molecular; Monitor; Nature; Non-Viral Vector; Nucleic Acids; Peptides; Phase; Phase Transition; Play; Polyethylene Glycols; Process; Research; Shapes; Synaptic Transmission; Synthesis Chemistry; System; Techniques; Temperature; Tertiary Protein Structure; Testing; Therapeutic; Tissues; Vesicle; Viral; Viral Proteins; base; design; folate-binding protein; improved; membrane model; molecular dynamics; monolayer; nanoparticle; novel; performance tests; plasmid DNA; polycation; public health relevance; receptor mediated endocytosis; research study; retinal rods; theories; trafficking; transgene expression; vinyl ether

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.

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。描述了一个跨学科的项目，这将扩大可用于加速这一根本性的重要过程的材料的范围。所提出的材料将作为掩蔽的非融合性化合物并入客体膜囊泡中，所述化合物在暴露于酸性或氧化环境时将变得融合-这是一种触发过程，在概念上类似于酸性内体内pH诱导的基于病毒蛋白的膜融合。初步实验已经指导了可裂解的乙烯基醚-PEG脂质的设计，其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相分离、可裂解的PEG脂质家族中安装可调不稳定性的乙烯基醚键。这些化合物将含有疏水性杆段，其一端被可裂解的亲水性乙烯基醚-PEG单元掩蔽，另一端通过相分离的氢键疏水嵌段锚定到膜上。平均场单链理论将用于指导PEG脂质的设计，所述PEG脂质将在活化之前保持分散，但在触发发生之后形成化学稳定的相分离状态。分子动力学模拟将被用来产生初始输入的平均场计算。然后测试该融合基因文库在PEG裂解和将未掩蔽的疏水杆结构域插入并置双层后在模型膜系统中促进膜融合的能力。将使用HPLC分析和基于荧光的测定来监测在酸性或氧化触发条件下PEG脂质裂解、膜脂质混合和囊泡内容物混合的速率。还将使用31 P NMR、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。将使用流式细胞术和激光共聚焦显微镜技术测定最有效的融合子在靶向细胞中实现质粒DNA货物的细胞质释放的能力，以通过受体介导的内吞作用内化这些载体。 公共卫生关系：该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。描述了一个跨学科的项目，这将扩大可用于加速这一根本性的重要过程的材料的范围。所提出的材料将作为掩蔽的非融合性化合物并入客体膜囊泡中，所述化合物在暴露于酸性或氧化环境时将变得融合-这是一种触发过程，在概念上类似于酸性内体内pH诱导的基于病毒蛋白的膜融合。将使用流式细胞术和激光共聚焦显微镜技术测定最有效的融合子在靶向细胞中实现质粒DNA货物的细胞质释放的能力，以通过受体介导的内吞作用内化这些载体。