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

基本信息

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
项目类别：
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

项目摘要

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）：拟议研究的主要目标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，该项目将扩大可用于加速这一至关重要的过程的材料范围。所提出的材料将作为掩蔽的非融合化合物掺入客体膜囊泡中，这些化合物在暴露于酸性或氧化环境时将变得融合——这一触发过程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。初步实验指导了可裂解乙烯基醚-PEG脂质的设计，该脂质在引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 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单元掩盖，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，该脂质在激活之前保持分散状态，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为建议的平均场计算生成初始输入。然后将测试该融合剂文库在 PEG 裂解并将未掩蔽的疏水杆结构域插入到相邻双层中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酸性或氧化触发条件下 PEG 脂质裂解、膜脂混合和囊泡内容物混合的速率。触发前后膜结构的物理表征也将使用 31P NMR、单层膜天平、C-TEM/C-SEM 和 DSC 进行。将使用流式细胞术和激光共聚焦显微镜技术对最有效的融合剂进行分析，以确定它们在细胞中影响质粒DNA货物的细胞质释放的能力，这些细胞旨在通过受体介导的内吞作用内化这些载体。公共健康相关性：拟议研究的主要目标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，该项目将扩大可用于加速这一至关重要的过程的材料范围。所提出的材料将作为掩蔽的非融合化合物掺入客体膜囊泡中，这些化合物在暴露于酸性或氧化环境时将变得融合——这一触发过程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。将使用流式细胞术和激光共聚焦显微镜技术对最有效的融合剂进行测定，以确定它们在细胞中影响质粒DNA货物的细胞质释放的能力，这些细胞旨在通过受体介导的内吞作用内化这些载体。