MOLECULAR EXCHANGE AND DEFECT FORMATION IN MEMBRANES

膜中的分子交换和缺陷形成

• 批准号: 2180195
• 负责人: DAVID NEEDHAM
• 金额: $ 17.42万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-12-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180195
• 关键词: adsorption; amphiphilicity; avidin; biophysics; cell membrane; elasticity; electric field; electroporation; erythrocyte membrane; erythrocytes; fluidity; fluorescent dye /probe; laboratory rat; lipid bilayer membrane; liposomes; lysophospholipids; membrane activity; membrane lipids; membrane permeability; micromanipulator; passive transport; surfactant; thermodynamics; viscosity; voltage /patch clamp

This research has two main components: (1) fundamental basic science experiments, aimed at understanding how and under what conditions surfactant-like molecules such as lysolipids exchange with lipid bilayers, form defects such as pores, modify the behavior of electropores, and ultimately disrupt the bilayer, and (2) direct clinical applications, with the goal of developing new and more rational protocols to provide higher yields for genetic transfection. With regard to the basis science aspect, the transport of surfactants will be characterized as they move to, from and within synthetic and natural lipid membranes. In Specific Aim #1 (SA1), we will investigate the sequence of steps involved in the molecular exchange of surfactant lipids with lipid bilayer vesicles. These steps include: transport through the aqueous media, intercalation into the outer lipid monolayer, intrabilayer transfer to the inner monolayer, and desorption from either bilayer interface. The amount and rate of surfactant exchange with the lipid bilayer will be measured by micropipet manipulation techniques in terms of the relative area change of single bilayer vesicles supported by micropipet suction. The role of a variety of conditions will be evaluated, ranging from: the concentration of surfactant; the rate of delivery of surfactant to the vesicle surface; the chemical nature of the surfactant (headgroup size, charge, attached polymer moieties like polyethylene glycol, as well as hydrocarbon chain length and saturation); and the chemical and physical nature of the bilayer (compressibility, gel or liquid crystalline phase, charged and polymeric headgroups). In this first SA, molecular interactions will also be studied between model ligands (avidin) and receptors (biotin) at the lipid bilayer surface. In SA2, two types of porous defect will be characterized, formed either by intercalation of certain surfactants (nanopores) or by the action of an electric field (micropores). Pore size and pore line tension will be measured as a function of the chemical nature and concentration of the exchanging surfactant. In SA3, the same experiments established in SAs 1&2 will be carried out on various model natural membranes, such as erythrocytes, erythrocyte ghosts and membrane vesicles in order to determine whether surfactant exchange and defect formation in natural biological membranes is similar to that observed in lipid bilayer membranes. Regarding the clinical application, gene transfection is now a burgeoning scientific and medical endeavor. In SA4, experiments will be conducted in our own laboratory and with BTX Inc., and two gene transfection research groups here at Duke. New strategies will focus on the use of electroporation to form resealable pores in surfactant-modified cell membranes through which genetic material can be selectively delivered to the interior of the cell. It is expected that this work will help researchers and clinicians to genetically manipulate cells for scientific study and to develop new anti-cancer and anti-HIV treatments.

这项研究有两个主要组成部分：（1）基础科学基础科学 实验，旨在了解如何以及在什么条件下 表面活性剂样分子，例如与脂质双层交换的溶酶体交换， 形式缺陷，例如孔，修改电孔的行为，然后 最终破坏双层，（2）直接临床应用， 制定新的和更合理的协议以提供更高的目标的目标 遗传转染的产量。 关于基础科学方面，表面活性剂的运输将 当它们从合成和自然中移动，从和内部移动时被描述 脂质膜。 在特定的目标＃1（SA1）中，我们将调查 表面活性剂脂质分子交换的步骤序列 与脂质双层囊泡。 这些步骤包括：通过 水性介质，插入到外脂质单层的插入 转移到内部单层，并从任一双层解吸 界面。 与脂质的表面活性剂交换的数量和速率 双层将通过微孔处理技术来测量 由支撑的单双层囊泡的相对面积变化 微孔吸力。 各种条件的作用将是 评估，范围：表面活性剂的浓度；速率 将表面活性剂递送到囊泡表面；的化学性质 表面活性剂（头组尺寸，充电，连接的聚合物部分） 聚乙烯乙二醇，以及烃链的长度和饱和度）； 以及双层的化学和物理性质（可压缩性，凝胶 或液晶相，充电和聚合头组）。 在这个 第一个SA，也将在模型之间研究分子相互作用 脂质双层表面的配体（Avidin）和受体（生物素）。 在 SA2，将表征两种类型的多孔缺陷，由 某些表面活性剂（纳米孔）的插入或通过 电场（微孔）。 孔径和孔线张力将是 根据化学性质和浓度的函数测量 交换表面活性剂。 在SA3中，在SAS中建立了相同的实验 1和2将在各种自然膜上进行，例如 红细胞，红细胞幽灵和膜囊泡 确定表面活性剂交换和自然中的缺陷形成 生物膜与脂质双层中观察到的膜相似 膜。 关于临床应用，基因转染现在是一种新兴 科学和医学努力。 在SA4中，将进行实验 我们自己的实验室和BTX Inc.以及两项基因转染研究 杜克的小组。 新策略将集中于使用 在表面活性剂修饰的细胞中形成可重新密封孔的电穿孔 可以选择性地传递遗传物质的膜 细胞的内部。 预计这项工作会有所帮助 研究人员和临床医生在遗传上操纵细胞以进行科学 研究并开发新的抗癌和抗HIV治疗。