Preparation and Properties of Vesicles with Highly Controlled Lipid Asymmetry

该奖项由材料研究部的生物材料计划授予斯托尼布鲁克的纽约州立大学，目的是利用不对称脂质囊泡研究膜的基本结构和功能。脂质不对称性是生物膜内外层脂质组成的差异，是许多细胞膜脂质双层的一个突出和关键特性。 由脂质双层组成的人工囊泡（脂质体）是已被证明是生物膜的宝贵模型的生物材料。使用环糊精，研究者的初步研究表明，通过脂质交换选择性地将脂质引入脂质囊泡的外单层来制备不对称脂质囊泡的可行性。本项目将系统地研究使用广泛的脂质控制脂质交换程度的条件，并将研究有关膜的重要基础问题。将确定环糊精提供最有效的脂质结合的条件，然后优化条件以实现最大脂质交换，同时保持不对称性。 第二个目标是研究控制不对称性的囊泡的生物学重要物理性质及其对膜结构的影响。要研究的第一个属性是作为脂质结构的函数的外小叶和内小叶中的脂质的物理状态之间的耦合。这种小叶间的偶联可以将信号从膜的外部传递到膜的内部。预计这些实验将区分脂质和蛋白质如何促进或抑制小叶间偶联的替代假设。要研究的第二个属性将是小叶之间的脂质横向扩散（脂质触发器），以及脂质结构和膜蛋白如何影响这一点。重要的是，既要定义条件，并确定可以使用的蛋白质，同时保持稳定的脂质不对称性，并了解什么样的膜蛋白序列影响横向扩散。该项目将通过培训研究生和本科生，对未来科学家，包括少数民族学生的职业发展产生广泛影响。他们将接受用于研究膜蛋白和脂质的各种生化技术的专门培训，包括强调荧光和荧光猝灭方法的光谱技术。 学生还将接受科学研究，写作和口语的适当行为的培训，并为生物/生物物理研究和/或教学领域的职业生涯做好准备。该项目应允许其他实验室轻松应用该方法，并对人类（和动物）健康产生重大影响，包括药物输送应用。本研究项目将开发需要不同化学组成的分子宽度层的纳米材料应用。活细胞被膜脂质（特殊脂肪分子）和蛋白质组成的膜包围。 这些膜控制营养物质进入细胞的吸收，以及细胞与环境之间的沟通。细胞膜非常复杂，由数千种不同的蛋白质和脂质组成。 人工膜囊泡由少量的膜脂和蛋白质组成，形成类似于细胞膜的包膜，是了解膜功能的重要工具。 天然膜具有两层膜脂质并且是不对称的，每层中具有不同类型的脂质，但是人工膜的实用性的关键限制是它们缺乏脂质不对称性。 最近开发的方法来制备不对称人工膜的研究人员将进行详细研究，使其能够与各种不同的脂质和蛋白质。 人工膜的使用将大大扩展膜的知识基础。 该项目还将对未来科学家，包括少数民族学生的职业发展产生广泛影响。在教育层面，研究生和本科生（包括通过与其他当地机构的联系）将接受研究，实验原理以及研究膜结构和功能的科学（包括光谱技术）的培训。 这些学生还将接受科学研究，科学写作和演讲的适当行为的培训，并为他们在科学研究和教学领域的职业生涯做好准备。

This award by the Biomaterials program in the Division of Materials Research to State University of New York at Stony Brook is to investigate basic structure and function of membranes using asymmetric lipid vesicles. Lipid asymmetry, a difference in the lipid composition in the inner and outer layers of a biological membrane, is a prominent and crucial property of the lipid bilayer of many cell membranes. Artificial vesicles (liposomes) composed of lipid bilayers are biomaterials that have proven invaluable models of biological membranes. Using cyclodextrins, preliminary studies by the investigator indicated the feasibility of preparing asymmetric lipid vesicles by selectively introducing lipids into the outer monolayer of a lipid vesicle via lipid exchange. This project will systematically study the conditions that control the extent of lipid exchange using a wide range of lipids, and will investigate important basic questions about membranes. Conditions that provide most efficient lipid binding by cyclodextrins will be identified, and then conditions will be optimized for maximal lipid exchange while maintaining asymmetry. The second goal is to study biologically-important physical properties of vesicles that control asymmetry and its impact on membrane structure. The first property to be studied is coupling between the physical state of lipids in the outer and inner leaflets as a function of lipid structure. This interleaflet coupling may transduce signals from the outside to the inside of membranes. These experiments are expected to distinguish between alternate hypotheses for how lipids and proteins promote or inhibit interleaflet coupling. The second property to be studied will be lipid transverse diffusion (lipid flip-flop) between leaflets, and how this is impacted by lipid structure and membrane proteins. It is important, both to define the conditions and identify proteins that can be used while maintaining stable lipid asymmetry, and to understand what membrane protein sequences affect transverse diffusion. This project will have a broad impact on career development of future scientists, including minority students, at the educational level by training both graduate and undergraduate students. They will receive specialized training in a wide variety of biochemical techniques used to study membrane proteins and lipid, including spectroscopic techniques that emphasize the fluorescence and fluorescence quenching approaches. The students will also be trained in proper conduct of scientific studies, writing and speaking, and preparing them for careers in field of biological/biophysical research and/or teaching. This project should allow facile applications of the methods by other labs, with applications that have a significant impact upon human (and animal) health, including drug delivery applications. Nanomaterial applications in which molecular-width layers with different chemical compositions are needed will be developed by this research project.Living cells are surrounded by membranes composed of membrane lipids (specialized fat molecules) and proteins. These membranes control the uptake of nutrients into cells, and the communication between cells and their environment. Cell membranes are very complex, consisting of thousands of types of different proteins and lipids. Artificial membrane vesicles, composed of small variety of membrane lipids and proteins which form an envelope similar to a cell membrane, are important tools for understanding how membranes function. Natural membranes have two layers of membrane-lipids and are asymmetric, with different types of lipids in each layer, but a key limitation in the utility of artificial membranes has been their lack of lipid asymmetry. The recently developed method to prepare asymmetric artificial membranes by the investigator will be studied in detail to allow it to be used with a wide variety of different lipids and proteins. The use artificial membranes will greatly expand the knowledge base on membranes. This project will also have a broad impact on career development of future scientists, including minority students. At the educational level, both graduate and undergraduate students (including via contacts with other local institutions) will be trained in the conduct of research, experimental principles, and the science of studying membrane structure and function including spectroscopic techniques. These students will also be trained in proper conduct of scientific studies, scientific writing and speaking, and preparing them for careers in field of scientific research and teaching.