Uncovering Fundamental Relationships Between Molecular Structure and Passive Cell Membrane Transport

揭示分子结构与被动细胞膜运输之间的基本关系

• 批准号: 1067021
• 负责人: Noah Malmstadt
• 金额: $ 24.01万
• 依托单位: University of Southern California
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1067021&HistoricalAwards=false
• 关键词: Uncovering; Fundamental; Relationships; Between; Molecular

1067021MalmstadtPassive transport through the cell membrane represents a major route by which drugs enter cells. It is the primary route by which orally delivered drugs enter systemic circulation. Environmental toxins can also enter the body passively. Understanding the mechanistic details of passive transport is essential to understanding how and why certain molecules make good drugs or dangerous toxins. Unfortunately, current approaches to measuring passive transport across membranes face severe limitations and have failed to yield reproducible results. This project applies a new approach to measuring passive transport that minimizes measurement artifacts by: 1.) Facilitating transient, rather than steady-state measurements, thereby minimizing the formation of transport artifacts and 2.) Allowing for complete characterization of the spatial concentration profile of a transported molecule, allowing for precise fitting of a transport model. This approach is based on the observation of the transport of molecules into giant unilamellar lipid vesicles (GUVs) via spinning-disc confocal microscopy (SDCM). SDCM allows for rapid imaging of the concentration profile on both sides of the GUV membrane at any instant in time. This, in turn, will allow researchers to establish the time course of the evolution of the concentration profile. Simple modeling of early experimental results shows that membrane permeability can be determined easily from transient concentration profile data. This technique can answer a series of important research questions: 1. What are the limits of SDCM in measuring passive transport? 2. What molecular properties control passive transport. 3. How do membrane composition and charge state modulate passive transport. The goal of this project is to develop and perfect a new technology for measuring how easily drugs and toxins can enter cells. This new technology will improve on existing techniques by providing researchers with real-time images of drug and toxin molecules crossing cell membranes. A detailed picture of the process of cell entry will be a valuable tool for designing effective next-generation drugs.Intellectual MeritThe proposed work develops new engineering tools to address central shortcomings of techniques currently used to measure the passive transport of molecules across lipid bilayer membranes. Most existing tools for observing passive transport can only access bulk concentrations at steady state, leading to highly limiting measurement artifacts. The tools developed here will allow for the transient observation of the full concentration profile, which will allow for precise measurement of the parameters that govern passive transport.These precise measurements will provide a foundation for a quantitative study of the relationship between molecular structure and membrane permeability. Two aspects of molecular structure will be investigated: the structure of molecules permeating the bilayer and the structure of the lipids that make up the bilayer itself. Molecules crossing the bilayer will be investigated by systematically varying lipophilicity, molecular weight, and hydrogen-bonding groups. Lipid molecules will be investigated by varying tail length, charge state, hydrogen bonding capacity, and membrane phase state. Developing a quantitative relationship between molecular structure and passive transport will facilitate mechanistic insight into this process. Such an insight is key to developing a comprehensive theory of how drug molecules enter cells.Broader ImpactsPassive transport across the cell membrane is of critical importance to how drugs behave in the body. Drugs that are able to pass through the cell membrane without activation of the cellular machinery have high oral bioavailability. A thorough mechanistic understanding of passive transport is essential to understanding how small molecules interact with the human body, a fundamental question with implications ranging from drug development to environmental toxicology. This research project is integrated with a comprehensive education and outreach plan that focuses on five areas: undergraduate research, laboratory module development for undergraduates, graduate curriculum development, outreach to high school students, and outreach to underrepresented graduate students. The primary objective of the high school outreach program is to facilitate intensive research experiences for students drawn from the diverse population of the Los Angeles Unified School District (LAUSD). The primary broader impact of this outreach program will be to provide unique lab-based experiential education opportunities to the LAUSD population, which contains a larger proportion of disadvantage students and minority groups that are underrepresented in science and engineering. The graduate level outreach program involves participation in a workshop at the Graduate Institute of the annual Society of Hispanic Professional Engineers conference.

1067021Malmstadtpassive通过细胞膜转运代表了药物进入细胞的主要途径。这是口服输送药物进入全身循环的主要途径。环境毒素也可以被动地进入身体。了解被动运输的机械细节对于了解某些分子如何制造好药物或危险毒素至关重要。 不幸的是，当前测量跨膜传输的方法面临严重的局限性，并且未能产生可重复的结果。该项目采用了一种新方法来测量被动传输，该方法通过：1。）通过：1。）促进瞬态而不是稳态测量，从而最大程度地减少了运输工件的形成和2.）允许完全表征运输分子的空间浓度，从而可以精确地拟合运输模型。这种方法是基于通过旋转盘共聚焦显微镜（SDCM）观察到分子传输到巨型Unilamelar脂质囊泡（GUV）的过程。 SDCM允许在任何瞬间及时对GUV膜两侧的浓度曲线进行快速成像。反过来，这将使研究人员能够建立浓度概况演变的时间过程。对早期实验结果的简单建模表明，可以从瞬态浓度谱数据中轻松确定膜渗透率。该技术可以回答一系列重要的研究问题：1。SDCM测量被动运输的限制是什么？ 2。哪种分子特性控制被动转运。 3。膜组成和电荷状态调节如何进行被动转运。该项目的目的是开发并完善一种新技术，用于测量药物和毒素可以进入细胞的容易程度。这项新技术将通过为研究人员提供越过细胞膜的药物和毒素分子的实时图像来改进现有技术。细胞输入过程的详细图片将是设计有效的下一代药物的宝贵工具。智能优点拟议的工作开发了新的工程工具，以解决目前用于衡量跨脂质双层膜的分子被动运输的技术的中心缺点。观察被动运输的大多数现有工具只能在稳定状态下访问大量浓度，从而导致高度限制的测量工件。此处开发的工具将允许对全浓度曲线进行短暂观察，这将允许精确测量控制被动传输的参数。这些精确的测量将为对分子结构和膜渗透性之间关系的定量研究提供基础。将研究分子结构的两个方面：渗透到双层的分子结构和构成双层本身的脂质的结构。穿越双层的分子将通过系统变化的亲脂性，分子量和氢键组来研究。脂质分子将通过不同的尾部长度，电荷状态，氢键能力和膜相状态来研究。在分子结构和被动转运之间建立定量关系将有助于对这一过程的机械洞察力。这种见解是开发一种关于药物分子如何进入细胞的综合理论的关键。在整个细胞膜上的影响范围内的交通运输对于药物在体内的行为至关重要。能够通过细胞膜的药物而不激活细胞机械的药物具有高口服生物利用度。对被动运输的彻底理解对于理解小分子如何与人体相互作用至关重要，这是一个基本问题，其含义从药物开发到环境毒理学。该研究项目与全面的教育和外展计划融合在一起，该计划的重点是五个领域：本科研究，针对本科生的实验室模块开发，研究生课程发展，向高中生的宣传以及向代表性不足的研究生推广。高中外展计划的主要目标是促进从洛杉矶统一学区（LAUSD）种群中吸引的学生进行深入的研究经验。该外展计划的主要更广泛的影响是为LAUSD人口提供独特的基于实验室的体验教育机会，其中包含更大比例的劣势学生和少数群体，这些学生和少数群体在科学和工程中的代表性不足。研究生级外展计划涉及参加西班牙裔专业工程师年度研究生研究所的研讨会。