Uncovering Fundamental Relationships Between Molecular Structure and Passive Cell Membrane Transport

通过细胞膜的被动转运是药物进入细胞的主要途径。它是口服给药进入体循环的主要途径。环境毒素也可以被动进入体内。了解被动转运的机理细节对于理解某些分子如何以及为什么产生好的药物或危险的毒素至关重要。不幸的是，目前测量跨膜被动转运的方法面临着严重的局限性，并且无法产生可重复的结果。该项目采用了一种新的方法来测量被动运输，通过以下方式将测量工件最小化：促进瞬态测量，而不是稳态测量，从而最大限度地减少运输工件的形成。允许完整的表征空间浓度分布的运输分子，允许精确拟合的运输模型。这种方法是基于通过旋转盘共聚焦显微镜（SDCM）观察分子进入巨型单层脂质囊泡（GUVs）的运输。SDCM允许在任何时刻对GUV膜两侧的浓度剖面进行快速成像。反过来，这将使研究人员能够确定浓度曲线演变的时间过程。对早期实验结果的简单建模表明，从瞬态浓度剖面数据可以很容易地确定膜的渗透率。该技术可以回答一系列重要的研究问题：1。SDCM测量被动输运的限制是什么？2. 什么分子性质控制被动运输？3. 膜组成和电荷状态如何调节被动输运。该项目的目标是开发和完善一种新技术，以测量药物和毒素进入细胞的容易程度。这项新技术将改进现有技术，为研究人员提供药物和毒素分子穿越细胞膜的实时图像。细胞进入过程的详细图像将是设计有效的下一代药物的有价值的工具。本研究开发了新的工程工具，以解决目前用于测量分子在脂质双分子层膜上被动运输的技术的主要缺陷。大多数现有的观测被动输运的工具只能获得稳定状态下的体积浓度，导致高度受限的测量伪像。这里开发的工具将允许对整个浓度剖面进行瞬态观察，这将允许对控制被动输运的参数进行精确测量。这些精确的测量结果将为分子结构与膜通透性关系的定量研究奠定基础。分子结构的两个方面将被研究：分子的结构渗透双分子层和构成双分子层本身的脂质结构。通过系统地改变亲脂性、分子量和氢键基团来研究穿过双层的分子。脂质分子将通过改变尾巴长度、电荷状态、氢键容量和膜相状态来研究。发展分子结构和被动转运之间的定量关系将有助于深入了解这一过程的机理。这种见解是发展药物分子如何进入细胞的综合理论的关键。更广泛的影响跨细胞膜的被动运输对药物在体内的表现至关重要。能够通过细胞膜而不激活细胞机制的药物具有高的口服生物利用度。深入了解被动转运的机制对于理解小分子如何与人体相互作用至关重要，这是一个涉及从药物开发到环境毒理学的基本问题。该研究项目与一个全面的教育和推广计划相结合，该计划侧重于五个领域：本科生研究、本科生实验室模块开发、研究生课程开发、向高中生推广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为来自洛杉矶联合学区（LAUSD）不同人口的学生提供深入的研究经验。这项扩展计划的主要影响将是为LAUSD人口提供独特的基于实验室的体验式教育机会，其中包含较大比例的弱势学生和少数群体，这些学生在科学和工程领域的代表性不足。研究生水平的拓展计划包括参加西班牙裔专业工程师协会年度会议研究生院的研讨会。

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.