Uncovering Fundamental Relationships Between Molecular Structure and Passive Cell Membrane Transport

1067021 Malmstadt通过细胞膜的被动转运是药物进入细胞的主要途径。它是口服药物进入体循环的主要途径。环境毒素也可以被动进入人体。了解被动转运的机制细节对于理解某些分子如何以及为什么成为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严重的限制，并且未能产生可重复的结果。该项目应用了一种新的方法来测量被动传输，通过以下方式最大限度地减少测量伪影：1）促进瞬态而不是稳态测量，从而最大限度地减少传输伪影的形成，以及2.）允许完整表征转运分子的空间浓度分布，允许精确拟合转运模型。这种方法是基于通过旋转圆盘共聚焦显微镜（SDCM）观察分子转运到巨大的单层脂质囊泡（GUV）中。SDCM允许在任何时刻对GUV膜两侧的浓度分布进行快速成像。反过来，这将使研究人员能够建立浓度分布演变的时间过程。早期实验结果的简单建模表明，膜渗透率可以很容易地确定从瞬态浓度分布数据。这种技术可以回答一系列重要的研究问题：1。SDCM在测量被动输运方面的局限性是什么？2.什么样的分子特性控制着被动转运。 3.膜成分和电荷状态如何调节被动转运。该项目的目标是开发和完善一种新技术，用于测量药物和毒素进入细胞的难易程度。这项新技术将通过为研究人员提供药物和毒素分子穿过细胞膜的实时图像来改进现有技术。详细的图片的过程中的细胞条目将是一个有价值的工具，设计有效的下一代drugs.Intellectual MeritThe拟议的工作开发新的工程工具，以解决中央的缺点，目前使用的技术来测量分子的被动运输跨脂质双层膜。大多数现有的工具，用于观察被动运输只能访问批量浓度在稳定状态下，导致非常有限的测量工件。这里开发的工具将允许瞬态观察的全部浓度分布，这将允许精确测量的参数，支配被动transport.These精确的测量将提供一个基础，定量研究分子结构和膜通透性之间的关系。将研究分子结构的两个方面：渗透双层的分子结构和构成双层本身的脂质结构。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长、电荷状态、氢键能力和膜相状态来研究。发展分子结构和被动运输之间的定量关系将促进对这一过程的机械洞察。这种认识是发展药物分子如何进入细胞的综合理论的关键。更广泛的影响穿过细胞膜的被动运输对药物在体内的行为至关重要。能够穿过细胞膜而不激活细胞机器的药物具有高口服生物利用度。对被动转运的彻底机械理解对于理解小分子如何与人体相互作用至关重要，这是一个涉及从药物开发到环境毒理学的基本问题。该研究项目与综合教育和推广计划相结合，重点关注五个领域：本科生研究，本科生实验室模块开发，研究生课程开发，高中生推广和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联合学区（LAUSD）的不同人口的学生密集的研究经验。这个推广计划的主要更广泛的影响将是提供独特的实验室为基础的体验式教育机会，以LAUSD人口，其中包含较大比例的弱势学生和少数群体，在科学和工程中代表性不足。研究生水平的推广计划包括参加西班牙裔专业工程师年会研究生院的研讨会。

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.