Interaction of Engineered Nanomaterials with Artificial Cell Membranes

在过去的五年中，人们对工程纳米材料的毒性这一与健康有关的问题越来越感兴趣。细胞有多种途径通过细胞膜摄取分子和颗粒来控制其内环境，包括高选择性的膜蛋白和多肽，以及蛋白质介导的内吞和吞噬作用。基于纳米颗粒（NP）的药物传递和分子成像应用将NP传递到细胞中，通常使用生化功能化来促进特定的信号传导和摄取。构成细胞膜的脂质双分子层被认为是离子和非功能化大分子无法穿透的，然而，流行病学研究表明，非功能化的NPs可以在某些条件下通过被动、无介导的途径穿过或破坏细胞膜，导致急性细胞毒性和细胞死亡。非介导的NP在细胞上的吸附和进入细胞的摄取尚不清楚。最近的研究集中在收集经验流行病学数据（如细胞对NP的吸收，对大鼠或鱼类等生物的毒性）或精确的NP表征（如大小，形状，聚集程度，电荷和表面化学）。然而，几乎不可能从这些测量结果过渡到对非介导的NP摄取进入细胞和双分子层破坏的机制的详细理解。需要评估纳米材料的生物利用度和毒性的定量测量，以便处理纳米技术对人类健康和环境的影响。知识价值：本研究的知识价值在于理解工程纳米材料破坏简化模型细胞膜（即脂质双分子层）并通过其进行被动运输的机制和条件。研究人员假设，在某些条件下，工程化的NPs可以被动地跨层转运，并在双分子层中引起纳米级缺陷，这在细胞毒性中起作用。纳米颗粒和脂质双层的相互作用是独特的，因为颗粒和膜具有几乎相同的长度尺度。更广泛的影响：对NP与脂质双层之间相互作用的基本理解具有潜在的变革性，因为它可能：(1)提高我们对工程NP和环境NP毒性的理解；(2)合理设计用于药物传递和生物医学/分子成像的良性NP；(3)产生高通量毒性检测方案；(4)基于证据的纳米材料监管与协议。将开发一个实验平台和方法，用于实时量化NP通过脂质膜的转运，作为NP和脂质性质以及物理化学环境的函数。将采用“自下而上”的方法，通过结合膜蛋白和糖脂形成人工糖萼来增加双分子层的复杂性。工程纳米颗粒在很大程度上不受监管，因为NP的运输、命运和毒性尚未得到充分评估。提出的研究重点是工程纳米材料与脂质双分子层的相互作用，脂质双分子层可以说是生命与环境之间最重要的界面。该提案解决了NP毒性问题，并对NP在医药、服装、化妆品等领域的生产、分销和应用的监管具有重要意义。作为拟议工作的一个组成部分，该计划旨在通过以下方式提高工程和物理科学研究生对纳米科学和技术的社会和伦理影响的认识：(1)开发一门关于纳米技术的社会和伦理影响的交叉课程；(2)在华盛顿特区组织为期两周的学生研讨会，探讨科学政策和文化。PI还将通过资助代表性不足的本科生研究人员来建立他对本科生研究的坚定承诺。

0932885PosnerOver the past five years, there has been a growing interest in the health-related issue of toxicity of engineered nanomaterials. Cells have various routes for uptake of molecules and particles through their cell membranes to control their internal environment including highly selective membrane proteins and peptides as well as protein mediated endocytosis and phagocytosis. Nano-particle (NP) based drug delivery and molecular imaging applications that deliver NP into cells typically use biochemical functionalization which promote specific signaling and uptake. The lipid bilayers that make up cellular membranes are believed to be impenetrable to ions and unfunctionalized macromolecules, however, epidemiological studies have shown that unfunctionalized NPs can, under some conditions, cross or disrupt the cell membrane through passive, unmediated routes causing acute cellular toxicity and cell death. The unmediated NP adsorption onto and the uptake into cells is poorly understood. Recent research focuses on either collection of empirical epidemiological data (e.g. uptake of NP by cells, toxicity to organisms such as rats or fish) or precise NP characterization (e.g. size, shape, degree of aggregation, charge, and surface chemistry). However, it is almost impossible to transition from these measurements to detailed understanding of the mechanisms responsible for unmediated NP uptake into cells and disruption of the bilayer. Quantitative measures of nanomaterial bioavailability and toxicity need to be assessed so that the impact of nanotechnology on human health and the environment can be addressed. Intellectual Merit: The intellectual merit of the proposed work is to understand the mechanisms and conditions under which engineered nanomaterials can cause disruption of, and passive transport through, simplified model cell membranes, namely lipid bilayers. The,investigators hypothesize that under some conditions engineered NPs can passively translocate across, and cause nanoscale defects in, bilayers which plays a role in cellular toxicity. The interaction of nanoparticles and lipid bilayers are unique because the particle and membranes have nearly the same length scale. Broader impact: Fundamental understanding of the interaction between NP and lipid bilayers is potentially transformative because it may: (1) improve our understanding of toxicity of engineered and environmental NP; (2) enable rational design of benign NP for delivery of drugs and biomedical/molecular imaging; (3) result in high-throughput toxicity testing protocols; and (4) evidence-based regulation and protocols of nanomaterials. An experimental platform and methods will be developed for quantifying the NP transport through lipid membranes in real time as a function of the NP and lipid properties and the physicochemical environment. A "bottom-up" approach will be employed to increase the complexity of the bilayer through incorporation of membrane proteins as well as glycolipids to form an artificial glycocalyx.Engineered nanoparticles are largely unregulated because the transport, fate, and toxicity of NP have not been adequately assessed. The proposed research focuses on the interactions of engineered nanomaterials with lipid bilayers, arguably the most important interface between life and the environment. This proposal addresses NP toxicity and has strong implications on the regulation of NP production, distribution, and application in medicine, clothing, cosmetics, etc. As an integral part of the proposed work, the PI aims to increase engineering and physical science graduate students' awareness of the societal and ethical implications of nano science and technology through: (1) development of a cross-listed graduate level course on the societal and ethical implications of nanotechnology; and (2) organization of a two week student workshop in Washington, DC which examines scientific policy and culture. The PI will also build upon his strong commitment to undergraduate research by funding underrepresented undergraduate researchers.