Study Protein-Nanomaterial Interactions and Their Impacts on Protein Activities

描述（由申请人提供）：由于纳米材料与生物物质的尺寸匹配，其大的比表面积及其无机核心材料，纳米材料与生物系统的相互作用不同于小分子或生物聚合物，我们以前了解的对象。随着人类越来越多地接触到以环境污染物或纳米医学形式存在的工程纳米材料（ENM），我们迫切需要更好地了解ENM在生物系统中的行为。 一旦ENM进入人体，它们就会遇到大量的蛋白质，并在其表面形成所谓的蛋白质“电晕”。ENM的生物学效应可以通过这种蛋白质“冠”来实现。例如，蛋白质冠可以携带生物系统可识别的特殊信号，从而控制ENM的行为，例如从循环中清除，渗透到膜屏障，运输到居住地，触发防御系统等。另一方面，吸附在ENM表面的蛋白质可以钝化其高表面活性，并防止产生自由基，这是纳米毒性的主要来源，但以结合蛋白质可能的构象和活性改变为代价。通过假设蛋白质-纳米材料相互作用与ENM在生物系统中的行为之间的直接联系，我们建议分析蛋白质冠的形成，整理其对ENM物理性质的依赖性，并开始探索结合ENM对蛋白质的潜在影响。我们已经开发了有效的相互作用研究工具，包括用于分离高亲和力蛋白质的流场流动分级和用于结合亲和力测量的毛细管电泳。利用这些工具，我们将筛选人血浆或细胞裂解物中对所选ENM（特异性目标1）具有高亲和力的蛋白质。这项研究将揭示ENM进入我们的身体或入侵细胞后冠层中存在的蛋白质。这些蛋白质在调控ENM在生物宿主中的行为和宿主对ENM进入的反应中发挥重要作用。同时，由于这些蛋白质的紧密接触，它们将成为ENM最可能作用的靶点。其次，我们将详细研究一组代表性蛋白质与所选纳米材料之间的相互作用（具体目标2），旨在更好地了解相互作用对ENM特性的一般依赖性，例如核心材料，尺寸，形状和表面功能化。最后，我们将从酶活性的改变方面评估蛋白质-纳米材料相互作用的结果（具体目标3）。 我们的研究将导致更好地了解纳米材料的生物效应和相互作用对ENM特性的一般依赖性，这可以指导纳米药物的设计，并确保纳米材料的安全实施。 公共卫生相关性：我们提出实验来验证我们的假设，即纳米材料的生物学行为可能是通过与血浆和细胞蛋白质的强烈相互作用来进行的。这种相互作用可以改变蛋白质构象，从而改变蛋白质功能。这项研究的结果可能会揭示纳米材料在生物宿主中的行为，纳米医学设计和纳米材料暴露控制所需的知识。

DESCRIPTION (provided by applicant): Due to the matching sizes of nanomaterials with biological substances, their large specific surface areas, and their inorganic core materials, nanomaterials interact with biological systems differently than small molecules or biopolymers, objects we have previous knowledge of. With the increasing exposure of human beings to engineered nanomaterials (ENM) in the form of environmental pollutant or nanomedicine, it is greatly demanded that we obtain better understanding of the behavior of ENM inside the biological systems. Once the ENM enters the human body, they encounter enormous amounts of proteins and form the so-called protein "corona" on their surface. The biological effects of ENM could then be carried out by this protein "corona". For example, the protein corona could bear special signals recognizable by biological systems and thus govern the behaviors of ENM, such as clearance from circulation, penetration to membranous barriers, transportation to residential sites, trigger of defense system, etc. On the other hand, proteins adsorbed onto ENM surface may passivate their high surface activity and prevent generation of free radicals, the main source of nanotoxicity, but at the expense of possible conformational and activity alteration to the bound proteins. By hypothesizing the direct link between protein-nanomaterial interaction and the behavior of ENM in biological systems, we propose to analyze the formation of protein corona, sort out its dependence on the physical properties of ENM, and initiate the exploration of potential effects of binding to ENM on proteins. We have developed effective tools for the interaction study, including flow-field flow fractionation for isolation of proteins of high affinity and capillary electrophoresis for binding affinity measurement. With these tools, we will screen for proteins in human plasma or cell lysate having high affinity to the selected ENM (Specific Aim 1). This study will reveal proteins present in the corona after ENM entering our body or invading cells. These proteins should play important roles in governing the ENM behaviors in their biological hosts and the host response to ENM entry. In the meanwhile, these proteins will be the most possible targets being affected by ENM because of the close contact. Secondly, we will study the interactions between a representative group of proteins and the selected nanomaterials in details (Specific Aim 2), aiming to obtain better understanding on the general dependence of interaction to the ENM properties, such as core materials, size, shape, and surface functionalization. Last, we will assess outcomes of protein-nanomaterial interaction from the aspect of alteration of enzyme activities (Specific Aim 3). Our study will lead to better understanding of the biological effects of nanomaterials and the general dependence of interaction on the ENM properties, which can guide the design of nanomedicines as well as ensure safe implementation of nanomaterials. PUBLIC HEALTH RELEVANCE: We propose experiments to test our hypothesis that the biological behavior of nanomaterials may be carried out via strong interactions with plasma and cellular proteins. Such interactions could alter the protein conformation and thus protein function. The outcome of the propose research may shed light on how the nanomaterials behave inside the biological host, knowledge required by nanomedicine design and nanomaterials exposure control.