Collaborative Research: Effects and transport of exopolymeric substances (EPS) on engineered nanoparticle (EN) into marine phytoplankton cells

工程纳米颗粒（ENs）的尺寸从1纳米到100纳米不等，其性能与具有相同化学成分的散装材料不同，被用于制造许多消费品，包括印刷涂料、洗涤剂、杀菌剂、涂料、化妆品、防晒霜、轮胎、计算机电子产品和药物输送。尽管这些材料的使用日益增长和广泛，但它们对人类健康和海洋生态系统健康的影响在很大程度上是未知的。ENs包括碳纳米管、含金属粒子、零价金属产品、树状大分子和量子点（QDs）。量子点不仅在生物成像、疾病诊断和治疗等方面有广泛的应用，而且其广泛的激发光谱、可调谐的发射波长和相当稳定的荧光也使其适合于机制研究。虽然对ENs的主要关注是其潜在的毒性（例如，产生活性氧的潜力），但拟议的研究将主要集中在海洋浮游植物细胞的运输步骤和机制上，这可能受到静电和疏水相互作用的影响。例如，ENs的相对亲水性是控制其到达沿海水域的能力以及生物利用度和生物吸收的关键因素之一。由于黄腐酸和外聚物（EPS）等陆生和水生天然有机物的乳化和表面活性剂特性，可以预期到达地表水的ENs更亲水。由海洋中的浮游植物和细菌分泌的EPS是富含多糖的阴离子胶体聚合物，在海洋凝胶、海洋雪和生物膜的形成，以及胶体和微量元素的清除和防止病毒感染方面发挥重要作用。同样的EPS特性也会影响生物体对EN的吸收和积累。基于对动物细胞有限的研究，ENs可以通过内吞作用（被摄取的物质逐渐被质膜包围，最终分离形成内吞囊泡）或其他与载体无关的过程，如表面粘附和被动扩散，进入细胞。在本研究中，将研究EPS对水生系统中EN稳定性的影响以及EN在海洋浮游植物膜中的转运机制。本文拟对以下假设进行验证：H1: EPS可显著影响水生环境中QD的稳定性，EPS可改变QD的附着和/或进入细胞的途径，其中EPS的相对疏水性起主要作用。H2：量子点不仅可以吸附在细胞表面，还可以内化和浓缩在某些藻类细胞器中；这反过来将决定它们对浮游植物的最终影响（是消除还是减轻潜在的损害）。H3: QDs可以通过胞吞作用进入没有明显细胞壁的浮游植物，而非囊泡相关过程可能是有细胞壁的浮游植物的主要进入机制。或者量子点不一定会进入细胞，但会刺激非特异性膜受体。H4：细胞内Ca2+信号在多种细胞反应的刺激偶联激活中起着普遍的关键作用。量子点与浮游植物细胞的潜在相互作用可能触发细胞内Ca2+升高，导致胞吐和/或内吞作用。为了验证这四种假设，将首先对五种不同的浮游植物进行筛选，以了解它们产生EPS和在细胞内摄取ENs的能力。在初步筛选后，将从5个物种中选择2个物种进行与量子点相互作用的机制研究，这将是选择的EN。EN的亚细胞分布以及EN和EPS通过Ca2+介导的分泌过程排出浮游植物细胞也将被检查。本研究结果不仅有助于我们了解ENs在水生系统中的行为和毒性，浮游植物的毒性反应，以及EPS的相对疏水性/亲水性在改变ENs稳定性和命运中的重要性，还将为制定ENs法规提供知识基础，并将加强TAMUG和UC Merced的博士后，研究生和本科生培训计划。它还将有助于提高科学、学术和地方社区对与这种发展中的技术有关的环境风险的认识。

0933137/0932404 Santschi/ Chin Engineered Nanoparticles (ENs), which range in size from 1 to 100 nm and have properties that are different from those of bulk materials of the same chemical composition, are used in the fabrication of numerous consumer goods, including printing paints, detergents, bactericides, coatings, cosmetics, sunscreens, tires, computer electronics, and drug delivery. In spite of their growing and widespread use, the impacts of these materials on human health and the health of ocean ecosystems are largely unknown. ENs include carbon nanotubes, metal containing particles, zero-valent metal products, dendrimers, and quantum dots (QDs). The proposed research will utilize QDs, as they not only have many applications in biological imaging, disease diagnostics, and therapeutics, but their broad excitation spectrum, tunable emission wavelength and quite stable fluorescence also make them suitable for mechanistic studies. While the major concern with ENs is in terms of their potential toxicity (e.g., the potential for producing reactive oxygen species), the proposed research will mainly focus on the transport steps and mechanisms into marine phytoplankton cells, which can be affected by both electrostatic and hydrophobic interactions. For example, the relative hydrophilicity of ENs is one of the key factors controlling their ability to reach coastal waters, as well as their bioavailability and biological uptake. Because of the emulsification and surfactant qualities of terrestrial and aquatic natural organic substances such as fulvic acids and exopolymeric substances (EPS), it can be expected that ENs that reach surface waters are more hydrophilic. EPS, secreted from phytoplankton and bacteria in the ocean are polysaccharide-rich anionic colloidal polymers that are important in the formation of marine gels, marine snow and biofilms, as well as in colloid and trace element scavenging and in providing protection against virus infection. The same EPS characteristics will also affect EN uptake and accumulation in organisms. Based on the limited research on animal cells, ENs can enter cells either by endocytosis (material to be ingested is progressively enclosed by the plasma membrane, which eventually detaches to form an endocytic vesicle) or by other non-vehicle related processes such as surface adhesion and passive diffusion. In this research, the effects of EPS on EN stability in aquatic systems and the mechanisms of EN transport across the marine phytoplankton membranes will be examined. The following hypothesis will be tested in the proposed research: H1: QD stability in the aquatic environment can be significantly influenced by EPS, which can alter the attachment and/or entry pathways of QDs into cells, with the relative the hydrophobicity of EPS playing a major role. H2: QDs may not only be adsorbed onto the cell surface but may also be internalized and concentrated in certain algal organelles; this in turn will determine their final effects (either elimination or amelioration of potential damages) on phytoplankton. H3: QDs may enter phytoplankton without an obvious cell wall through endocytosis while non-vesicle related processes may be the main mechanisms of entry in phytoplankton with cell walls. Or QDs may not necessarily enter cells, but stimulate nonspecifically membrane receptors. H4: Intracellular Ca2+ signaling plays a universal critical role in stimulus coupling of activation in a broad variety of cell responses. The potential interactions of QDs with phytoplankton cells could trigger intracellular Ca2+ elevation leading to exocytosis and/or endocytosis. In order to test these four hypotheses, five different phytoplankton species will be screened first for their ability to produce EPS and to uptake ENs intracellularly. After an initial screening, two of the five species will be chosen for further mechanistic research on their interactions with QDs, which will be the EN of choice. EN subcellular distribution as well as the excretion of ENs and EPS out of the phytoplankton cells via a Ca2+-mediated secretion process will also be examined. Besides contributing to our knowledge of ENs' behavior and toxicity in aquatic systems, toxicity responses of phytoplankton, and importance of relative hydrophobicity/hydrophilicity of EPS in changing stability and fate of ENs, the outcome of this research will also contribute to the knowledge base for policy-making of ENs regulations, and will enhance training programs at TAMUG and UC Merced for postdoctoral fellows, graduate and undergraduate students. It will also contribute to increased awareness in the scientific, academic and local communities of the environmental risks associated with this developing technology.