Fundamental Molecular Studies of Complex Fluids for Preservation of Biological Systems

威斯康星大学麦迪逊分校的胡安·J·德·巴勃罗《用于保存生物系统的复杂流体的基础分子研究》一文指出，蛋白质、细胞或组织等不稳定的生物系统通常必须长时间储存。这通常是通过将感兴趣的体系冻结在含有各种添加剂的水溶液中来实现的，其目的是将形成冰晶造成的损害降至最低。在某些情况下，还可以冷冻干燥所产生的配方，从而产生无需冷藏即可储存的产品。在大多数情况下，很大一部分生物分子或细胞在冻融或冷冻干燥过程中无法存活；回收率很低，对最终产品的成本和可用性有很大影响。这些添加剂或“保护剂”的一些例子是由二甲基亚砜(DMSO)和乙二醇提供的，这两种物质都有不良的副作用。在实践中，保护剂配方通常是通过代价高昂的反复试验过程来构思的。而且，虽然关于它们的功能已经出现了一些一般性的想法，但保护剂分子在冷冻和干燥过程中保持活性的确切机制还不是很清楚。在这项工作中，PI建议对各种保护剂分子在溶液和无水保护剂基质中对蛋白质稳定性的作用进行系统和全面的研究。这项工作主要集中在双糖的研究上，近年来，双糖显示出有效保存生物系统的希望。PI还研究了相同的保护剂如何在溶液和干燥基质中稳定脂质双层膜。该项目包括建模和实验部分。计算方面将优先于实验(将不进行X射线结构分析、核磁共振和量热工作)。在模型水平上，PI使用分子模拟来阐明保护剂的作用机制，并确定使某些保护剂分子比其他保护剂分子更有效的主要结构属性。这将需要开发新的模拟方法，以便于研究远离和接近玻璃化转变点的大型复杂分子(水、双糖、电解质和寡肽)的系综。在实验水平上，PI打算表征各种保护剂和模型蛋白质溶液和玻璃的热物理性质，并将这些与保存效果和我们的模拟结果联系起来。等电点可以表征在保护剂和不同水量存在下脂双分子层的平衡热力学性质。这些数据将与模拟结果进行比较，并将被用来证明他和其他人关于细胞保存的观察结果是合理的。这项研究的更广泛的影响是，它将导致对蛋白质和细胞在溶液和保护剂溶质存在下的稳定性的更深入的理解。它还将生成预测模型和实验热力学性质和传输系数数据，用于合理设计和优化保存过程。更重要的是，它有可能导致保护剂分子和配方的改进，从而允许食品、药品和生物医学产品的长期储存。这项研究的潜在教育影响被认为是很高的，因为PI有让学生参与他的研究的记录，包括代表性不足的群体。

Juan J. de Pablo, U of Wisconsin - Madison"Fundamental Molecular Studies of Complex Fluids for Preservation of Biological Systems"Labile biological systems such as proteins, cells, or tissue must often be stored for prolonged periods of time. This is generally achieved by freezing the system of interest in an aqueous solution containing various additives, whose purpose is to minimize the damage that arises from the formation of ice crystals. In some cases, it is also possible to freeze-dry the resulting formulation, thereby resulting in a product that can be stored without a need for refrigeration. In most cases, a large fraction of the biomolecules or cells do not survive the freeze-thawing or freeze-drying processes; the rates of recovery are low and contribute significantly to the cost and availability of the resulting product.Some examples of these additives, or "protectants ", are provided by dimethylsulfoxide (DMSO) and ethylene glycol, both of which have undesirable side effects. In practice, protectant formulations are generally conceived through a costly trial-and-error process. And, while some general ideas have emerged regarding their function, the precise mechanisms by which protectant molecules act to preserve viability during freezing and drying are not well understood.In this work the PI proposes to conduct a systematic and comprehensive study of the role of various protectant molecules on the stability of proteins in solution and in anhydrous, protectant matrices. The work is largely focused on the study of the disaccharides, which in recent years have shown promise for effective preservation of biological systems. The PI also examines how the same protectants act to stabilize lipid bilayer membranes in solution and in dry matrices.The project includes both modeling and experimental components. Computational aspects will take priority over experimental (X-ray structure analysis, NMR and calorimetry work will not be performed). At the modeling level, the PI uses molecular simulations to elucidate the mechanisms by which protectants work, and to identify the main structural attributes that render some protectant molecules more effective than others. This will require that new simulation methods be develop to facilitate the study of large ensembles of complex molecules (water, disaccharides, electrolytes and oligopeptides) both remote and near the glass transition point. At the experimental level, the PI intends to characterize the thermophysical properties of various protectant and model-protein solutions and glasses, and to relate these to preservation efficacy and to the results of our simulations. The PI may characterize the equilibrium thermodynamic properties of lipid bilayers in the presence of protectants and varying amounts of water. These data will be compared to simulation results, and will be used to rationalize his observations and those of others regarding the preservation of cells.The broader impact of the research is that it will lead to a deeper understanding of protein and cell stability in solution and in the presence of protectant solutes. It will also generate predictive models and experimental thermodynamic-property and transport-coefficient data for rational design and optimization of preservation processes. More importantly, it has the potential to result in improved protectant molecules and formulations that will permit long-term storage of food, pharmaceutical, and biomedical products. The potential educational impact of the research is considered high since the PI has a record of involving students, including underrepresented groups, in his research.