Collaborative Research: Real time Chemical Imaging of Nanoparticle Templated Tubulin-Polymerization

纳米粒子可以设计成各种大小、形状和功能。该项目将研究纳米颗粒与特定细胞蛋白质的相互作用，这些蛋白质对生命过程非常重要，并将为癌症等疾病开发有效的治疗方法。PI将专注于微管，这是一种中空蛋白质圆柱体，赋予细胞结构刚性，使其具有运动性，并作为细胞内运输的管道。这些材料的聚合和解聚能力影响细胞过程，几十年来一直是广泛研究的主题。了解如何控制这些蛋白质结构的破坏可以降低细胞结构的刚性，并成为癌症治疗的目标。纳米颗粒介导的抑制微管蛋白聚合的报道已有简要报道，然而，普遍的机制仍缺乏了解。这些知识可以进一步加深科学理解，为设计新的疗法铺平道路。在这个项目中，PI将从生化和结构的角度研究纳米颗粒对微管(和其他细丝)聚合的影响。为了识别和定量评估聚合过程中纳米颗粒与微管蛋白的化学结合，PI将利用红外光谱、机器学习工具和其他生化技术。在分析能力方面，测量设备、成像和数据分析的综合工具包将是研究分子与环境相互作用的宝贵资源，这些相互作用可能扩展到其他生物系统。这一知识可能会对理解基于纳米颗粒的新型癌症疗法产生广泛的影响。在教育和外展活动方面，合作的私人投资机构将为大一新生(理科和非理科专业的学生)开发一个探索性课程，通过在小班中加强与教师的互动来加强他们的教育，并学习纳米技术的研究。该团队将通过大学范围的项目，为早期职业生涯和代表性不足的学生的教育做出具体贡献。在这个项目中产生的算法和数据将被用作从高中到专业水平的教育活动的基础。该项目技术方法的核心是认识到，通过应用分子光谱和成像技术，完全了解纳米颗粒对微管聚合的影响是可能的。通过这一项目，PIS将开发用于微管蛋白聚合研究的最佳方法，研究具有各种化学和表面特性的纳米材料。PI将利用红外光谱的优势，利用成像技术的最新进展，开发一种空间和时间分辨的方法，揭示微管动力学的分子细节，并定量评估纳米颗粒在微管聚合中的作用。该项目包括与红外透明、经济高效的微流控平台相结合的新兴红外测量技术，该平台可以在空间和时间上精确控制生化环境。利用这一平台，PI将研究微管与不同组成、浓度、大小和表面功能的纳米颗粒结合时的生化、动力学和结构表现。聚合程度将使用荧光分析进行分析。凝胶电泳研究将用于评估聚合程度，磷酸盐释放途径将被分析以获得可能的机理洞察。纳米粒子的微流体辅助红外成像将被用来研究调制的微管形成，以揭示完全不同的二级结构阵列的表现，这可能是由于在聚合过程中选定的单体单元粘合在一起的倾向引起的。纳米颗粒与聚合和干燥的微管孵育的红外图像将揭示微管与聚集蛋白质的纳米颗粒光谱特征。主要的技术贡献是一种高通量红外成像方法，用于研究微流控连续流动混合装置中纳米颗粒不存在和存在时的蛋白质聚合，并验证结果，以便于了解纳米颗粒与蛋白质的相互作用。所获得的数据将进一步用统计和机器学习方法进行调查。PI将致力于确保所有级别的学生都可以使用这里开发的方案，使他们有机会利用纳米技术了解微管蛋白动力学的基本原理，并激发他们开发新癌症疗法的创造力。此外，学生将参与专门的项目，了解这些领域未来的职业机会。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Nanoparticles can be designed in a variety of sizes, shapes, and functionalities. This project will research the interaction of nanoparticles with specific cellular proteins that are important for life processes and to develop effective therapies for diseases such as cancer. The PIs will focus on microtubules, which are hollow protein cylinders that impart structural rigidity to cells, enable motility, and serve as conduits for intracellular transport. The ability of these materials to polymerize and depolymerize affects cellular processes and has been the subject of extensive research interest over decades. Understanding how to control the disruption of these protein structures can reduce cellular structural rigidity and serve as a target for cancer therapy. Nanoparticle-mediated inhibition of tubulin polymerization has been reported briefly; however, a general mechanistic understanding is still lacking. Such knowledge can further scientific understanding and pave the way to designing novel therapies. In this project the PIs will study the influence of nanoparticles on the polymerization of microtubules (and other filaments) both from a biochemical and structural perspective. To identify and quantitatively evaluate the chemical association of the nanoparticles with tubulin during polymerization, the PIs will utilize infrared spectroscopy, machine learning tools and other biochemical techniques. In terms of analytic ability, the integrated toolkit of measurement devices, imaging, and data analysis will be a valuable resource to investigate molecular-environmental interactions that may be expanded to other biological systems. This knowledge could have widespread implications for the comprehension of nanoparticle-based novel cancer therapeutics. For educational and outreach activities, the collaborating PIs will develop an exploratory program course intended for freshman (science and non-science majors) to enhance their education through greater interaction with faculty in small classes and to learn about research in nanotechnology. The team will specifically contribute to the education of early career and underrepresented students via university-wide programs. The algorithms and data generated during this project will be used as a basis for educational activities from the high school to professional levels. At the core of this project's technical approach is the recognition that a complete understanding of the influence of nanoparticles on microtubules polymerization is possible by applying molecular spectroscopy and imaging. Through this project the PIs will develop best methods to use for tubulin polymerization studies with nanomaterials having various chemical and surface properties. The PIs will harness the advantages of infrared spectroscopy by using recent advances in imaging technology to develop a spatially and temporally resolved approach that illuminates molecular details of microtubules dynamics and quantitatively evaluates the role of nanoparticles in tubulin polymerization. The project includes emerging infrared measurement technology coupled to an infrared transparent, cost-effective microfluidic platform that accurately controls biochemical environments spatiotemporally. Using this platform, the PIs will investigate the biochemistry, kinetics, and structural manifest of microtubules upon association with nanoparticles of varying composition, concentration, size, and surface functionalities. The extent of polymerization will be analyzed using a fluorescence assay. Gel electrophoresis studies will be used to assess the extent of polymerization and the phosphate release pathway will be analyzed to obtain possible mechanistic insight. Microfluidics-assisted infrared imaging of the nanoparticles will be employed to study the modulated microtubules formation to reveal manifestation of a completely different array of secondary structures that may arise from the propensity of select monomer units to adhere together during polymerization. Infrared images of the nanoparticles incubated with polymerized and dried microtubules will reveal nanoparticle spectral signatures of microtubules with aggregated proteins. The main technical contribution is a high-throughput IR imaging method to study protein polymerization in the absence and presence of nanoparticles in the microfluidic continuous flow mixing device and validate the results to facilitate understanding of the nanoparticle-protein interaction. The obtained data will further be investigated with statistical and machine learning approaches. The PIs will work to ensure that the protocols developed here are available to students of all levels, giving them an opportunity to understand the fundamentals of tubulin dynamics with nanotechnology and stimulating their creativity to develop new cancer therapeutics. In addition, the students will participate in dedicated projects, learning about future career opportunities in these fields.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.