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

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.