Diffusing Colloidal Probes of Biospecific Interactions and Biological Interfaces

[摘要] Weak, biospecific and non-specific macromolecular interactions play a major role in fundamental biological process such as cell adhesion, motility, and signaling and in medicine, macromolecules have been integrated into materials for drug delivery and tissue engineering applications. Direct measurements capable of sensitively detecting weak, specific and non-specific macromolecular interactions at interfaces are needed to better understand their function in biological systems and utility in biomaterials.In this dissertation, diffusing colloidal probe microscopy (DCPM) was used to make direct, quantitative measurements of colloidal interactions mediated by weak, specific protein-carbohydrate interactions and interactions with the surface of live cells mediated by specific and non-specific macromolecular interactions. In addition, models were developed capable of predicting colloid and surface interactions mediated by specific biomolecular interactions with direct input of binding affinities for further characterization of experimental results. To study reversible protein-carbohydrate interactions, biospecific adsorption of the sugar-binding protein, Concanavalin A (ConA), to dextran-modified colloidal particles was visualized using fluorescent confocal microscopy and colloidal association dynamics mediated by ConA-dextran interactions were quantified using optical video microscopy. These results demonstrate how aggregation kinetics in colloidal dispersions can be reversibly tuned with competitive ConA-glucose interactions. Direct connections were established between the observed aggregation kinetics and net particle-particle interactions as a function of ConA and glucose concentration. To model this system, Monte Carlo simulations were developed to model bulk and interfacial biomacromolecular binding. With direct input of dissociation constants, particle-particle interactions were predicted for a range of binding affinities and protein concentrations. Dark field video microscopy was used to study interactions between live cells and colloidal particles functionalized with natural and synthetic macromolecules all with relevance to bioengineering. With combined real-time particle tracking and cell boundary determination, particle trajectories can be monitored in relation to their distance from cell surfaces. Particle-cell surface binding lifetimes and potentials of mean force were measured for colloids functionalized with polyethylene glycol (PEG), bovine serum albumin (BSA), dextran, and hyaluronic acid (HA). With the modeling tools developed for colloidal interactions mediated by biospecific binding, the interactions of targeted drug delivery nanoparticles with cell surfaces were characterized for target membrane proteins with varying binding affinities and expression levels. The findings from this work provide a basis for in-depth characterization of biomolecular interactions and biophysical properties of cell surfaces with a combination of experimental and modeling techniques.

[发布机构] Johns Hopkins University

[效力级别] proteins

[关键词] Colloids;proteins;carbohydrates;cells;specific binding;Chemical & Biomolecular Engineering