Protein-Biomaterial Interactions
It has been suggested that it is possible to influence the cellular response to implanted biomaterials, especially at early interaction times, by controlling the orientation, conformation, and composition of the initial layer of proteins that adsorb to the material upon implantation. One portion of my research has focused on developing three mechanisms for controlling protein orientation and conformation for applications in biomaterials and biosensors. These three mechanisms include specific protein-protein interaction driven orientation, specific protein-substrate interaction driven orientation, and charge-driven protein orientation.
The first mechanism, specific protein-protein interaction driven orientation, was probed by comparing the cellular adhesion properties of two bone proteins, bone osteopontin (OPN) and bone sialoprotein (BSP) when they were specifically bound to collagen type I or randomly adsorbed to control substrates. During natural bone formation cells first lay down a collagenous matrix composed of primarily type 1 collagen. After this collagen network is formed, non-collagenous proteins bind to the collagen matrix and then the matrix is mineralized to form mature bone. Both OPN and BSP have been implicated in this process and are known to have specific binding interactions with collagen. All of the substrates were prepared with identical amounts of adsorbed OPN or BSP as determined by radiolabeled adsorption isotherms. This allows for a direct comparison of the cell binding ability of these proteins on all of the surfaces examined. In this study it was determined that OPN has a favorable orientation imparted to it for cell binding when it is specifically bound to collagen I. At the same time, it was found that the cellular adhesion properties of BSP are a direct result of the conformational flexibility of the protein. When directly compared to each other, it was determined that OPN is more important than BSP for cellular adhesion to collagen.
The second mechanism for controlling protein orientation is that of specific protein-substrate interaction driven orientation. In this study OPN and BSP were specifically bound to hydroxyapatite (HAP), a mineral that mimics the main component of bone. The results of this work indicate that BSP has a more favorable orientation or conformation than OPN for cellular adhesion to HAP. However, this positive effect on cell binding is eliminated when the surface roughness of the underlying HAP substrate becomes too great.
The final mechanism is charge-driven protein orientation. This mechanism has been used to control the orientation of antibodies for biosensor applications and osteopontin for cell adhesion by others. I utilized this mechanism to compare the roles that both orientation and conformation have on the cellular adhesion properties of adsorbed vitronectin (VN), a naturally labile protein. The orientation of VN was controlled with charged self-assembled monolayers and the conformation was varied with changes in pH. In this study it was found that conformation plays a greater role than orientation in the cell binding properties of VN. Additionally, the maximum cell binding was obtained under conditions with an acidic pH as typical of that found in a wound healing environment.
Through these fundamental studies I have been able to elucidate an understanding of when orientation or conformation will dominate the bioactivity of a protein. There is a trade-off between the conformational flexibility of the protein structure in the area surrounding the bioactive site of interest and the location of the bioactive site relative to the site of orientation modulation. At the same time, it has been shown that it is possible to control the orientation and conformation of adsorbed proteins using these three mechanisms for biomaterials applications.
Super-low Fouling Surface Coatings
The control of non-specific protein adsorption is useful throughout a number of industries including biomedical materials, biosensors, and ship coating applications. Recent advances in this field have suggested that surface coatings developed from a mixture of charged components that maintain an overall neutral surface charge, greatly reduce the non-specific protein adsorption. My recent research efforts have focused on using atom transfer radical polymerization (ATRP) to graft zwitterionic and mixed charge monomers to surfaces to test their non-fouling properties.
The main focus of this research has been to develop and test a novel class of polymeric brushes formed by ATRP from mixed solutions of monomers with various charged groups. The polymer brushes were characterized by atomic force microscopy (AFM) and electron spectroscopy for chemical analysis (ESCA) and the protein adsorption to these surfaces was measured using a surface plasmon resonance biosensor. In this work it was determined that in order to achieve super-low fouling surfaces, the monomers must be well mixed on the surface in equal ratios. Additionally, the reaction conditions must be closely monitored in order to achieve this desired surface ratio. Further work is on-going to determine the importance of the polymer brush thickness on its non-fouling properties.
Research Directions
One portion of my proposed plan of research is centered on the development of biomimetic materials for tissue engineering applications. In order to develop tissue engineering scaffolds or biomaterial coatings that closely resemble native tissue, an understanding of the role that specific proteins play in specific tissues must be developed. One way to develop this understanding is to break down native tissues into their components and characterize the interactions of specific proteins with each component at the molecular level. This detailed functional information can be combined with recombinant protein technology and nanoscale processing techniques to develop 3-dimensional biomimetic implants with molecular level control. It will result in scaffolds which have superior properties to traditional scaffolds because they closely mimic the native tissue they are replacing.
In my future research, I would also like to work on the development of an immobilized, bioactive cell-ligand substrate for use in an SPR sensor. The goal of this work would be to immobilize cancer cell ligands to a sensor platform in a way that allows them to maintain their native biological properties. This substrate would rapidly advance the development of specific ligand targeting drugs by providing an array-based diagnostics sensor for the analysis of novel ligand-targeting drug delivery vehicles in a rapid and quantitative method. Healthy, normal cell ligands would also be included in the array to analyze the targeting specificity of the drug delivery vehicles to cancer cells versus normal cells.