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To date, protein adsorption is an unavoidable response to implanted biomaterials. When proteins interact with materials, adverse biological events such as thrombus formation and inflammation can occur and challenge device efficacy. Protein adsorption is influenced by various material and surface properties which can be modified in efforts to alter the protein-material interactions and the subsequent cellular response. There is a need for simple modifications of commonly used biomaterials and the effect of these modifications on (1) material properties (2) proteins and (3) cells is important to study. In this work, the effect of modifying polydimethylsiloxane (PDMS) and its interactions with fetuin-A are studied for potential immunomodulatory properties. PDMS modifications are achieved by altering the ratio of PDMS formulations to simply and effectively control elastic modulus, and by coating PDMS with polydopamine (PDA), a molecule commonly used as a bioglue. Surface characterization confirmed that altering the PDMS formulation changed the elastic modulus without affecting surface wetting properties. Minor changes in surface roughness via atomic force microscopy and surface chemistry via x-ray photoelectron spectroscopy were detected on some samples, and the deposition of PDA was confirmed. Protein adsorption studies provided quantitative and qualitative data on fetuin-A interactions. It was determined that fetuin-A adsorption was influenced by the PDMS formulations, and that the preferential adsorption changed when adsorbed from a competitive environment. Following modification of samples with adsorbed fetuin-A, the inflammatory effects of fetuin-A were investigated by measuring the concentration of pro- and anti-inflammatory cytokines in response to modified and unmodified samples. Data suggest that elastic modulus influences cytokine secretion at certain timepoints, a result of varied protein adsorption amounts and orientations in response to material stiffness. The addition of a PDA layer demonstrated the potentially cytokine mitigating effect of PDA cell interactions and protein immobilization when compared to unmodified PDMS samples. / Thesis / Master of Applied Science (MASc)

Gold nanoparticles have received significant interest recently due to their utility in biomedical imaging and therapy. Nanoparticles which exhibit intense extinction in the near infrared (NIR) region, where blood and tissue absorb light weakly, are of great interest as contrast agents for biomedical imaging applications. While strong NIR extinction often requires sizes greater than ~20-30 nm, effective clearance from the body to avoid toxic accumulation necessitates sizes below ~6 nm. Moreover, effective clearance depends upon lack of adsorption of serum proteins in the bloodstream onto the particles. Herein, this conflict is addressed by assembling sub-5 nm gold nanoparticles into clusters with controlled size and morphology, in order to provide intense NIR extinction. Furthermore, the surfaces of the primary gold nanoparticles are designed such that the particles avoid the adsorption of any serum proteins. Binary ligand monolayers of anionic citrate and appropriate amounts of either cationic lysine or zwitterionic cysteine are synthesized to completely prevent serum protein adsorption from undiluted fetal bovine serum. A mechanism is proposed whereby the zwitterionic tips which are present on both the lysine and cysteine ligands limit the interactions between serum proteins and the "buried" charges on the nanoparticle surfaces. These primary nanoparticles are subsequently assembled into biodegradable nanoclusters via "quenched assembly", wherein nanoclusters are assembled and subsequently quenched by the adsorption of a biodegradable polymer on the cluster surface. The sizes of completely reversible "quenched equilibrium" nanoclusters formed from gold nanoparticles coated with a mixture of lysine and citrate are tuned from 20 nm to 40 nm, and nanocluster size is semi-quantitatively predicted by a free-energy model. Additional control over nanocluster size and extinction is demonstrated by adding NaCl, which is shown to decrease the polymer adsorption on the clusters and thus decrease polymer bridging interactions. This nanocluster formation platform is extended to nanospheres capped with citrate and the thiolated, zwitterionic cysteine ligand. A general paradigm is presented whereby the sizes and optical properties of biodegradable gold nanoclusters formed from nanospheres which do not adsorb any serum proteins are tuned via control over van der Waals, electrostatic, depletion, and polymer bridging colloidal interactions. / text

Investigations of specific and nonspecific interactions of biomolecules at liquid/solid interfaces are presented. To investigate specific multivalent ligand-receptor interactions, bivalent antibodies and haptens bound to solid supported membrane were used as models for ligand-receptor coupling. Novel microfabrication strategies, which included spatially addressed bilayer arrays and heterogeneous microfluidic assays, in conjunction with total internal reflection microscopy, was employed to achieve this goal. These high throughput techniques allow thermodynamic data of binding interactions to be acquired with only a few microliters of analyte and superior signal to noise. The results yield both the first and second dissociation constant for bivalent IgG antibodies with membrane bound hapten molecules. Studies were conducted both as a function of hapten density and cholesterol content in the membrane. Another research area of this dissertation is the molecular level description of nonspecific adsorption and displacement of the model protein, fibrinogen, onto hydrophilic surfaces. Techniques such as atomic force microscopy, immunochemical assays, fluorescence microscopy, and vibrational sum frequency spectroscopy were employed to probe this system. The results demonstrate that the protein's αC domains play the critical role. When fibrinogen is adsorbed to a hydrophilic surface via these moieties, its displacement rate in the presence of human plasma is approximately 170 times faster than when these domains are not in direct surface contact. Even more significantly, spectroscopic studies show evidence for highly aligned Arg and Lys residues interacting with the negatively charged substrate only when the αC domains make direct surface contact. The interfacial ordering of these residues appears to be the hallmark of a weak and labile electrostatic attraction between the substrate and the adsorbed macromolecule.

In order to measure the thickness of a protein layer on a structured surface of silicon rubber, we have used ellipsometry and Fourier transform infrared (FTIR)-spectroscopy. The aim was to determine whether this type of measurement method can be used on protein layers or not. By hot-embossing a specific pattern of micrometre-sized pillars was created on the surface of the silicon rubber, which then was exposed to a phosphate buffer solution (PBS) containing human serum albumin (HSA) protein. FTIR measurements confirmed that proteins had attached to the surface. Ellipsometric studies were made and even though the protein layer was too thin to be measured, a simulation was made that revealed that a protein layer needs to be at least 1,5 nm to be measured properly with this method. We can also see that the protein molecules can get out of the solution, to find their way into the small pits of the samples.

<p> An experimental method for monitoring the adsorption of proteins in real time has been developed and is the topic of this thesis.</p> <p> Proteins were radioactively labeled using 125I and injected at a constant flow rate into a well stirred glass cell containing buffer and the adsorbing surface in particle form. The buffer contained in the cell was gradually displaced by the protein solution. NaI(TI) detectors, coupled to a multichannel analyzer and high voltage power supply, were placed at the exit of the cell to monitor the radioactivity of the bulk solution. Adsorption of the protein was determined by depletion of protein in the bulk solution.</p> <p> A process to treat the surface of the cell and the glass tubing so as to prevent adsorption was developed and implemented prior to the flow experiments. This procedure involved exposing the cell and glass tubing to a 1 mg/mL fibrinogen solution for two hours at room temperature. The cell and tubing were then placed in an oven for 10 minutes at 70° C. This procedure (referred to as the thermal treatment procedure) resulted in a non-adsorbing protein layer that appeared, from extensive experiments, to be irreversibly attached to the surface of the cell and tubing.</p> <p> The adsorption of fibrinogen from a single component system, as well as from plasma, was studied using the experimental arrangement described above. Concentrations of fibrinogen in isotonic Tris, for the single component system, ranged from 40 mg/mL to 300 mg/mL. It was found from these experiments that an initial, fairly rapid, adsorption of protein took place. Later the adsorption of protein slowed significantly.</p> <p> The adsorption and subsequent desorption of fibrinogen from diluted plasma was also studied. Dilutions ranged from 1% to 20%. The results from these experiments are consistent with the model proposed by Vroman for plasma surface interactions.</p> / Thesis / Master of Engineering (MEngr)

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