Papers

AVS2000 Session BI-WeA: Non-fouling Surfaces

Wednesday, October 4, 2000 2:00 PM in Room 202

Wednesday Afternoon

Start	Invited?	Item
2:00 PM		BI-WeA-1 Fundamental Studies of Self-Assembling Monolayers as Model Systems for Biological Interfaces G. Hähner (ETH Zurich Switzerland) The first level of concern of interactions between proteins and synthetic surfaces deals with non-specific adsorption: that is, adsorption reflecting hydrophobic or electrostatic interaction. Chemically modified Scanning Force Microscope (SFM) probes allow it to study such interactions with surfaces separately and hence to mimic the different contributing forces to protein-surface interaction independently. Poly(ethylene glycol) (PEG) has been recognized for a long time for its outstanding protein resistant properties. The force between oligo(ethylene glycol) terminated (self-assembled) monolayers and proteins, however, depends on the conformation of the adsorbed molecules and parameters of the environment. We have studied the interaction between differently functionalized (charged and hydrophobic) SFM probes and oligo(ethylene glycol) terminated surfaces depending on the length of the ethylene glycol part, the cation in the electrolyte solution and the ion concentration of the aqueous environment.
2:40 PM		BI-WeA-3 Protein Adsorption and Cellular Responses to Polysaccharide Coatings S.L. McArthur (CSIRO Molecular Science and CRCERT, Australia); P.G. Hartley (CSIRO Molecular Science, Australia); K.M. McLean, G. Johnson, M.L. Jenkins, H.J. Griesser (CSIRO Molecular Science and CRCERT, Australia) There is increasing evidence that the cellular response to a biomedical implant is determined by the presence of specific proteins at the interface. There are a number of protein/surface interactions that influence such adsorption events. In this study we assess the roles of steric and electrostatic interactions on the protein adsorption characteristics of a range of cell supporting and cell-resistant polysaccharide surfaces. Polysaccharides were grafted onto highly anionic, flat, radio frequency glow discharge (rfgd) coatings with or without secondary graft supporting interlayers. Polysaccharides of differing functionality and charge density (aminodextran, carboxymethyldextran and oxidised dextran) were used. The properties of the resulting surfaces were assessed using XPS, streaming potential and AFM imaging and colloid probe force measurements. The impact of surface properties on protein adsorption was also monitored using XPS, ELISA and Surface-MALDI-MS techniques. The results of these studies indicate that protein adsorption occurs regardless of steric interactions provided an electrostatic attraction exists between the protein and the surface. To illustrate this point, surfaces were engineered which were expected to display a low affinity for cell adhesive proteins. In vitro cell studies on such surfaces showed minimal cell attachment and growth which was in marked contrast to the same polysaccharide surfaces with the proteins covalently attached. In this instance, cell attachment and growth was observed. These studies demonstrate the role of specific protein adsorption in the mediation of cellular responses on polysaccharide surfaces.
3:00 PM		BI-WeA-4 Film Formation of Self-assembled Monolayers of Thiol-modified Polyethylene Glycol on Gold S. Tokumitsu, S. Herrwerth, W. Eck, M. Himmelhaus, M. Grunze (Universität Heidelberg, Germany) The adsorption kinetics of self-assembled monolayer (SAM) of thiol-modified polyethylene glycol (PEG: 2000 dalton) on polycrystalline gold surface in dilute solution has been studied by using ellipsometry, infrared reflection absorption spectroscopy (IRRAS), X-ray photoemission spectroscopy (XPS) and second harmonic generation (SHG). Thickness and structure of the finally obtained layer exhibit strong dependence on the solvent. In-situ SHG monitoring of the headgroup adsorption of PEG from 50 µM DMF solution shows rapid coverage increase up to 44% within the first 10 min of immersion, followed by a slowly proceeding adsorption step. Final coverage is reached after a total immersion time of about 2 hours. On the other hand, ellipsometry reveals that film thickness drastically increases after 1-2 hours of immersion. These results indicate a restructuring of the PEG moiety from an amorphous to a crystalline-like phase at that time. The results for film thickness and structure obtained by in-situ and ex-situ experiments will be discussed in detail and finally a phenomenological model of the adsorption process will be presented.
3:20 PM		BI-WeA-5 Synthesis and Characterization of Functionalized Polymerizable Diacetylene Containing Thiol Monolayers N.R. Holcomb (Agilent Technologies); D.G. Castner (University of Washington); D.W. Grainger (Colorado State University) Self-assembled structures have been used to tailor the surface character of materials: enhance adhesion, lubrication, catalysis, and molecular/cell immobilization and recognition in biotechnology.^1,2 Utilization of polymeric materials in the self-assembling monolayer can increase the thermal and mechanical strength of the films formed from monomeric materials.³ The use of polymeric blocks within self-assembled films has shown that there is added stability toward solvent attack.^4,5 We report the synthesis and characterization of new surface functional diacetylene thiols of the formula: R-(CH2)n-C?C-C?C-(CH2)m-SH where n=m=9, and R=fluoro alkyl, or oligoethylene glycol. These materials were synthesized via multistep schemes featuring an asymmetric Cadiot-Chodkiewicz coupling to create the functional diacetylenes from pairs of alkynes. Self-assembled monolayers of these organic molecules are characterized with polarized FTIR, ellipsometry, contact angle, XPS and TOF-SIMS. Films were formed from diacetylene thiols containing contrasting surface functional groups to provide a triethylene glycol non-biofouling surface⁶ and a low surface energy fluorinated surface.³ ¹ K. M. McClary, D. W. Grainger, Biomaterials, 20, 1999, 2435-2446 ² K. M. McClary, D. W. Grainger J. Biomed. Mater. Res., in press (2000). ³ Ebert, R.; Laschewsky, A.; Ringsdorf, H.; J. Am. Chem. Soc. 1985, 107, 4134 ⁴ Sun, F.; Grainger, D.W.; J. Polym Sci. Polym. Chem. Ed. 1993, 31, 172 ⁵ Sun, F.; Castner, D.G.;Grainger, D.W.; Langmuir. 1993, 9, 3200 ⁶ Pertsin, A. J.; Grunze, M.; Garbuzova, I. A. J. Phys. Chem. B 1998, 102, 4918-4926.
3:40 PM		BI-WeA-6 Modification of Metal Oxide Surfaces for Biosensor and Biomaterial Applications Based on Assembled, Functionalized Poly(L-lysine)-g-poly(ethylene glycol) M. Textor, J. Vörös, R. Hofer, D. Elbert (ETH Zurich, Switzerland) Poly(L-lysine) grafted with poly(ethylene oxide) (PLL-g-PEG) is a polycationic block copolymer that spontaneously assembles as a monolayer at negatively charged metal oxide surfaces such as those formed by titanium oxide, tantalum oxide or niobium oxide. The interaction with the negatively charged surface is shown to be electrostatic through the terminal amine groups of the poly(L-lysine) side chains charged positively at pH below 9. The surfaces have been characterized ex situ using X-ray photoelectron spectrocopy, time-of-flight secondary ion mass spectrometry and reflection-absorption infrared spectroscopy. The planar optical waveguide (grating coupler) technique was used in situ both to monitor in real time the assembly process at the metal oxide waveguide surface, as well as to determine the degree of non-specific adsorption when exposed to serum. The degree of protein resistance was found to depend on the PLL-g-PEG coverage, on the grafting ratio between lysine monomer units and PEG side chains, and on the molecular weight of the PEG used. Using optimized polymer architectures, very low values of serum adsorption could be achieved, typically below the detection limit of our optical waveguide instrument (1 ng/cm2). The surfaces remain protein-resistant in flowing buffer solution at least up to 7 days. Functionalized PLL-g-PEG molecules were synthesized with functional groups such as biotin at the terminal position of the PEG side chains. The functionality of these polymer layers on optical waveguide chips was investigated using a model assay with streptavidin binding, followed by the adsorption of biotinylated recognition units and targeting of proteins such as IgG. This new polymeric interface is shown to have an excellent potential for future applications both in the area of bioaffinity sensor to control specific and non-specific adsorption and for implants such as stents.
4:00 PM		BI-WeA-7 Design and Characterization of Specific Biorecognition Interfaces using Derivatized Poly(L-lysine)-grafted-poly(ethylene glycol) Monolayers L.A. Ruiz-Taylor, T.L. Martin, M. Heidecker, P. Indermuhle, P. Wagner (Zyomyx, Inc.) Control of interfacial events such as specific recognition versus non-specific protein adsorption is a major issue in biotechnological applications. In diagnostic assays or biomaterial devices, non-specific binding events can often be the limiting factor towards higher detection sensitivity or implant integration, respectively. In this study, we report the design of interfacial polymers that have the ability to spontaneously adsorb to negatively charged surfaces under physiological pH and efficiently repel non-specific protein adsorption while providing PEG tethered functional/active sites for specific biomolecule recognition. As a model system, we synthesized biotin-derivatized poly(L-lysine)-grafted-poly(ethylene glycol) copolymers, PLL-g-(PEG)(1-x)(PEG-biotin)x, where x varies from 0.1 to 1. XPS was used to characterize the properties and the organization of the monolayers formed on titanium dioxide. Molecular recognition properties were investigated using radiolabelled streptavidin alone and within complex protein mixtures. We showed that the system allows the specific recognition of streptavidin, that the extent of the recognition is not influenced by the presence of other proteins and that streptavidin-horseradish peroxidase displays enzymatic activity on the modified surfaces. Finally, we used the PLLPEG-biotin copolymer system in conjunction with microfluidic patterning techniques to provide micron-size features with specific protein recognition separated by areas preventing non-specific binding, as shown by AFM and fluorescence microscopy.
4:20 PM		BI-WeA-8 Analysis of Protein Absorption on PEG-covered Silica Surfaces by ATR-FTIR N.A. Alcantar, A. Stacy, J. Au (University of California, Santa Barbara); T.L. Kuhl (University of California, Davis); E.S. Aydil, J.N. Israelachvili (University of California, Santa Barbara) The most desirable characteristic of biomaterials is their capability to reject protein adhesion because non-specific adsorption of proteins to a surface of an artificial material enhances atypical development of cells. Surfaces covered with polyethylene glycol (PEG, OH-(CH₂-CH₂-O)_n-H) have been shown to enhance protein rejection, nonimmunogenecity and nonantigenicity. In order to produce a generic biocompatible surface coating, we developed and analyzed a direct method for grafting PEG onto amorphous water plasma activated silica surfaces or films. In this paper, we investigated the biocompatibility of this PEG coating by measuring its ability to resist protein adsorption with attenuated total internal reflection Fourier transform infrared (ATR-FTIR) spectroscopy. PEG-coated silica surfaces, water plasma treated silica surfaces, and bare silica films were all exposed to several concentration solutions of fibrinogen and human serum albumin (HSA) at 37°C and pH=7.4 (imitating physiological conditions). We measured the protein adhesion to each of these three surfaces. We found that the surface covered with PEG had very little protein adsorption. Conversely, the bare silica surface has relatively high amounts of adsorbed protein. The surface treated with water plasma (but no polymer) adsorbed some proteins falling in between the bare silica and PEG-coated surfaces. PEG-covered silica coatings can be applied to protect diverse materials having different chemistries and shapes.
4:40 PM		BI-WeA-9 Combining Polymer Chemistry and Photolithography to Manipulate Gene Expression and Protein Synthesis K.E. Healy (University of California, Berkeley); J.H. Collier, C.H. Thomas (Northwestern University); C. Sfeir (OHSU); S.L. Golledge, D.G. Castner (University of Washington) Materials that actively regulate the response of mammalian cells are designed to act via a combination of biomolecular recognition processes and device microarchitecture. We have developed methods that incorporate photolithography, organosilane chemistry, photoinitiated polymerization, and peptide chemistry to create surfaces that control the spatial distribution, projected area, and nuclear shape of mammalian cells. Interfacial interpenetrating polymer networks (IPNs) were synthesized by sequential photoinitiated free-radical polymerization of a thin layer of polyacrylamide followed by a secondary photoinitiation step using poly(ethylene glycol)-based monomers to create the network. Characterization of the IPNs by contact angle goniometry, spectroscopic ellipsometry, XPS, and static SIMS has confirmed the formation of an interfacial IPN ~ 20nm thick. These IPNs prevent protein adsorption and cell adhesion and therefore represent an excellent surface to control the spatial distribution of either biological macromolecules, cells, or viruses. In one application, materials with patterned surface chemistry could serve as templates for the organization of tissue structure surrounding medical devices, which would theoretically influence their biocompatibility. To address this hypothesis, the nuclear shape of mammalian cells was controlled on microfabricated substrata with reigospecific chemistry. Protein synthesis and expression at the mRNA level and were altered by changing the shape of the cell nucleus. Our data supports the concept of “architectural” transcription factors that promote gene expression based on optimal stress within the nuclear matrix transduced by the cytoskeleton.
5:00 PM		BI-WeA-10 Investigation of Protein Interactions with Poly (Ethylene Glycol) Modified Liposomes J.L. Brash, M.E. Price (McMaster University, Canada) Liposomes have considerable potential as drug delivery vehicles. However unmodified liposomes are rapidly removed from the circulation by the reticuloendothelial system. This is believed to be initiated by adsorption of plasma proteins. Modification of liposomes with poly(ethylene glycol) (PEG) has been shown to increase their lifetime in vivo. Although there is some information on protein interactions with conventional liposomes, there is little if any on PEG-modified liposomes. In this study liposome interactions with fibrinogen in buffer and with plasma have been investigated. Sucrose-loaded large unilamellar liposomes were prepared. Dry phospholipid films (unmodified or modified with phosphatidylethanolamine-conjugated PEG (PE-PEG)) were hydrated with sucrose buffer followed by extrusion through two stacked 100 nm polycarbonate membranes. Liposome size distribution was estimated by dynamic light scattering (DLS) with diameters in the range of 135 ± 8 nm. Liposomes were incubated for 3 h in Tris-buffered 125I-fibrinogen solutions. The mixtures were centrifuged and the radioactivity of liposome pellet determined. Liposomes were also incubated in plasma and bound proteins identified by SDS solubilization followed by gel electrophoresis and immunoblotting using antibodies to some 20 plasma proteins. Fibrinogen adsorption was found to increase as solution concentration increased, with no apparent plateau. Adsorbed amounts decreased with incorporation of PEG and with increasing MW of PEG in the range 500-5000. The gels and immunoblots showed that the unmodified and PEG-modified liposomes adsorbed most of the proteins probed for. The protein patterns (relative amounts of each, degradation, activation) were similar.