Biomedical subjects

Hans J Griesser

Publications and source records attributed to Hans J Griesser.

12 recordsLinked to original sources

PubMed2006

High salt stability and protein resistance of poly(L-lysine)-g-poly(ethylene glycol) copolymers covalently immobilized via aldehyde plasma polymer interlayers on inorganic and polymeric substrates.

The electrostatic adsorption onto charged surfaces of comb copolymers comprising a polyelectrolyte backbone and pendent PEG side chains, such as poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), has in previous studies provided protein-repellent thin coatings, particularly on metal oxide surfaces. A drawback of this approach is, however, the instability of such adsorbed layers under extreme pH values or high ionic strength. We have overcome this limitation in the present study by covalently immobilizing PLL-g-PEG copolymers onto aldehyde plasma-modified substrates. Silicon wafers, optical waveguide chips, and perfluorinated ethylene-co-propylene (FEP) polymer substrates were first coated with a thin plasma polymer layer using a propionaldehyde plasma, followed by covalent immobilization of PLL-g-PEG via reductive amination between amine groups of the PLL backbone with aldehyde groups on the plasma-deposited interlayer. The stability in high salt media and the protein resistance of different molecular architectures of immobilized PLL-g-PEG layers were quantitatively investigated by XPS, an optical waveguide technique (OWLS), and ToF-SIMS. The adsorption of bovine serum albumin was found to be below the detection limit (<2 ng/cm(2)), as for electrostatically adsorbed PLL-g-PEG layers. However, after 24 h of exposure of covalently immobilized layers of PLL-g-PEG to high ionic strength buffer (2400 mM NaCl), no significant change in the protein resistance was observed, whereas under the same conditions electrostatically adsorbed PLL-g-PEG coatings lost their protein resistance. Moreover, covalent immobilization via an aldehyde plasma interlayer enabled the application of PLL-g-PEG layers onto substrates such as FEP onto which electrostatic binding is not possible. These findings create a generic platform for the covalent immobilization of PLL-g-PEG onto a wide variety of substrates.

Adsorption↗

PubMed2006

Colloid probe AFM investigation of interactions between fibrinogen and PEG-like plasma polymer surfaces.

Interaction forces between surfaces designed to be protein resistant and fibrinogen (Fg) were investigated in phosphate-buffered saline with colloid probe atomic force microscopy. The surfaces of the silica probes were coated with a layer of fibrinogen molecules by adsorption from the buffer. The technique of low-power, pulsed AC plasma polymerization was used to make poly(ethylene glycol) (PEG)-like coatings on poly(ethylene teraphthalate) by using diethylene glycol vinyl ether as the monomer gas. The degree of PEG-like nature of the films was controlled by use of a different effective plasma power in the chamber for each coating, ranging from 0.6 to 3.6 W. This produced a series of thin films with a different number of ether carbons, as assessed by X-ray photoelectron spectroscopy. The interaction force measurements are discussed in relation to trends observed in the reduction of fibrinogen adsorption, as determined quantitatively by (125)I radio-labeling. The plasma polymer coatings with the greatest protein-repelling properties were the most PEG-like in nature and showed the strongest repulsion in interaction force measurements with the fibrinogen-coated probe. Once forced into contact, all the surfaces showed increased adhesion with the protein layer on the probe, and the strength and extension length of adhesion was dependent on both the applied load and the plasma polymer surface chemistry. When the medium was changed from buffer to water, the adhesion after contact was eliminated and only appeared at much higher loads. This indicates that the structure of the fibrinogen molecules on the probe is changed from an extended conformation in buffer to a flat conformation in water, with the former state allowing for stronger interaction with the polymer chains on the surface. These experiments underline the utility of aqueous surface force measurements toward understanding protein-surface interactions, and developing nonfouling surfaces that confer a steric barrier against protein adsorption.

Journal Article↗

PubMed2006

Thin calcium phosphate coatings on titanium by electrochemical deposition in modified simulated body fluid.

Adherent and optically semitransparent thin calcium phosphate (CaP) films were electrochemically deposited on titanium substrates in a modified simulated body fluid at 37 degrees C. Coatings deposited by using periodic pulsed potentials showed better adhesion and better mechanical properties than coatings deposited with use of a constant potential. Scanning electron microscopy was used to study the morphology of the coatings. The coatings displayed a polydispersed porous structure with pores in the range of a few nanometers to 1 mum. Furthermore, X-ray diffractometry and the O(1s) satellite peaks in X-ray photoelectron spectroscopy indicated that the coatings possessed a similar surface chemistry to that of natural bone minerals. These results were confirmed by inductively coupled plasma optical emission spectrometry, which yielded a Ca:P ratio of 1.65, close to that of hydroxyapatite. Contact mode atomic force microscopy (AFM) showed the average thickness of the coatings was in the order of 200 nm. Root-mean-square (RMS) roughness values, also derived by AFM, were shown to be much higher on the titanium-CaP surfaces in comparison with untreated titanium substrates, with RMS values of about 300 and 110 nm, respectively. Cell culture experiments showed that the CaP surfaces are nontoxic to MG63 osteoblastic cells in vitro and were able to support cell growth for up to 4 days, outperforming the untreated titanium surface in a direct comparison. These easily prepared coatings show promise for hard-tissue biomaterials.

Adhesiveness↗

PubMed2006

Two-dimensional patterning of thin coatings for the control of tissue outgrowth.

Control of the precise location and extent of cellular attachment and proliferation, and of tissue outgrowth is important in a number of biomedical applications, including biomaterials and tissue engineered medical devices. Here we describe a method to control and direct the location and define boundaries of tissue growth on surfaces in two dimensions. The method relies on the generation of a spatially defined surface chemistry comprising protein adsorbing and non-adsorbing areas that allow control over the adsorption of cell-adhesive glycoproteins. Surface modification was carried out by deposition of thin acetaldehyde and allylamine plasma polymer coatings on silicon wafer and FEP substrates, followed by grafting of a protein resistant layer of poly(ethylene oxide). Spatially controlled patterning of the surface chemistry was achieved by masking during plasma polymerization. XPS and AFM were used to provide evidence of successful surface modifications. Adsorption of the extracellular matrix protein collagen I followed by tissue outgrowth experiments with bovine corneal epithelial tissue for up to 21 days showed that two-dimensional control over tissue outgrowth is achievable with our patterning method over extended time frames. The method promises to be an effective tool for use in a number of in vitro and in vivo applications.

Adsorption↗

PubMed2005

Effects of ionic strength and surface charge on protein adsorption at PEGylated surfaces.

PEGylated Nb2O5 surfaces were obtained by the adsorption of poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) copolymers, allowing control of the PEG surface density, as well as the surface charge. PEG (MW 2 kDa) surface densities between 0 and 0.5 nm(-2) were obtained by changing the PEG to lysine-mer ratio in the PLL-g-PEG polymer, resulting in net positive, negative and neutral surfaces. Colloid probe atomic force microscopy (AFM) was used to characterize the interfacial forces associated with the different surfaces. The AFM force analysis revealed interplay between electrical double layer and steric interactions, thus providing information on the surface charge and on the PEG layer thickness as a function of copolymer architecture. Adsorption of the model proteins lysozyme, alpha-lactalbumin, and myoglobin onto the various PEGylated surfaces was performed to investigate the effect of protein charge. In addition, adsorption experiments were performed over a range of ionic strengths, to study the role of electrostatic forces between surface charges and proteins acting through the PEG layer. The adsorbed mass of protein, measured by optical waveguide lightmode spectroscopy (OWLS), was shown to depend on a combination of surface charge, protein charge, PEG thickness, and grafting density. At high grafting density and high ionic strength, the steric barrier properties of PEG determine the net interfacial force. At low ionic strength, however, the electrical double layer thickness exceeds the thickness of the PEG layer, and surface charges "shining through" the PEG layer contribute to protein interactions with PLL-g-PEG coated surfaces. The combination of AFM surface force measurements and protein adsorption experiments provides insights into the interfacial forces associated with various PEGylated surfaces and the mechanisms of protein resistance.

Adsorption↗

PubMed2005

Relationship between interfacial forces measured by colloid-probe atomic force microscopy and protein resistance of poly(ethylene glycol)-grafted poly(L-lysine) adlayers on niobia surfaces.

Adsorbed layers of "comb-type" copolymers consisting of PEG chains grafted onto a poly(l-lysine) (PLL) backbone on niobium oxide substrates were studied by colloid-probe AFM in order to characterize the interfacial forces associated with coatings of varying architectures (PEG/PLL ratios and PEG chain lengths) and their relevance to protein resistance. The steric and electrostatic forces measured varied substantially with the architecture of the PLL-g-PEG copolymers. Varying the ionic strength of the buffer solutions enabled discrimination between electrostatic and steric-entropic contributions to the net interfacial force. For high PEG grafting densities the steric component was most prominent, but at low ionic strengths and high grafting densities, a repulsive electrostatic surface force was also observed; its origin was assigned to the niobia charges beneath the copolymer, as insufficient protonated amine groups in the PLL backbone were available for compensation of the oxide surface charges. For lower grafting densities and lower ionic strengths there was a substantial attractive electrostatic contribution arising from interaction of the electrical double layer arising from the protonated amine groups, with that of the silica probe surface (as under low ionic strength conditions, the electrical double layer was thicker than the PEG layer). For these PLL-g-PEG coatings the net interfacial force can thus be a markedly varying superposition of electrostatic and steric-entropic contributions, depending on various factors. The force curves correlate with protein adsorption data, demonstrating the utility of AFM colloid-probe force measurements for quantitative analysis of surface forces and how they determine interfacial interactions with proteins. Such characterization of the net interfacial forces is essential to elucidate the multiple types of interfacial forces relevant to the interactions between PLL-g-PEG coatings and proteins and to advance interpretation of protein adsorption or repellence beyond the oversimplified steric barrier model; in particular, our data demonstrate the importance of an ionic-strength-dependent minimum PEG layer thickness to screen the electrostatic interactions of charged interfaces.

Adsorption↗

PubMed2003

Immobilization and surface characterization of NeutrAvidin biotin-binding protein on different hydrogel interlayers.

For a number of potential applications, it is desirable to immobilize avidin class molecules onto solid supports and exploit their ability to bind biotinylated molecules with high affinity. NeutrAvidin molecules were surface immobilized in various ways. In this study, NeutrAvidin was covalently attached by carbodiimide chemistry onto carboxyl groups of polyacrylic acid and carboxymethyl-dextran hydrogel interlayers. A third strategy involved the affinity "docking" of NeutrAvidin onto a biotinylated poly(ethylene glycol) interlayer. These three interlayers were selected for their low nonspecific binding of proteins, which was expected to minimize surface binding of NeutrAvidin by nonspecific interfacial adsorption. X-ray photoelectron spectroscopy (XPS) analyses allowed detailed characterization of the multilayer fabrication steps. An ELISA assay was used to measure NeutrAvidin activity, which varied with the surface immobilization route. Atomic force microcopy (AFM) force measurements showed that the hydrogel interlayer contributed to a repulsive force and verified the specific interaction between biotinylated AFM tips and the NeutrAvidin surfaces. When a solution of free biotin was injected into the AFM liquid cell, the force curve changed substantially and became identical to that recorded between surfaces carrying no NeutrAvidin, indicating that the free solution biotin had displaced NeutrAvidin proteins off the PEG-biotin layer.

Avidin↗

PubMed2003

A comparison of biological coatings for the promotion of corneal epithelialization of synthetic surface in vivo.

PURPOSE: To investigate the effect of a range of biological coatings on corneal epithelialization of a synthetic polymer surface in vivo. METHODS: Eight diverse biological factors (collagen I, collagen III, collagen IV, laminin, fibronectin, endothelial extracellular matrix, hyaluronic acid, and chondroitin sulfate) were coated individually onto the surface of polycarbonate membranes with a pore size of 0.1 micro m. The coated membranes were implanted on the anterior cornea of adult cats and were clinically assessed for rapidity and extent of and persistence of epithelial overgrowth. The membranes with persistent epithelial attachment were examined histologically by immunohistochemistry and routine light and electron microscopy. RESULTS: Collagen I, collagen IV, and laminin consistently enhanced migration and attachment of corneal epithelial cells in vivo. Multiple-layered epithelium over the collagen I-, collagen IV-, and laminin-coated membranes was demonstrated histologically. The collagen I-coated membranes performed best, in that they showed greater stratification and differentiation of the epithelium. Formation of basement membrane and adhesion complexes over the collagen I-coated membranes was detected by immunohistochemistry and electron microscopy up to 9 weeks after implantation. Membranes coated by fibronectin, endothelial extracellular matrix, hyaluronic acid, and chondroitin sulfate did not support persistent epithelial overgrowth. Compromised biostability of these coatings was mostly likely associated with postsurgical reactions of the host corneal tissue. CONCLUSIONS: A biologically modified polymer can support migration and adhesion of corneal epithelial cells in vivo. The collagen I-modified surface exhibited the most promising performance, both clinically and histologically.

Animals↗

PubMed2002

Immobilized liposome layers for drug delivery applications: inhibition of angiogenesis.

Liposomes were immobilized onto the surface of perfluorinated polymer tape samples and tissue culture polystyrene well-plates using a multilayer immobilization strategy. In the first step, a thin interfacial bonding layer with surface aldehyde groups was deposited from a glow discharge struck in acetaldehyde vapour. Polyethylenimine was then covalently bound onto the aldehyde groups by reductive amination, followed by covalent binding of NHS-PEG-biotin molecules onto the surface amine groups by carbodiimide chemistry. Next, NeutrAvidin protein molecules were bound onto the PEG-biotin layer. Finally, liposomes containing PEG-biotinylated lipids were docked onto the remaining binding sites of the surface-immobilized NeutrAvidin molecules. AFM was used to image surface-bound liposomes and revealed a density well below close packing. The release characteristics of the surface-bound liposomes were measured by the fluorescence intensity changes of carboxyfluorescein upon release. Liposomes filled with sodium orthovanadate were surface immobilized and used in two in vitro angiogenesis assays. Marked differences compared to various control samples were observed, demonstrating the utility of drug-filled, surface-bound liposomes for evoking localized, controlled biological host responses proximal to an implanted biomedical device.

Angiogenesis Inhibitors↗

PubMed2002

Effects of cloud-point grafting, chain length, and density of PEG layers on competitive adsorption of ocular proteins.

The effects of pinning density, chain length, and 'cloud point' (CP) versus non-CP grafting conditions have been studied on the ability of polyethylene glycol (PEG) layers to minimize adsorption from a multicomponent (lysozyme, human serum albumin (HSA), IgG and lactoferrin) protein solution. Methoxy-terminated aldehyde-PEG (M-PEG, MW 5000) and dialdehyde-PEG (PEG(ald)2, MW 3400) were grafted by reductive amination onto two surfaces of different amine group density, generated by radiofrequency glow discharge (r.f.g.d.) deposition of n-heptylamine (HA) (low density) or allylamine (AlA) (high density) r.f.g.d. polymer layers. The PEG graft density was varied also by increasing the temperature and salt (K2SO4) content of the grafting solution; it reached a maximum at the CP of the PEGs. The CP reaction conditions were critical for producing PEG layers capable of minimizing protein adsorption. X-ray photoelectron spectroscopy (XPS) showed that under these conditions, PEG(ald)2 produced a thick linear PEG layer, most likely by aldol condensation. Protein adsorption was assessed using XPS and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) in the surface mode (Surface-MALDI-MS). Coatings grafted at non-CP conditions showed some protein adsorption, as did the HA/M-PEG layer grafted at the CP. On the other hand, no protein adsorption was detected on the HA/PEG(ald)2, AlA/M-PEG, and AlA/PEG(ald)2 surfaces when grafted at the CP. Thus, the effects of pinning density and chain length are interrelated, but the key factor is optimization of PEG chain density by use of the CP conditions, provided that a sufficient density of pinning sites exists.

Adsorption↗

PubMed2002

Ultrasensitive probing of the protein resistance of PEG surfaces by secondary ion mass spectrometry.

The highly sensitive surface analytical techniques X-ray photoelectron spectroscopy (XPS) and time-of-flight static secondary ion mass spectrometry (ToF-SIMS) were used to test the resistance of poly(ethylene glycol) (PEG) coatings towards adsorption of lysozyme (LYS) and fibronectin (FN). PEG coatings were prepared by grafting methoxy-terminated aldehyde-PEG (MW 5000 Da) onto two amino-functionalised surfaces with different amine group densities, generated by radio frequency glow discharge polymerisation of n-heptylamine and allylamine. Grafting was performed at the lower critical solution temperature to maximise the graft density of the PEG chains. XPS showed that the grafted density of PEG chains was slightly higher on the allylamine surface. XPS detected no adsorption of either protein on either PEG coating. ToF-SIMS analysis, on the other hand, found, in the positive ion spectra, minute but statistically significant signals assignable to amino acid fragment ions from both proteins adsorbed to the lower density PEG coating and from LYS but not FN on the higher density PEG coating. Negative ion spectra contained relatively more intense protein fragment ion signals for the lower density PEG coating but no changes assignable to adsorbed proteins on the higher density PEG coating. These results demonstrate the importance of utilising highly sensitive techniques to study protein adsorption on surfaces intended to be protein resistant, and that both positive and negative ion ToF-SIMS spectra should be acquired to probe for possible very low levels of protein adsorption.

Adsorption↗

PubMedDate not supplied

Characterization of surface-immobilized layers of intact liposomes.

Surface-immobilized liposome layers are of interest for various potential applications such as localized drug delivery, but their characterization is challenging. We have employed an AFM method and fluorescent dye release to analyze anchored liposomes. In addition, we studied whether the liposomes are surface-bound solely via specific interaction (NeutrAvidin/biotin) or whether physisorptive binding also plays a role. Liposomes containing PEG-biotin lipids were affinity bound to NeutrAvidin molecules which had been immobilized onto solid supports via three different hydrogel interlayers. After liposome docking, approaching the surface with a colloid probe mounted onto an AFM cantilever showed considerable compression behavior, consistent with expectation based on intact, deformable liposomes but not lipid bilayers, thus showing that disruption of liposomes did not occur upon immobilization onto these support surfaces. Plastic deformation suggestive of liposome disruption on compression was not observed. The kinetics of fluorescent dye release also demonstrated that intact liposomes had been successfully immobilized onto all three supports. Blocking surface-immobilized NeutrAvidin molecules with excess biotin in solution before exposure to liposomes showed that the docking of liposomes was dependent largely but not exclusively on biotin-NeutrAvidin affinity binding, with evidence for some nonspecific physisorption, as the extent of liposome binding onto blocked NeutrAvidin surfaces was appreciably lower than for unblocked surfaces but not zero. Finally, consecutive addition of further NeutrAvidin and liposome layers enabled fabrication of multilayers, and this was clearly seen in AFM compressibility and fluorescent dye release measurements.

Avidin↗