Surface-MALDI mass spectrometry in biomaterials research.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been used for over a decade for the determination of purity and accurate molecular masses of macromolecular analytes, such as proteins, in solution. In the last few years the technique has been adapted to become a new surface analysis method with unique capabilities that complement established biomaterial surface analysis methods such as XPS and ToF-SSIMS. These new MALDI variant methods, which we shall collectively summarize as Surface-MALDI-MS, are capable of desorbing adsorbed macromolecules from biomaterial surfaces and detecting their molecular ions with high mass resolution and at levels much below monolayer coverage. Thus, Surface-MALDI-MS offers unique means of addressing biomaterial surface analysis needs, such as identification of the proteins and lipids that adsorb from multicomponent biological solutions in vitro and in vivo, the study of interactions between biomaterial surfaces and biomolecules, and identification of surface-enriched additives and contaminants. Surface-MALDI-MS is rapid, experimentally convenient, overcomes limitations in mass resolution and sensitivity of established biochemical techniques such as SDS-PAGE, and can in some circumstances be used for the quantitative analysis of adsorbed protein amounts. At this early stage of development, however, limitations exist: in some cases proteins are not detectable, which appears to be related to tight surface binding. This review summarizes ways in which Surface-MALDI-MS methods have been applied to the study of a range of issues in biomaterials surfaces research.

Enhancing hepatocyte adhesion by pulsed plasma deposition and polyethylene glycol coupling.

Decreased hepatocyte adhesion to polymeric constructs limits the function of tissue engineered hepatic assist devices. We grafted adhesion peptides (RGD and YIGSR) to polycaprolactone (PCL) and poly-L-lactic acid (PLLA) in order to mimic the in vivo extracellular matrix and thus enhance hepatocyte adhesion. Peptide grafting was done by a novel technique in which polyethylene glycol (PEG)-adhesion peptide was linked to allyl-amine coated on the surface of PCL and PLLA by pulsed plasma deposition (PPD). Peptide grafting density, quantified by radio-iodinated tyrosine in YIGSR, was 158 fmol/cm(2) on PLLA and 425 fmol/cm(2) on PCL surfaces. The adhesion of hepatocytes was determined by plating 250,000 hepatocytes/well (test substrates were coated on 12 well plates) and quantifying the percentage of adhered cells after 6 h by MTT assay. Adhesion on PCL surfaces was significantly enhanced (p < 0.05) by both YIGSR (percentage of adhered cells = 53 +/- 7%) and RGD (53 +/- 12%) when compared to control surfaces (31 +/- 8%). Hepatocyte adhesion on PLLA was significantly (p < 0.05) enhanced on PLLA-PEG-RGD surfaces (76 +/- 14%) compared to control surfaces (42 +/- 19%) and more (68 +/- 25%) but not statistically significant (p = 0.15) on PLLA-PEG-YIGSR surfaces compared to control surfaces. These results indicate that hepatocyte adhesion to PCL and PLLA based polymeric surfaces can be enhanced by a novel adhesion peptide grafting technique using pulsed plasma deposition and PEG cross-linking.

Pulsed plasma deposition of allylamine on polysiloxane: a stable surface for neuronal cell adhesion.

Allylamine was pulse-plasma polymerized onto a hydrophobic polysiloxane substrate to create cell adhesion surfaces for cell culture that would not require pretreatment with polylysine, could be sterilized via autoclaving, and could be re-used for several culture cycles. We investigated two different plasma deposition protocols at 200 W RF power: (1) a duty cycle of 3 ms on and 5 ms off; and (2) a cycle of 3 ms on and 45 ms off. Control surfaces were unmodified polysiloxane, activated polysiloxane via flaming, and flamed polysiloxane further modified with poly-D-lysine (PDL). The different surfaces were characterized with XPS analysis, water contact angle, and cell adhesion and growth using dissociated murine embryonic spinal tissue. We found that both the amine content of the 3/45 duty cycle surface and the wettability was higher than that of the 3/5 surface. Also, spinal cord cells were better dispersed 24 h after seeding on the 3/45 surface, suggesting a difference in early adhesion dynamics. However, the networks on the two types of modified surfaces revealed no obvious morphological differences after 2 weeks in vitro. The stability of allylamine-decorated surfaces after autoclaving was high with only minor changes in wettability and nitrogen content. Cell growth on such surfaces after autoclaving was comparable to that found on flamed polysiloxane, freshly modified with PDL. Allylamine surfaces were still usable as cell growth substrates after three autoclaving cycles, 4 weeks under warm culture medium, and simple cleaning procedures, indicating the achievement of a long-lasting modification that did not require the repeated use of PDL before each seeding.

Studies of peptide binding to allyl amine and vinyl acetic acid-modified polymers using matrix-assisted laser desorption/ionization mass spectrometry.

Previous studies have shown that increases in surface-peptide binding affinity result in decreases in peptide matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) ion signals. The present work demonstrates that, with appropriate corrections for peptide ionization efficiency under MALDI conditions, relative surface-peptide binding affinities can be assayed using the MALDI MS methodology. Peptides with a range of pI values are allowed to interact with amine-modified and carboxylic acid-modified polymer surfaces (produced by pulsed radio-frequency plasma polymerization of allyl amine and vinyl acetic acid) in buffered solutions of neutral pH. Because of the net positive and negative charges associated with the peptides and surfaces in solution, both electrostatic and hydrophilic interactions play a role in the surface-peptide interaction. Consistent with expectations, the peptide MALDI ion signals for peptides with net negative charges in solution are smaller than those for peptides with net positive charges in solution when the peptides are allowed to interact with positively charged surfaces. A reversal of the relative peptide MALDI ion signal intensities is observed when the same peptides are allowed to interact with negatively charged surfaces. Cumulatively, the results demonstrate that even modest changes in surface-peptide interactions can be comparatively probed by MALDI mass spectrometry.

Effects of protein-surface interactions on protein ion signals in MALDI mass spectrometry.

The influence of polymer surface-protein binding affinity on protein ion signals in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is examined. The surfaces of poly(vinylidene fluoride) and poly(ethylene terephthalate) polymer substrates are modified by pulsed rf plasma deposition of allylamine. By varying the on/off duty cycle of the pulsed rf plasma, the polymer substrate surfaces are coated with thin films having varying densities of surface amine groups. The varying surface amine density is shown to lead to systematic changes in the surface binding affinity for the 125I-radiolabeled peptides angiotensin I and porcine insulin. Unlabeled angiotensin I and porcine insulin are then deposited on the pulsed rf plasma-modified substrates and analyzed by MALDI mass spectrometry. The experimental approach involves applying the peptide to the modified polymer surface in an aqueous phosphate-buffered saline solution and allowing the peptide solution to dry completely under ambient conditions. Subsequently, the MALDI matrix alpha-cyano-4-hydroxycinnamic acid in methanol and 10% trifluoroacetic acid in water are added to the peptide-coated modified polymer surfaces. The results of these studies demonstrate that, for the sample preparation method employed, increases in the surface peptide binding affinity lead to decreases in the peptide MALDI ion signal.

Stability of plasma-polymerized allylamine films with sterilization by autoclaving.

The stability of plasma-polymerized allylamine films with autoclaving sterilization cycles was investigated. Polymerized films were deposited under pulsed plasma conditions using two different duty cycles to provide surfaces having different initial amino group concentrations. The film properties were analyzed by XPS and water contact angle measurements before and after autoclaving. The reactions of these surfaces with trifluoroacetic anhydride provided quantitation of the amino surface concentrations before and after autoclaving. In general, the plasma-polymerized films exhibit good stability vis à vis the autoclaving process, with relatively high retention of the surface amino groups. The results of this work are of specific value with respect to tissue culture studies in which surface modifications involving the introduction of amino groups have been shown to have high efficacy in promoting cell growth. The results obtained suggest that the simple one-step plasma treatment process is a viable alternative to the more cumbersome surface modification procedures currently employed to introduce amino groups in these tissue culture studies.

Fibrinogen adsorption and host tissue responses to plasma functionalized surfaces.

The physical and chemical characteristics of material surfaces are thought to play important roles in biomaterial-mediated tissue responses. To understand the importance of discrete biomaterial chemical characteristics in modifying host tissue responses, we constructed surfaces bearing different functional groups using radio frequency glow discharge plasma polymerization. Surfaces evaluated included those having high concentrations of -OH, -NH2, -CF3, and siloxyl groups. These surfaces and polyethylene terephthalate controls were used to assess the importance of particular physicochemical characteristics in surface:protein:cell interactions both in vitro and in vivo. The results obtained show that surface functionalities do significantly affect both the adsorption and "denaturation" of adsorbed fibrinogen (which is an important mediator of inflammatory responses to biomaterial implants). In addition, these surfaces provoke different degrees of acute inflammatory responses. Interestingly, the amounts of "denatured" fibrinogen that spontaneously accumulate on the individual surfaces correlate closely with the extent of biomaterial-mediated inflammation. These results suggest that surfaces that tend to "irreversibly" bind fibrinogen prompt greater acute inflammatory responses. Unexpectedly, all test surfaces except those bearing a siloxyl group engender relatively similar biomaterial-mediated fibrotic responses. Thus surface functionalities alone may not be sufficient to affect subsequent fibrotic responses.

Reduced protein adsorption on plastics via direct plasma deposition of triethylene glycol monoallyl ether.

The direct plasma-induced deposition of tri(ethylene glycol) monoallyl ether is reported. RF plasma polymerization of this monomer was carried out under both continuous wave (CW) and pulsed plasma operation. The major focus of this work was optimization of the degree of retention of the C-O-C bonds of the starting monomer during the deposition process. This successfully was accomplished using low RF power during the CW runs and low RF duty cycles during the pulsed plasma experiments. Spectroscopic analysis of the plasma films revealed a strong dependence of film composition on the RF power and duty cycles employed. In particular, an unusually high level of film chemistry compositional control was demonstrated for the pulsed plasma studies, with film composition varying in a steady, progressive fashion with sequential changes in the ratios of plasma on to plasma off times. This film chemistry controllability is demonstrated despite the relatively low volatility of the starting monomer. The utility of this plasma deposition approach in introducing polyethylene oxide (PEO) structures on solid substrates was evaluated via protein adsorption studies. Radiolabeled bovine albumin adsorption was studied on plasma-modified poly(ethylene teraphthalate) (PET) substrates. Dramatic reductions in both initial adsorption and retention of this protein were observed on PET samples having maximal PEO content relative to its adsorption on untreated PET surfaces. Good stability and adhesion of the plasma films to the underlying PET substrates were observed, as evidenced from prolonged immersion of plasma-treated surfaces in aqueous solution. Overall, the results obtained from the present work provide additional support for the utility of one-step plasma process to reduce biological fouling of surfaces via deposition of PEO surface units.

Pulsed plasma discharge polymer coatings.

The authors studied a pulsed radiofrequency glow discharge polymer film deposition method (pRFGD) on polyethylene terephthalate (PET), silicon (Si), and potassium chloride (KCl) surfaces, with the aim of better controlling film uniformity and homogeneity. A pulse generator was used to control a conventional 13.56 MHz RFGD circuit to provide plasma on and off times throughout a wide range of duty cycles. Starting monomers included fluorocarbon monomers (C8F16O and C3F6O) and more conventional unsaturated monomers [acrylonitrile (C3H3N) and vinyl trimethyl silane (C5H12Si)]. With each of these monomers progressive, large scale changes in the molecular structure of the plasma deposited films were noted with systematic variations in the RF duty cycle. Film characterizations were performed using electron spectroscopy for chemical analysis (ESCA) and fourier transform infrared (FTIR) analysis. In the case of fluorocarbon (FC) films, systematically decreasing plasma on time at a constant off time resulted in enhanced CF2 and CF3 content compared with that seen with the less highly fluorinated groups. There was virtually no oxygen atom incorporation in the FC films obtained from the oxygen containing monomers. Overall, a dramatic decrease in cross-linking of the FC polymer films was observed with decreasing RF duty cycles. A highly ordered Teflon-like structure was obtained for the lowest duty cycles. In the silane experiments, a systematic variation in the ratio of Si-H/Si-CH3 groups was observed, with this ratio increasing as the RF duty cycle decreased. Experiments with C3H3N revealed an increasing surface density of -CN groups with decreasing RF duty cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular surface tailoring of biomaterials via pulsed RF plasma discharges.

A pulsed RF plasma glow discharge is employed to demonstrate molecular level controllability of surface film deposits. Molecular composition of plasma deposited films is shown to vary in a significant manner with the RF duty cycle. Three fluorocarbon monomers are used to illustrate the process. All three exhibit a trend towards increased surface CF2 content with decreasing pulsed RF duty cycle, including exclusion of oxygen. Significant variations in carbon-fluorine surface functionalities are obtained over a controllable range of film thickness. Film growth rate measurements reveal the occurrence of surface reactions during significant portions of the off portion of the duty cycle. Albumin adsorption on fluorocarbon-treated PET films is unchanged from PET controls for a 100-fold range of bulk concentrations and 60-fold range of adsorption times. However, increased retention of albumin is observed following incubation with protein-denaturing sodium dodecyl sulfate solution, the retention decreasing with increasing bulk concentration of albumin. The increased retention of albumin suggests the treated surfaces may have promise as biocompatible materials.

Spin-labeled analogues of ATP, ADP and AMP: substitutes for normal nucleotides in biochemical systems.

The different roles and effectiveness of adenosine monophosphate, diphosphate and triphosphate labeled at the 6 position of the purine ring with 2,2,6,6-tetramethylpiperidine-1-oxyl in reactions catalyzed by Escherichia coli glutamine synthetase (GS) have been investigated. Our results show that the spin-labeled ATP (Tempo-ATP) serves as a substrate in the glutamine synthesis reaction and in the adenylation of E. coli glutamine synthetase catalyzed by ATP: glutamine adenylyl transferase (ATase) with essentially the same effectiveness as normal ATP. In another reaction (gamma-glutamyltransferase), Tempo ADP serves as an effector with a Km of 9.4 . 10(-8) M compared to 1.2 . 10(-8) M for the normal ADP, while covalently bonded Tempo-AMP serves as a modifier on the catalytic properties of E. coli glutamine synthetase just as the covalently bonded normal AMP does. The dissociation constants between the labeled nucleotides, Mn2+, Mg2+ and Ca2+ are in the same order of magnitude as the binding constants for those cations and the corresponding normal nucleotides. Our findings indicate that the spin-labeled nucleotides are good substitutes for the normal nucleotides in the biochemical systems studied.

Epsilon-adenylylated glutamine synthetase: an internal fluorescence probe for enzyme conformation.

A fluorescent derivative of ATP, epsilon-ATP, was used to adenylylate glutamine synthetase (EC 6.3.1.2) from Escherichia coli enzymatically. The epsilon-adenylylated enzyme exhibits similar catalytic properties and inhibitor susceptibility to those of the naturally adenylylated enzyme. The fluorescence properties of the epsilon-adenosine and of tryptophan residues of the enzyme were used to study ligand-induced conformational changes involving alterations of the tryptophan regions and the adenylylation site of the protein. Binding of Mn(2+) to the epsilon-adenylylated enzyme is accompanied by a decrease of epsilon-adenosine fluorescence as compared to the effect observed for the Mg(2+) binding. An ADP binding study shows that at low ADP concentration, ADP causes enhancement of the tryptophan fluorescence only, reflecting the binding to unadenylylated subunits; and at high ADP concentration, ADP causes not only enhancement of the fluorescence, but also a quenching of the fluorescence of enzyme-bound epsilon-AMP, reflecting binding to the adenylylated subunits. Dissociation constants calculated from these fluorescence changes agree well with those determined from binding studies of ADP to adenylylated and unadenylylated enzymes. Binding of the feedback inhibitor, alanine, to Mn(2+)-dependent glutamine synthetase causes enhancement of the epsilon-AMP fluorescence, from which a dissociation constant of 1.5 mM was calculated for the inhibitor. The fluorescence changes observed due to ligands binding suggest that Mg(2+) and Mn(2+) stabilize different conformational states of the enzyme.