Matthew S. Wagner

PEO-like plasma polymerized tetraglyme surface interactions with leukocytes and proteins: in vitro and in vivo studies

Polyethylene oxide (PEO) surfaces reduce non-specific protein and cell interactions with implanted biomaterials and may improve their biocompatibility. PEO-like polymerized tetraglyme surfaces were made by glow discharge plasma deposition onto fluorinated ethylene propylene copolymer (FEP) substrates and were shown to adsorb less than 10 ng/cm2 of fibrinogen in vitro. The ability of the polymerized tetraglyme surfaces to resist leukocyte adhesion was studied in vitro and in vivo. Polymerized tetraglyme and FEP were implanted subcutaneously in mice and removed after 1 day or 4 weeks. Histological analysis showed a similar degree of fibrous encapsulation around all of the 4-week implants. Darkly stained wells were present in the fibrous tissues at the tissue-material interface of both FEP and tetraglyme. Scanning electron micrographs showed that in vivo macrophage adhesion to polymerized tetraglyme was much higher than to FEP. After 2-hour contact with heparinized whole blood, polymorphonuclear leukocyte (PMN) adhesion to polymerized tetraglyme was much higher than to FEP, while platelet adhesion to polymerized tetraglyme was lower than to FEP. When PMNs isolated from blood were suspended in 10% autologous plasma, cell adhesion to polymerized tetraglyme was higher than to FEP; however when the cells were suspended in heat inactivated serum, cell adhesion to FEP was higher than to polymerized tetraglyme. The surface chemistry of polymerized tetraglyme did not change after 2-hour blood contact, but displayed nitrogen functional groups after 1-day implantation and became slightly degraded after 4-week implantation. The surface chemistry of FEP did not change significantly after blood contact or implantation. Loosely bound proteins such as fibrinogen on polymerized tetraglyme may contribute to the adhesion of PMNs and macrophages and ultimately to fibrous encapsulation (the foreign body response) around the implants.

Characterization of adsorbed protein films by time of flight secondary ion mass spectrometry

Time of flight secondary ion mass spectrometry (ToF-SIMS) is a useful technique in the study of adsorbed protein films because of its high surface sensitivity and chemical selectivity. However, the protein mass spectra generated by ToF-SIMS are complex fragmentation patterns of a polymer consisting of 20 different monomers (i.e., amino acids). Principal component analysis (PCA) was implemented to classify several reference positive ion protein spectra according to protein and substrate type. Furthermore, the positive ion 74/102 and 120/130 SIMS intensity ratios, radiolabeled experiments, and PCA were used to track the relative surface concentrations of bovine serum albumin and bovine fibronectin in a binary adsorption experiment. In all cases, the combination of ToF-SIMS and PCA proved capable in classifying proteins by their type (in the case of pure protein spectra) and relative surface concentration (in the case of the binary protein spectra).

Limits of detection for time of flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS): detection of low amounts of adsorbed protein

Characterization of biomaterial surfaces requires analytical techniques that are capable of detecting a wide concentration range of adsorbed protein. This range includes detection of low amounts of adsorbed protein (<10 ng/cm2) that may be present on non-fouling biomaterials. X-ray Photoelectron Spectroscopy (XPS) and Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) are surface sensitive techniques capable of detecting adsorbed proteins. We have investigated the lower limits of detection of both XPS and ToF-SIMS on four model substrates each presenting unique challenges for analysis by XPS and ToF-SIMS: mica, poly(tetrafluoroethylene), allyl amine plasma polymer and heptyl amine plasma polymer. The detection limit for XPS ranged from 10 ng/cm2 of fibrinogen (on mica) to 200 ng/cm2 (on allyl amine plasma polymers). The detection limit for ToF-SIMS ranged from 0.1 ng/cm2 of fibrinogen to 100 ng/cm2, depending on the substrate and data analysis. Optimal conditions provided detection limits between 0.1 ng/cm2 and 15 ng/cm2 on all of the substrates used in this study. While both techniques were shown to be effective in detecting protein, the sensitivity of both XPS and ToF-SIMS was shown to be dependent on substrate surface chemistry and the organization of the adsorbed protein film. This study specifically highlights the applicability of ToF-SIMS in the characterization of low level protein adsorption.

Characterizing multicomponent adsorbed protein films using electron spectroscopy for chemical analysis, time-of-flight secondary ion mass spectrometry, and radiolabeling: capabilities and limitations

Characterization of complex adsorbed protein films is a critical aspect of biomaterials science, particularly in understanding the in vivo response to biomaterials. The surface analysis techniques electron spectroscopy for chemical analysis (ESCA) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) are particularly suited to the analysis of complex adsorbed protein films due to their wide applicability to a variety of materials. We have investigated the applicability of ESCA for studying the structure of adsorbed serum and plasma protein layers. ESCA was able to monitor the thickness of the adsorbed protein film. Due to its chemical specificity, ToF-SIMS was used to estimate the composition of the plasma and serum protein layers by comparison of their spectra with the spectra of single protein films. The limit of detection of ToF-SIMS for the plasma protein fibrinogen was determined by comparison with independent radiolabeled fibrinogen adsorption measurements. While ToF-SIMS was able to determine some qualitative trends in the composition of the plasma protein films as a function of adsorption time, the detection limit of the minor components in multicomponent adsorbed protein films ultimately limits the ability of ToF-SIMS to quantify the composition of these films. However, both ESCA and ToF-SIMS can provide useful information on adsorbed plasma protein films without further sample treatment. This study outlines the strengths and weaknesses of ESCA and ToF-SIMS for studying multicomponent adsorbed plasma protein films.

Inhibition of monocyte adhesion and fibrinogen adsorption on glow discharge plasma deposited tetraethylene glycol dimethyl ether

Monocytes and macrophages play important roles in host responses to implanted biomedical devices. Monocyte and macrophage interactions with biomaterial surfaces are thought to be mediated by adsorbed adhesive proteins such as fibrinogen and fibronectin. Non-fouling surfaces that minimize protein adsorption may therefore minimize monocyte adhesion, activation, and the foreign body response. Radio-frequency glow discharge plasma deposition (RF-GDPD) of tetraethylene glycol dimethyl ether (tetraglyme) was used to produce polyethylene oxide (PEO)-like coatings on a fluorinated ethylene-propylene (FEP) surface. Electron spectroscopy for chemical analysis (ESCA) and static time of flight secondary ion mass spectrometry (ToF-SIMS) were used to characterize the surface chemistry of tetraglyme coating. Fibrinogen adsorption to the tetraglyme surface was measured with 125I-labeled fibrinogen and ToF-SIMS. Adsorption of fibrinogen to plasma deposited tetraglyme was less than 10 ng cm-2, a 20-fold decrease compared to untreated FEP or tissue culture polystyrene (TCPS). Monocyte adhesion to plasma deposited tetraglyme was significantly lower than adhesion to FEP or TCPS. In addition, when the surfaces were preadsorbed with fibrinogen, fibronectin, or blood plasma, monocyte adhesion to plasma deposited tetraglyme after 2 h or 1 day was much lower than adhesion to FEP. RF-GDPD tetraglyme coating provides a promising approach to make non-fouling biomaterials that can inhibit non-specific material-host interactions and reduce the foreign body response.

Quantitative time-of-flight secondary ion mass spectrometry for the characterization of multicomponent adsorbed protein films

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is ideal for the characterization of adsorbed proteins due to its chemical specificity and surface sensitivity. We have employed ToF-SIMS and multivariate analysis to determine the surface composition of adsorbed protein films from binary mixtures, blood serum, and blood plasma. Good correlation between ToF-SIMS data and independent radiolabeling studies was achieved for binary mixtures, though these results depended on the substrate. Qualitative insight into the composition of the serum and plasma protein films was obtained via comparison to standard single protein film spectra. ToF-SIMS and multivariate analysis were able to measure the surface composition of multicomponent adsorbed protein films.

Characterization of adsorbed protein films using time-of-flight-secondary ion mass spectrometry and multivariate analysis

The complexity of the mass spectra obtained by static time-of-flight-secondary ion mass spectrometry (ToF-SIMS) demands high-throughput data analysis techniques that rapidly process and interpret the resulting data. We have used ToF-SIMS to analyze adsorbed protein films. Positive ion mass spectra from different protein films are challenging to differentiate due to the absence of unique, identifying peaks between the different spectra. Therefore, the multivariate pattern recognition techniques principal component analysis (PCA) and linear discriminant analysis (LDA) have been employed to differentiate the spectra of different proteins and understand the major sources of variation in these spectra. Because of its supervised nature, LDA enhanced discrimination between groups and classification of unknowns when compared with PCA. However, PCA was able to provide better information on the sources of variation in the data set. Both PCA and LDA are important in the analysis of static ToF-SIMS spectra from organic samples.