Rena M. Cornelius

Protein adsorption to polyethylene glycol modified liposomes from fibrinogen solution and from plasma.

Unmodified and polyethylene glycol (PEG) modified neutral and negatively charged liposomes were prepared by freeze-thaw and extrusion followed by chromatographic purification. The effects of PEG molecular weight (PEG 550, 2000, 5000), PEG loading (0-15 mol%), and liposome surface charge on fibrinogen adsorption were quantified using radiolabeling techniques. All adsorption isotherms increased monotonically over the concentration range 0-3 mg/ml and adsorption levels were low. Negatively charged liposomes adsorbed significantly more fibrinogen than neutral liposomes. PEG modification had no effect on fibrinogen adsorption to neutral liposomes. An inverse relationship was found between PEG loading of negatively charged liposomes and fibrinogen adsorption. PEGs of all three molecular weights at a loading of 5 mol% reduced fibrinogen adsorption to negatively charged liposomes. Protein adsorption from diluted plasma (10% normal strength) to four different liposome types (neutral, PEG-neutral, negatively charged, and PEG-negatively charged) was investigated using gel electrophoresis and immunoblotting. The profiles of adsorbed proteins were similar on all four liposome types, but distinctly different from the profile of plasma itself, indicating a partitioning effect of the lipid surfaces. alpha2-macroglobulin and fibronectin were significantly enriched on the liposomes whereas albumin, transferrin, and fibrinogen were depleted compared to plasma. Apolipoprotein AI was a major component of the adsorbed protein layers. The blot of complement protein C3 adsorbed on the liposomes suggested that the complement system was activated.

Surface analysis methods for characterizing polymeric biomaterials.

Surface properties have an enormous effect on the success or failure of a biomaterial device, thus signifying the considerable importance of and the need for adequate characterization of the biomaterial surface. Microscopy techniques used in the analysis of biomaterial surfaces include scanning electron microscopy, transmission electron microscopy, atomic force microscopy, and confocal microscopy. Spectroscopic techniques include X-ray photoelectron spectroscopy, Fourier Transform infrared attenuated total reflection and secondary ion mass spectrometry. The measurement of contact angles, although one of the earlier techniques developed remains a very useful tool in the evaluation of surface hydrophobicity/hydrophilicity. This paper provides a brief, easy to understand synopsis of these and other techniques including emerging techniques, which are proving useful in the analysis of the surface properties of polymeric biomaterials. Cautionary statements have been made, numerous authors referenced and examples used to show the specific type of information that can be acquired from the different techniques used in the characterization of polymeric biomaterials surfaces.

Identification of proteins adsorbed to hemodialyser membranes from heparinized plasma

The protein layers formed during contact of plasma with hemodialysis membranes were studied. Dialysers having membranes of cellulose acetate (CA), saponified cellulose ester (SCE), cuprophane (CUP), polymethylmethacrylate (PMMA), and polyacrylonitrile (PAN) were used. Heparinized human plasma was recirculated through the dialysers for four hours. They were then rinsed and the proteins adsorbed to the membranes were eluted with 2% SDS. The yields of protein from the different membranes increased in the order: PMMA < CA < SCE < CUP < PAN. This is the probable order of increasing hydrophilicity. SDS-PAGE and Western blots were performed on the dialyser eluates. The blots were positive for most of the twenty proteins tested for. There were some interesting differences in adsorption patterns among the different membrane materials, notably for high molecular weight kininogen (HMWK), plasminogen and the C3 component of complement. HMWK was intact in the eluates from CA, CUP and SCE, whereas on PMMA and PAN there was evidence of cleavage, suggesting that activation of the contact phase of coagulation was more extensive on the latter two materials. Intact plasminogen was visible on all the blots. However, low molecular weight fragments were visible in the PAN eluates, suggesting activation of the fibrinolytic pathway. Low molecular weight fibrinogen fragments eluted from PAN membranes support this conclusion. C3 was visible in the blots obtained for all membrane materials, and the data suggest that complement is activated by all the membranes. A C3 fragment at about 30 kD (possibly C3d) was seen in the blots for the cellulosic membranes but not for PMMA or PAN.

Towards practical soft X-ray spectromicroscopy of biomaterials

Scanning transmission X-ray microscopy (STXM) is being developed as a new tool to study the surface chemical morphology and biointeractions of candidate biomaterials with emphasis on blood compatible polymers. STXM is a synchrotron based technique which provides quantitative chemical mapping at a spatial resolution of 50 nm. Chemical speciation is provided by the near edge X-ray absorption spectral (NEXAFS) signal. We show that STXM can detect proteins on soft X-ray transparent polymer thin films with monolayer sensitivity. Of great significance is the fact that measurements can be made in situ, i.e. in the presence of an overlayer of the protein solution. The strengths, limitations and future potential of STXM for studies of biomaterials are discussed.

X-ray spectromicroscopy of immiscible polymer blends: Polystyrene-poly(methyl methacrylate)

Abstract Spun cast thin films of blends of low and high molecular weight mono-disperse polystyrene (PS) and poly(methyl methacrylate) (PMMA) with nominal compositions ranging from 66/33 wt.%/wt.% (w/w) up to 10/90 w/w PS/PMMA have been studied, as-made and after annealing. Two synchrotron-based X-ray microscopies — scanning transmission X-ray microscopy (STXM) and X-ray photoemission electron microscopy (X-PEEM) — as well as several variants of atomic force microscopy (AFM) were used to probe the composition and morphology of the bulk and surface of these blends. The chemical sensitivities and spatial resolutions of these three techniques are compared. All samples are observed to have a PS signal in the C 1s X-ray absorption spectrum of the surface of the PMMA-rich domains as measured in the X-PEEM. A continuous thin PS layer is not expected at a PMMA surface since neither polymer should wet the other at thermodynamic equilibrium. The likely origin of this PS surface signal is from a bimodal distribution of PS domain sizes with the PS signal arising from domains at the surface which are smaller than the ∼200-nm resolution of the X-PEEM. High resolution AFM and STXM provide direct evidence for this explanation.

Measurement of protein adsorption to gold surface by radioiodination methods: suppression of free iodide sorption

The adsorption of albumin and fibrinogen to gold metal using proteins radiolabeled with 125I is reported. Previous studies indicated that the interaction of the metal surface with free 125I− ion, present in trace amounts during protein adsorption experiments, resulted in substantial uptake, thus giving erroneously high estimates of adsorbed protein. Different approaches were investigated either to suppress the sorption of 125I− ion to gold or to differentiate between the sorption of labeled protein and 125I−. In the latter case an appropriate correction to the radioactivity on the surface should allow a valid estimate of protein adsorption. It was found that the addition of small amounts of nonradioactive iodide to the protein solution effectively suppresses the binding of 125I− ion (present in trace amounts relative to nonradioactive iodide) to the gold surface. An alternative approach involving pre-exposure to sodium iodide was not effective in suppressing the sorption of 125I− to gold. It was also found that treatment of gold surface with SDS is not 100% efficient in eluting adsorbed protein.

MEASUREMENT OF PROTEIN ADSORPTION TO METALS USING RADIOIODINATION METHODS : A CAVEAT

Abstract The adsorption of fibrinogen and albumin, measured by radiolabelling with 125 I , from single protein solutions to four metal surfaces (gold, silver, titanium and copper) is reported. The measured amounts of adsorbed protein were found to be erroneously high due to interactions of residual free 125 I -iodide ion, present in the protein solution, with the metal surfaces. The “apparent” adsorption was particularly high on gold and silver. Treatment of the protein-adsorbed metal surfaces with 2% aqueous sodium dodecyl sulfate (SDS) removed the protein, but left the iodide ion attached. Adsorbed amounts estimated from the radioactivity of the SDS-eluates were significantly lower than those estimated from total surface radioactivity. These results suggest that caution should be used when interpreting data on the adsorption of 125 I -labelled proteins to metal surfaces, since artifacts can arise due to metal-iodide ion interactions.

Measurement of protein adsorption kinetics by an in situ, “real-time,” solution depletion technique

Abstract An experimental method, which provides real-time in situ data, was used to study protein adsorption. Proteins (fibrinogen or immunoglobulin G) were radioactively labeled with 125I and added in small amounts to the appropriate solution under study, either single protein in buffer or plasma. This protein solution was injected at a constant flow rate into a well-mixed cell containing a suspension of spherical soda-lime glass beads. The buffer contained in the cell was gradually displaced by the protein solution. NaI(T1) solid scintillation detectors, coupled to a high voltage power supply and a multichannel analyzer, were placed at the exit of the cell to monitor the radioactivity of the bulk solution. Adsorption of the protein was determined via depletion of protein in the bulk solution prior to attainment of steady state. The method may thus be considered a solution depletion technique. For fibrinogen as a single protein in buffer, a period of rapid initial adsorption was observed, followed by slower adsorption and attainment of constant surface coverage. The period of rapid initial adsorption rate decreased and the initial adsorption rate increased with increasing fibrinogen feed concentration. The maximum coverage observed was about 0.35 μ/cm2. The effects of diffusion and adsorption kinetics on the initial stages of fibrinogen surface binding were simulated using the appropriate form of the diffusion equation coupled with Langmuir adsorption kinetics. Based on comparisons between the simulated and real data it was determined that the principal adsorption rate limitations were due to protein supply and the kinetics of surface binding. Diffusion effects were shown to be negligible in this well-stirred system. The simulation may also be used to design experiments and fit mathematical adsorption models to data for the adsorption of any solute in a similar system. Adsorption of fibrinogen from blood plasma was found to be transient, thus confirming, essentially in real time and in situ, the so-called “Vroman effect,” which has been observed with non-in situ methods. Adsorption maxima occurred at times which depended on the concentration of plasma fed to the cell and which decreased with increasing concentration. This trend is in agreement with accepted mechanisms of the Vroman effect. Adsorption of IgG from plasma was also found to be transient and the peaks occurred at times shorter than those for fibrinogen, again in agreement with the “principles” of competitive adsorption as reflected in the Vroman effect.

Identification of apolipoprotein A-I as a major adsorbate on biomaterial surfaces after blood or plasma contact

Apolipoprotein A-I is the major protein component of HDL. It is reported in this communication that apo A-I is a significant component of the protein layer adsorbed from blood or plasma to a variety of biomaterial surfaces, including hydrophobic and hvdrophilic polymers, liposomes, a heparinized surface, and a polysulfone hemodialyzer membrane. Evidence in support of this conclusion from SDS-PAGE and immunoblots of proteins eluted from the surfaces after blood or plasma contact is presented. Whether the presence of apo A-I on these surfaces results from the uptake of the free protein or HDL particles remains to be determined. It appears that apolipoprotein A-I and/or HDL deposition may be an important effect in blood-biomaterial interactions generally, and one that has been largely overlooked.

X-ray Spectromicroscopy Study of Protein Adsorption to a Polystyrene-Polylactide Blend

Synchrotron-based X-ray photoemission electron microscopy (X-PEEM) was used to study the adsorption of human serum albumin (HSA) to polystyrene-polylactide (40:60 PS-PLA, 0.7 wt %) thin films, annealed under various conditions. The rugosity of the substrate varied from 35 to 90 nm, depending on the annealing conditions. However, the characteristics of the protein adsorption (amounts and phase preference) were not affected by the changes in topography. The adsorption was also not changed by the phase inversion which occurred when the PS-PLA substrate was annealed above T(g) of the PLA. The amount of protein adsorbed depended on whether adsorption took place from distilled water or phosphate buffered saline solution. These differences are interpreted as a result of ionic strength induced changes in the protein conformation in solution.