Lipoprotein interactions with a polyurethane and a polyethylene oxide-modified polyurethane at the plasma-material interface.

Lipoproteins [high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL)] are present in blood in relatively high concentrations, and, given their importance in cardiovascular disease, the interactions of these species with blood contacting biomaterials and their possible role in thrombogenesis is of interest. In the present communication, quantitative data on the adsorption of apolipoprotein AI, apolipoprotein AII (the main protein components of HDL), and apolipoprotein B (the main protein component of LDL and VLDL), as well as the lipoproteins themselves from plasma to a biomedical grade polyurethane (PU) with and without a copolymer additive that contains polyethylene oxide (PEO) segments, were investigated. Adsorption from some binary solutions was also studied. Significant quantities of the apolipoproteins were found to adsorb from plasma to the PU, while adsorption to the PEO material was more than 90% lower, demonstrating strong protein resistance of the latter material. In contrast, significant quantities of the lipoproteins were found to adsorb to the PEO as well as to the PU material. From these and previously published results, it is concluded that the protein layer formed on the PU surface from plasma (and by extension from blood) contains apolipoproteins and lipoproteins in addition to other plasma proteins; the layer formed on the PEO surface, however, appears to contain minimal quantities of plasma proteins (including free apolipoproteins) but significant quantities of lipoproteins.

Surface modification of poly(dimethylsiloxane) with a covalent antithrombin-heparin complex for the prevention of thrombosis: use of polydopamine as bonding agent.

A modified poly(dimethyl siloxane) (PDMS) material is under development for use in an extracorporeal microfluidic blood oxygenator designed as an artificial placenta to treat newborn infants suffering from severe respiratory insufficiency. To prevent thrombosis triggered by blood-material contact, an antithrombin-heparin (ATH) covalent complex was coated on PDMS surface using polydopamine (PDA) as a "bioglue". Experiments using radiolabelled ATH showed that the ATH coating on PDA-modified PDMS remained substantially intact after incubation in plasma, 2% SDS solution, or whole blood over a three day period. The anticoagulant activity of the ATH-modified surfaces was also demonstrated: in contact with plasma the ATH-coated PDMS was shown to bind antithrombin (AT) selectively from plasma and to inhibit clotting factor Xa. It is concluded that modification of PDMS with polydopamine and ATH shows promise as a means of improving the blood compatibility of PDMS and hence of the oxygenator device.

Interfacial interactions of apolipoprotein AI and high density lipoprotein: overlooked phenomena in blood-material contact.

Apolipoprotein AI (apo AI) is the major protein component of high density lipoprotein (HDL), and represents ∼1% of the total protein content of plasma. In previous work, apo AI was identified as a major component of the protein layers adsorbed from plasma to biomaterials having a wide range of surface properties. Notwithstanding such indications of the major contribution of lipoprotein interactions, these phenomena have been largely overlooked in the blood-contacting biomaterials area. In this communication, detailed quantitative data on the adsorption of apo AI to typical "biomedical" segmented polyurethane (PU) are reported. Using radiolabeled apo AI, adsorption levels from buffer and plasma were found to be ∼0.5 and ∼0.2 µg/cm(2), respectively. Albumin adsorption from plasma was comparable at about ∼0.17 µg/cm(2). Since, it is unknown how much of the adsorbed apo AI is associated with HDL versus how much is in the free state, the corresponding molar quantities cannot be determined with certainty. However, if it is assumed that all of the adsorbed apo AI is free, the molar quantities adsorbed from plasma were in the ratio apo AI:albumin = 2.77, compared with 0.00063 in plasma. Immunoblot data showed similar trends with respect both to the variation in adsorbed quantity with plasma concentration and to the relative adsorbed quantities of apo AI and albumin. These data show unequivocally the very strong surface activity of apo AI and suggest that a key future focus for blood compatibility research should be lipoprotein interactions.

Immunoblot analysis of proteins associated with self-assembled monolayer surfaces of defined chemistries.

Intact and fragmented proteins, eluted from self-assembled monolayer (SAM) surfaces of alkanethiols of different chemistries (-CH3, -OH, -COOH, -NH2), following exposure to human plasma (HP) or human serum (HS), were examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques. The SAM surfaces were incubated for 1 h with 10% (v/v) sterile-filtered, heat-inactivated (h.i.) HS or 1% (v/v) sterile-filtered h.i. HP preparations [both in phosphate buffered saline (PBS)]. Adsorbed proteins were eluted using 10% SDS/2.3% dithioerythritol for characterization of protein profiles. The type of incubating medium may be an important determinant of adsorbed protein profiles, since some variations were observed in eluates from filtered versus control unfiltered h.i. 10% HS or 1% HP. Albumin and apolipoprotein A1 were consistently detected in both filtered h.i 10% HS and 1% HP eluates from all SAM surfaces and from control tissue culture-treated polystyrene (TCPS). Interestingly, Factor H and Factor I, antithrombin, prothrombin, high molecular weight kininogen (HMWK), and IgG were present in eluates from OH, COOH, and NH2 SAM surfaces and in eluates from TCPS but not in eluates from CH3 SAM surfaces, following exposure to filtered h.i. 10% HS. These results suggest that CH3 SAM surfaces were the least proinflammatory of all SAM surfaces. Overall, similar trends were observed in the profiles of proteins eluted from surfaces exposed to filtered 10% HS or 1% HP. However, the unique profiles of adsorbed proteins on different SAM surface chemistries may be related to their differential interactions with cells, including immune/inflammatory cells.

Biological properties of photocrosslinked alginate microcapsules.

An alternative form of gene therapy using recombinant cell lines delivering therapeutic products encapsulated in alginate hydrogel has proven effective in treating many murine models. The lack of long-term capsule stability has led to a new strategy to reinforce the microcapsules with a photopolymerized interpenetrating covalent network of N-vinylpyrrolidone (NVP) and sodium acrylate. Here the properties for potential application in gene therapy are reported. In assessing potential toxicity of the unpolymerized residues, HPLC showed that even after 1 week of washing, no toxic monomers could be detected. Their ability to sustain cell growth was monitored with growth of the encapsulated cells in vitro and in vivo. Although the initial photopolymerization caused significant cell damage, the cells were able to recover normal growth rates thereafter. After implanting into mice, the NVP-modified capsules showed a high level of biocompatibility as measured by hematological and biochemical functional tests. There was also no difference in the amount and type of plasma proteins adsorbing to the NVP-modified and the classical alginate capsules, thus indicating their similar biological compatibility. Both in vitro and in vivo tests confirmed that the NVP-modified capsules were more resistant to osmotic stress than the alginate microcapsules. Furthermore, when applied to the treatment of a murine model of human cancer by delivering encapsulated cells secreting angiostatin, the NVP-modified microcapsules suppressed tumor growth as successfully as the regular alginate microcapsules. In conclusion, the covalently modified microcapsules have shown a high level of biocompatibility, safety, increase in stability, and clinical efficacy for use as immunoisolation devices in gene therapy.

Interactions of antithrombin and proteins in the plasma contact activation system with immobilized functional heparin.

The interactions of antithrombin (AT) and the contact phase clotting factors with two commercially available heparinized surfaces are reported. The Carmeda (CBAS) and Corline surfaces along with controls (a sulfonated polyethylene surface and a CBAS analog in which the heparin used was devoid of specific AT-binding sequences) were exposed to human plasma. Adsorbed proteins were eluted and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The CBAS and Corline surfaces adsorbed large amounts of AT, whereas adsorption on the controls was negligible. Immunoblots for the four contact phase clotting factors indicated less contact activation on the CBAS and Corline surfaces than on the controls. Determination of adsorbed functional AT using a FXa inhibition assay showed that the CBAS surface adsorbed about 4 times as much AT as the Corline surface. Adsorption of AT to the control surfaces was minimal. Assays for adsorbed FXII and FXIIa based on kallikrein generation showed that all four surfaces adsorbed similar amounts of FXII. However, on the controls, most of the FXII was in activated form, whereas on the CBAS and Corline surfaces very little activation occurred.

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.

Effects of reuse and bleach/formaldehyde reprocessing on polysulfone and polyamide hemodialyzers.

The surface features, morphology, and blood interactions of fibers from pristine, bleach/formaldehyde reprocessed, and reused Fresenius Polysulfone High Flux (Hemoflow F80B) hemodialyzers and Gambro Polyflux 21S Polyamide hemodialyzers have been studied. SEM images of fibers from both hemodialyzer types revealed a dense skin layer on the inner surface and a relatively thick porous layer on the outer surface. The 21S polyamide support layer consisted of interconnected highly porous structures. Environmental scanning electron microscopy and atomic force microscopy images of both membrane types showed alterations in morphology due to reprocessing and reuse; however the changes were more marked for the 21S polyamide dialyzers. Fluorescence microscopy images showed only minimal fluorescence associated with the fibers after patient use and reprocessing, suggesting that blood derived deposits were removed by processing. The protein layers formed on pristine and reused hemodialyzer membranes during clinical use were studied using SDS-PAGE and immunoblotting. Before bleach/formaldehyde treatment, protein layers of considerable amount and complexity were found on the blood side of singly and multiply used dialyzers. Proteins adsorbed on the dialysate side were predominantly in the molecular mass region below 30 kDa. However, some higher molecular mass proteins were detected on the dialysate side of the 21 S polyamide dialyzers. Very little protein was detected on dialyzers that were treated with bleach/formaldehyde after dialysis, regardless of whether they had been used/reprocessed once or 12 times.

Adsorption of proteins from infant and adult plasma to biomaterial surfaces.

The hemostatic mechanism of the newborn is immature. In general, the clotting times in screening tests are prolonged, the coagulation factors are low, and the coagulation inhibitors (with the exception of alpha-2-macroglobulin) are low. Recognizing that many of the proteins present in infant plasma are at low levels, it is of interest to determine if, following exposure to artificial surfaces, the profile of adsorbed proteins is different for infant versus adult plasma. The question of whether differences in protein profiles could lead to differences in thromboembolic episodes associated with the use of central venous catheters (or other blood-contacting devices) in infant versus adult subjects also is relevant. To address these issues, the adsorption of proteins from pooled infant plasma and pooled normal adult plasma to three different polymer surfaces (polyvinyl chloride, PVC; polymethyl methacrylate, PMMA; and polyethylene oxide-modified polyurethane, PEO-PU) was studied using SDS-PAGE and immunoblotting techniques. The total amount of protein adsorbed to each surface also was determined. It was found that the PMMA and PVC surfaces adsorbed considerably more protein than the PEO-PU surface. Furthermore, the amount of protein adsorbed to the PMMA and PVC surfaces from infant plasma was significantly less than that adsorbed from adult plasma. No such difference was seen for the protein-repellent PEO-PU surface. The immunoblot responses of proteins bound to the PMMA and PVC surfaces from infant plasma were, in general, weaker than those bound from adult plasma. It is likely that these differences were due to decreased protein levels in infant plasma.

Effects of heat/citric acid reprocessing on high-flux polysulfone dialyzers.

The surface features, morphology, and tensile properties of fibers obtained from pristine, reprocessed, and reused Fresenius Polysulfone High-Flux (Hemoflow F80A) hemodialyzers have been studied. Scanning electron microscopy of the dialyzer fibers revealed a dense skin layer on the inner surface of the membrane and a relatively thick porous layer on the outer surface. Transmission electron microscopy and atomic force microscopy showed an alteration in membrane morphology due to reprocessing and reuse, or to a deposition of blood-borne material on the membrane that is not removed with reprocessing. Fluorescent microscopy images also showed that a fluorescent material not removed by heat/citric acid reprocessing builds up with continued use of the dialyzers. The tensile properties of the dialyzer fibers were not affected by the heat/citric acid reprocessing procedure. The protein layers formed on pristine and reused hemodialyzer membranes during clinical use were also studied using sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting. A considerable amount of protein was found on the blood side of single and multiple use dialyzers. Proteins adsorbed on the dialysate side of the membrane were predominantly in the molecular weight region below 30 kDa. Little protein was detected on the membranes of reprocessed hemodialyzers.