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Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
A new hydrophilic polymer for biomaterial coatings with low protein adsorption a
b
James A. Braatz , Aaron H. Heifetz & Clifton L. Kehr
c
a
W. R. Grace & Co.-Conn. Research Division, 1 Departments of Mammalian Cell Research b
W. R. Grace & Co.-Conn. Research Division, 1 Departments of Mammalian Cell Research c
Designed Polymers Research, 73 79 Route 32, Columbia, MD 21044, USA Published online: 02 Apr 2012.
To cite this article: James A. Braatz , Aaron H. Heifetz & Clifton L. Kehr (1992) A new hydrophilic polymer for biomaterial coatings with low protein adsorption, Journal of Biomaterials Science, Polymer Edition, 3:6, 451-462, DOI: 10.1163/156856292X00439 To link to this article: http://dx.doi.org/10.1163/156856292X00439
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low
new
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protein
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biomaterial
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adsorption
JAMES A. BRAATZ1, AARON H. HEIFETZ1 and CLIFTON L. KEHR2 W. R. Grace & Co.-Conn. Research Division, 1 Departmentsof Mammalian Cell Research and 2 Designed Polymers Research, 73 79Route 32, Columbia, MD 21044, USA Received 10 September 1991;accepted 14 December 1991 Abstract-BIOPOL® polyurethane polymers, an extension of the HYPOL®Polymer series of foamable hydrophilicpolymers, have been developedwhich exhibit improved performance for selectedbiomedical applications. Membersof the BIOPOL polyurethane polymer series, with molecularweightsin the range of 7000 to 30000, are larger moleculesthan HYPOL polymers (MW < 3000) and produce hydrogels, rather than foams, when mixed with water. The prototype material in this series, BIOPOL XP-5, is a liquid prepolymer which chain extends in water and forms a hydrogel which can contain >85% water. The time required for polymerizationwith water was dependent on the prepolymer water ratio. This : prepolymerwas coated onto silica and medical grade tubing and then cured in place with water to form a stable coating which was resistant to non-specificprotein binding. In addition, soluble, isocyanate-free forms of the prepolymerwere tested for toxicity and shown to produce no adverse effects when injected intravenouslyinto mice or when applied to a chickenchorioallantoicmembrane. BIOPOL polymerscan be useful in applications where protein adsorption is an undesirable event. Key words: BIOPOL®polyurethane polymers; HYPOL®polymers; protein adsorption; polymer coated surfaces; biomedicalcoatings. INTRODUCTION of proteins to surfaces is a critically important phenomenon in a Adsorption variety of disciplines [ 1 ] .For medical devices in particular, contact of prosthetics with body fluids leads to protein adsorption, and contact with blood leads to cascade activation events, including thrombosis, which can be fatal. Complement activation during hemodialysis is well known and its clinical significance can not be underestimated [2]. The interaction of proteins with surfaces is a complex process dependent upon the nature of the individual proteins, their presentation in combination with other proteins, concentration, time, temperature, etc., all of which may influence the final state of the absorbed protein. Subsequent cell-surface interactions mediated by adsorbed proteins are influenced by the manner in which proteins adsorb to a surface, and the regions of the proteins which remain accessible in the aqueous environment [3, 4]. Despite progress in these areas it is clear that there is a need for improved biomaterials for these applications. Here we describe a polyurethane prepolymer which when coated onto a variety of surfaces, can be cured with water to form a stable, non-leaching layer which is highly protein resistant. The polymer is non-toxic and is therefore well suited for medical coating applications. The polymers used in this study were selected from the family of HYPOL and BIOPOL polyurethane prepolymers (Organic Chemicals Division, W. R. Grace & Co.-Conn., prepolymers that are Lexington, MA). Like other polyurethane 451
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available [5], these materials are essentially oligomeric polyether commercially polyols which are end capped with a diisocyanate. An isocyanate-terminated polyurethane prepolymer reacts with water through the following well-known reaction scheme [6]:
In the case of a water-soluble, isocyanate-terminated prepolymer, when dissolved in water, the reactive solvent will be present in extreme excess relative to isocyanate. The amine which forms reacts with isocyanate much faster than does water, such of that crosslinking through urea groups is favored over chain termination to amine. isocyanate primary BIOPOL prepolymers have low isocyanate content, and the amount of CO2 formed when mixed with water is insufficient to generate a foam. The resulting material is a crosslinked polyether network with a hard segment content generally below 10 wt %. HYPOL prepolymers, on the other hand, have relatively higher isocyanate contents, and can generate sufficient CO2 during reaction with water to form a foam in the presence of appropriate surfactants used in polyurethane foam formation [7]. For the materials evaluated in this study, however, carbon dioxide evolution is minimal, thus eliminating rapid and extensive gas bubble formation, thereby leading to formation of a hydrogel. These crosslinked hydrogels may be considered to be elastomeric polyurethaneurea polymers consisting of hard segment and soft segment components [6]. For HYPOL and BIOPOL prepolymers, which are used commercially to form crosslinked hydrogels and foams, the polyether soft segment is water soluble and therefore composed almost completely of poly(ethylene oxide); further, the polyol consists of a mixture of di- and trifunctional component species to provide crosslinking. MATERIALS AND METHODS Materials BIOPOL XP-5 is a polyetherurethane (PEU) prepolymer prepared by end-capping a water-soluble polyether polyol with an aliphatic diisocyante. The polyol was a single branch copolymer of ethylene oxide (75%) and propylene oxide (25%) with terminal hydroxyl groups. Each of the hydroxyl groups was reacted with isophorone diisocyanate to yield an isocyanate-terminated prepolymer [8]. The prepolymer is soluble in water, and when mixed with 5 parts by weight of water will form a The prepolymer is soluble in a hydrogel after 30-60 min at room temperature. variety of organic solvents and will not react to form a gel if the solvents are free of water. To minimize side reactions of the isocyanate group, aprotic solvents are generally used to prepare stable solutions. HYPOL prepolymers used in this study include commercially available HYPOL 2000, which is a product of the reaction between poly(ethylene glycol) (MW = 1000), and toluene diisocyanate [9], and an experimental prepolymer trimethylolpropane HYPOL rather than aromatic X6100, which contains designated aliphatic isocyanate end groups.
453 Table 1. Model prepolymersprepared from this study
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PEG = poly(ethyleneglycol); PPG = poly(propyleneglycol). Other water-soluble polyurethane prepolymers were made as model compounds by reacting diisocyanate with polyol under nitrogen at 70-75°C until the isocyanate equivalent weight of the prepolymer, determined by reaction with standard dibutylamine and back titration of the excess base with standard hydrochloric acid [10], indicated a complete reaction with hydroxyl groups. Table 1 lists the model isocyanate prepolymers prepared for this study. Arteriovenous blood tubing (3.17 mm i.d.) and Matrex® Silica, 500 A, 30,um, were from Amicon Division, W. R. Grace & Co.-Conn., Beverly, MA. Teflon,, (2 mm i.d.) and Silicone (1.5 mm i.d.) tubing were from Cole-Parmer Instrument Company, Chicago, IL. A series of poly(ethylene glycols) (Mn = 200, 400, 1000, 1450, 3350 and 8000; were obtained from single, narrow peaks by gel permeation chromatography) the Sigma Chemical Co., St. Louis, MO. Poly(ethylene oxides) (100000 and 4 x 106MW, by viscosity), poly(propylene glycol) and poly(ethylene oxide-propylene PA. Jeffamines (water-soluble oxide) were from Polysciences, Inc., Warrington, aliphatic polyether primary di- and tri-functional amines) were obtained from The Jefferson Chemical Co., a subsidiary of Texaco Inc., Bellaire, TX. Pluracol V7 (7000 MW ) and Pluracol V10 (22 000 MW ) are viscous, liquid, water-soluble branched poly(ethylene oxide-propylene oxide) polymers with terminal hydroxyl groups obtained from BASF Wyandotte Corporation, Parsippany, NJ. Biotinylated protein A (1.6 mg/ml) and glucose oxidase-avidin D (1.0 mg/ml) were obtained from Accurate Chemical and Scientific Corp., Westbury, NY. These were supplied in 0.01 M sodium phosphate, 0.25M NaCI, pH 7.6 with 15 mg/ml BSA as stabilizer and 0.01 Vo thimerosal as preservative. Horseradish peroxidase type VI = 3.1, where pure crystalline peroxidase has a (275 units/mg; A403 nm/A275 nm ratio of 3.0), Tween 20 and ABTS were obtained from Sigma Chemical Company, St. Louis, MO. Chicken serum was obtained from GIBCO, Grand Island, NY. Polyclonal rabbit anti-BSA was from Miles Scientific, Naperville, IL. Bovine hemoglobin prepared from washed, lysed and dialyzed erythrocytes was obtained from Sigma Chemical Company. Methods Silicas were coated with hydrophilic Coating silica with hydrophilic prepolymers. prepolymers by three different procedures referred to as suspension, evaporation and filtration. For suspension and evaporation coatings, prepolymers were dissolved in an aprotic organic solvent (previously dried with molecular sieves to < 0.1 1 (Vo of water), such as acetone, methylene chloride or acetonitrile, to a concentration
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454 (w/v). To 5.0 g silica in a clean flask (previously heated 17 h at 80°C to remove moisture), 50 ml of 5% prepolymer in organic solvent was added then shaken gently on a platform shaker. For evaporative coatings, the solvent was allowed to For the suspension coatings, the flask was sealed to prevent evaporate completely. 72 h of shaking, the coating step was terminated by after evaporation. Generally, filtration and washing of the silica with the same solvent. The silicas were immersed in excess water at room temperature for 17 h to cure the polymer and react excess isocyanates. The silicas were collected by filtration, washed with water, then dry isopropanol, and dried in a vacuum oven at 40°C. Filtration coating was performed by continuously passing the polymer solution over the silica in a fritted glass filtering funnel until all the solvent had evaporated. The filtration-coated silica was then treated as the other coated silicas after polymer coating was completed. The extent of polymer coating on silica was determined by thermal gravimetric analysis. One hundred milligram samples were heated for 5 h at 800°C, during which time the polymer completely burned off the underlying silica support, which itself was stable at this temperature. The loss of weight after heating was determined and expressed as a percentage of the original weight. Stability of polymer coatings on silica were in some cases determined by thermal gravimetric analysis after subjecting the coated silica beds to alternating high and low pH washes using 4 x 25 ml of 0.05M sodium phosphate, pH 8.0, followed by 4 x 25 ml of 0. 1 Olotrifluoroacetic acid in 60/40 2-propanol/water. Determining of protein binding to silica. Silica (0.5 g) was mixed with 10 ml of a solution of bovine hemoglobin in PBS (1-10 mg/ml) then slowly mixed on a rotary mixer for 1 hour at room temperature, then 17 h at 4°C. Although significant binding of hemoglobin to bare silica occurs in 10-15 min, these extended incubation times were selected to provide maximum opportunity for the adsorption of protein. The silica was collected by filtration and washed with PBS until a total of 50 ml of filtrate was collected. Hemoglobin content of the filtrate was determined by the dye-binding procedure of Bradford [11] and by absorbance at 410 nm. The amount of protein bound to the silica was calculated by difference. Hemoglobin was chosen as the model to assess protein adsorption to silica due to its ease of quantitation by visible light absorption and because small amounts bound to a white silica bed ( < 1 mg/gm) are visually apparent (see Fig. 2). Furthermore, hemoglobin is a very surface active protein, more so than fibrinogen, and therefore is a good model for these studies [12]. Coating tubing with hydrophilic polymers. Six inch lengths of tubing were coated by filling with a 5% (w/v) solution of prepolymer in dry isopropanol and allowing it to stand for 1 h at room temperature. The tube was then drained, dried in a vacuum oven, then immersed in water for 17 h at room temperature to completely react the isocyanate groups and crosslink the polymer. The coated tubing was then air dried for 24 h. Indirect evidence that polymer was coated to the tubing was obtained by the observation that these surfaces became less adsorptive toward protein (see below). ELISA. The ELISA of protein binding to tubing using a modified EL/&4. Determination described in this section was modelled after a similar assay previously used to quantitate a tumor antigen [13]. Reagent dilutions and protein concentrations found
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455 optimal in the previous work were employed in this study. BSA was selected as the test protein since antibody to BSA, which is required in the assay, was readily available. The antibody was tested at various dilutions ranging from 1 : 250 to 1 : 1000 to obtain an optimal response. Detergent was included in the wash buffers to help eliminate loosely bound protein. Coated and control tubings were treated with a solution of BSA (10 mg/ml in PBS) for 2 h at room temperature. These conditions were selected to provide ample opportunity for binding to occur. They were then washed with PBS containing 0.5% Tween 20 (this is referred to as 'PT buffer') to remove unbound protein. The tubes were then filled with the following solutions, in sequence, which were allowed to remain in the tubes for the indicated times. Between each addition of solution, the previous contents were removed from the tubes by filling and draining 4 times with with PT buffer: (1) polyclonal rabbit anti-BSA, diluted 1 : 1000 with PT buffer containing 5% chicken serum (this is referred to as 'PTCS'); 1 h; (2) biotinylated protein A, diluted 1 : 100 in PTCS; 1 h; (3) glucose oxidase avidin-D, diluted 1: 200 with PT buffer; 15 min; and (4) enzymesubstrate solution which is comprised of 12.5 ml ABTS/PBS (6.05 gm sodium + 1.65 sodium gm phosphate, dibasic, heptaphosphate, monobasic, monohydrate, hydrate, + 40 mg ABTS + 2.5 gm sodium cacodylate dissolved in 250 ml water) + 1.5 ml of 18070 ?3-D-glucose + 0.5 ml peroxidase (Type VI, 20 mg/100 ml). The enzyme-substrate solution was removed and replaced every 15 min and its A plot of absorbance vs time provided absorbance at 410 nm was determined. a relative indication of the amounts of protein which adsorbed to the surface of the tubes. Since the initial challenge with test protein was at a concentration of 10 mg/ml, and since a direct assay of this type can detect levels of protein in the here provides a very sensitive, relative, ng/ml range, the assay as performed of the estimate protein bound to the tubing. although non-quantitative aqueous solutions of polymer were injected Toxicological tests. Isocyanate-free intravenously into mice which were then monitored for change in body weight. In Table 4, experiment 1, groups of 8 mice were given single, 200 ,ul intravenous injections of a 2007o solution of methanol-capped prepolymer in Dulbecco's PBS on day 0. The weight of each animal was recorded on days 0 and 18. For experiment 2, groups of 8 mice were given four injections of 100,u1 of a 20% solution of methanolcapped prepolymer in Dulbecco's PBS on days 0, 3, 6 and 9. The weight of each animal was recorded on days 0 and 13. The chicken chorioallantoic membrane (CAM) is a sensitive system for detecting angiogenic activity associated with various molecules [14]. Delivery systems such as ELVAX (poly(ethylene-vinyl acetate)), agarose, etc. are used to store and slowly release the test substance to the membrane surface. BIOPOL polymer hydrogels have been tested in this model at Angiogen, Inc., Baltimore MD, for inherent inflammatory potential. BIOPOL polymer hydrogels were applied to days 6 and 8 CAMs which were then observed for any inflammatory response. It was noted that if the hydrogels were incompletely hydrated before application to the membrane, that localized desiccation occurred which resembled an inflammatory response. RESULTS Polymers can be coated onto a wide variety of surfaces and then used to determine the relative extent of protein binding afforded by such coatings. Table 2 lists the
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Table 2. Prevention of nonspecificprotein binding to silica by various polyethers
Silica was coated with each polymer by the addition of 50 ml of 2.5070 (w/v) polymer in methylene chloride to 5.0 gm silica using the evaporation technique described in the Methods section. Hemoglobin was measured in the liquid phase by a dye binding method and by its absorbance at 410 nm as describedin Methods. EO = ethylene oxide; PO = propylene oxide; PPO = poly(propylene oxide); PEOPO = poly(ethyleneoxide - propylene oxide); n.d. = not determined. ability of various polyethers to prevent hemoglobin binding after solutions of these polymers are coated onto a silica surface. In general, these results show that polyethers are more effective as their molecular weights and relative ethylene oxide content increases. It is likely that the ineffectiveness of the lower molecular weight forms is related to an overall weaker interaction with the silica, leading to detachment of the polymer from the surface. The larger forms, however, are more stably bound. The Pluracol V7 and V 10 polyols, for example, are bound to the silica surface tightly enough that they are not removed by an aqueous wash. Several different prepolymers, all isocyanate-terminated, were evaluated for their relative abilities to prevent protein binding in this manner. Increasing amounts of
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Figure 1. Reductionof protein binding to silicaby polymercoatings. Polymers were coated onto 500 A, 35,um silica by evaporation from methylenechloride. The values on the abscissa denote the amount of polymer presentedin solution to the silica. Hemoglobin, 10ml of 1 mg/ml in PBS, was mixed with I gm silica for one hour. Protein remainingin the supernatant was determinedby both dye binding and visible light absorption as described in Methods. prepolymers were added to a constant amount of silica then tested for hemoglobin binding. Figure 1 shows the result of this study which indicates relatively poor performance by prepolymers prepared from low molecular weight PEGs (PEG 1000) terminated with either aliphatic (M3) or aromatic (HYPOL 2000) isocyanates. The most effective prepolymers, BIOPOL XP-5 and M2, were prepared from larger polyols and were terminated with aliphatic isocyanates. An example of the appearance of the silica beds after exposure to hemoglobin is shown in Fig. 2. To investigate whether the prepolymer has any advantage over a large molecular weight polyol for the purpose of preventing protein binding when coated on silica, both coatings were subjected to more severe washing conditions. As shown in Table 3, polyol coated on silica will not withstand alternating high and low pH washes as evidenced by the loss of coating determined by thermal gravimetric analysis. The BIOPOL XP-5/silica on the other hand was essentially unaffected by the same washing procedure. Other surfaces were evaluated for their ability to retain a coating of BIOPOL XP-5 and become more protein resistant. The presence of coating was determined functionally by quantitating protein adsorption using a modified enzyme immunoassay. Three different types of tubing were coated with the prepolymer and tested for protein adsorption. Figure 3, panels A-C, shows the results obtained using and Silicone TeflonO, Tygon tubing, respectively. In all cases, nonspecific protein to the internal surfaces of the tubings was greatly reduced by the coating. adsorption The coatings thus formed were stable to flushing with large volumes of water as determined by the protein binding immunoassay. Toxicological testing has been performed on the BIOPOL XP-5 polymers used in these studies. In the toxicology tests described here, there were no free isocyanate groups present in the materials. These were removed either by capping the terminal isocyanates with methanol to form soluble derivatives, or by forming
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Figure 2. Hemoglobin binding to silica beds coated with various polymers. Silica was coated with limitingamounts of polymerthen treated with 10ml of 2.5 mg/ml bovinehemoglobinfor 24 h. The silica beds were washed with PBS. Samples were coated with: (A) Polymer `M1', 0.05 gm/gm; (B) Polymer `M1', 0.1gm/gm; (C) BIOPOL XP-5, 0.05 gm/gm; (D) BIOPOL XP-5, 0.1 gm/gm; (E) None (bare silica);(F) Polymer `M1',0.05 gm/gm; (G) BIOPOL XP-5, 0.05 gm/gm; (H) HYPOL 2002,0.05 gm/gm; (I) HYPOL X6100,0.05 gm/gm; (J) None (bare silica).SamplesA-E and F-J wererun at different times. insoluble hydrogels by reacting with water. As shown in Table 4, part A, soluble, forms of the prepolymer caused no adverse effects when injected isocyanate-free into mice. Table 4, part B indicates that no inflammatory reaction was intravenously noted when BIOPOL XP-5 was placed in direct contact with the chicken chlorioallantoic membrane. Likewise, water-insoluble hydrogel forms of the polymer showed no effect on kidney epithelial cells in culture, and were non-inflammatory in rabbit cornea implants [15]. Table 3. Stability of silica-polymercoatings
" After coating, the samples indicated were washed with alternating high and low pH solutions as describedin Methods to remove looselybound polymer from the silica surface.
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Figure 3. Protein binding to tubing coated with BIOPOL XP-5. Tubings were coated using 5% prepolymer in dry isopropanol for one hour, air dried then cured with water. Protein binding was assessed using a modified ELISA which involved incubations with: (a) BSA; (b) polyclonal antiserum to BSA; (c) biotinylated protein A; (d) glucose oxidase-avidin D; and (e) horseradish peroxidase + glucose + ABTS. (A) Protein binding to Teflon"'tubing. (B) Protein binding to Siliconetubing. Two sets of control (uncoated) and polymer-coatedSiliconetubes were tested. One set ( + BSA)was treated with all assay reagents, the other set ( - BSA)had the initial treatment with BSA omitted. (C) Protein binding to arteriovenous blood tubing.
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Table 4. Toxicologicalevaluation of BIOPOL* polymers
DISCUSSION Polyethylene oxides are well known as protein compatible molecules when coated or grafted onto surfaces [16]. Both protein and platelet adsorption to PEO-surfaces have been shown to be reduced by PEO chains when attached to the surface at one end of the molecule. Adsorption was demonstrated to be inversely proportional to the length of the chain, with 100 monomer units providing minimal protein adsorption, and essentially no platelet attachment [ 17] . At that chain length, when attached by one end to a surface, NMR studies indicated molecular mobilities similar to PEO free in solution. It was thought that this mobility at the surface prevented protein adsorption by preventing stagnation and subsequent adhesion. More recently, a theoretical study of the protein resistance of terminally-attached PEO chains was performed [18, 19]. PEO was considered as attached to a hydrophobic surface. Steric repulsion between PEO chains and approaching protein were calculated, along with hydrophobic and van der Waals attractions between the protein and the surface. High surface density and long chain PEOs were found to favor protein resistance, with the surface density having the more pronounced influence. of polyethylene glycols with silica has been studied and its The interaction practical use evaluated on chromatographic supports. The interaction of PEG with silica is believed to be due to hydrogen bond formation between surface silanol
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461 groups and the alternate ether oxygens of the polyether [20]. A combination of interactions has also been proposed [21]. hydrogen bonding and hydrophobic PEG has been For chromatographic used to minimize protein-silica supports, but can leach from the support and contaminate the molecule of interactions, interest [22, 23]. The covalent attachment of PEG to the support should prevent leaching. In fact, PEG has been bonded to silica and used to perform hydrophobic interaction chromatography (HIC), since it exhibits weaker interactions than other HIC supports and allows elution of biomolecules using aqueous solvent systems [24, 25]. The weak hydrophobic character of polyethylene oxide has been previously discussed [26]. The BIOPOL polymers described in this report are largely comprised of polyether subunits, and their protein compatibility when applied as a surface coating may be attributed to one or more of the arguments presented above. The prototype and most thoroughly studied in the series is BIOPOL XP-5. As an isocyanate it is stable indefinitely at room temperature if kept free of moisture. prepolymer Solutions of the prepolymer can also be stored if prepared in dry, aprotic solvents such as acetonitrile, acetone, methylene chloride, etc. When dissolved in water at a hydrogel forms after 30-60 min, which is transparent appropriate concentrations, and has a water content of > 85%, depending on processing conditions. In the work reported here, the hydrophilic BIOPOL XP-5 prepolymer, being branched and hence multifunctional, may lie flat on a silica surface if the ether with Once the associate silanol oxygens groups through hydrogen bonding. is set on the surface and the solvent water removed, organic curing prepolymer should join some of the ends of the polymers in urea linkage, thus stabilizing the coating. Alternatively, bonding between isocyanate groups and surface hydroxyl groups may occur, resulting in urethane linkages which would fix the polymer directly to the surface. The greater stability of crosslinked coatings was demonstrated in Table 3 in which it was shown that alternating high and low pH washes removed precursor polyol coatings, but not those from cured prepolymer. This directly demonstrates the advantage of the prepolymer. In addition, the relative ineffectiveness of the low molecular weight polyethers shown in Table 2 was due to their weak association with the silica surface rather than being an probably intrinsic property of these materials. We have in fact observed, but not reported here, that several of the smaller polyethylene glycols used were in fact easily removed by water from the silica surface. Thus their inability to prevent binding was likely due to their inability to remain attached to the support. The protein adsorption data reported in Table 2 were obtained by use of a dye binding techwas confirmed by an independent nique. The accuracy of these measurements the values obtained based on absorbance on some of the same Thus analysis samples. at 410 nm agree well with the dye binding results. Multiple methods of analysis were also applied in the experiment shown in Fig. 1. The results presented were values obtained using the dye binding method, and were again in excellent agreement with values obtained by visible light absorption. Verification of the presence of polymer on the silica surfaces was obtained for the samples of Fig. 1 coated with BIOPOL XP-5. As increasing amounts of polymer were added in the coating solutions, thermal gravimetric analysis demonstrated increasing polymer coating levels which ranged from 5.75?/0 on the lowest sample to 51.18% on the highest.
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462 Direct evidence for the presence of polymer on a silica surface was obtained by Diffuse Reflectance Infrared spectroscopy. Addition of polymer to the silica produced an increase in the C-H stretch region (2865 cm-1) and a peak at 1717 cm-1 corresponding to urethane linkages. There was also a dramatic change in the O-H and N-H stretch regions. In addition to the surfaces reported here, a variety of other surfaces can be coated with the prepolymer and rendered protein resistant. This can be useful where nonspecific protein adsorption is a critical concern, such as in membranes for filtration or diagnostic assays [27, 28]. In particular, medical products which come in direct contact with blood or other body fluids may benefit from improved protein demonstrated in several systems shown in resistance. The excellent biocompatibility Table 4 also lends itself favorably for these applications. Other products, such as biosensor probes, may likewise benefit from decreased protein binding, since it may lead to better signal-to-noise responses. Reduction of protein adsorption in other systems, such as medical devices for implantation or direct blood contact, may also be worth evaluating. REFERENCES 1.J. D. Andrade, in: Surface and Interfacial Aspects of Biomedical Polymers, Vol. 2, p. 1, J. D. Andrade (Ed.). Plenum Press, New York (1985). 2. A. Agostoni and M. Gardinali, J. Biomater. Appl. 4, 102(1989). 3. T. A. Horbett and J. L. Brash, in: Proteins at Interfaces, Physicochemicaland BiochemicalStudies, p. 1, M. J. Comstock (Ed.). American Chemical Society, Washington, DC (1987). 4. J. D. Andrade, S. Nagaoka, S. Cooper, T. Okano and S. W. Kim, Trans. Am. Soc. Art. Int. Org. 33, 75 (1987). 5. J. H. Saunders and K. C. Frisch, in: Polyurethanes: Chemistry and Technology,Vol. 1, pp. 11, 315-317 and Vol. 2, pp. 8-49, IntersciencePublishers, New York (1962and 1964). 6. M. D. Lelah and S. L. Cooper, Polyurethanes in Medicine. CRC Press, Boca Raton (1986). 7. L. L. Wood, J. Cellular Plastics, 12, 285 (1976). 8. J. A. Braatz and C. L. Kehr, U.S. Patent Number 4,886,866. 9. F. J. Hartdegen and W. E. Swann, U.S. Patent Number 4,237,229. 10. S. Siggia, in: Quantitative OrganicAnalysis via Functional Groups, p. 559, John Wiley, New York (1963). 11. M. M. Bradford, Anal. Biochem. 72, 248 (1976). 12. T. A. Horbett, P. K. Weathersbyand A. D. Hoffman, J. Bioeng. 1, 61 (1977). 13. J. A. Braatz, D. T. Hua and G. L. Princler, J. Natl. Cander Inst. 72, 841 (1984). 14. J. Folkman, Cancer Res. 46, 467 (1986). 15. J. A. Braatz, A. H. Heifetz and C. L. Kehr, Am. Soc. Art. Int. Org. Abst. 20, 23 (1991). 16. E. W. Merrill and E. W. Salzman, J. Am. Soc. Art. Int. Org. 6, 60 (1983). 17. S. Nagaoka, Y. Mori, H. Tanzawa, Y. Kikuchi,F. Inagaki, Y. Yokota and Y. Noishiki, J. Am. Soc. Art. Int. Org. 10, 76 (1987). 18. S. I. Jeon, J. H. Lee, J. D. Andrade and P. G. DeGennes,J. Colloid Int. Sci. 142, 149(1991). 19. S. J. Jeon and J. D. Andrade, J. Colloid Int. Sci. 142, 159 (1991). 20. E. Killman and K. Winter, AngewandteMakromol. Chem. 43, 53 (1975). 21. J. Rubio and J. A. Kitchener, J. Colloid Int. Sci. 57, 132 (1976). 22. K. Marcinka, Acta Virol. 16, 52 (1972). 23. H. Engelhardt and D. Mathes, Chromatographia 14, 325 (1981). 24. J.-P. Chang, Z. E. Rassi and C. Horvath, J. Chromatog. 319, 396 (1985). 25. J.-P. Chang and J. G. An, Chromatographia 25, 350 (1988). 26. P. M. Claesson, R. Kjellander, R. Stenius and H. K. Christenson, J. Chem. Soc. Faraday Trans. I 82, 2735 (1986). 27. M. E. Parham and K. E. Milligan, U.S. Patent Number 4,787,976. 28. M. E. Parham and J. L. Rudolph, U.S. Patent Number 4,794,090.
Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
A new hydrophilic polymer for biomaterial coatings with low protein adsorption a
b
James A. Braatz , Aaron H. Heifetz & Clifton L. Kehr
c
a
W. R. Grace & Co.-Conn. Research Division, 1 Departments of Mammalian Cell Research b
W. R. Grace & Co.-Conn. Research Division, 1 Departments of Mammalian Cell Research c
Designed Polymers Research, 73 79 Route 32, Columbia, MD 21044, USA Published online: 02 Apr 2012.
To cite this article: James A. Braatz , Aaron H. Heifetz & Clifton L. Kehr (1992) A new hydrophilic polymer for biomaterial coatings with low protein adsorption, Journal of Biomaterials Science, Polymer Edition, 3:6, 451-462, DOI: 10.1163/156856292X00439 To link to this article: http://dx.doi.org/10.1163/156856292X00439
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A
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low
new
hydrophilic
protein
polymer
for
biomaterial
coatings
with
adsorption
JAMES A. BRAATZ1, AARON H. HEIFETZ1 and CLIFTON L. KEHR2 W. R. Grace & Co.-Conn. Research Division, 1 Departmentsof Mammalian Cell Research and 2 Designed Polymers Research, 73 79Route 32, Columbia, MD 21044, USA Received 10 September 1991;accepted 14 December 1991 Abstract-BIOPOL® polyurethane polymers, an extension of the HYPOL®Polymer series of foamable hydrophilicpolymers, have been developedwhich exhibit improved performance for selectedbiomedical applications. Membersof the BIOPOL polyurethane polymer series, with molecularweightsin the range of 7000 to 30000, are larger moleculesthan HYPOL polymers (MW < 3000) and produce hydrogels, rather than foams, when mixed with water. The prototype material in this series, BIOPOL XP-5, is a liquid prepolymer which chain extends in water and forms a hydrogel which can contain >85% water. The time required for polymerizationwith water was dependent on the prepolymer water ratio. This : prepolymerwas coated onto silica and medical grade tubing and then cured in place with water to form a stable coating which was resistant to non-specificprotein binding. In addition, soluble, isocyanate-free forms of the prepolymerwere tested for toxicity and shown to produce no adverse effects when injected intravenouslyinto mice or when applied to a chickenchorioallantoicmembrane. BIOPOL polymerscan be useful in applications where protein adsorption is an undesirable event. Key words: BIOPOL®polyurethane polymers; HYPOL®polymers; protein adsorption; polymer coated surfaces; biomedicalcoatings. INTRODUCTION of proteins to surfaces is a critically important phenomenon in a Adsorption variety of disciplines [ 1 ] .For medical devices in particular, contact of prosthetics with body fluids leads to protein adsorption, and contact with blood leads to cascade activation events, including thrombosis, which can be fatal. Complement activation during hemodialysis is well known and its clinical significance can not be underestimated [2]. The interaction of proteins with surfaces is a complex process dependent upon the nature of the individual proteins, their presentation in combination with other proteins, concentration, time, temperature, etc., all of which may influence the final state of the absorbed protein. Subsequent cell-surface interactions mediated by adsorbed proteins are influenced by the manner in which proteins adsorb to a surface, and the regions of the proteins which remain accessible in the aqueous environment [3, 4]. Despite progress in these areas it is clear that there is a need for improved biomaterials for these applications. Here we describe a polyurethane prepolymer which when coated onto a variety of surfaces, can be cured with water to form a stable, non-leaching layer which is highly protein resistant. The polymer is non-toxic and is therefore well suited for medical coating applications. The polymers used in this study were selected from the family of HYPOL and BIOPOL polyurethane prepolymers (Organic Chemicals Division, W. R. Grace & Co.-Conn., prepolymers that are Lexington, MA). Like other polyurethane 451
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available [5], these materials are essentially oligomeric polyether commercially polyols which are end capped with a diisocyanate. An isocyanate-terminated polyurethane prepolymer reacts with water through the following well-known reaction scheme [6]:
In the case of a water-soluble, isocyanate-terminated prepolymer, when dissolved in water, the reactive solvent will be present in extreme excess relative to isocyanate. The amine which forms reacts with isocyanate much faster than does water, such of that crosslinking through urea groups is favored over chain termination to amine. isocyanate primary BIOPOL prepolymers have low isocyanate content, and the amount of CO2 formed when mixed with water is insufficient to generate a foam. The resulting material is a crosslinked polyether network with a hard segment content generally below 10 wt %. HYPOL prepolymers, on the other hand, have relatively higher isocyanate contents, and can generate sufficient CO2 during reaction with water to form a foam in the presence of appropriate surfactants used in polyurethane foam formation [7]. For the materials evaluated in this study, however, carbon dioxide evolution is minimal, thus eliminating rapid and extensive gas bubble formation, thereby leading to formation of a hydrogel. These crosslinked hydrogels may be considered to be elastomeric polyurethaneurea polymers consisting of hard segment and soft segment components [6]. For HYPOL and BIOPOL prepolymers, which are used commercially to form crosslinked hydrogels and foams, the polyether soft segment is water soluble and therefore composed almost completely of poly(ethylene oxide); further, the polyol consists of a mixture of di- and trifunctional component species to provide crosslinking. MATERIALS AND METHODS Materials BIOPOL XP-5 is a polyetherurethane (PEU) prepolymer prepared by end-capping a water-soluble polyether polyol with an aliphatic diisocyante. The polyol was a single branch copolymer of ethylene oxide (75%) and propylene oxide (25%) with terminal hydroxyl groups. Each of the hydroxyl groups was reacted with isophorone diisocyanate to yield an isocyanate-terminated prepolymer [8]. The prepolymer is soluble in water, and when mixed with 5 parts by weight of water will form a The prepolymer is soluble in a hydrogel after 30-60 min at room temperature. variety of organic solvents and will not react to form a gel if the solvents are free of water. To minimize side reactions of the isocyanate group, aprotic solvents are generally used to prepare stable solutions. HYPOL prepolymers used in this study include commercially available HYPOL 2000, which is a product of the reaction between poly(ethylene glycol) (MW = 1000), and toluene diisocyanate [9], and an experimental prepolymer trimethylolpropane HYPOL rather than aromatic X6100, which contains designated aliphatic isocyanate end groups.
453 Table 1. Model prepolymersprepared from this study
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PEG = poly(ethyleneglycol); PPG = poly(propyleneglycol). Other water-soluble polyurethane prepolymers were made as model compounds by reacting diisocyanate with polyol under nitrogen at 70-75°C until the isocyanate equivalent weight of the prepolymer, determined by reaction with standard dibutylamine and back titration of the excess base with standard hydrochloric acid [10], indicated a complete reaction with hydroxyl groups. Table 1 lists the model isocyanate prepolymers prepared for this study. Arteriovenous blood tubing (3.17 mm i.d.) and Matrex® Silica, 500 A, 30,um, were from Amicon Division, W. R. Grace & Co.-Conn., Beverly, MA. Teflon,, (2 mm i.d.) and Silicone (1.5 mm i.d.) tubing were from Cole-Parmer Instrument Company, Chicago, IL. A series of poly(ethylene glycols) (Mn = 200, 400, 1000, 1450, 3350 and 8000; were obtained from single, narrow peaks by gel permeation chromatography) the Sigma Chemical Co., St. Louis, MO. Poly(ethylene oxides) (100000 and 4 x 106MW, by viscosity), poly(propylene glycol) and poly(ethylene oxide-propylene PA. Jeffamines (water-soluble oxide) were from Polysciences, Inc., Warrington, aliphatic polyether primary di- and tri-functional amines) were obtained from The Jefferson Chemical Co., a subsidiary of Texaco Inc., Bellaire, TX. Pluracol V7 (7000 MW ) and Pluracol V10 (22 000 MW ) are viscous, liquid, water-soluble branched poly(ethylene oxide-propylene oxide) polymers with terminal hydroxyl groups obtained from BASF Wyandotte Corporation, Parsippany, NJ. Biotinylated protein A (1.6 mg/ml) and glucose oxidase-avidin D (1.0 mg/ml) were obtained from Accurate Chemical and Scientific Corp., Westbury, NY. These were supplied in 0.01 M sodium phosphate, 0.25M NaCI, pH 7.6 with 15 mg/ml BSA as stabilizer and 0.01 Vo thimerosal as preservative. Horseradish peroxidase type VI = 3.1, where pure crystalline peroxidase has a (275 units/mg; A403 nm/A275 nm ratio of 3.0), Tween 20 and ABTS were obtained from Sigma Chemical Company, St. Louis, MO. Chicken serum was obtained from GIBCO, Grand Island, NY. Polyclonal rabbit anti-BSA was from Miles Scientific, Naperville, IL. Bovine hemoglobin prepared from washed, lysed and dialyzed erythrocytes was obtained from Sigma Chemical Company. Methods Silicas were coated with hydrophilic Coating silica with hydrophilic prepolymers. prepolymers by three different procedures referred to as suspension, evaporation and filtration. For suspension and evaporation coatings, prepolymers were dissolved in an aprotic organic solvent (previously dried with molecular sieves to < 0.1 1 (Vo of water), such as acetone, methylene chloride or acetonitrile, to a concentration
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454 (w/v). To 5.0 g silica in a clean flask (previously heated 17 h at 80°C to remove moisture), 50 ml of 5% prepolymer in organic solvent was added then shaken gently on a platform shaker. For evaporative coatings, the solvent was allowed to For the suspension coatings, the flask was sealed to prevent evaporate completely. 72 h of shaking, the coating step was terminated by after evaporation. Generally, filtration and washing of the silica with the same solvent. The silicas were immersed in excess water at room temperature for 17 h to cure the polymer and react excess isocyanates. The silicas were collected by filtration, washed with water, then dry isopropanol, and dried in a vacuum oven at 40°C. Filtration coating was performed by continuously passing the polymer solution over the silica in a fritted glass filtering funnel until all the solvent had evaporated. The filtration-coated silica was then treated as the other coated silicas after polymer coating was completed. The extent of polymer coating on silica was determined by thermal gravimetric analysis. One hundred milligram samples were heated for 5 h at 800°C, during which time the polymer completely burned off the underlying silica support, which itself was stable at this temperature. The loss of weight after heating was determined and expressed as a percentage of the original weight. Stability of polymer coatings on silica were in some cases determined by thermal gravimetric analysis after subjecting the coated silica beds to alternating high and low pH washes using 4 x 25 ml of 0.05M sodium phosphate, pH 8.0, followed by 4 x 25 ml of 0. 1 Olotrifluoroacetic acid in 60/40 2-propanol/water. Determining of protein binding to silica. Silica (0.5 g) was mixed with 10 ml of a solution of bovine hemoglobin in PBS (1-10 mg/ml) then slowly mixed on a rotary mixer for 1 hour at room temperature, then 17 h at 4°C. Although significant binding of hemoglobin to bare silica occurs in 10-15 min, these extended incubation times were selected to provide maximum opportunity for the adsorption of protein. The silica was collected by filtration and washed with PBS until a total of 50 ml of filtrate was collected. Hemoglobin content of the filtrate was determined by the dye-binding procedure of Bradford [11] and by absorbance at 410 nm. The amount of protein bound to the silica was calculated by difference. Hemoglobin was chosen as the model to assess protein adsorption to silica due to its ease of quantitation by visible light absorption and because small amounts bound to a white silica bed ( < 1 mg/gm) are visually apparent (see Fig. 2). Furthermore, hemoglobin is a very surface active protein, more so than fibrinogen, and therefore is a good model for these studies [12]. Coating tubing with hydrophilic polymers. Six inch lengths of tubing were coated by filling with a 5% (w/v) solution of prepolymer in dry isopropanol and allowing it to stand for 1 h at room temperature. The tube was then drained, dried in a vacuum oven, then immersed in water for 17 h at room temperature to completely react the isocyanate groups and crosslink the polymer. The coated tubing was then air dried for 24 h. Indirect evidence that polymer was coated to the tubing was obtained by the observation that these surfaces became less adsorptive toward protein (see below). ELISA. The ELISA of protein binding to tubing using a modified EL/&4. Determination described in this section was modelled after a similar assay previously used to quantitate a tumor antigen [13]. Reagent dilutions and protein concentrations found
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455 optimal in the previous work were employed in this study. BSA was selected as the test protein since antibody to BSA, which is required in the assay, was readily available. The antibody was tested at various dilutions ranging from 1 : 250 to 1 : 1000 to obtain an optimal response. Detergent was included in the wash buffers to help eliminate loosely bound protein. Coated and control tubings were treated with a solution of BSA (10 mg/ml in PBS) for 2 h at room temperature. These conditions were selected to provide ample opportunity for binding to occur. They were then washed with PBS containing 0.5% Tween 20 (this is referred to as 'PT buffer') to remove unbound protein. The tubes were then filled with the following solutions, in sequence, which were allowed to remain in the tubes for the indicated times. Between each addition of solution, the previous contents were removed from the tubes by filling and draining 4 times with with PT buffer: (1) polyclonal rabbit anti-BSA, diluted 1 : 1000 with PT buffer containing 5% chicken serum (this is referred to as 'PTCS'); 1 h; (2) biotinylated protein A, diluted 1 : 100 in PTCS; 1 h; (3) glucose oxidase avidin-D, diluted 1: 200 with PT buffer; 15 min; and (4) enzymesubstrate solution which is comprised of 12.5 ml ABTS/PBS (6.05 gm sodium + 1.65 sodium gm phosphate, dibasic, heptaphosphate, monobasic, monohydrate, hydrate, + 40 mg ABTS + 2.5 gm sodium cacodylate dissolved in 250 ml water) + 1.5 ml of 18070 ?3-D-glucose + 0.5 ml peroxidase (Type VI, 20 mg/100 ml). The enzyme-substrate solution was removed and replaced every 15 min and its A plot of absorbance vs time provided absorbance at 410 nm was determined. a relative indication of the amounts of protein which adsorbed to the surface of the tubes. Since the initial challenge with test protein was at a concentration of 10 mg/ml, and since a direct assay of this type can detect levels of protein in the here provides a very sensitive, relative, ng/ml range, the assay as performed of the estimate protein bound to the tubing. although non-quantitative aqueous solutions of polymer were injected Toxicological tests. Isocyanate-free intravenously into mice which were then monitored for change in body weight. In Table 4, experiment 1, groups of 8 mice were given single, 200 ,ul intravenous injections of a 2007o solution of methanol-capped prepolymer in Dulbecco's PBS on day 0. The weight of each animal was recorded on days 0 and 18. For experiment 2, groups of 8 mice were given four injections of 100,u1 of a 20% solution of methanolcapped prepolymer in Dulbecco's PBS on days 0, 3, 6 and 9. The weight of each animal was recorded on days 0 and 13. The chicken chorioallantoic membrane (CAM) is a sensitive system for detecting angiogenic activity associated with various molecules [14]. Delivery systems such as ELVAX (poly(ethylene-vinyl acetate)), agarose, etc. are used to store and slowly release the test substance to the membrane surface. BIOPOL polymer hydrogels have been tested in this model at Angiogen, Inc., Baltimore MD, for inherent inflammatory potential. BIOPOL polymer hydrogels were applied to days 6 and 8 CAMs which were then observed for any inflammatory response. It was noted that if the hydrogels were incompletely hydrated before application to the membrane, that localized desiccation occurred which resembled an inflammatory response. RESULTS Polymers can be coated onto a wide variety of surfaces and then used to determine the relative extent of protein binding afforded by such coatings. Table 2 lists the
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Table 2. Prevention of nonspecificprotein binding to silica by various polyethers
Silica was coated with each polymer by the addition of 50 ml of 2.5070 (w/v) polymer in methylene chloride to 5.0 gm silica using the evaporation technique described in the Methods section. Hemoglobin was measured in the liquid phase by a dye binding method and by its absorbance at 410 nm as describedin Methods. EO = ethylene oxide; PO = propylene oxide; PPO = poly(propylene oxide); PEOPO = poly(ethyleneoxide - propylene oxide); n.d. = not determined. ability of various polyethers to prevent hemoglobin binding after solutions of these polymers are coated onto a silica surface. In general, these results show that polyethers are more effective as their molecular weights and relative ethylene oxide content increases. It is likely that the ineffectiveness of the lower molecular weight forms is related to an overall weaker interaction with the silica, leading to detachment of the polymer from the surface. The larger forms, however, are more stably bound. The Pluracol V7 and V 10 polyols, for example, are bound to the silica surface tightly enough that they are not removed by an aqueous wash. Several different prepolymers, all isocyanate-terminated, were evaluated for their relative abilities to prevent protein binding in this manner. Increasing amounts of
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Figure 1. Reductionof protein binding to silicaby polymercoatings. Polymers were coated onto 500 A, 35,um silica by evaporation from methylenechloride. The values on the abscissa denote the amount of polymer presentedin solution to the silica. Hemoglobin, 10ml of 1 mg/ml in PBS, was mixed with I gm silica for one hour. Protein remainingin the supernatant was determinedby both dye binding and visible light absorption as described in Methods. prepolymers were added to a constant amount of silica then tested for hemoglobin binding. Figure 1 shows the result of this study which indicates relatively poor performance by prepolymers prepared from low molecular weight PEGs (PEG 1000) terminated with either aliphatic (M3) or aromatic (HYPOL 2000) isocyanates. The most effective prepolymers, BIOPOL XP-5 and M2, were prepared from larger polyols and were terminated with aliphatic isocyanates. An example of the appearance of the silica beds after exposure to hemoglobin is shown in Fig. 2. To investigate whether the prepolymer has any advantage over a large molecular weight polyol for the purpose of preventing protein binding when coated on silica, both coatings were subjected to more severe washing conditions. As shown in Table 3, polyol coated on silica will not withstand alternating high and low pH washes as evidenced by the loss of coating determined by thermal gravimetric analysis. The BIOPOL XP-5/silica on the other hand was essentially unaffected by the same washing procedure. Other surfaces were evaluated for their ability to retain a coating of BIOPOL XP-5 and become more protein resistant. The presence of coating was determined functionally by quantitating protein adsorption using a modified enzyme immunoassay. Three different types of tubing were coated with the prepolymer and tested for protein adsorption. Figure 3, panels A-C, shows the results obtained using and Silicone TeflonO, Tygon tubing, respectively. In all cases, nonspecific protein to the internal surfaces of the tubings was greatly reduced by the coating. adsorption The coatings thus formed were stable to flushing with large volumes of water as determined by the protein binding immunoassay. Toxicological testing has been performed on the BIOPOL XP-5 polymers used in these studies. In the toxicology tests described here, there were no free isocyanate groups present in the materials. These were removed either by capping the terminal isocyanates with methanol to form soluble derivatives, or by forming
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Figure 2. Hemoglobin binding to silica beds coated with various polymers. Silica was coated with limitingamounts of polymerthen treated with 10ml of 2.5 mg/ml bovinehemoglobinfor 24 h. The silica beds were washed with PBS. Samples were coated with: (A) Polymer `M1', 0.05 gm/gm; (B) Polymer `M1', 0.1gm/gm; (C) BIOPOL XP-5, 0.05 gm/gm; (D) BIOPOL XP-5, 0.1 gm/gm; (E) None (bare silica);(F) Polymer `M1',0.05 gm/gm; (G) BIOPOL XP-5, 0.05 gm/gm; (H) HYPOL 2002,0.05 gm/gm; (I) HYPOL X6100,0.05 gm/gm; (J) None (bare silica).SamplesA-E and F-J wererun at different times. insoluble hydrogels by reacting with water. As shown in Table 4, part A, soluble, forms of the prepolymer caused no adverse effects when injected isocyanate-free into mice. Table 4, part B indicates that no inflammatory reaction was intravenously noted when BIOPOL XP-5 was placed in direct contact with the chicken chlorioallantoic membrane. Likewise, water-insoluble hydrogel forms of the polymer showed no effect on kidney epithelial cells in culture, and were non-inflammatory in rabbit cornea implants [15]. Table 3. Stability of silica-polymercoatings
" After coating, the samples indicated were washed with alternating high and low pH solutions as describedin Methods to remove looselybound polymer from the silica surface.
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Figure 3. Protein binding to tubing coated with BIOPOL XP-5. Tubings were coated using 5% prepolymer in dry isopropanol for one hour, air dried then cured with water. Protein binding was assessed using a modified ELISA which involved incubations with: (a) BSA; (b) polyclonal antiserum to BSA; (c) biotinylated protein A; (d) glucose oxidase-avidin D; and (e) horseradish peroxidase + glucose + ABTS. (A) Protein binding to Teflon"'tubing. (B) Protein binding to Siliconetubing. Two sets of control (uncoated) and polymer-coatedSiliconetubes were tested. One set ( + BSA)was treated with all assay reagents, the other set ( - BSA)had the initial treatment with BSA omitted. (C) Protein binding to arteriovenous blood tubing.
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Table 4. Toxicologicalevaluation of BIOPOL* polymers
DISCUSSION Polyethylene oxides are well known as protein compatible molecules when coated or grafted onto surfaces [16]. Both protein and platelet adsorption to PEO-surfaces have been shown to be reduced by PEO chains when attached to the surface at one end of the molecule. Adsorption was demonstrated to be inversely proportional to the length of the chain, with 100 monomer units providing minimal protein adsorption, and essentially no platelet attachment [ 17] . At that chain length, when attached by one end to a surface, NMR studies indicated molecular mobilities similar to PEO free in solution. It was thought that this mobility at the surface prevented protein adsorption by preventing stagnation and subsequent adhesion. More recently, a theoretical study of the protein resistance of terminally-attached PEO chains was performed [18, 19]. PEO was considered as attached to a hydrophobic surface. Steric repulsion between PEO chains and approaching protein were calculated, along with hydrophobic and van der Waals attractions between the protein and the surface. High surface density and long chain PEOs were found to favor protein resistance, with the surface density having the more pronounced influence. of polyethylene glycols with silica has been studied and its The interaction practical use evaluated on chromatographic supports. The interaction of PEG with silica is believed to be due to hydrogen bond formation between surface silanol
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461 groups and the alternate ether oxygens of the polyether [20]. A combination of interactions has also been proposed [21]. hydrogen bonding and hydrophobic PEG has been For chromatographic used to minimize protein-silica supports, but can leach from the support and contaminate the molecule of interactions, interest [22, 23]. The covalent attachment of PEG to the support should prevent leaching. In fact, PEG has been bonded to silica and used to perform hydrophobic interaction chromatography (HIC), since it exhibits weaker interactions than other HIC supports and allows elution of biomolecules using aqueous solvent systems [24, 25]. The weak hydrophobic character of polyethylene oxide has been previously discussed [26]. The BIOPOL polymers described in this report are largely comprised of polyether subunits, and their protein compatibility when applied as a surface coating may be attributed to one or more of the arguments presented above. The prototype and most thoroughly studied in the series is BIOPOL XP-5. As an isocyanate it is stable indefinitely at room temperature if kept free of moisture. prepolymer Solutions of the prepolymer can also be stored if prepared in dry, aprotic solvents such as acetonitrile, acetone, methylene chloride, etc. When dissolved in water at a hydrogel forms after 30-60 min, which is transparent appropriate concentrations, and has a water content of > 85%, depending on processing conditions. In the work reported here, the hydrophilic BIOPOL XP-5 prepolymer, being branched and hence multifunctional, may lie flat on a silica surface if the ether with Once the associate silanol oxygens groups through hydrogen bonding. is set on the surface and the solvent water removed, organic curing prepolymer should join some of the ends of the polymers in urea linkage, thus stabilizing the coating. Alternatively, bonding between isocyanate groups and surface hydroxyl groups may occur, resulting in urethane linkages which would fix the polymer directly to the surface. The greater stability of crosslinked coatings was demonstrated in Table 3 in which it was shown that alternating high and low pH washes removed precursor polyol coatings, but not those from cured prepolymer. This directly demonstrates the advantage of the prepolymer. In addition, the relative ineffectiveness of the low molecular weight polyethers shown in Table 2 was due to their weak association with the silica surface rather than being an probably intrinsic property of these materials. We have in fact observed, but not reported here, that several of the smaller polyethylene glycols used were in fact easily removed by water from the silica surface. Thus their inability to prevent binding was likely due to their inability to remain attached to the support. The protein adsorption data reported in Table 2 were obtained by use of a dye binding techwas confirmed by an independent nique. The accuracy of these measurements the values obtained based on absorbance on some of the same Thus analysis samples. at 410 nm agree well with the dye binding results. Multiple methods of analysis were also applied in the experiment shown in Fig. 1. The results presented were values obtained using the dye binding method, and were again in excellent agreement with values obtained by visible light absorption. Verification of the presence of polymer on the silica surfaces was obtained for the samples of Fig. 1 coated with BIOPOL XP-5. As increasing amounts of polymer were added in the coating solutions, thermal gravimetric analysis demonstrated increasing polymer coating levels which ranged from 5.75?/0 on the lowest sample to 51.18% on the highest.
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462 Direct evidence for the presence of polymer on a silica surface was obtained by Diffuse Reflectance Infrared spectroscopy. Addition of polymer to the silica produced an increase in the C-H stretch region (2865 cm-1) and a peak at 1717 cm-1 corresponding to urethane linkages. There was also a dramatic change in the O-H and N-H stretch regions. In addition to the surfaces reported here, a variety of other surfaces can be coated with the prepolymer and rendered protein resistant. This can be useful where nonspecific protein adsorption is a critical concern, such as in membranes for filtration or diagnostic assays [27, 28]. In particular, medical products which come in direct contact with blood or other body fluids may benefit from improved protein demonstrated in several systems shown in resistance. The excellent biocompatibility Table 4 also lends itself favorably for these applications. Other products, such as biosensor probes, may likewise benefit from decreased protein binding, since it may lead to better signal-to-noise responses. Reduction of protein adsorption in other systems, such as medical devices for implantation or direct blood contact, may also be worth evaluating. REFERENCES 1.J. D. Andrade, in: Surface and Interfacial Aspects of Biomedical Polymers, Vol. 2, p. 1, J. D. Andrade (Ed.). Plenum Press, New York (1985). 2. A. Agostoni and M. Gardinali, J. Biomater. Appl. 4, 102(1989). 3. T. A. Horbett and J. L. Brash, in: Proteins at Interfaces, Physicochemicaland BiochemicalStudies, p. 1, M. J. Comstock (Ed.). American Chemical Society, Washington, DC (1987). 4. J. D. Andrade, S. Nagaoka, S. Cooper, T. Okano and S. W. Kim, Trans. Am. Soc. Art. Int. Org. 33, 75 (1987). 5. J. H. Saunders and K. C. Frisch, in: Polyurethanes: Chemistry and Technology,Vol. 1, pp. 11, 315-317 and Vol. 2, pp. 8-49, IntersciencePublishers, New York (1962and 1964). 6. M. D. Lelah and S. L. Cooper, Polyurethanes in Medicine. CRC Press, Boca Raton (1986). 7. L. L. Wood, J. Cellular Plastics, 12, 285 (1976). 8. J. A. Braatz and C. L. Kehr, U.S. Patent Number 4,886,866. 9. F. J. Hartdegen and W. E. Swann, U.S. Patent Number 4,237,229. 10. S. Siggia, in: Quantitative OrganicAnalysis via Functional Groups, p. 559, John Wiley, New York (1963). 11. M. M. Bradford, Anal. Biochem. 72, 248 (1976). 12. T. A. Horbett, P. K. Weathersbyand A. D. Hoffman, J. Bioeng. 1, 61 (1977). 13. J. A. Braatz, D. T. Hua and G. L. Princler, J. Natl. Cander Inst. 72, 841 (1984). 14. J. Folkman, Cancer Res. 46, 467 (1986). 15. J. A. Braatz, A. H. Heifetz and C. L. Kehr, Am. Soc. Art. Int. Org. Abst. 20, 23 (1991). 16. E. W. Merrill and E. W. Salzman, J. Am. Soc. Art. Int. Org. 6, 60 (1983). 17. S. Nagaoka, Y. Mori, H. Tanzawa, Y. Kikuchi,F. Inagaki, Y. Yokota and Y. Noishiki, J. Am. Soc. Art. Int. Org. 10, 76 (1987). 18. S. I. Jeon, J. H. Lee, J. D. Andrade and P. G. DeGennes,J. Colloid Int. Sci. 142, 149(1991). 19. S. J. Jeon and J. D. Andrade, J. Colloid Int. Sci. 142, 159 (1991). 20. E. Killman and K. Winter, AngewandteMakromol. Chem. 43, 53 (1975). 21. J. Rubio and J. A. Kitchener, J. Colloid Int. Sci. 57, 132 (1976). 22. K. Marcinka, Acta Virol. 16, 52 (1972). 23. H. Engelhardt and D. Mathes, Chromatographia 14, 325 (1981). 24. J.-P. Chang, Z. E. Rassi and C. Horvath, J. Chromatog. 319, 396 (1985). 25. J.-P. Chang and J. G. An, Chromatographia 25, 350 (1988). 26. P. M. Claesson, R. Kjellander, R. Stenius and H. K. Christenson, J. Chem. Soc. Faraday Trans. I 82, 2735 (1986). 27. M. E. Parham and K. E. Milligan, U.S. Patent Number 4,787,976. 28. M. E. Parham and J. L. Rudolph, U.S. Patent Number 4,794,090.
