In vitro assessment of interaction of blood with model surfaces: Acrylates and methacrylates

In Vitro Assessment of Interaction of Blood with Model Surfaces: Acrylates and Methacrylates DESTINY BRIER-RUSSELL, EDWIN W. SALZMAN, AND JACK LINDON Departments of Surgery, Harvard Medical School and Beth Israel Hospital, Boston, Massachusetts 02215

ROBERT HANDIN Departments of Medicine, Harvard Medical School, Peter Bent Brigham Hospital, Boston, Massachusetts 02115

AND

EDWARD W. MERRILL, 1 ALl K. DINCER, AND JIANN-SHING WU Chemical Engineering Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received April 4, 1980; accepted October 29, 1980 Polymers synthesized from alkyl acrylates and alkyl methacrylates (alkyl = methyl, ethyl, propyl . . . . , lauryl) were deposited from solution as coatings on surfaces of glass beads packed into columns. Surfaces were exposed to citrated human blood, in some cases after prior incubation with platelet-poor plasma (PPP). The average platelet recovery index, ? (? = effluent platelet count/ influent platelet count), was determined. For both polyacrylates and polymethacrylates, ~ decreased with increasing number of carbons in the alkyl group, being highest for alkyl = methyl. The degree of secretion of platelet constituents varied directly with length of the alkyl side chain. Incubation with PPP increased ?. Results are interpreted in terms of hydrophilic-hydrophobic balance of the polymer surfaces and plasma protein adsorption.

INTRODUCTION

The contact of artificial materials with blood in prosthetic devices frequently leads to thrombembolic complications and to consumption of platelets and labile protein coagulation factors. The aspects of surface chemistry that govern these interactions are poorly understood. There is little agreement as to which properties of a material are critical in dictating surface-platelet interactions, in part because prior studies have often examined only commercially available

1 Address all communications to: Professor E. W. Merrill, Room 66-562, Massachusetts Institute of Technology, Cambridge, Mass. 02139.

materials with insufficient regard to the possibility of contamination of the surface with mold-release agents, waxes or polishes, cross-linking initiators or terminators, plasticizers, or other additives. Among the more widely used polymers are the acrylates and the methacrylates, whose physical properties are suitable for use in a variety of practical applications. We studied a series of highly purified homopolymers prepared from acrylate and methacrylate monomers, varying in the length of the alkyl side chains, in a model system which permitted quantitative assessment of the interaction with blood platelets of the surfaces of these homopolymers deposited from solution onto glass beads. 311

Journal of Colloidand Interface Science, Vol.81, No. 2, June 1981

0021-9797/81/060311-08502.00/0 Copyright© 1981by AcademicPress, Inc. All rightsof reproductionin any formreserved.

312

BRIER-RUSSELLET AL. METHODS

Preparation of Polymers All monomers were obtained from Polyscience, Inc. as materials 99%+ pure by gas chromatography. In the acrylate series, the following monomers were polymerized as homopolymers: methylacrylate, ethylacrylate, propylacrylate, n-butylacrylate, n-hexylacrylate, n-decylacrylate, and laurylacrylate. In the methacrylate series, the following monomers were converted to homopolymers: methylmethacrylate, ethylmethacrylate, propylmethacrylate, n-butylmethacrylate, amylmethacrylate, isodecylmethacrylate, and laurylmethacrylate. The solvent for all the polymerization runs was chromatographygrade methylethyl ketone (MeK), which constituted 60% by volume of the reaction mixture, the remainder being monomer/polymer. The initiator was azoisobutyronitrile (AIBN) obtained from Aldrich Chemical Company (Milwaukee, Wisc.). Instead of removing the free-radical inhibitor added to the monomer (0.24 wt% based on total solution), we allowed for titration of it by AIBN, thereby delaying, to varying extents, the onset of polymerization. Our polymerizations were carried out, generally to about 79% conversion under the vapor pressure of the solvent, in sealed 25-ml scintillation vials in a thermostated water bath at 60°C. The reaction mixture was quenched by adding it to cold methanol or methanol/water, which caused precipitation of the homopolymers, leaving in the supernatant phase residual monomer, initiator, and initiator fragments. The precipitated polymer was redissolved in MeK, reprecipirated in methanol or methanol/water, and dried under argon.

Preparation of Glass Bead Columns Disposable polyethylene columns (0.8 by 20.5 cm) obtained from Kontes Corporation (Vineland, N. J.) were cut in 5 cm lengths. A single large siliconized glass bead (3 mm Journal of Colloid and Interface Science, Vol. 81, No. 2, June 1981

diameter) was placed at the bottom of the column. Thoroughly washed (24 hr in sulfuric-acid-chromic-acid cleaning solution, rinsed, soaked 24 hr in 2 M NaOH, and rinsed with distilled water until pH was neutral) glass beads (Potter Industries) 0.35 to 0.500 mm with a number average of 0.43 mm diameter were poured dry into the columns. The column bed volume was 2.5 ml, and its void volume was approximately 0.6 ml. The surface area of the beads was about 200 cm 2.

In-situ Coating of Glass Bead Columns with Polymer The homopolymers, after the purification steps described above, were placed in chloroform to form 1% (w/v) solutions. Each solution was drawn up vertically through the packed column from a reservoir under vacuum applied through a syringe, with observation of the column of beads by transillumination to assure that no air bubbles were entrained. When the meniscus of the solution had risen above the top of the bed of beads, the vacuum was broken and the solution in the column was allowed to drain back under gravity into a receiver. After a drainage time of approximately 30 sec, flow of argon was commenced through each of the columns at a rate of approximately 5 cma/ min, the columns being held at a temperature of approximately 30°C. The argon sweep was continued for 3 days to insure total removal of all solvent. The columns were then capped at the ends and stored at room temperature until used.

Exposure of Blood to Polymer Surface Using a modification of methods described elsewhere (1), blood was taken six times from each of six donors and was anticoagulated with sodium citrate (3.8 mg/ml final cone.). At least a 7-day interval was observed between phlebotomies. Each experiment tested six bead columns simultaneously (three pairs

BLOOD/ACRYLATES,METHACRYLATES of polymer surfaces with and without plasma precoating). Pyrogen-free sterile saline (70 ml, 0.154M) was pumped from below through each of the six columns at 1.0 ml/ min. One of each pair of columns was exposed to 5.0 ml of fresh, autologous plateletpoor plasma (PPP; platelet count < 5000/ mm 3) at 0.25 ml/min, followed by an additional 12 ml of saline (1.0 ml/min). After imipramine (0.025 mM) was added to citrated blood which had previously been incubated for 1 hr at 37°C with [14C]serotonin, six 12ml syringes were filled with blood and placed in a Harvard pump. Three 2.0-ml blood samples were set aside as controls. The whole blood (kept at 37°C) was pumped through the columns at 0.25 ml/min. Four sequential 1.0-ml aliquots were collected from each column after discarding the first millimeter that emerged from the efferent tubing.

Testing of Effluent Samples and the Control Blood Samples Platelet counts on control samples and each of the four aliquots from each column were done using a Technicon platelet counter (Technicon Corp., Tarrytown, N. Y.). The control samples and one pooled sample (0.335 ml from each of the four fractions) for each column was centrifuged for PPP, which was then frozen and stored for further analysis. Duplicate PPP samples were counted for 14C in a liquid scintillation counter. After a 40fold dilution, each sample of plasma was assayed for/3-thromboglobulin (/3-TG) levels (2) by RIA with a kit supplied by Amersham/ Searle (IM.88, Arlington Heights, Ill.). Platelet-factor-4 (PF4) determinations were performed by the method of Handin et al. (3).

Statistical Analysis Data organization and analysis were performed on the Prophet system, a national computer resource sponsored by the Chemical/Biological Information Handling Program, National Institutes of Health.

313

RESULTS

Characterization of Polymers 1. Determination of Tg. Following exhaustive drying under argon, a small sample of the bulk polymer was submitted to differential scanning calorimetry (DuPont II) and scanned over the range 150 to 400°K. The glass transitions Tg and, in certain cases, side-chain melting point Tm, were thereby determined as peaks in the conventional manner. 2. Purity of the polymer, molecular weight. Homopolymer samples were dissolved (1%, w/v) in chromatography-grade chloroform. Samples were injected into a

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series of polymers plotted vs length of the alkyl side chain (A) or glass transitiontemperature(T~)(B). For code of abbreviations, see Table I. For PLA, Tgis an approximate estimate. Journal of Colloid and Interface Science, Vol. 81, No. 2, June 1981

314

BRIER-RUSSELL

Waters Associates AC/GPC 204 Chromatograph, using five Styragel columns in series (106, 105, 104, 103, 102). Lack of oligomeric or micromolecular contaminants was confirmed by absence of peaks at such positions in the chromatograms. For most of the polymers, the polydispersity index, Mw//f/n, was around 2.2 and the number average molecular weight was approximately 60,000. For the acrylate and methacrylate monomers studied in this work, the solvent transfer coefficient, Cs, is small enough so that in all of these polymers it is estimated that not more than 1 out of 10 polymer chain ends could contain a fragment derived from the solvent (methylethyl ketone).

3. Evidence for completeness of coating.

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Journal of Colloid and Interface Science, Vol. 81, No. 2, June 1981

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columns packed with the glass spheres were coated with polymer solutions of concentration equal to that used here (1% by weight for 1, 2, 3, 4, and 5 passes), followed by intermediate total evaporation of the solvent (chloroform) under dry argon at room temperature. Scanning electron microscopy (SEM) demonstrated that, even after the first coating, a continuous layer of polymer was deposited as a film around each of the spherical glass beads, this coating rounding gently at the point of contact between contiguous beads. As none of the polymers studied is significantly water absorbent, no problem was encountered in swelling and buckling of the polymer coatings on the beads.

BLOOD/ACRYLATES, METHACRYLATES

Reactivity with Platelets Platelet retention. The platelet recovery

index (r) for each of four successive aliquots of blood issuing from each column was determined by dividing the platelet count in the effluent blood by the platelet count of the control (unexposed) blood. A number near 0.0 indicated a high degree of platelet retention by the column, and a number near 1.0 indicated that there was little platelet interaction with the surface. Three-way analysis of variance with replication established that the variation of ? from aliquot to aliquot was insignificant compared to variation among blood donors. In the results presented below, a twice averaged value of platelet recovery index, ?, is used. Mathematically stated: d=6 a=5

= ¼"1/6 Y, ra,d, a=2 d=l

where a is the number of the aliquot, d is the number of donors.

315

It is this averaged value that is shown as the ordinate in Figs. 1-3, plotted against alkyl chain length (Fig. IA, 2A, 3A), or against glass transition Tg (Figs. 1B, 2B, 3B). Together, these figures show that ~ is maximum for the shortest alkyl chains (methyl) and decreases as chain length increases; i.e., that ~ increases as Tg increases, being maximum at the highest Tg of each series, and that the acrylate series are approximately equivalent to the methacrylate at equal chain length but are very much blander than the methacrylate series (higher ?) when compared at equal Tg over the range of overlap o f Tg.

The rank order of these 14 polymers in the two series is shown in Table I. Platelet secretion. A measure of platelet reactivity with polymer surfaces is the extent of platelet secretion induced by the encounter, i.e., levels of serotonin, fl-thromboglobulin (fi-TG), and platelet factor 4 (PF4) in the effluent plasma. The extent of release of serotonin (5HT) from platelets previously labeled with [14C]5HT is ex-

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X = p < 0.05 (Duncan's multiple-range test). For example, ? of PBMA is significantly different from PAMA but not from PBA. No te. P means poly-; LMA, lauryl methacrylate; DA, n-decylacrylate; HA, n-hexylacrylate; AMA, n-amylmethacrylate; BA, n-butylacrylate; DMA, n-decylmethacrylate; PMA, n-propylmethacrylate; BMA, n-butylmethacrylate; LA, laurylacrylate; PA, n-propylacrylate; EMA, ethylmethacrylate; MMA, methylmethacrylate; EA, ethylacrylate; MA, methylacrylate. Journal of Colloid and Interface Science, Vol. 81, No. 2, June 1981

316

BRIER-RUSSELL ET AL.

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pressed as a ratio of cpm in effluent plasma to cpm in control plasma (the higher the value, the greater the release of 5HT). 5HT ratios less than 1.0 are real and are probably the result of continued uptake of 5HT by platelets retained in the column, which is incompletely blocked by imipramine added to the blood before the experiment. Figure 4A shows the secretion of [14C]5HT; Fig. 4B presents data for fl-TG secretion, and Fig. 4C, for PF4 for the methacrylate series. There is a correlation with length of the alkyl side chain, which is best demonstrated by the fl-TG results. The correlation is analogous to that obtained with ?. Data for the acrylate series (not shown) are similar.

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Chain length FIG. 4A. Secretion of 5-hydroxytryptamine (serotonin; 5HT) by platelets exposed to the methacrylate series of polymers, plotted vs alkyl side-chain length. Results are expressed as a ratio of t4C cpm in the effluent plasma to 14C cpm in a control sample. FIG. 4B./3-Thromboglobulin (~-TG) secretion plotted vs alkyl side-chain length. FIG. 4C. Platelet-factor-4 (PF-4) secretion plotted vs side-chain length. Journal of Colloid and Interface Science, Vol. 81, No. 2, June 1981

that preliminary exposure of polymethylacrylate to platelet-poor plasma reduces the reactivity of the polymer with platelets upon subsequent exposure to whole blood in vitro (4) or in vivo (5). This behavior is not shared by other polymers, including polyurethanes and polystyrene. Passivation by preincubation with plasma proved to be a feature of all the acrylate and methacrylate series studied (Figs. 5A and 5B). All members of the methacrylate series achieved approximately the same extent of platelet reactivity when subjected to such treatment, and so did the acrylates, although in the latter case the blandness of PMA and PEA prior to plasma pretreatment limited the increment in platelet reactivity produced by plasma pretreatment. DISCUSSION

The search for a unifying theoretical base for the development of thromboresistant surfaces has led to consideration of a long list of properties to account for the consequences of blood-surface interaction, including water wettability, surface charge, surface free energy, topography or roughness, the balance between hydrophobic and hydrophilic groups, regular order of the structure,

317

BLOOD/ACRYLATES, METHACRYLATES

chain mobility, crystallinity, the capacity for ionic and hydrogen bonding and hydrophobic interactions, and the presence of specific chemical groups at the surface. At present, there is no general agreement as to which chemical or physical properties determine the behavior of artificial materials in contact with blood. This study examined two similar polymer series, the acrylates and methacrylates, modified by various alkyl side-chain substitutions which thereby changed the polymer Tg and the possible extent of hydrophobic interactions. We found a striking inverse correlation between the platelet reactivity and the glass transition temperature of the polymers as well as a direct correlation of the reactivity with platelets and length of the polymer alkyl side chain. Preliminary exposure of all these polymers to plasma was observed to decrease the reactivity of the surface with platelets upon subsequent exposure to whole blood. The glass transition temperature is an expression of the temperature above which the polymer main chain (backbone) becomes rubbery and mobile and loses stable van der Waals associations with nearby atomic groups. That molecular rigidity rather than backbone mobility favors passivity towards platelets, which would be one interpretation of these results, seems contrary to intuition. It is more likely that the correlation of platelet reactivity with Tg reflects two parallel phenomena having no causal relationship. A detailed examination of platelet reactivity with segmented polyurethanes (SPU) (6) showed that increasing hydrophilicity resuited in decreased reactivity with platelets, so long as the polymer structure did not include formation of hydrogen bonds or other polar interactions. It is likely that an increase in the side-chain length of the acrylates and methacrylates increased their capacity for hydrophobic interactions, presumably with adsorbed plasma proteins, which might account for increased platelet reactivity to these polymers. There is no obvious gen-

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Chain length FIG. 5A. Fassivation of polymers of the acrylate series by preliminary exposure to platelet-poor plasma (PPP) before whole blood; ? vs alkyl-sidc-chain length. FIG. 5B. Effect of PPP pretreatrnent on the mcthacrylate series.

eral relationship between Tg and a polymer's capacity for hydrophobic interactions, so the correlation of platelet reactivity to Tg may be an epiphenomenon. In any case, neither the side-chain length nor Tg is itself the sole determinant of thromboresistance, for the acrylate and methacrylate series do not lie on the same regression line in these experiments. Not all polymers become less reactive with platelets if exposed to plasma prior to blood, as the acrylates and methacrylates do. Of 20 varieties of SPU studied, not one showed this behavior (6). Polystyrene was also unaffected by plasma pretreatment (4). In studies of methylacrylate/styrene coJournal of Colloid and Interface Science, Vol. 81, No. 2, June 1981

318

BRIER-RUSSELL ET AL.

p o l y m e r s (7), the r e s p o n s e to p l a s m a pretreatment paralleled the relative methylacrylate fraction of the c o p o l y m e r . The p h e n o m e n o n of p l a s m a passivation presumably involves selective adsorption of specific plasma proteins by methacrylate and acrylate surfaces or else a particular alteration of adsorbed protein once resident on the surface, but the nature of this effect is unknown. The roughly parallel b e h a v i o r of platelet retention in the bead columns and platelet secretions of serotonin,/3-thromboglobulin, and platelet factor 4 indicates that variations in reactivity of the different p o l y m e r s have effects that extend b e y o n d simple platelet adhesion. Retention of platelets m a b e a d column m a y result f r o m adhesion of single platelets to the beads or f r o m formation of platelet aggregates. The latter p h e n o m e n o n is m o r e closely associated with secretion of platelet constituents. The implications of this relationship are discussed in detail elsewhere (1).

Journal of Colloidand Interface Science, Vol.81, No. 2, June1981

ACKNOWLEDGMENTS This work was supported by Grants HL 20079 and HL 25066 of the NHLBI and by Grant RR-01032 from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health. The advice of Professor David Waugh and Dr. Bernard Ransil is gratefully acknowledged. REFERENCES 1. Lindon, J. N., Rodvien, R., Brier, D., Greenberg, R., Merrill, E., and Salzman, E. W . , J. Lab. Clin. Med. 92, 904 (1978). 2. Ludlam, C. A., Moore, S., Bolton, A. E., Pepper, D. S., and Cash, J. D., Thromb. Res. 6, 543 (1975). 3. Handin, R. I., McDonough, M., and Lesch, M., J. Lab. Clin. Med. 91, 340 (1978). 4. Salzman, E. W., Lindon, J., Brier, D., and Merrill, E. W . , Proc. N. Y. Acad. Sci. 283, 114 (1977). 5. Lindon, J. N., Collins, R. E. C., Coe, N. P., Jagoda, A., Brier-Russell, D., Merrill, E. W., and Salzman, E. W., Circ. Res. 46, 84 (1980). 6. Sa Da Costa, V., Brier-Russell, D., Trudel, G., Wolfe, L., Waugh, D., Salzman, E. W., and Merrill, E. W . , J. Colloid Interface Sci. 76, 594 (1980). 7. Merrill, E. W., Meeting of the American Institute of Chemical Engineers, November 17-20, 1980, Chicago, Illinois, paper #266.