adsorption of complement protein c3 at polymer surfaces

JOURNAL OF COLLOID AND INTERFACE SCIENCE 179, 163–172 (1996)ARTICLE NO. 0198

Adsorption of Complement Protein C3 at Polymer Surfaces

MARTIN MALMSTEN,* ,1 BO LASSEN,* JERKER WESTIN,† CARL-GUSTAF GOLANDER,‡ROLF LARSSON,† AND ULF R. NILSSON†

*Institute for Surface Chemistry, P.O. Box 5607, S-114 86 Stockholm, Sweden; †Department of Clinical Immunology and Transfusion Medicine,University Hospital, S-751 85 Uppsala, Sweden; and ‡Pharmacia AB Pharmaceuticals Uppsala, S-751 82 Uppsala, Sweden

Received July 3, 1995; accepted September 15, 1995

e.g., the liver and spleen, and problems related to extracorpo-The adsorption of C3 at poly(methyl methacrylate) (PMMA) real therapy (1–5).

and poly(styrene) (PS) surfaces was investigated with in situ ellip- The complement system, which is a major contributor tosometry and compared to that at (hydrophilic and negatively

the undesired effects mentioned above, directs its effectscharged) silica and (hydrophobic) methylated silica. The adsorp-against, e.g., biomaterials as a manifestation of a generaltion of C3 at PMMA was higher than that at PS, while the adsorbedphysiological role to detect and destroy various foreign enti-layer thickness was the same for the two surfaces. For both PMMAties, such as invading materials, microorganisms, and para-and PS the adsorbed layer thickness (10 { 2 nm) correspondssites (6) . Complement activation may proceed through ei-rather closely to that of end-on oriented C3 molecules. The adsorp-

tion of C3 at PMMA and PS was found to be intermediate between ther the classical or alternative Pathways. Classical pathwaythat at silica and methylated silica, although the adsorbed layer activation is typically initiated by immunoglobulins (IgGthickness was similar for all surfaces. The competitive adsorption and IgM) and complement component C1q. This leads toamong C3, human serum albumin (HSA), and factor B was inves- adsorption and activation of complement component C3 ontigated with ellipsometry and total internal reflection fluorescence the target surface. Alternative pathway activation as definedspectroscopy (TIRF). Addition of HSA after C3 preadsorption

on biological target surfaces involves an initial nonspecificresulted in fractional C3 desorption for both PMMA and PS. Fac-deposition of low levels of C3b, which subsequently triggerstor B deposition at PS after preadsorption of C3 and blockinga proteolytic activation cascade of the system. Both path-with HSA was found to be largely due to specific binding to C3/ways promote the assembly of target-bound enzymes whichC3b, while in the case of PMMA, factor B was largely accumulatedcleave C3 into its biologically active fragments, C3a andthrough passive (displacement) adsorption. q 1996 Academic Press, Inc.

Key Words: adsorption; C3; complement; complement activa- C3b. A covalent interaction is triggered between a thiolestertion; ellipsometry; polymer; poly(methyl methacrylate) ; poly(sty- group exposed on C3b and nucleophilic (e.g., hydroxyl orrene) . amino) surface groups related to molecules such a proteins,

carbohydrates, or lipids. Complex mechanisms of regulationof the binding of C3b and its participation in the formation

INTRODUCTION of a C3-cleaving enzyme (C3 convertase) are recognizedand include host cell membrane proteins (complement re-

Polymers are frequently used in a number of biomedical ceptor 1, membrane cofactor protein, decay accelerating fac-applications including drug delivery, biomaterials, solid tor) and plasma proteins (B, D, H, I) . This regulatory systemphase diagnostics, and extracorporeal therapy (1, 2) . In most constitutes the essential mechanism for the recognition func-of these applications the polymer surface interacts with a tion of the alternative pathway.complex mixture of proteins. The initial adsorption from the In view of the apparent importance of complement activa-protein mixture and the resulting adsorbed layer structure tion in a variety of biomedical applications involving poly-and composition initiates complex biochemical /biophysical mer materials, as well as the importance of the interactionprocesses which determine the biological response to the between complement components and surfaces, the authorspresence of the surface. Examples of such responses include undertook the present study. In particular, since C3 plays afrequently observed inflammations related to the use of bio- central role in both the classical and alternative pathwaysmaterials, premature removal of colloidal drug carriers from we investigate the state of adsorption (adsorbed amount,circulation in bloodstream and subsequent accumulation in, adsorbed layer structure and formation, competitive adsorp-

tion) of this protein at poly(methyl methacrylate) (PMMA)1 To whom correspondence should be addressed. and poly(styrene) (PS), two polymers frequently used in

163 0021-9797/96 $18.00Copyright q 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

AID JCIS 4041 / 6g0d$$$221 03-11-96 11:22:10 coidas AP: Colloid

164 MALMSTEN ET AL.

biomedical applications. In doing so we use the techniques were then kept in ethanol until use. Just prior to use, theslides were rinsed twice successively with ethanol and water,in situ ellipsometry (7) and total internal reflection fluores-

cence spectroscopy (TIRF) (8) . followed by drying with filtered nitrogen (Aga, Sweden).Methylated silica surfaces were prepared from silica surfacesby double rinsing subsequently with water, ethanol, and tri-EXPERIMENTALchloroethylene (pro analysi, Merck), followed by treatment

Materials with 0.1 wt% Cl2(CH3)2Si (Merck) in trichloroethylene for90 min (11). Finally, they were rinsed four times in trichlo-

Water was purified by a Milli-RO 10PLUS unit (Millipore roethylene and ethanol. This procedure rendered the slidesCorpn., U.S.A.) , including depth filtration, carbon adsorp- hydrophobic, with an advancing and receeding contact angletion and decalcination preceeding reverse osmosis. Subse- of 957 and 887, respectively. They were then stored in ethanolquently, it was led through a Milli-Q PLUS185 unit (UV until use.light (185 and 254 nm)) and a Q-PAK unit consisting of an Polymer-coated surfaces were prepared by spin-coatingactive carbon unit, a mixed bed ion exchanger, an Organex onto silica surfaces. This was performed by dissolving thecartridge, and a final 0.22-mm Millipak 40 filter. (pure) polymers, i.e., poly(methyl methacrylate) (PMMA;

Complement factor 3 (C3) and factor B were prepared as from intraoccular lenses, Pharmacia, Sweden) and polysty-described previously (9, 10). C3 has a molecular weight of rene (PS; from microtitre plates, NUNC, Denmark) in tolu-approximately 185 kD and is composed of two disulphide ene (0.8 and 0.6 wt% for PMMA and PS, respectively) .linked polypeptide chains of 75 and 110 kD (6). In keeping The solution was spun at 4,000 rpm for 30 sec, whereafterwith its central role in complement activation C3 serum the surfaces were dried at 557C in vacuum for one minute.concentration (1000–1350 mg/liter ; 1 mg/liter Å 1 ppm) This procedure resulted in polymer films with an approxi-is much greater than that of other complement proteins. Fac- mate thickness of about 30 { 5 nm, as obtained from ellip-tor B has a molecular weight of 90 kD and a serum concen- sometry.tration of 180–250 mg/liter (6) . Human serum albumin For the TIRF experiments, microscope slides from boron(HSA), containing fatty acids, but globulin-free, lyophi- silicate were used instead of oxidized silicon slides. Thelized, and crystallized, was obtained from Sigma Chemical reason for this is that while ellipsometry requires highlyCo., USA. reflective surfaces, TIRF requires transparent ones. How-

For the TIRF experiments, the labeling of HSA and factor ever, the surface regions of these materials are essentiallyB was carried out with fluorescein isothiocyanate (FITC; identical from both a physical (wettability, charge) and aIsomer I, Molecular Probes, USA). The labeled proteins chemical (presence of silanol groups) point of view. Further-were purified from unbound FITC by dialysis. The molar more, identical coating procedures were used for the tworatio of FITC to protein was approximately unity. The effect materials.of the labeling on the interfacial behavior of HSA was pre-viously investigated by surface tension measurements using Methodsthe pendant drop method, and no effects were found on the

Ellipsometrysurface tension up to 1.5 bound FITC groups per proteinmolecule (48). Ellipsometry measurements were all performed in situ by

Chemicals for the buffer preparations were of analytical means of null ellipsometry (7) , using an automated Rudolphgrade and used without further purification. thin-film ellipsometer, type 436, controlled by a personal

computer. A xenon lamp filtered to 4015 A was used as theSurfaceslight source. A thorough description of the experimentalsetup has been given previously (12). Prior to adsorption,Silica surfaces were prepared from polished silicon slides

(p-type, boron-doped, resistivity 7–13 Vcm; Okmetic, Fin- the ellipsometry measurements require determination of thecomplex refractive index of the substrate (7) . In the caseland). In short, these were oxidized thermally in pure and

saturated oxygen, followed by annealing and cooling in of a layered substrate such as oxidized silicon a correctdetermination of the adsorbed layer thickness and mean re-argon flow to generate an oxide layer thickness of about 30

nm. The slides were then cleaned in a mixture of 25% fractive index requires determination of the silicon bulk com-plex refractive index (N2 Å n2 0 ik2) as well as of theNH4OH, 30% H2O2, and H2O (1:1:5, by volume) at 807C

for 5 min, followed by cleaning in a mixture of 32% HCl, thickness (d1) and the refractive index (n1) of the oxidelayer. This is done by measuring the ellipsometric parame-30% H2O2, and H2O (1:1:5, by volume) at 807C for 5 min.

The slides were then rinsed twice with water and ethanol. ters c and D in two different media, e.g., air and buffer.From the two sets of c and D, n2 , k2 , d1 , and n1 can beThis procedure rendered the surfaces hydrophilic, with a

water–air contact angle of less than 107. The silica slides determined separately (12–14). The methyl layer is ne-


165ADSORPTION OF COMPLEMENT PROTEIN C3

glected for the methylated silica surfaces (14). [Electropho-retic studies suggest such silanization only adds 5 A or lessto the double layer surface of shear (15, 16). Similar resultswere obtained with ellipsometry.]

For the polymer surfaces, a five-layer optical model, in-volving also the thickness (dxx) and refractive index (nxx)of the spun polymer film, has to be applied (17). This wasdone in the following way: A two ambient media (air andbuffer) measurement was made on the bare silica surface,yielding information on N2 , d1 , and n1 (vide supra) [Thesurfaces used in these measurements were not used further.]Measurements were then made on the polymer-coated sub-strate, allowing the thickness and refractive index of thepolymer film to be determined. The procedure was pre-

FIG. 1. Adsorbed amount (G) of C3 (30 ppm) at PMMA (circles) andviously found to give similar results as the use of measure- PS (triangles) from 0.01 M phosphate buffer, 0.15 M NaCl, pH 7.5.ments of c and D in three different media (air, water, andbuffer) and a simultaneous determination of all six un-

a 488-nm argon ion laser (Spectra-Physics, USA) of 0.16knowns (17). All measurements were performed by four-W was used. The flow cell used consists of a dove-tail prism,zone null ellipsometry in order to reduce effects of opticala silicon rubber gasket, which also constitutes the flow cham-component imperfections (7) .ber, and a metal support. The surface to be studied wasAfter optical analysis of the bare substrate surface, theattached to the dove-tail prism by a refractive index matchingprotein solution was added to the cuvette, and the values ofliquid (glycerol, BDH, England), whereafter the flow cellc and D recorded. [The adsorption was monitored in onewas assembled and mounted in the apparatus. The fluores-zone, since the four-zone procedure is time-consuming andcence detection system consists of a lens system, a mono-since corrections for component imperfections had alreadychromator (Jobin Yvon, France), a photomultiplier tubebeen performed.] The maximal time-resolution between two(Hamamatsu, Japan and Jobin Yvon, France), a preamplifiermeasurements is 3–4 s. Stirring was performed by a mag-(Stanford Research Systems, Inc., USA), and a photonnetic stirrer at about 300 rpm. [No effect of stirring rate iscounter (Model SR400, Stanford Research System, Inc.,observed in this range.]USA). To improve the signal-to-noise ratio, the instrumentFrom c and D, the mean refractive index (n f ) and averageis equipped with an optical chopper for lock-in amplifyingthickness (del ) of the adsorbed layer were calculated numeri-(Model SR540, Stanford Research Systems, Inc., USA). Tocally according to an optical four-layer model for silica andovercome the adverse effect of bleaching of the fluorophore,methylated silica, and a five-layer model for PMMA and PSthe sample was only illuminated during measurement, and(vide supra) (7, 12–14, 17). [Error propagation in this typethe laser intensity kept to a minimum.of analysis has been discussed previously (13).] The adsorbed

The TIRF experiments were initiated by rinsing the cellamount (G) was calculated according to de Feijter (18), usingwith buffer solution. A buffer solution containing 0.1 mg/a refractive index increment (dn/dc) of 0.18 cm3/g, 0.18 cm3/ml sodium fluorescein (BDH, England) was then introducedg, and 0.187 cm3/g for C3, factor B, and HSA, respectively.into the cell. By this procedure, the instrument could beDue to the refractive index increment similarity for these pro-properly aligned, at the same time as an internal standardteins the ellipsometry measurements for the mixed protein lay-was introduced. Subsequently, the cell was rinsed with bufferers will provide the total adsorbed amount. It was previouslysolution until no bulk fluorescence could be detected. Aftershown that both adsorbed amounts and adsorbed layer thick-this, the adsorption experiment could be initiated. [Althoughnesses obtained with ellipsometry agree well with those ob-the mass transport to the surface (mainly diffusion overtained with other methods for proteins, polymers, and surfac-an unstirred layer) is somewhat different in the TIRF andtants at model surfaces (13, 14, 19–21).ellipsometry experiments, the two types of experiments arequite comparable, since we focus on the long-term adsorp-TIRFtion behavior.]

TIRF (total internal reflection fluorescence spectroscopy)(8) was used to selectively observe the adsorption/desorp- RESULTS AND DISCUSSIONtion of HSA or factor B in the mixed protein systems. Inshort, the TIRF apparatus consists of a light source, a flow The adsorption of complement component C3 at PMMA

and PS is shown in Fig. 1. As can be seen, a limiting ad-cell, and a fluorescence detection system. As the light source,


166 MALMSTEN ET AL.

FIG. 2. Adsorbed layer thickness (del , circles) and mean adsorbed layer refractive index (n f , triangles) of C3 (30 ppm) adsorbed at PMMA (a) andPS (b) from 0.01 M phosphate buffer, 0.15 M NaCl, pH 7.5.

sorbed amount of 1.0 mg/m2 and 0.7 mg/m2 was obtained is reached after less than a few hundred seconds for bothPMMA and PS. This indicates that at PMMA and PS initiallyfor PMMA and PS, respectively. [These values correspond

to plateau values in the respective adsorption isotherm.] adsorbing C3 molecules do not ‘‘spread’’ at the surface.Instead, molecules oriented end-on seem to attach immedi-These adsorbed amounts are smaller than those expected for

a close packed layer. From the C3 molecular dimensions ately, with little conformational or orientational change. Thisis different from the behavior of C3 at both silica and methyl-(vide infra) an adsorbed amount of about 5 mg/m2 and 26

mg/m2 is expected for a close-packed layer of molecules ated silica (vide infra) .The adsorption of C3 at (hydrophilic and negativelyadsorbed side-on and end-on, respectively. Clearly, the ad-

sorbed layer is not close packed, which is also found from charged) silica and (hydrophobic) methylated silica isshown in Fig. 3. Contrary to the adsorption of, e.g., fibrino-direct measurements of the adsorbed layer refractive index

(vide infra) . Furthermore, at both PMMA and PS saturation gen, IgG, and HSA, the adsorption of C3 is much higher atsilica than at methylated silica (21, 23). This finding mayadsorption was reached after about 2000 s. The adsorbed

layer structure was further investigated by measuring the indicate that hydrophobic interactions between the proteinand the surface do not provide a strong driving force foradsorbed layer thickness (del ) and mean refractive index

(n f ) . As shown in Fig. 2, an adsorbed layer thickness of 10 adsorption. [Note, however, that a low adsorbed amount notnecessarily implies a weak macromolecule–surface interac-{ 2 nm was observed for both PMMA and PS. This indicates

that C3 adsorbs in a similar conformation and orientation at tion (cf, e.g., polyelectrolyte adsorption at oppositelycharged surfaces at low electrostatic screening (49).] Thethe two surfaces. Previous small-angle neutron and X-ray

scattering results indicate that C3 in aqueous solution has a high adsorption at silica, on the other hand, indicates thateither the surface negative charge or the surface hydroxylstructure which could best be approximated with an elliptical

cylinder of 11-nm length and cross-section semi-axes of 5.6 groups give rise to a strong driving force for adsorption. [Itwas previously found that proteins with a net positive charge,and 2.1 nm (22). Thus, the ellipsometrically obtained thick-

nesses indicate that at both PMMA and PS, C3 molecules e.g., lysozyme (21), and proteins rich in basic amino acids,e.g., apolipoprotein B (24), adsorb more extensively at silicaoriented end-on are present in the adsorbed layer, although

coexistence of other orientations cannot be excluded from than at methylated silica.] Another interesting feature seenfrom Fig. 3a is that while the adsorption at silica is ratherthe present results.

Since the adsorbed amount of C3 is slightly higher at slow, that at methylated silica is very fast. The differencesin the adsorbed amount and the adsorption kinetics suggestPMMA than at PS, while the adsorbed layer thickness is

similar for the two surfaces, the adsorbed layer protein con- different adsorption mechanisms at the two surfaces.As can be seen from Fig. 3b, the adsorbed C3 layer reachescentration is higher at the former surface. Using the bulk

refractive index increment, we find that the average adsorbed a saturation thickness of 12–15 nm on both silica and meth-ylated silica. [The larger scattering in the data obtained forlayer protein concentration is 0.10 and 0.07 g/cm3 for

PMMA and PS, respectively. An interesting feature of Fig. C3 at methylated silica is due to the much lower adsorbedamount at this surface (Fig. 3a) .] In analogy to PMMA and1 and 2 is that while both the adsorbed amount and the

adsorbed layer mean refractive index increase during the PS, this indicates that at both silica and methylated silicaC3 molecules adsorbed end-on are present in the adsorbedfirst 2000 s of adsorption, a limiting adsorbed layer thickness



FIG. 3. (a) Adsorbed amount (G) , (b) adsorbed layer thickness (del ) , and (c) mean adsorbed layer refractive index (n f ) of C3 (Ceq Å 60 ppm) atsilica (open circles) and methylated silica (filled circles) from 0.01 M phosphate buffer, 0.15 M NaCl, pH 7.5.

layer, although coexistence of other orientations cannot be analogous to the behavior at PMMA and PS, and results ina lower adsorbed amount, but in a faster adsorption.excluded from the present results.

Since the adsorbed amount of C3 at silica is much higher From the considerations above, we conclude that C3 ad-sorbs at PMMA and PS in an amount intermediate betweenthan that at methylated silica, while the adsorbed layer thick-

ness is similar at the two surfaces, the average protein con- that at (hydrophilic and negatively charged) silica and (hy-drophobic) methylated silica. Since the wettability ofcentration in the adsorbed layer is higher at the former sur-

face. This is illustrated in Fig. 3c, showing the mean ad- PMMA (ua É 65–757) (25, 26) and PS (ua É 80–907) (26,27) is intermediate between that for silica (ua õ 107) andsorbed layer refractive index during the adsorption. Using

the bulk refractive index increment, we find that the average methylated silica (ua Å 957) , as is the charge density, anadsorption intermediate between that at silica and methylatedprotein concentration in the adsorbed layer is about 0.23 and

0.03 g/cm3 for silica and methylated silica, respectively. silica is expected. Furthermore, since PMMA and PS resem-ble methylated silica more than silica it is expected that theSimilar to the adsorbed amount, the mean adsorbed layer

refractive index at methylated silica reaches its saturation adsorbed amount at these surfaces resemble that at the for-mer more than that at silica. The finding of a higher amountvalue at adsorption times less than a few hundred seconds,

while for silica both the adsorbed amount and the mean C3 adsorbed at PMMA than at PS may indicate that thePMMA negative surface charge, apart from the hydrophobicadsorbed layer refractive index increase up to about 3000 s.

These findings indicate that at silica C3 molecules are rather character of the surface, may be a contributing factor to theC3 adsorption at this surface. [The PMMA surface carriesmobile and can pack closer over time, resulting in a higher

adsorbed amount and a slower adsorption. At methylated a negative surface charge due to fractional hydrolysis toform methacrylic acid residues (26, 28).]silica, on the other hand, adsorbed C3 molecules appear to

be essentially ‘‘immobilized’’, as packing density does not There have been few previous studies of the independentadsorption of C3 at well defined surfaces. However, Elwing etincrease with time after a few hundred seconds. This is partly


168 MALMSTEN ET AL.

layers at PMMA and PS containing at least a fraction ofmolecules adsorbing essentially end-on, it undergoes confor-mational changes at both surfaces. The magnitude of theadsorption isotherm hysteresis is related to the entropy gen-erated on adsorption. Although we have no detailed informa-tion on the adsorption isotherm hysteresis in the presentsystems, the finding of a more limited desorption of C3 atPMMA than at PS may indicate either different or moreextensive conformational changes at PMMA.

The adsorption of several serum proteins at PMMA andPS was studied previously by Hasegawa and Kitano (26).Using TIRF, these authors found a significant adsorptionat both PMMA and PS. Furthermore, the adsorption wasessentially irreversible since scarcely any of the protein ad-FIG. 4. Total adsorbed amount difference (DGtot ) on rinsing (100 mlsorbed was removed on rinsing with buffer (26). A limitedover 10 min initiated at (a)) after preadsorption of C3 at PMMA (circles)

and PS (triangles) for 30 min. The adsorption was performed from 0.01 or slow desorption at PMMA was found for BSA by ChengM phosphate buffer, 0.15 M NaCl, pH 7.5. et al. (36). These results are thus in qualitative agreement

with the present findings of a largely irreversible adsorptionat PMMA and PS.al. studied serum complement deposition at methylated silica,

using ellipsometry, and found significant (9 mg/m2) anti-C3 A study of the competitive protein adsorption at polymersurfaces in C3-containing systems was prompted by our at-antibodies deposition after preincubation with human sera (29).

Elwing et al. also found that C3 adsorbed more extensively at tempts to test the feasability of analysing with ellipsometryand TIRF the alternative pathway activating potential of ad-methylated silica than at silica (at most about 4 and 2 mg/m2,

respectively) (30), and that preadsorption of IgG caused a sorbed C3. Therefore, after the rinsing step discussed in theprevious paragraph, HSA (200 ppm) was added and thepronounced increase in adsorption of serum proteins or anti-

C3 (31). On the other hand, Lui and Elwing found that the change in the total adsorbed amount followed. As can beseen in Fig. 5, the HSA addition results in a reduction iniC3b content in sera contacted with hydrophilic glass was much

higher than that in sera contacted with hydrophobic glass (32). the total adsorbed amount for both PMMA and PS. Thischange in the total adsorbed amount indicates the presence ofNilsson-Ekdahl et al. studied the complement activation at ra-

dio frequency (rf) plasma modified polystyrene and found that competitive adsorption processes. In order to obtain furtherinformation on this we performed TIRF experiments, whereC3 deposition depended on the surface characteristics (27).

Hydroxyl functional surfaces displayed a higher degree of C3 HSA was fluorescence-labelled with FITC, whereas the pre-adsorbed C3 was unlabelled. From Fig. 6, we see that atdeposition than methyl functional ones. Furthermore, carboxyl

functional surfaces resulted in a higher C3 deposition than PS. addition of HSA, the fluorescence intensity increases rapidly.Untreated PS micro titre plates were found by Golander et al.to display a significant C3 adsorption (33). C3 adsorption atPS latex particles was found also by Norman et al., althoughno attempts were made to quantify the adsorption (34). Al-though the experimental conditions vary rather extensively inthese studies, and although these reports involved complicatedsystems, precluding a detailed comparison, they support ourfinding of the ability of both PS, PMMA, silica, and methylatedsilica to appreciably adsorb C3.

On dilution with buffer after C3 adsorption at PMMA andPS for 30 min, there is a slight desorption for both surfaces(Fig. 4) , although the effect is slightly more pronounced forPS than for PMMA. This indicates that for both surfaces butPMMA in particular C3 is quite firmly attached regardingdilution. It was previously shown that adsorption isotherm

FIG. 5. Total adsorbed amount difference (DGtot ) on addition of HSAhysteresis behavior on cycling the equilibrium concentration(200 ppm added at (a)) after preadsorption of C3 at PMMA (circles) and

necessitates an entropy generation during adsorption, most PS (triangles) for 30 min and subsequent rinsing (100 ml over 10 min).likely achieved by an interfacial conformational change The adsorption was performed from 0.01 M phosphate/5 mM barbiturate

buffer, 0.15 M NaCl, pH 7.5.(35). Thus, we conclude that although C3 forms adsorbed



on addition of factor B, whereas essentially no change inthe total adsorbed amount was observed for PMMA.

An example of the effects of passive factor B adsorptionis shown in Fig. 8a. While the adsorption of factor B atmethylated silica after the treatment described above is com-parable to that found for PMMA and PS, the adsorbedamount increase on addition of factor B is quite pronouncedin the case of silica. This increase in the adsorbed amountis achieved at an essentially constant adsorbed layer thick-ness, by only increasing the adsorbed layer protein concen-tration (Fig. 8b).

Contrary to hydrophobic surfaces such as methylated sil-ica, many proteins, and in particular HSA, do not adsorb

FIG. 6. Fluorescence intensity versus time. C3 (30 ppm) was adsorbed extensively at silica (21). More importantly, although HSAat PMMA (circles) and PS (triangles) from 0.01 M phosphate buffer, 0.15 is quite effective in ‘‘blocking’’ hydrophobic surfaces fromM NaCl, pH 7.5, followed by rinsing (100 ml over 10 min) with 0.01 M further protein adsorption through an irreversible adsorptionphosphate/5 mM barbiturate buffer, 0.15 M NaCl, pH 7.5, and addition (at

involving grand conformational changes (37, 39, 40), thistime zero) of fluorescence labeled HSA (200 ppm). After an adsorptionis not the case for hydrophilic surfaces such as silica (41).time of 2000 s, rinsing was initiated. The equilibrium concentration after

rinsing was less than 1 ppm. Thus, at silica preadsorbed HSA is easily displaced by pro-teins adsorbing with a higher surface affinity. Generally, acomplete exchange cascade initiated by the adsorption and

Thus, particularly for PMMA, HSA adsorption occurs. Since subsequent displacement of HSA, usually referred to as thethe total adsorbed amount decreases somewhat during this Vroman effect, is observed for hydrophilic surfaces (42–process this means that a fraction of the preadsorbed C3 45). The notion of the high factor B adsorption at silicamolecules are desorbed at HSA adsorption. [Unfortunately, being an effect of displacement of particularly HSA, but alsosince C3 is quite labile (6) , FITC labeling of this protein C3, is further supported by Fig. 8c, showing that the factorwas precluded and the amount C3 present in the mixed ad- B adsorption is roughly twice as high in the absence thansorbed layer could therefore not be followed directly in a in the presence of preadsorbed C3. Thus, these results willsimilar manner as for HSA.] A further interesting aspect of be discussed no further.these experiments is that at dilution after HSA adsorption, While the passive adsorption of factor B at silica is quitethere is a further reduction in the total adsorbed amount (not pronounced that at methylated silica, PMMA, and PS isshown) at the same time as there is a reduction in the amountHSA adsorbed (Fig. 6) . In particular, there is a completedesorption of HSA in the case of PS, while at PMMA, afraction of the HSA is irreversibly adsorbed. Once more,this indicates that although the adsorbed layer structure, andin particular the adsorbed layer thickness, is comparable forPMMA and PS, C3 and HSA adsorb differently at these twosurfaces.

After preadsorption of C3, rinsing, HSA adsorption, anda second rinsing step, factor B was added. Factor B is partici-pating in complement activation through complex formationwith C3/C3b. The specific binding of factor B to pread-sorbed C3/C3b thus constitutes a measure on the degree ofC3 activation. Consequently, we investigated the adsorptionof factor B to the system described above. Analogous tothe standard procedure in immunoassays, HSA was used to FIG. 7. Total adsorbed amount difference (DGtot ) on addition of factor‘‘block’’ surface regions not covered by C3 (37, 38), B (60 ppm) after adsorption of C3 (30 ppm) at PMMA (circles) and PS

(triangles) followed by rinsing (100 ml over 10 min), adsorption of HSAthereby preventing the non-specific adsorption of factor B,(200 ppm) and a second rinsing (100 ml over 10 min). The adsorption ofwhich would otherwise preclude detailed interpretation offactor B was performed from 0.01 M phosphate/5 mM barbiturate buffer,

the degree of C3 activation. The results from these experi- 1 mM NaCl2 , 0.15 M NaCl, pH 7.5, while the other steps were carried outments are illustrated in Fig. 7. As can be seen the total in the absence of NiCl2 , and the C3 adsorption also in the absence of

barbiturate.adsorbed amount at PS increases somewhat (É0.1 mg/m2)


170 MALMSTEN ET AL.

FIG. 8. (a) Total adsorbed amount difference (DGtot ) on addition of factor B (60 ppm) after adsorption of C3 (30 ppm) at silica (open circles) andmethylated silica (filled circles) followed by rinsing (100 ml over 10 min), adsorption of HSA (200 ppm), and a second rinsing (100 ml over 10 min).Shown also is (b) the adsorbed layer thickness (del , circles) and mean adsorbed layer refractive index (n f , triangles) on addition of factor B, as well as(c) the total adsorbed amount difference in the presence (circles) and absence (triangles) of C3 preadsorption (silica) . The adsorption of factor B wasperformed from 0.01 M phosphate/5 mM barbiturate buffer, 1 mM NiCl2 , 0.15 M NaCl, pH 7.5, while the other steps were carried out in the absenceof NiCl2 , and the C3 adsorption also in the absence of barbiturate.

much lower, at least judging from the total adsorbed amount comparison between Figs. 9a and 9b we infer that at themost 30% of the factor B accumulated at PMMA in theincrease observed on factor B addition (Fig. 7 and 8). In

order to obtain further information on the relative importance presence of preadsorbed C3 is due to specific binding, whilefor PS the corresponding value is about 85%. Thus, it ap-of passive adsorption and specific binding of factor B, we

performed two sets of experiments, where PMMA and PS pears that although the amount of factor B accumulating atthe surface after C3 adsorption is comparable for PMMAsurfaces where pretreated with either C3 followed by HSA

or HSA alone, whereafter the interfacial accumulation of and PS, the latter surface generates a much higher degreeof specific binding of factor B.factor B fluorescence labelled with FITC was studied with

TIRF. [The partial C3 desorption caused by HSA is expected In the complement activation cascade factor B binds to C3/C3b in a 1:1 complex. From the amount C3 preadsorbed weto result in a decrease in the amount factor B bound.] As

can be seen from Fig. 9, factor B adsorbs/binds at both would therefore expect the maximum amount factor B boundto be 0.5 and 0.35 mg/m2 for PMMA and PS, respectively.PMMA and PS surfaces both in the absence and presence

of preadsorbed C3. In the presence of preadsorbed C3, the [Estimates were made based on the molecular weight of C3.]From the considerations in preceeding paragraphs we infer thatamount factor B accumulated at the surface is approximately

the same for PMMA and PS (Fig. 9a) . In the absence of only a very small fraction of the adsorbed C3 molecules areaccessible to form these complexes at PMMA, while for PSpreadsorbed C3, on the other hand, the amount of factor B

accumulated at the surface is reduced for both PMMA and the corresponding value is equal or less than 25%.From the findings of the present investigation it appearsPS, but particularly so for the latter surface (Fig. 9b). From



FIG. 9. (a) Fluorescence intensity versus time. C3 (30 ppm) was adsorbed at PMMA (circles) and PS (triangles) from 0.01 M phosphate buffer,0.15 M NaCl, pH 7.5, followed by rinsing (100 ml over 10 min) with 0.01 M phosphate/5 mM barbiturate buffer, 0.15 M NaCl, pH 7.5, and additionof HSA (200 ppm). After an adsorption time of 2000 s, rinsing with 0.01 M phosphate/5 mM barbiturate buffer, 1 mM NiCl2 , 0.15 M NaCl, pH 7.5,was initiated, whereafter fluorescence labeled factor B (60 ppm) was added (at time zero). For PMMA and PS, rinsing after factor B adsorption wasinitiated at (a) and (b), respectively. (b) Same as in Fig. 9a but with no C3 preadsorption.

that although C3 adsorbs more extensively at PMMA than drophobic ) methylated silica. The adsorption of C3 atPMMA and PS was intermediate between that at silicaat PS the degree of specific binding is more extensive for

PS than for PMMA. [Note, however, that even the passive and methylated silica, and the adsorption at PMMAslightly higher than that at PS. The adsorbed layer thick-adsorption of factor B is essentially irreversible, since little

desorption is observed on rinsing (Fig. 9) .] For the latter ness, corresponding to that of end-on oriented C3 mole-cules, was the same for all surfaces. Ellipsometry andmaterial, little C3 activation is observed despite rather exten-

sive passive adsorption of C3. Indeed, this may be one con- total internal reflection fluorescence spectroscopy ( TIRF)showed that addition of HSA after C3 preadsorption re-tributing factor to the success of PMMA in biomaterial

applications. sulted in fractional C3 desorption for both PMMA andPS. Factor B accumulated at PS after preadsorption of C3The adsorption of C3 at PS has previously been studied

indirectly by immuno-assay techniques. For example, Nils- and blocking with HSA was found to be largely due tospecific binding to C3/C3b, while in the case of PMMA,son et al. found that when PS was incubated with human

serum C3 was deposited at the surface by both passive ad- factor B was largely accumulated through passive (dis-placement ) adsorption. The occurence of the specific fac-sorption and specific binding dependent on activation of the

classical and alternative pathways (46). Furthermore, Nils- tor B binding ability of adsorbed C3 supports the pre-viously proposed molecular mechanism underlying the al-son et al. studied conformational epitopes of C3 at PS and

concluded that deposition on PS is a combined effect of ternative pathway activating function observed for C3 atPS (46) .passive adsorption and active binding, triggered by the com-

plement activation sequences (47). The present results, al-though not directly comparable with the results obtained by ACKNOWLEDGMENTSNilsson et al., tend to support and validate these previous

Annika Dahlman is thanked for help with the surface preparations. Thisstudies. Therefore, continued studies combining ellipsometrywork was financed by Astra Arcus, Karlshamns LipidTeknik, Nycomedand TIRF with assay techniques seem promising for theImaging, Pharmacia Biosensor, Pharmacia Hospital Care, Pharmacia Phar-

further analysis of a possible early step which initiates alter- maceuticals, the Swedish Dairies’ Association (SMR), and the Swedishnative pathway activation by non-proteolytic contact activa- National Board for Industrial and Technical Development (NUTEK).tion of C3.

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adsorption of complement protein c3 at polymer surfaces

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