Langmuir 1996,11, 984-989
984
Structural Changes of Bovine Serum Albumin upon Adsorption to Modified Fluoropolymer Substrates Used for Neural Cell Attachment Studies Evan J. Bekos,t John P. Ranieri,* Patrick Aebischer,$ Joseph A. Gardella, Jr.,*vt and Frank V. Bright*>? Department of Chemistry, Natural Sciences and Mathematics Complex, State University of New York at Buffalo, Buffalo, New York 14260-3000, and Division of Surgical Research, Centre Hospitalier Universitaire de Lausanne, Lausanne University Medical School, 101 1 Lausanne, Switzerland, and Section of Artificial Organs, Biomaterials, and Cellular Technology, Brown University, Providence, Rhode Island 02912 Received January 20, 1994. I n Final Form: December 1, 1994@ In this work, we report changes in steady-state fluorescence spectra for dansylatedbovine serum albumin (FEP) (BSA) on its adsorption to a series of modified poly(tetrafluoroethy1ene-co-hexafluoropropylene) surfaces. Specifically, dansylated BSA (BSA-Dan)is adsorbed to unmodified FEP, radio frequency glow discharge plasma modified FEP (resulting in an hydroxylated surface, FEP-OH), and FEP-OH that is subsequently treated with y-(aminopropy1)triethoxysilane(resulting in an aminated surface). The emission spectra of the most strongly adsorbed BSA-Dan is red shifted ('45 nm) relative to native BSA-Dan. The emission spectrum of BSA-Dan adsorbed on amine-modified FEP is red shifted by an additional 15 nm relative to BSA-Dan adsorbed on unmodified FEP. These results suggest that the microenvironment surroundingthe dansylreporter group is altered significantlyon BSA adsorption and the microenvironments are different for BSA-Dan adsorbed to the various FEP-based surfaces. The emission spectrum of BSADan adsorbed to the amine-modified FEP surface resembled that of chemically denatured BSA-Dan on unmodified FEP. This is consistent with adsorption leading to a conformational change that is similar to the one induced by chemical denaturation. These results, suggesting conformational changes in the adsorbed BSA, provide insight into our previously published results showing selective cellular attachment of mouse neuroblastoma cells (NB2a)and rat endothelial cells onto the amine-modified FEP only in the presence of adsorbed BSA (J. Biomed. Mater. Res 1993,27, 917).
Introduction The structure and function of proteins adsorbed a t liquidsolid interfaces are of great importance in many biological areas.1-6 Protein-surface interactions clearly influence and control a large number ofprocesses occurring at an interface, includingmaterial-blood interactions and cell a d h e ~ i o n . ~Of J course, proteins can undergo conformational changes when adsorbed to a surface, and this may lead to different biochemical and biophysical responses from the biointerface. In turn, different substrate chemistries will, in principle, have a profound affect on the adsorbed protein and in turn its chemistry. Thus, if one can attach different terminal functionalities to a substrate, one can systematically study how interfacial chemistries affect protein structure and function. Our groups have been working with a series of hydroxylated and aminated FEP (poly{tetrafluoroethyleneco-hexafluoropropylene)) surfaces (FEP-x)~-" that are
* Authors to whom all correspondence should be addressed. University of New York at Buffalo.
* Lausanne University Medical School a n d Brown University.
@Abstractpublished in Advance ACS Abstracts, February 15, 1995. (1)Andrade, J. D. Principles In Protein Adsorption. In Surface and Interfacial Aspects of Biomedical Polymers, uol. 2, Protein Adsorption; Plenum Press: New York, 1985. (2) Soderquist, M. E.; Walton, A. G. J . Colloid Interface Sci. 1980, 75, 386. (3) Sevastianov, V. I. Crit. Rev. Biocompat. 1988,4, 109. (4) Baier, R. E.; Meyer, A. E.; Natiella, J. R.; Natiella, R. R.; Carter, J. M. J . Biomed. Mater. Res. 1984, 18, 337. (5) Horbett, T. A.; Brash, J. L. Proteins at Interfaces: Current Issues and Future Prospects. In Proteins a t Interfaces, Physiochemical and Biochemical Studies; ACS Symposium Series 343; American Chemical Society: Washington DC, 1987. (6) Norde, W.; Lyklema, J . J . Biomater. Sci., Polym. Ed.1991,2,183. (7) Vargo, T. G.; Gardella, J. A., Jr.; Meyer, A. E.; Baier, R. E. J . Polym. Sei. Polym. Chem. 1991,29, 555.
unique because they retain many of the physicochemical properties ofthe parent FEP and offer a substantial degree of interfacial tunability, and the surface chemistry can be localized to specific domains on the substrate (Le., the material can be patterned).' The hydroxylated FEP (FEPOH) is prepared using a radio frequency glow discharge (RFGD) plasma p r o c e s ~ . This ~ , ~ surface is subsequently modified with y-(aminopropy1)triethoxysilane(APTES)to yield a n interface possessing primary amine functionalit^.^^^ To date, these materials have beenused as platforms for biosensorslOJ1and as substrates for cell attachment studies.12 Most recently, Ranieri et al. reported that these FEP-x surfaces had the unique ability to either promote or inhibit cellular attachment as a function of surface modification and preadsorbed proteins.12 As a n example, selective cellular attachment was achieved on an amine-modified FEP surface (FEP-APS) only in the presence of preadsorbed bovine serum albumin (BSA). Unmodified FEP and FEP-OH did not support cell attachment with preadsorbed BSA, yet cells indiscriminately attached to all three substrates when preadsorbed with fibronectin.12 This result is especially intriguing because BSA is often used to passivate surfaces and inhibit cellular attachment.13 Additionally, the BSA adsorption behavior was (8) Hook, D. J.; Vargo, T. G.; Gardella, J. A., Jr.; Litwiler, K. S.; Bright, F. V. Langmuir 1991, 7, 142. (9) Vargo, T. G.; Thompson, P. M.; Gerenser, L. J.; Valentini, R. F.; Aebischer, P.; Hook, D. J.; Gardella, J. A,, Jr. Langmuir 1992,8, 130. (10) Bright, F. V.; Litwiler, K. S.; Vargo, T. G.; Gardella, J. A., Jr. Anal. Chim. Acta 1992,262, 323. (11)Li, M.; Pacholski, M. L.; Bright, F. V.App1. Spectrosc. 1994,48, 630. (12)Ranieri, J . P.; Ballamkonda, R.; Jacob, J.; Vargo, T. G.; Gardella, J. A., Jr.; Aebischer, P. J . Biomed. Mater. Res. 199S, 27, 917. (13)Horbett, T. A. Colloids and Surfaces B: Blointerfaces 1994,2, 225.
0743-746319512411-0984$09.00/0 0 1995 American Chemical Society
Adsorption of BSA on Fluoropolymer Substrates substrate dependent. BSA adsorbed in higher concentrations and more strongly to FEP-APS than either FEP and FEP-OH.12 Together these results lead Ranieri et al. to propose that (1)the FEP-x surfaces induced different degrees of structural change in the adsorbed BSA and (2) when BSA was adsorbed to FEP-APS it assumed a conformation that promoted cellular attachment. However, no evidence was provided to argue the degree or extent of these supposed conformational changes in the adsorbed BSA. Previous work on substrata-protein-cell interactions has focused on known cell adhesive molecules on derivatized silica. For example, Lewandowska et al. studied the response of murine fibroblasts and human neuroblastoma cells on derivatized silica surfaces after fibronectin had been preadsorbed.14J5 These authors reported that the amount of adsorbed fibronectin was similar on all surfaces; however, the cellular response was greatest on the amine-modified silica. Conformational differences of the adsorbed fibronectin were invoked in order to explain the variation in cell a t t a ~ h m e n t . ’ ~ In agreement with these results, Iuliano et al. observed that bovine aortic endothelial cells exhibited greater spreading, were more adherent under sheer stress, and formed better contacts to unmodified silica than octadecylmodified silica after fibronectin preconditioning.16 Iuliano, et al. used acrylodan-labeled fibronectin (Fn-Ac) and fluorescence spectroscopy to observe shifts in the steadystate spectra of Fn-Ac on adsorption to the different silica substrates.16 In both cases, unmodified silica and octadecyl-modified silica, the emission spectra shifted to higher wavelengths on adsorption relative to native Fn-Ac in Trisbuffered saline. The emission spectrum for Fn-Ac on unmodified silica red shifted a n additional 35 nm relative to Fn-Ac on octadecyl-modified silica. These results indicate the microenvironment surrounding the acrylodan probe had become more “polar” on Fn-Ac adsorption and even more polar when Fn-Ac adsorbed to unmodified silica. The results do correlate a fibronectin structural change to cell response, although the authors do not discuss the broadness or overlapping shoulders found in the emission spectra, which may be due to excited-state relaxation processes or several microenvironments per sample. However, the working hypothesis is that fibronectin is absorbed in a range of orientationslconformations, some of which are better suited for promoting cell attachment, most likely by making the cell binding domain (RGD)more or less accessible. Grinnell and Feld first reported on structural changes in surface-adsorbed fibr0ne~tin.l~ These authors concluded that different orientations of adsorbed fibronectin on polystyrene were responsible for differences in attachment of baby hamster kidney cells. In this paper we investigate the effects of different FEP substrate chemistries (i.e., native, -OH, -NH2) on the structure of adsorbed BSA and correlate the apparent BSA structural to our previous cell attachment results.12 Steady-state fluorescence spectroscopy is used to follow structural changes in the adsorbed BSA. In the current study, BSA is labeled with the environmentally sensitive probe (i.e., reporter group) dansyl. Dansyl has been widely used for fluorescently labeling proteins and its photophysics are known.1a-21Moreover, recent work by Wang (14)Lewandowska, K.;Balachander, N.; Sukenik, C. N.; Culp, L. A. J . Cell Physiol. 1989,141, 334. (15)Lewandowska, K.;Pergament, E.; Sukenik, C. N.; Culp, L. A. J . Biomed. Mater. Res. 1992,26, 1343. (16)Iuliano; D.J.;Saavedra, S. S.;Truskey, G. A. J . Biomed. Mater. Res. 1993, 27,1103. (17) Grinnell F.; Feld, M. K. J . Biomed. Mater. Res. 1981,15, 363. (18)Haugland, R. P. Handbook ofFluorescenceProbes and Research Chemicals; Molecular Probes: Eugene, OR, 1992.
Langmuir, Vol. 11, No. 3, 1995 985
OH
\..PF
0
F
n
RFGD PLASMA
OH
\.,,++
H
\.P
OH
Quick Dip in ca. 1% APTESlbarane
Figure 1. Simplified reaction sequence for FEP conversion t o FEP-OH and FEP-APS. In the first step FEP is hydroxylated using a radio frequency glow discharge plasma in a hydrogen/ methanol atmosphere.Asilane couplingagent, y-(aminopropy1)triethoxysilane, is then polymerized on to the surface from hexane (R = -C3He-). and Bright20,21 have reported on the detailed photophysics of dansylated BSA (BSA-Dan) used in this work.
Materials and Preparation The followingchemicalswere used: dansyl chloride(Molecular Probes);dimethylformamide(DMF),urea (98%),y-(aminopropy1)triethoxysilane (APTES)and guanidine hydrochloride (GdsHCl) (99%) (Aldrich); hexane and methanol (Fisher);bovine serum albumin (BSA) (United States Biochemical); dialysis tubing (cellulose membrane) (Sigma). Poly(tetrafluoroethy1ene-cohexafluoropropylene) (FEP) was donated by DuPont. FEP films (0.002 in. thick) were all ultrasonically cleaned in hexane and methanol prior to use. All RFGD plasma modifications were carried out using the apparatus and protocol described previo~sly.~-~ RFGD plasma-modified FEP (FEP-OH) were ultrasonically cleanedin methanol and dried before beingexposed to protein solution. NickelTEM grids (LaddResearchIndustries) were usedto mask the FEPduring the RFGDtreatment.8,9Aminemodified FEP (FEP-APS)films were producedby reacting RFGD modified FEP(FEP-OH)W ~ ~ ~ A P T The E S FEP-OH . ~ , ~ substrates were dipped in 1%APTES in hexane (approximately 5 s) and then ultrasonically washed,first in hexane and then in methanol. The resulting surface, FEP-APS (aminopropylsilane), has been previouslycharacterized using electron spectroscopyfor chemical analysis, secondaryion mass spectrometry,infrared spectroscopy, and fluorescence spectroscopy.8-10The modification procedure is summarized in Figure 1. Dansyl-labeled BSA was prepared following the procedure described previously by Wang and Bright.20The final dansyl-to-BSAratio was 0.6.20 Methods BSA-Dan was adsorbed onto the FEP-x substrates at 37 “C for 24 h. The wet samples were supported on a (19)Weber, G. Biochem. J . 1962, 51, 145. (20)Wang, R.;Bright, F. V. J . Phys. Chem. 1993,97, 4231. (21)Wang, R.;Bright, F. V. J.Phys. Chem. 1993,97, 10872.
Bekos et al.
986 Langmuir, Vol. 11, No. 3, 1995
0)
i
d:
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:
E:
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450
500
550
600
Emission Wavelength (nm) Figure2. Steady-statefluorescencespectra ofBSA-Dansorbed t o FEP (upper panel) and FEP-APS (lower panel). The substrates were incubated in 50 pM BSA-Dan (PBS) for 24 h at 37 "C. The first spectrum was obtained after the substrates were removed from the BSA-Dan solution (no rinsing). The individual samples were then washed in PBS and an emission spectrum was obtained after each washing ( l x to 6x1.
fused silica plate and placed in the excitation path within the fluorometer sample chamber. Steady-state fluorescence spectra were obtained with a SLM-AMINCO48000 MHF spectrofluorometer using a Xe arc lamp as the excitation source and a modified sample chamber. The sample chamber was modified by replacing the cuvette holder with a horizontal sample holder that was designed to hold thin polymer films vertically. The sample holder can be moved in the x , y, andz direction allowing for optical alignment. An excitation wavelength of 340 nm (8 nm bandpass) was used to excite the dansyl moiety and emission was monitored with a monochromator between 390 and 590 nm (8 nm bandpass). All emission spectra were background subtracted and corrected for detector and monochromator transmission nonlinearities.
Results Initially, the FEP-x surfaces were incubated in a solution containing 50 pM BSA-Dan. An emission spectrum of BSA-Dan adsorbed to the FEP-x surfaces was obtained. The substrata were subsequently washed with PBS and another emission spectrum was obtained. The washing and spectra acquisition process was repeated 6 times. Typical series of emission spectra for BSA-Dan adsorbed onto FEP and FEP-APS are shown in Figure 2. Two
features merit special mention. First, the emission spectra for BSA-Dan adsorbed onto FEP, FEP-APS, and FEP-OH (not shown but similar to FEP and FEP-APS) all show a similar decrease in emission intensity and red shift on repeated PBS washings. The initially high fluorescence intensity is thus clearly caused by the presence of strongly and loosely adsorbed BSA-Dan. The most weakly adsorbed BSA is effectively removed with each successive PBS wash (particularly with the first wash), leaving the most strongly adsorbed BSA-Dan at the surface. Second, there is a gradual red shift in the dansyl emission profile on removal of the loosely adsorbed BSA-Dan. The red shift indicates that the average microenvironments sensed by the dansyl reporter group is changed after each wash. Specifically, the average microenvironment sensed by the dansyl probe is becoming more hydrophilic.18 This result suggests that the average conformation of the more strongly sorbed BSA differs significantly from that of the loosely bound and native BSA. This result is not unpre~edented.l~,~~-~' Figure 3 illustrates the effect of washing number and substrate chemistry on the BSA-Dan fluorescence maximum. The fluorescence maximum of BSA-Dan in PBS is 435 f 1 nm and is used as a benchmark. Inspection of these data show several interesting features. First, prior to any washing steps, the emission maximum of BSADan on all FEP-x surfaces is 450 f 3 nm. As discussed above, the emission from an initial unwashed surface is a result of the combined fluorescence from all BSA-Dan at the surface (Le., strongly and weakly adsorbed). This result suggests that the FEP-x surface has a profound affect on all forms of BSA associated with the surface. Second, by the third or fourth wash the emission spectra become coincident within a given system. At this point only the most strongly and irreversibly adsorbed BSA molecules remain at the interface. Thus, the observed fluorescenceis from the truly interfacial BSA-Dan. Third, the "final" spectra on the three substrates are quite different from one another. This demonstrates that the individual substrate chemistries affect BSA differently. Fourth, there is no observable fluorescence from BSADan on FEP-OH after the sixth rinse. This result is consistent with our previous work12 and suggests that BSA is least strongly adsorbed to the FEP-OH surface. Fifth, BSA-Dan fluorescenceis observed on FEP and FEPAPS even after six washes. This suggests that BSA is (22)Garrison, M. D.; Iuliano, D. J.; Saavedra, S. S.;Truskey, G. A.; Reichert, W. M. J . Colloid Interface Sci. 1992,148, 415. (23)Watkins, R. D.; Robertson, C. R. J . Biomed. Mater. Res. 1977, 11, 915. (24)Walton, A.G.; Maenpa, F. C. J . Colloid Interface Sci. 1979, 72, 265. (25)Sonderquist, M. E.; Walton, A. G. J . Colloid Interface Sci. 1980, 75, 386. (26)Burghardt, T.P.; Axelrod, D. Biophys. J . 1981,33, 455. (27)Van Wagenen, R. A.; Rockhold, S.; Andrade, J . D. In Biomaterials: Interfacial Phenomena and Applications; Cooper, S. L., Peppas, N. A., Eds.; ACS Syposium Series 199;American Chemical Society: Washington DC, 1982. (28)Rockhold, S. A.;Quinn, R. D.; Van Wagenen, R. A.; Andrade, J . D.; Reichert, W. M. J. Electroanal. Chem. 1983,150, 261. (29)Cheng, Y.L.;Darst, S. A.: Robertson, C. R. J . Colloid Interface Sci. 1987, l i 8 , 212. (30)Reichert, W. M.CRC Crit. Reu. Biocompat. 1989,5, 173. (31)Andrade. J. D.: Hladv. " , V.: Wei.. A. P.: Golander. C. G. Croat. Chem. Acta 1990,63,527. (32)Andrade, J. D.; Hlady, V. J . Biomater. Sci., Polym. Ed. 1991,2, 161. (33)Andrade, J . D.;Hlady, V.; Wei, A. P.; Ho, C. H.; Lea, A. S.;Jeon, S. I.; Lin, Y. S.; Stroup, E. Clin. Mater. 1992, 11, 67. (34)Andrade, J. D.; Hlady, V.; Wei, A. P. Pure Appl. Chem. 1992,64, 1777. (35)Walker, D.S.;Garrison,M. D.; Reichert, W. M.J. Colloid Interface Sci. 1993, 157, 41. (36)Hlady, V.; Andrade, J. D. Colloids Su$. 1988,32, 359. (37)Hlady, V.; Andrade, J. D. Colloids Su$. 1989,42, 85. I
Adsorption of B S A on Fluoropolymer Substrates
Langmuir, Vol. 11,No.3, 1995 987 m 1.2
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Figure 3. Effects ofwashingwith PBS on the observed emission maximum of BSA-Dan (50 pM) adsorbed to various FEP-x surfaces. The emission maximum for native BSA-Dan in PBS solution is 435 nm. Symbols: FEP (O),FEP-OH (v),and FEP-
APS
(VI.
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more strongly adsorbed to these interfaces. Sixth, the samples were purposely excited directly by the incident light and not by a n evanescent wave created a t the FEPBSA interface, as in a total internal reflectance fluorescence (TIRF) experiment.22~26~29~36~37 Therefore, the fluorescence from individual BSA-Dan molecules is weighted equally, unlike the TIRF experiment where the molecules closest to the surface are preferentially excited. Finally, the emission spectra associated with the most strongly adsorbed BSA-Dan on FEP-APS is clearly red shifted by 15 nm relative to the spectra obtained on unmodified FEP (Figure 3). That is, the dansyl probe in BSA-Dan adsorbed to FEP-APS senses a significantly more dipolar environment when compared to BSA-Dan on FEP. Hence, the structure of BSA adsorbed to the two surfaces is indeed quite different. Moreover, the fluorescence from BSADan on FEP-APS is more dissimilar to the spectrum obtained for native BSA-Dan in buffer, suggesting the structure of BSA on FEP-APS is also less like native BSA. Ranieri et al. used a 0.025% (5 pM) BSA solution for all their work;12 therefore, one might question if a 10-fold increase in the BSA concentration affects the conformation of adsorbed BSA. To address this issue, we incubated FEP and FEP-APS with 5 pM BSA-Danfollowing the same procedures used in the cell response studies.12 Prior to obtaining a n emission spectrum, the substrates were washed twice with PBS while still in the substrate mounts. The recovered fluorescence emission spectra represent the strong, irreversibly adsorbed BSA (Figure 4, upper panel) and are, within our experimental uncertainty, indistinguishable from those of strongly adsorbed BSADan observed in Figure 2. The 15-nm red shift is also reproducible a t lower BSA concentrations. These results demonstrate that the observed spectral shifts are not a consequenceof the BSA concentration; they reflect surfaceinduced changes in the actual BSA structure. In a n effort to determine the origin of the change in the BSA conformation on adsorption to FEP-x surfaces, we investigated BSA-Dan that was first chemicallydenatured prior to adsorption. Native BSA-Dan was adsorbed to these same FEP surfaces, as described previously, t o serve as a control. For denatured BSA-Dan, BSA-Dan was
e 0.2
=
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400
450
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Figure 4. Recovered steady-statefluorescencespectra for BSADan adsorbed in various forms on FEP and FEP-APS: (upper panel) native BSA-Dan on FEP and FEP-APS: (lower panel) native and denatured BSA-Dan on FEP. All substrates were incubated in 5 pM BSA-Dan for 24 h at 37 "C and washed with PBS. mixed 1:l with 8 M Gd-HC1before adsorption to the FEP. The concentration of BSA-Dan was maintained a t 5 pM, the pH was kept constant at 7.4, and the substrates were incubated for 24 h a t 37 "C. All substrates were rinsed with PBS prior to obtaining emission spectra. The results from these experiments are summarized in Figure 4. In the upper panel we present the normalized steady-state emission spectra for native BSA-Dan adsorbed to FEP and FEP-APS. The 15nm shift is maintained and readily apparent. The bottom panel presents the recovered emission spectra for native and chemically-denatured BSA-Dan adsorbed to FEP. The emission spectrum of adsorbed, denatured BSA-Dan is clearly red shifted relative to adsorbed, native BSA-Dan. The emission spectrum from adsorbed, denatured BSA-Dan on FEP is similar to that obtained for native BSA-Dan on FEP-APS (upper panel). Thus, it would appear that native BSA adsorbed to FEP-APS and denatured BSA adsorbed to FEP are, from the perspective of the dansyl report group, structurally similar. Discussion Many researchers have reported proteins changingtheir conformation on adsorption to different surfaces. For example, Hlady and Andrade used a n environmentally sensitive fluorescent probe, 1-anilinonaphthalene-8-sulfonate (ANS), physisorbed to BSA and the intrinsic fluorescence from the tryptophan residues to study conformational changes of BSA upon adsorption to silica and derivatized silica surface^.^^^^^ They reported that
988 Langmuir, Vol. 11, No. 3, 1995
Bekos et al.
the microenvironment of the average tryptophan residues (there are two in BSA) and the ANS binding sites changed when BSA was adsorbed to a silica ~ u r f a c e . ~ Upon ~?~’ adsorption the tryptophan emission blue shifted and the A N S spectrum red shifted when compared to native BSA in solution. This suggested that upon adsorption the conformation of BSA changes in such a way that it “hides” the two tryptophan residues in a hydrophobic region and exposes the ANS binding regions to bulk water or polar amino acid residues. However,the inherent difficultywith interpreting these results is that one has to contend with emission from several fluorescent centers simultaneously and one cannot ensure that the physisorbed ANS is actually in the same location within the protein architecture on adsorption. Garrison et al. significantly improved on this situation when they labeled BSA with the fluorescent probe acrylodan (Ac)and studied the Ac fluorescencewhen BSAAc was adsorbed to alkyl silane-modifiedglass coverslips.22 The advantage to using Ac is that it reacts selectively to free thiol groups and BSA has only one free thiol, cys-34 of loop 1in domain I. It was observed that as the surface became more hydrophobic (unmodified glass < -C1 < -CIS) the emission spectra blue shifted. This result has been interpreted in terms of the Ac binding site experiencing a more hydrophobic environment, as BSA adsorbs to the substrate. The authors suggested that the Ac binding site is exposed or perhaps in contact with the alkane surface. These emission studies show that the conformation of adsorbed BSA changes from its bulk conformation and that different surface chemistries affect further the conformation of adsorbed BSA. Additional evidence for structural changes of proteins a t surfaces is drawn from work using time-resolved fluorescence spectroscopy, which report changes in the fluorescent intensity decay kinetics upon protein adsorption or covalent attachment to different s u r f a c e ~ . ~ ~ - ~ l As stated in the Introduction, this work was prompted by the selective cellular attachment of mouse neuroblastoma cells (NB2a) and rat endothelial cells to FEP-APS &er BSA adsorption12where selective cellular attachment to FEP-APS was dependent upon the presence of preadsorbed BSA. When the substrates were preadsorbed with fibronectin, BSNfibronectin mixtures, or fetal bovine serum (consisting of 90% albumin), cells would attach indiscriminately. The cells were plated from serum-free media. These conditions were tested with nonpatterned and patterned surfaces. With patterned surfaces consisting of adjacent FEP and FEP-APS regions and preadsorbed with BSA, cells would only attach to the FEP-APS BSA domains. Patterned surfaces preadsorbed with a B S N fibronectin mixture or fetal bovine serum actually attenuated the selective cellular attachment to the APS regions. Finally, cells attached everywhere on patterned surfaces when preadsorbed with fibronectin and also to the patterned surface with no preadsorbed protein. Other researchers have reported that cells attach selectively to aminated regions patterned next to hydrophobic domains on glass substrates when the cells were plated from serum-containing media.42,43However, this
+
(38)Rainbow, M. R.; Atherton, S. A.; Eberhart, R. C. J. Biomed. Mater. Res. 1987,21,539. (39)Suci, P.;Hlady, V. Colloids Surf. 1990,51,89. (40)Fukumura, H.; Hayashi, K. J . Colloid Interface Sci. 1990,135, 435. (41)Crystall, B.;Rumbles, G.; Smith, T. A.; Phillips, D. J. Colloid Interface Sci. 1993,155,247. (42) Kleinfeld,D.;Kahler,K. H.;Hockberger, P.E. J. Neurosci. 1988, 8. - , 4098. (43) Britland, S.;Clark, P.; Connolly, P.;Moores, G. E X ~Cell . Res. 1992,198,124.
selective cellular attachment was lost if serum was removed from the plating media, as cells would attach equally well to either amine or alkane patterned regions. Ranieri et al. realized the uniqueness of their results,12 in that the FEP-based materials used here behave quite differently. Furthermore, BSA, which has long thought to serve as a surface passivating protein,13 actually promoted a selective cellular response12and demonstrated a significantly stronger binding character to FEP-APS. The character of the BSA binding forces was tested using radioiodinated BSA (1251-BSA)and the strength of binding was challenged with sodium dodecyl sulfate. One possible explanation for these results is that the FEP-APS surfaces effect a conformational change of the adsorbed BSA and the particular BSA conformation at FEP-APS promotes attachment of Nb2a cells. These proposed differences in BSA conformation can be attributed to a combination of hydrophobic and ionic forces existing a t the interface of BSA and the positively charged FEP-APS ~ u r f a c e . l * ” * Whereas, ~~ in the case of FEP and FEP-OH, only hydrophobic forces were responsible for the adsorption of BSA.1$44,45 Taking this one step further, the complex structure of BSA can be simplified by describing it as consisting of three domain~.~1-33?~6 Andrade et ~ 1 . proposed ~ ~ 3 a “tennis ball” model, in which the three domains line up like three tennis balls within a cylinder. The N-terminus is contained in domain I and the C-terminus is contained in domain 111. The domains differ in electrostatic charge, pH characteristics, and overall stability. At pH 7 (pH used in the protein adsorption and cell response studies),12the net charge on BSA is -18.’ However, each of the three domains has a distinct electrostatic charge at pH 7: domain I is -10, domain I1 is -8, and domain I11 is 0. Using the domain approach, Andrade et al. outlined how BSA adsorption might proceed at nominally positive, negative, and hydrophobic surface^.^^^^^ At pH 7 on a positively charged surface (e.g., FEP-APS), the negatively charged domains I and I1 would preferentially bind to the surface. These domains are less stable than domain I11 and it would be expected that the adsorbed BSA would be more denatured than on the other surfaces. At a hydrophobic surface (FEP),a side on adsorption process may occur with domain I11 due to its electrostatic neutrality. There is also a possibility of the loop 1region in domain I of BSA, which too is nonpolar, adsorbing preferentially to FEP. Significant denaturation will occur on a hydrophobic surface, especially with increased residence time, however, not to the same extent as on the positively charged surface.3l?33 Although previous work has shown that proteins do change their conformation at a n interface, few have presented cellular response studies along with any of the aforementioned protein adsorption results.16 Thus, it is not clear if many of the previously reported conformational changes in actuality affected cellular adhesion. Our results suggest that there are conformational changes in adsorbed BSA that actually influence cell adhesion at the FEP-APS surface. Our steady-state fluorescence data indirectly show that there are structural changes in BSA on adsorption to the various FEP substrates. A possible explanation for the observed results is that BSA denatures to varying extents on FEP, FEP-OH, and FEP-APS surfaces. Such a scenario is in-line with the BSA adsorption model proposed by Andrade et a1.31r33 The positively charged FEP-APS would be expected t o denature the adsorbed BSA to a greater ~
~~
(44)Norde, W.; Mac Ritchie, F.; Nowicka, G.;Lyklema, J. J.Colloid Interface Sci. 1986, 112,447. (45)Morrissey, B.W.Ann. N . Y. Acad. Sci. 1977,283,50. (46)Peters, T.Adv. Protein Chem. 1986,97,161.
Adsorption of BSA on Fluoropolymer Substrates Table 1. Emission Maxima of FEP-APS-Dansyland Dansyl Exposed to Different SolventdConditions
sample/solvent emission maximum ( n m P 477 FEP-APS-dansyl, dry 478 FEP-APS-dansyl, HzO wetted 481 FEP-APS-dansyl, methanol wetted 480 FEP-APS-dansyl, BSA adsorbed BSA-Dan in PBS buffer 435 perfluoro(methy1)cyclohexane
439
dimethyl sulfoxide (DMSO) 450 462 cyclohexane hexane 464 water 505 methanol 540 "he average ofthree separate experiments. * Uncertainty 5 6% RSD. extent than native FEP. The increased denaturation of BSA on the APS surface could result in turn in a structural change within the BSA that leads to cellular attachment. For example, one can envision unfolding of the BSA a-helical structure (55% a-helix in native state)47such that dansyl residue senses a different microenvironment and certain portions of the BSA protein become available and possess the ability to promote/allow cellular attachment. Unfortunately, BSA does not contain any known cell adhesion sequences (RGD, YIGSR, IKVAV, etc.); however, this does not preclude the possibility of FEPAPS adsorption leading to the exposure of unknown cell receptors, normally sheltered within the BSA interior. Another explanation for the fluorescence results is the reorganization of BSA structure on the surface resulting in direct fluorescent probe-surface interactions (dansylFEP-x), as Garrison et al. suggested in their work.22 To test this scenario, dansyl was covalently bound to the primary amine on the FEP-APS surface and its fluorescence spectra were recorded dry and in the presence of water, methanol, and BSA. The resulting spectral contours were coincident with one another, the only differences being in emission intensity. Table 1 summarizes these results along with the emission maxima of dansyl in a series of solvents ranging from extremely nonpolar to extremely polar. The perfluorinated solvents were used to simulate the native FEP surface. These results (47)Schechter,E.;Bluot, E. R. Proc. Natl. Acad. Sci. U.S.A. 1964, 51, 695.
Langmuir, Vol. 11, No. 3, 1995 989 demonstrate that the dansyl probe on the adsorbed BSADan is sensing environmental changes within the BSA architecture caused by surface adsorption and not directly sensing the FEP-x surface. Finally, it is reasonable to question the inherent uncertainty associated with the location of the dansyl residues.20,21Dansyl is selective for amine residues's and BSA has over 80 primary amines. However, previous timeresolved fluorescenceresults from our laboratory20s21 show that there are only two distinct sites labeled under our experimental conditions. Thus, although the dansyl labeling scheme is far from ideal, it is not prohibitively complicated.
Conclusions Interfacial fluorescence spectroscopy of BSA sorbed to three FEP-x surfaces has shown (1) the structure is different from its native form in bulk solution, (2) its structure changes upon adsorption in response to the different surface chemistries, and (3)the structure of BSADan most strongly associated with the FEP-x surface is a function of surface chemistry and not dependent upon the concentration of the adsorbing BSA solution. These results parallel recent work from our group on cell adhesion to FEP-APS surfaces pretreated with BSA and support our original supposition12that mouse NB2a and rat endothelial cell attachment to FEP-APS is a result of conformational changes in the adsorbed BSA. We are currently using our time-resolved interfacial fluorescence t e c h n i q ~ e s ~ ~to - ~better O understand the behavior of BSA (native, denatured, and fragments thereof7 adsorbed to these intriguing fluoropolymer interfaces.
Acknowledgment. This work was supported in part by the National Science Foundation (DMR-9303032 to J.A.G. and CHE-9300694 to F.V.B.). The authors also thank Run Wang and Upvan Narang for their assistance in setting up the initial fluorescence studies. LA9400691 (48)Bright, F. V . Appl. Spectrosc. 1993,47,1152. (49)Bright, F.V . ;Wang, R.; Li,M.; Dunbar, R. A. Immunomethods 1993.3. 104. (50) Lundgren,J. S.; Bekos,E. J.;Wang,R.; Bright,F. V.Ana1.Chem. 1994,66,2433.
