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Surface Engineering and Surface Analysis of a Biodegradable Polymer with Biotinylated End Groups Fiona E. Black, Mark Hartshorne, Martyn C. Davies, Clive J. Roberts, Saul J. B. Tendler, Philip M. Williams, and Kevin M. Shakesheff* Laboratory of Biophysics and Surface Analysis, School of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, U.K.
Scott M. Cannizzaro, Irene Kim, and Robert Langer Department of Chemical Engineering, Massachusetts Institute of Technology, 45 Carleton Street, Cambridge, Massachusetts 02139 Received March 31, 1998. In Final Form: January 4, 1999 In the design of advanced polymeric biomaterials there is a need to tailor the surface chemistry of the biomaterial to elicit beneficial interactions with cells and biomolecules. To facilitate the fabrication of complex biomaterial surfaces, we have previously described the synthesis and application of a poly(lactic acid)-poly(ethylene glycol) block copolymer (PLA-PEG) with the biotinylated PEG end groups (final polymer termed PLA-PEG-biotin). This polymer is biodegradable and resistant to nonspecific protein adsorption, and the biotin moiety allows surface chemical engineering to be achieved using avidin-biotin interactions. Here, we describe a detailed surface analysis of this polymer using X-ray photoelectron spectroscopy and surface plasmon resonance analysis. This analysis has revealed that the avidin-biotin surface engineering strategy is a rapid method of immobilizing biomolecules at biomaterial surfaces under aqueous conditions. The surface engineering generates a specific and high-density change in surface structure. The effect of PLA segment molecular weight and the influence of the surfactant on the nature of the surface engineering has been determined with the objective of proving that the extent of specific ligand immobilization is controllable and resilient to surface stabilization by surfactants.
Introduction A major goal in the design of new polymeric biomaterials is to control the interactions of biomolecules and living cells with biomaterial surfaces.1-3 Examples of advanced biomaterial applications that require such control include tissue engineering4,5 and site-specific drug delivery.6-7 Current aims of tissue engineering include the regeneration of damaged tissues, such as nerves,8 and the stimulation of bioartificial organ growth, for example, the liver.9 Site-specific drug delivery aims to utilize biomaterial-cell interactions to alter the distribution of drugs within the body.10 Control over biomaterial-cell interactions can be achieved by engineering the surfaces of biomaterials.1-3 An important example of this principle is given by the design of tissue-engineering templates that present RGD peptide sequences at their surfaces.11-14 These peptides promote the adhesion of cells via integrin receptors and, (1) Hubbell, J. A. Bio/technology 1995, 13, 565. (2) Ratner, B. D. J. Mol. Recog. 1996, 9, 617. (3) Elbert, D. L.; Hubbell, J. A. Annu. Rev. Mater. Sci. 1996, 26, 365. (4) Langer, R. Ann. Biomed. Eng. 1995, 23, 101. (5) Langer, R.; Vacanti, J. P. Science 1993, 260, 920. (6) Brindley, A.; Davis, S. S.; Davies, M. C.; Watts, J. F. J. Colliod Interface Sci. 1995, 23, 101. (7) Coombes, A. G. A.; Tasker, S.; Lindbald, M.; Holmgren, J.; Hoste, K.; Toncheva, V.; Schacht, E.; Davies, M. C.; Illum, L.; Davis, S. S. Biomaterials 1997, 18, 1153. (8) Schmidt, C. E.; Shastri, V. R.; Vacanti, J. P.; Langer R. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8948. (9) Michalopoulos, G. K.; DeFrances, M. C. Science 1997, 276, 6066. (10) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Science 1994, 263 1600. (11) Lin, H.-B.; Garcı´a-Echeverria, C.; Asakura, S.; Sun, W.; Mosher, D. F.; Cooper, S. L. Biomaterials 1992, 13, 905. (12) Ruoslahti, E. Annu. Rev. Cell Develop. Biol. 1996, 12, 697.
therefore, stimulate cell spreading and growth. The RGD peptide sequence represents one of a growing number of ligand motifs that interact with receptors to generate desirable changes in cell behavior. The main approach to immobilizing ligand motifs on biomaterial surfaces is covalent attachment to reactive polymer side chains.15 Covalent attachment strategies are not ideal for all biomaterial types because many biodegradable polymers lack reactive side groups. We have previously described an alternative method of surface engineering that uses the molecular recognition between avidin and biotin as a foundation for the attachment of ligands to polymer surfaces.16 As a basis for this surface engineering, we synthesized a biotinylated biodegradable polymer, PLA-PEG-biotin (Figure 1). The three components of this polymer each possess advantageous properties. The PLA component is a biodegradable poly(Rhydroxy ester) that provides structural integrity to fabricated biomaterials during the period of their empolyment.17 Biodegradation of the PLA component ensures that the biomaterial is metabolized and eliminated by the body after its function has been accomplished. The PEG block acts as a hydrophilic, protein-resistive component.18 (13) Barrera, D. A.; Zylstra, E.; Lansbury, P. T.; Langer, R. Macromolecules 1993, 115, 11010. (14) Cook, A. D.; Hrkach, J. S.; Gao, N. N.; Johnson, I. M.; Pajvani, U. B.; Cannizzaro, S. M.; Langer, R. J. Biomed. Mater. Res. 1997, 35, 513. (15) Drumheller, P. D.; Hubbell, J. A. Surface Immobilization of Adhesion Ligands for Investigations of Cell-Substrate Interactions; In The Biomedical Engineering Handbook; Bronzino, J. D., Ed.; CRC Press: Boca Raton, FL, 1995; Chapter 106, p 1583. (16) Cannizzaro, S. M.; Padera, R. F.; Langer, R.; Rogers, R.; Black, F. E.; Davies, M. C.; Tendler, S. J. B.; Shakesheff, K. M. Biotechnol. Bioeng. 1998, 58, 529. (17) Coombes, A. G. A.; Meikle, M. C. Clin. Mater. 1994, 17, 35.
10.1021/la9803575 CCC: $18.00 © 1999 American Chemical Society Published on Web 03/27/1999
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Figure 1. Structure of PLA-PEG-biotin.
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motif that can be biotinylated can be presented at the surface of PLA-PEG-biotin-based biomaterials, and precise control over the density of surfaces composed of mixtures of motifs can be achieved. A key component in the successful development of any surface-engineering strategy is the accurate analysis of surface properties. This paper describes the use of X-ray photoelectron spectroscopy (XPS) and surface plasmon resonance analysis (SPR) to investigate the composition of the surface layer of a range of biotinylated PLA-PEG copolymers, with varying PLA segment molecular weights. The effect of varying PEG surface concentrations on the comparatve kinetics of avidin binding to these polymers has been determined. In addition, the effect of exposing PLA-PEG-biotin surfaces to the surfactant poly(vinyl alcohol) (PVA) on the surface-engineering process has been measured. PVA and other polymeric surfactants are used in biomaterial fabrication to lower the surface energy of newly formed surfaces.21 An ideal postfabrication surface modification should be successful after PVA-biomaterial interactions have occurred. This would allow advanced biomaterials, such as microparticles and scaffolds, to be prepared using surfactants and then undergo a final surface-engineering step to attach ligand motifs. Methods
Figure 2. Schematic diagram showing the surface engineering of PLA-PEG-biotin to produce a cell-adhesive surface.
It is included to reduce nonspecific interactions between the biomaterial and the living environment (e.g., protein adsorption) and has been shown to reduce protein immunogenicity.19 Finally, the biotin moiety allows facile surface engineering using aqueous solutions of avidin.20 Avidin possesses a tetrameric structure with four binding sites for biotin. It binds to the biotin at the end of the PEG chains using one of these sites. The other free binding sites are then available for the attachment of biotinylated ligand motifs. This surface-engineering strategy is shown schematically in Figure 2. When compared to the conditions used in covalent coupling statergies, avidin-based surface engineering of PLA-PEG-biotin has the advantage of being rapidly completed in a mild aqueous environment, with simple washing and purification steps. It, therefore, may eliminate the potential of damage to ligands. In addition, any (18) Lea, A. S.; Andrade, J. D.; Hlady, V. Colloids Surf., A 1994, 93, 349. (19) Marshall, D.; Pedley, R. B.; Boden, R.; Melton, R. G.; Begent, R. H. J. Br. J. Cancer 1996, 73, 565. (20) Diamandis, E. P.; Chistopoulos, T. K. Clin. Chem. 1991, 375, 625.
The PLA-PEG-biotin polymers were synthesized as described previously with L-lactide.16 Three PLA-PEG-biotin polymers were prepared with a PLA block Mw of 22562, 9261, and 3242, as determind by NMR. For all these polymers the PEG block had a Mw of 3800. A nonbiotinylated PLA-PEG block copolymer was synthesized as a control material. L-lactide was polymerized from the free hydoxyl end group of monomethyl ether PEG (Mw 3000) (Shearwater Polymer Inc., Huntsville, Alabama). The Mw of the PLA block of the PLA-PEG polymer was 17861 producing an identical PLA-PEG molecular weight ratio (3.6:1) to the PLAPEG-biotin materials with a 22562 Mw PLA block. For XPS studies the polymers were spin cast at 2000 rpm using 200 µL of a 3 mg/mL chloroform (HPLC grade) solution on to silicon wafer squares (Exsil, Alfreton, Derbyshire, U.K.). All spectra were obtained using a VG Escalab Mk2 electron spectrometer (VG Scientific, East Grinstead, Sussex, U.K.) employing monochromatic Al KR (1486.7 eV) X-rays. The X-ray gun was operated at 200 W and the electron takeoff angle to the spectrometer is 35° unless otherwise stated. A wide scan over 500 eV was obtained as well as narrow scans of 20 eV in the regions of the carbon and oxygen 1s envelopes. The SPR instrument (Johnson & Johnson Clinical Diagnostics, Buckinghamshire, U.K.) was of a Kretschmann configuration with a monochromatic laser source of 780 nm wavelength. Polymer samples for SPR analysis were prepared on glass sensor slides; these were coated with an approximately 50 nm silver layer on one surface. Polymeric material was spin-coated onto the silver surface from a 3 mg/mL chloroform solution using a spinning rate of 2000 rpm and a aliquot size of 100 µL. Slides were prepared directly prior to SPR analysis. A polymer concentration of 3 mg/mL gave structurally continuous films that were thin enough to generate minima in refected light intensity at incident angles within the sensitivity range of the SPR instrument. This minimum in light intensity is termed the SPR angle (φSPR) (in millidegrees; mDA) and its change with time was recorded throughout each SPR experiment. The aqueous medium for SPR analysis was dibasic phosphate buffer (10 mM, pH 7.4). The flow of the medium and injection of the samples was electronically regulated by in-house software controlling rheodyne type, six-port valves. The flow rate remained constant at 0.240 mL/min. After each completed injection, a buffer wash period of 300 s was carried out to remove all molecules not adsorbed to the surface. All experiments were carried out at (21) Shakesheff, K. M.; Evora, C.; Soriano, I.; Langer, R. J. Colloid Interface Sci. 1997, 185, 538.
Biodegradable Polymer with Biotinylated End Groups ≈34.4 °C. SPR measurement of avidin binding was performed using avidin solutions of 5 × 10-7 M (Sigma, Poole, Dorset, UK.) in 10 mM dibasic phosphate buffer, pH 7.4. Surfactant adsorption studies used 80% mole hydrolyzed PVA (85000 Mw) (Sigma). The binding of avidin to polymer films was determined in 1350 s experiments, which included a buffer wash. A flowing phosphate buffer was introduced over the spin-cast polymer film slide for 120 s to obtain a stable baseline. Then, 1 mL of the avidin solution was introduced over the polymer surface followed by a buffer wash period. For experiments investigating the effect of PVA adsorption, an extra step was included where 1 mL of the PVA solution, either 1.0% or 0.1% w/v concentration, was flowed over the slide followed by a buffer wash, prior to the avidin injection as described above. All results were taken from experiments replicated at least seven times.
Results and Discussion XPS Analysis. Elemental surface analysis of all the PLA-PEG-biotin polymers displayed only peaks indicative to the polymer, showing the absence of common contaminants such as poly(dimethylsiloxane). In addition, the absence of peaks from Si atoms proved that complete films of at least 10 nm thick had formed over the substrate surface. C(1s) region scans of the three polymers, with differing PLA block molecular weights, are displayed in Figure 3. Deconvolution of this C(1s) data reveal the presence of four carbon group types. Peaks 1-3 (at binding energies of 285.00, 286.98, and 289.06 eV) are characteristic of the PLA block as described previously.22 Peak 4 (286.45 eV) is generated by ether carbon groups of the PEG block.23 The ratios of areas of peaks 1-3 are close to the theoretical 1:1:1 value for the PLA block. Minor deviations, no greater than 4%, were seen for polymers with PLA Mw of 3242 and 22562, due to hydrocarbon contamination. It is clearly evident from Figure 3 that the distinctive difference in surface chemistry between the three polymers is the increased contribution from peak 4 (i.e., the PEG component) as the PLA block molecular weight decreases. As the molecular weight of the PLA block decreases from 22562 to 9261 and 3242 the percentage of ethylene glycol contribution to the C(1s) spectra increases from 16.1% to 32.70% and 57.40%, respectively. As the biotin moieties are attached to the PEG end group, an increase in ethylene glycol units at the surface suggests an increase in biotin available for the surface immobilization of avidin. SPR Analysis. Effect of Polymer Composition on Avidin Binding. Figure 4 shows a typical SPR trace obtained during the adsorption of avidin to PLA(22562)-PEGbiotin. The surface immobilization of avidin generates an increase in φSPR. The saturation of the biotin binding sites occurs rapidly as demonstrated by the plateauing of the φSPR rate of increase within 70 s of avidin solution exposure to the polymer surface. The magnitude of the increase in φSPR can be used to compare the avidin binding capacity of the three PLAPEG-biotin polymers. PLA-PEG-biotin surfaces, composed of polymers with PLA molecular weights of 22562, 9261, and 3242, generated φSPR shifts of 180 ( 14, 256 ( 11, and 440 ( 54, respectively. The increase in φSPR reflects increased levels of PEG at the surface and hence biotin with decreasing PLA molecular weight. This demonstrates that the surface density of avidin, immobilized on the polymer surface, can be controlled by changing the PLA (22) Davies, M. C.; Short, R. D.; Khan, M. A.; Watts, J. F.; Brown, A.; Eccles, A. J.; Humphrey, P.; Vickerman, J. C.; Vert, M. Surf. Interface Anal. 1989, 14, 115. (23) Beamson, G.; Briggs, D. High-Resolution XPS of Organic Polymers, The Scienta ESCA300 Database; J. Wiley & Sons Ltd.: Chichester, U.K., 1992; p 84.
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molecular weight. It is interesting to note that the increase in the value of φSPR measured for the polymers with a PLA weight of 9261 and 3242 is larger than that obtained for a surface composed of a N-([3-biotinamido]hexyl)-3′[2′pyridyldithio]propamide self-assembled monolayer, which gave an avidin binding shift of 198 ( 16 mDA.24 This suggests that the amount of avidin binding to these polymers is greater than the amount of binding to a twodimensional array of biotin. A possible explanation for this effect is that the hydrophilic nature of PEG allows the block to act as an effective spacer molecule. The PEG component could form a three-dimensional hydrophilic surface region with a larger surface area than a selfassembled monolayer surface. In analyzing the extent of avidin immobilization on the PLA-PEG-biotin surfaces, it is important to differentiate between specific molecular recognition between the biotin and avidin molecules and nonspecific interactions between the polymer substrate and the protein. To assess the extent of nonspecific protein-to-polymer interactions, an avidin sample was prepared with all biotin-binding sites occupied by free biotin molecules. This blocked avidin was then exposed to the surface of a PLA(22562)-PEG-biotin film. The increase in φSPR was only 24 ( 8 mDA as compared to 180 ( 14 mDA, when unblocked avidin was used, indicating that specific interactions are the dominant mechanism of avidin immobilization. Further evidence of the minor role of nonspecific interactions was provided by SPR analysis of the immobilization of avidin to a nonbiotinylated PLA-PEG with an identical PLA:PEG molecular weight ratio to PLA(22562)-PEG-biotin. This PLA-PEG system generated a similar small increase in φSPR of only 30 ( 5 mDA, confirming nonspecific interactions are not the major component of the recognition events for the biotinylated polymers. Effect of PVA Adsorption on Avidin Binding. Surfactant adsorption to biodegradable surfaces is utilized as a mechanism of lowering surface energy during biomaterial fabrications. Surfactant chemistry can be tailored to create beneficial surface properties that improve biomaterial functioning and biocompatibility, for example, by reducing nonspecific protein adsorption. However, surfactant adsorption processes can limit the ability to undertake further surface engineering, for example, because of the masking of attachment sites for cell-adhesive ligands. To assess the resilience and retention of activity of the avidinbased surface-engineering strategy after surfactant adsorption, a series of SPR experiments were performed to measure avidin binding to surfaces pre-exposed to PVA. We first investigated the effect of exposing PLA-PEGbiotin surfaces to aqueous solutions of PVA. The data in Figure 5 show the change in φSPR caused by flowing either a 0.1 or 1% PVA solution over surfaces PLA(22562)-PEGbiotin. Both concentrations produced similar final increases in φSPR of approximately 95 mDa. However, it is apparent that the 1% PVA solution produces a very large initial increase of approximately 200 mDA and that this value decreased as the PVA solution in the flow cell of the SPR was replaced with phosphate buffer. The large initial increase in φSPR is due to the refractive index of the 1% PVA solution in the liquid above the surface and, therefore, is not indicative of a permanent surface interaction. The close agreement in the final φSPR increase for both PVA solution concentrations indicates that the maximum amount of surfactant adsorption possible, to this PLAPEG-biotin surface, has occurred. Indeed, when referring to the avidin φSPR shifts after adsorption of the two different (24) Heaton, R. J., unpublished work.
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Figure 3. C(1s) region data for PLA, PEG, and the PLA-PEG-biotin polymers with differing PLA block molecular weights. Deconvolution of peaks is shown. (i) Data courtesy of S. L. McGurk.
PVA concentrations, very similar values are observed in each case. The adsorption of PVA to this hydrophilic polymer brush suggests that it forms an incomplete steric boundary, allowing the interpolation of the PVA molecules within the PEG surface layer. The impact of such a mixed interfacial zone may be in the masking of biotin binding sites at the PEG end groups.
The data in Figure 6 show the full SPR data for PVA exposure followed by the adsorption of avidin on the PLA(22562)-PEG-biotin surface. The data shows that avidin immobilization still occurs after PVA adsorption. The average increase in φSPR caused by avidin exposure to PLA(22562)-PEG-biotin, after PVA adsorption, was found to be 143 ( 21 mDA, which represents a reduction of 26%
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Figure 4. SPR trace showing the interaction of avidin with PLA(22562)-PEG-biotin. (I) Point of injection of avidin. (B) Buffer wash starts. Table 1. SPR Angle Shifts for Avidin Binding to the Polymer Surfaces φSPR increase polymer
no PVA
0.1% PVA
1.0% PVA
PLA(22562)-PEG-biotin PLA(9261)-PEG-biotin PLA(3242)-PEG-biotin PLA-PEG
180 ( 14 264 ( 13 440 ( 54 30 ( 5
143 ( 21 183 ( 20 358 ( 40 29 ( 6
132 ( 19 182 ( 26 285 ( 29 24 ( 3
Conclusion
Figure 5. SPR trace showing the interaction of PVA with PLA(22562)-PEG-biotin. (a) 1.0% PVA. (b) 0.1% PVA. (I) Point of injection of PVA. (B) Buffer wash starts.
Figure 6. SPR trace showing the interaction of 0.1% PVA and avidin with PLA(22562)-PEG-biotin. (Ip) point of injection of PVA. (B) Buffer wash starts. (Ia) Point of injection of avidin.
over the φSPR increase on surfaces prepared in the absence of PVA. The results in Table 1 show equivalent experimental results for all the PLA-PEG-biotin polymers studied in this work. While all polymers demonstrate a reduction in avidin binding after PVA adsorption, the extent of avidin binding remains very significant. These findings suggest that the interpenetration of PVA into the PEG barrier, necessary for the formation of tissueengineering scaffolds, masks some binding sites (up to 30%) but that sufficient biotin binding sites are retained in the outer zone of the polymer brush.
Surface analysis has a key role to play in the design of novel biomaterials because for many advanced biomaterial applications the surface properties determine the nature of cellular interactions. Here, we have shown that XPS and SPR can be utilized in a complementary manner to obtain a detailed surface analysis of a novel biodegradable polymer intended to undergo surface engineering to generate biomimetic scaffolds. The XPS analysis showed from the deconvolution of the C(1s) envelopes that both PLA and PEG molecules were present at the surface. The extent of PEG contribution was governed by PLA molecular weight. As the bulk PLA molecular weight decreased, the amount of PEG at the surface increased. These data infer an increase in biotin at the surface, as this is bound to the PEG end group. SPR analysis of the range of PLA-PEG-biotin systems revealed that the immobilization of avidin at the surface, under aqueous conditions, is rapid and specific. SPR analysis confirmed XPS predictions that increased levels of specific avidin-biotin interactions at the surface occurred with decreasing PLA molecular weight, through the increase in the PEG concentration at the interface. The ability to control the extent of avidin binding by altering the length of the PLA portion of the system provides the opportunity to obtain a range of different surface densities of active groups for further surface engineering. The adsorption of PVA, a model surfactant to the surface of the system, has been shown to give a finite surface coverage probably due to the interpolation of the molecules within the PEG polymer brush. While PVA adsorption reduces the level of binding of avidin to the surface, the avidin-based surface-engineering strategy is resilient enough to still give a high level of specific avidin binding. LA9803575
