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Controlling Biological Interactions with Poly(lactic acid) by Surface Entrapment Modification Robin A. Quirk, Martyn C. Davies, Saul J. B. Tendler, Weng C. Chan, and Kevin M. Shakesheff* School of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom Received October 26, 2000. In Final Form: January 23, 2001 Poly(ethylene glycol) (PEG)-modified poly(lactic acid) (PLA) systems were created by physically entrapping the modifying species at the PLA surface. The surface characterization and biological performance of these materials are described. This modification strategy is performed by reversible gelation of the PLA surface following exposure to a solvent/nonsolvent mixture. PEG is then able to diffuse into the swollen surface region, before it is collapsed by the addition of more nonsolvent. This results in the localized physical entrapment of the diffused material. We have demonstrated by high-resolution X-ray photoelectron spectroscopy that control over the PEG surface density may be achieved by using predetermined process conditions, such as a particular solvent/nonsolvent ratio or a set polymer treatment time, and that surface coverage of around 75% is possible. Cell adhesion studies have shown that even in serum-containing media PEG entrapment will prevent attachment, with a 95% reduction in cell number compared to unmodified PLA. This modification strategy was also used to coentrap both PEG and poly(L-lysine)-RGD within the PLA surface region. The attachment of cells to this material shows that the entrapment approach may be used to create highly selective biomaterial surfaces that are able to prevent unwanted cell or protein adhesion yet actively promote specific cellular interaction.
Introduction Surface modification with poly(ethylene glycol) (PEG) is a well-established strategy for rendering biomaterials cell or protein resistant,1,2 thus reducing complications such as biofouling and thrombus formation on contact with biological fluids.3-5 Unfortunately, the poor mechanical properties of PEG prevent its use as a standalone biomaterial for many applications, and therefore modification strategies are often used to adapt wellestablished polymers (e.g., biodegradable poly(ester)s such as poly(lactic acid) (PLA);6-8 structures of PLA and PEG are shown in Figure 1). The incorporation of PEG into the biomaterial structure is usually achieved through either surface immobilization (e.g., grafting9 or adsorption of a surfactant derivative10) or synthesis of a bulk polymer material featuring PEG domains (e.g., copolymers8 or cross-linked networks6,11). A more sophisticated modification strategy emerging is the creation of highly specific material interfaces through the presentation of cell adhesion motifs over a background of nonadhesive hydrophilic polymer.12-15 Short * Corresponding author. E-mail: kevin.shakesheff@ nottingham.ac.uk. Tel: +44 115 951 5104. Fax: +44 115 951 5110. (1) Griffith, L. G. Acta Mater. 2000, 48, 263. (2) Elbert, D. L.; Hubbell, J. A. Annu. Rev. Mater. Sci. 1996, 26, 365. (3) Suggs, L. J.; West, J. L.; Mikos, A. G. Biomaterials 1999, 20, 683. (4) Elbert, D. L.; Hubbell, J. A. Chem. Biol. 1998, 5, 177. (5) Deible, C. R.; Petrosko, P.; Johnson, P. C.; Beckman, E. J.; Russell, A. J.; Wagner, W. R. Biomaterials 1998, 19, 1885. (6) Han, D. K.; Hubbell, J. A. Macromolecules 1997, 30, 6077. (7) Sheth, M.; Kumar, R. A.; Dave´, V.; Gros, R. A.; Mccarthy, S. P. J. Appl. Polym. Sci. 1997, 66, 1495. (8) Huh, K. M.; Bae, Y. H. Polymer 1999, 40, 6147. (9) Gombotz, W. R.; Guanghui, W.; Horbett, T. A.; Hoffman, A. S. J. Biomed. Mater. Res. 1991, 25, 1547. (10) Dunn, S.; Coombes, A. G. A.; Garnett, M. C.; Davis, S. S.; Davies, M. C.; Illum, L. J. Controlled Release 1997, 44, 65. (11) Bearinger, J. P.; Castner, D. G.; Healy, K. E. J. Biomater. Sci., Polym. Ed. 1998, 9, 629. (12) Hern, D. L.; Hubbell, J. A. J. Biomed. Mater. Res. 1998, 39, 266. (13) Griffith, L. G.; Lopina, S. Biomaterials 1998, 19, 979.
Figure 1. Chemical structures of PLA and PEG.
peptide sequences such as isoleucine-lysine-valinealanine-valine (IKVAV)16 and arginine-glutamic acidaspartic acid-valine (REDV)17 have been shown to interact with specific cell types via integrin receptors (neurites and endothelial cells, respectively), adhere to them, and possibly stimulate their phenotypic expression. Other receptor-ligand interactions, such as the hepatocytegalactose response, may also be employed to target a particular cell type at the exclusion of others. In a biological environment, these receptor-mediated interactions are of most benefit if nonspecific cell and protein interference is therefore silenced through the additional presence of PEG. We have previously reported a surface engineering strategy for the immobilization of modifying polymer species at the PLA interface, demonstrated by the incorporation of PEG and poly(L-lysine) (PLL).18 On the basis of a method reported by Desai and Hubbell,19,20 this (14) Park, A.; Wu, B.; Griffith, L. G. J. Biomater. Sci., Polym. Ed. 1998, 9, 89. (15) Carlisle, E. S.; Mariappan, M. R.; Nelson, K. D.; Thomes, B. E.; Timmons, R. B.; Constantinescu, A.; Eberhart, R. C.; Bankey, P. E. Tissue Eng. 2000, 6, 45. (16) Tashiro, K.; Sephel, G. C.; Weeks, B.; Sasaki, M.; Martin, G. R.; Kleinman, H. K.; Yamada, Y. J. Biol. Chem. 1989, 264, 16174. (17) Hubbell, J. A.; Massia, S. P.; Desai, N. P.; Drumheller, P. D. Bio/Technology 1991, 9, 568. (18) Quirk, R. A.; Davies, M. C.; Tendler, S. J. B.; Shakesheff, K. M.; Macromolecules 2000, 33, 258.
10.1021/la001509a CCC: $20.00 © 2001 American Chemical Society Published on Web 04/05/2001
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straightforward solution technique was shown to achieve high densities of the modifying materials without the requirement for the synthesis of new chemical entities or changes to the bulk characteristics of the starting material. The surface presentation of the polymers was achieved by exposing PLA to a solvent/nonsolvent system (2,2,2trifluoroethanol/water), which causes a reversible gelation of the polymer surface, into which either the PEG or PLL could diffuse. These modifying polymers were then entrapped by exposing the system to a large excess of the PLA nonsolvent (water). We were previously able to demonstrate control over the amount of PLL incorporated into the PLA surface.18 In this paper, we report the dependence of PEG surface density on the treatment conditions and the distribution of the modifier as a function of depth beneath the PLA surface. The biological relevance of this modification strategy is also demonstrated by studying cell adhesion to various PEG-modified PLA surfaces in serum-enriched media. Serum is often required for optimal cell culture conditions, and serum proteins are found in biological fluids. The adsorption of nonspecific adhesion molecules from serum can therefore be problematic, especially when precise surface interactions are targeted through biomolecular modification of a material. The potential for creating high specificity materials by using the surface engineering strategy to coentrap PEG and adhesion peptide-conjugated PLL (PLL-RGD) was therefore also investigated. Materials and Methods Surface Entrapment Modification of PLA. PLA disks (Alkermes, MW 12 845) of approximately 1.3 cm diameter and 0.3 mm thickness were prepared by melt-pressing the supplied material between clean glass surfaces. The surface-modifying solution was prepared by first dissolving PEG (Polysciences Inc., MW 18 500 or Sigma, MW 6000) in water and then adding 2,2,2trifluoroethanol (TFE, Aldrich) to the solution. Typically, the resultant mixture consisted of 50% w/v PEG and 10% v/v TFE, unless stated otherwise. Surface engineering of the PLA disks was performed by immersion in 1 mL of this solvent/nonsolvent system for a given time period, before adding a large excess of nonsolvent (∼20 mL water) to entrap the PEG material. The films were then removed from this solution, washed in water for 30 min, and then dried in a desiccator overnight. Coentrapment of PEG and poly(L-lysine)-GRGDS (PLL-RGD, synthesized within the laboratory using a previously reported protocol21) was attempted using a 50% w/v PEG, 10% w/v PLL-RGD, and 10% v/v TFE solution. X-ray Photoelectron Spectroscopy (XPS) Analysis. XPS spectra were obtained using a Scienta ESCA300 instrument (RUSTI, CLRC Daresbury, U.K.), which is described in detail elsewhere.22,23 Samples were analyzed using a monochromated Al KR X-ray source, a low energy electron flood gun for charge compensation, and a takeoff angle of 45° relative to the sample surface (unless otherwise stated). The C1s core level peaks presented in this paper were acquired using a pass energy of 150 eV and a 0.5 mm slit width. The spectra were analyzed using Scienta WinESCA software. Curve fitting was performed using a linear background, a Gaussian peak shape, and an initial full width at half-maximum (fwhm) of 1.15 eV. To obtain best fits, the fwhm values were later allowed to vary by (0.1 eV. Cell Seeding Studies. Culture and seeding of the bovine aortic endothelial cells (BAEs) used to study cell adhesion to the (19) Desai, N. P.; Hubbell, J. A. Biomaterials 1991, 12, 144. (20) Desai, N. P.; Hubbell, J. A. Macromolecules 1992, 25, 226. (21) Quirk, R. A.; Chan, W. C.; Davies, M. C.; Tendler, S. J. B.; Shakesheff, K. M. Biomaterials 2001, 22, 865. (22) Beamson, G.; Briggs, D.; Davies, S. F.; Fletcher, I. W.; Clark, D. T.; Howard, J.; Gelius, U.; Wannberg, B.; Baltzer, P. Surf. Interface Anal. 1990, 15, 541. (23) Gelius, U.; Wannberg, B.; Baltzer, P.; Fellner-Feldegg, H.; Carlsson, G.; Johansson, C. G.; Larsson, J.; Mu¨nger, P.; Vegerfors, G. J. Electron Spectrosc. Relat. Phenom. 1990, 52, 747.
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Figure 2. Example XPS C1s scans of (A) unmodified PLA and (B) PEG-modified PLA (50% w/v PEG, 10% v/v TFE, 6 h immersion time). modified PLA surfaces have also been described previously.21 In brief, BAEs were grown to near confluence using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% v/v fetal bovine serum, antibiotics, and glutamine. Before use, the cells were incubated with Cell Tracker green CMFDA (5chlorofluorescein diacetate) fluorescent stain (Molecular Probes), as per the manufacturer’s protocol, to enhance their detection following seeding onto the substrate surface. After the modified PLA disks were placed in a 24 well plate and UV sterilized for 15 min, BAEs were seeded at a density of 20 000 mL-1 and incubated at 37 °C/5% CO2 for 5 h. Any unadhered cells were removed by washing the disks three times with fresh media, and fluorescence images were then taken using a Leica inverted microscope (model DMIRB/E). Ten random images (10× magnification) were taken per surface, with each sample being repeated in triplicate.
Results and Discussion XPS Analysis. The C1s XPS core level spectra were used to determine the resultant surface concentrations of PEG following the entrapment process (surface being defined as a function of the sampling depth of the XPS instrument). The XPS data as displayed in Figure 2 show the C1s peak fits for PLA and PEG-modified PLA. As expected from the monomer structure, unmodified PLA produces a core level spectrum consisting of three peaks of comparable area. The C-O and CO2 carbon groups can be distinguished by increased shifts in binding energy of ∼2 and 4 eV relative to the C1s methyl group (C-C-H) at 285 eV. Following surface entrapment of PEG, an additional C1s ether peak (C-O) appears at ∼286.3 eV, the specific energy shift providing an opportunity for calculating the relative intensities of the two polymers. Table 1 displays C1s data obtained following a range of process variations. Surface adsorption of the modifying material is poor, resulting in a 0.1:1 PEG/PLA monomer ratio (50% w/v PEG, 0% v/v TFE, 24 h). Entrapment appears to provide a straightforward mechanism for the surface immobilization of PEG, as the concentration is greatly enhanced by inclusion of TFE into the PLA immersion mixture. We have previously demonstrated that the amount of PLL entrapped at the PLA surface can be controlled by varying the solvent/nonsolvent ratio, the treatment time, and/or the concentration of the surface-
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Table 1. C1s Peak Fit Data, Illustrating Effect of Varying Process Conditions on Surface PEG Incorporation entrapment conditions (% w/v PEG/time/% v/v TFE)
C-C-H
unmodified PLA (0/0/0) TFE-treated PLA (0/10 min/10) PEG adsorption (50/24 h/0) 6 h entrapment (50/6 h/10) 24 h entrapment (50/24 h/10) 2 min entrapment (10/2 min/50) 24 h entrapment, 6K MW PEG (50/24 h/10)
34.4 39.4 38.3 29.4 15.1 25.4 49.9
% peak area C-O CO2 32.8 30.6 28.5 22.0 10.0 23.2 22.8
32.8 30.0 27.0 19.2 7.2 22.2 19.1
C-O [PEG]
PEG/PLA monomer ratio
% PEG surface coverage
6.2 29.4 67.7 27.2 8.2
0.1:1 0.6:1 3.1:1 0.6:1 0.1:1
9 37 76 37 9
Figure 3. C1s scans showing the effect of sample treatment time on surface PEG incorporation (50% w/v PEG, 10% v/v TFE).
modifying agent in solution.18 The effect of exposure time of PLA to the solvent/nonsolvent on the amount of material entrapped is illustrated by the C1s scans in Figure 3. A 6 h exposure of PLA to the partial solvent resulted in a PEG surface coverage of 37%, a value which can be approximately doubled by extending the process to a 24 h period. As two identical carbon atoms contribute to the signal of each PEG monomer unit, a doubling in the surface density is observed in the XPS data as a 4-fold increase in the relative C-O PEG peak area. An increase in the TFE content of the mixture and a reduction in the amount of PEG in solution (50% w/v PEG was not soluble at higher TFE ratios) enabled the study of short exposure times to the solution (10% w/v PEG, 50% v/v TFE, 2 min contact time). Results indicate that a short-exposure surface engineering approach may be viable, with an observed ethylene glycol to lactic acid ratio of around 0.6:1 being achieved (Table 1). The entrapment of a lower molecular weight PEG (6000 MW) was relatively unsuccessful (PEG/PLA monomer ratio ) 0.1:1). Although the PEG chain length is approximately one-third of that previously used (and therefore the corresponding PEG signal will be two-thirds less for an equivalent entrapment efficiency), accounting for these changes would generate a PEG surface coverage equivalent to 27% using this molecular weight, compared with 76% for 18 500 MW PEG. It is thought that this effect is due to shorter polymer chains being less effectively entrapped because of limited anchorage within the PLA network. XPS spectra of PEG-modified PLA were also recorded from two sampling depths using takeoff angles of 10° and 45° relative to the sample surface. The analysis depth of XPS is described by 3λ sin θ, and therefore a lower takeoff angle (θ) results in a more surface-sensitive analysis. There is an approximately exponential decrease in signal with depth,24 with the most penetrative spectral information originating from around 8-10 nm beneath the actual sample surface. Spectra recorded using 10° and 45° takeoff (24) Cumpson, P. J. J. Electron Spectrosc. Relat. Phenom 1995, 73, 25.
Figure 4. Angle-resolved C1s data of PEG-modified PLA, acquired using 10° and 45° takeoff angles: (A) PEG adsorption (50% w/v PEG, 0% v/v TFE, 24 h) and (B) PEG entrapment (50% w/v PEG, 10% v/v TFE, 24 h).
angles therefore represent sampling depths that are at the most 17% and 71% of this maximum depth, respectively. Adsorption of PEG (50% w/v, 0% v/v TFE, 24 h), although minimal, was shown to result in a signal that was localized at the PLA surface (Figure 4A). A 10° XPS spectrum revealed a PEG/PLA monomer ratio of 0.14:1, compared with the similar ratio of 0.10:1 on increase of the sampling depth. This suggests that there is minimal surface modification and that the interfacial PEG layer is very thin or patchy. Such an observation is expected, as penetration of the modifying material beneath the PLA surface is highly unlikely to occur without the creation of the reversible gel-layer. Depending on the size of the gellayer generated by exposure to the solvent/nonsolvent system, it could be predicted that entrapment modification would result in a surface-localized or uniform PEG signal following analysis at the two sampling depths used. This was not found to be the case (Figure 4B), with the surfacesensitive spectrum revealing a lower concentration of PEG (PEG/PLA monomer ratio ) 1.6:1) compared with that probing deeper beneath the sample surface (PEG/PLA monomer ratio ) 3.1:1). This may be explained by extended treatment times enabling PEG to penetrate deep within the PLA material. On collapse of the swollen gel-layer, there is less opportunity for PLA chains near the surface to entangle material because of the shorter anchorage chain length. There is also subsequently less resistance to the leaching out of poorly entrapped material. Another contributing factor may be a surface enrichment effect of
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Figure 6. Percentage cell adhesion to PLA surfaces (relative to unmodified PLA) (n ) 3).
Figure 5. Fluorescence microscopy images of BAE cell attachment to various PLA surfaces: (A) untreated PLA, (B) TFE-treated PLA (10% v/v TFE, 5 min), (C) PEG adsorption (50% w/v PEG, 0% v/v TFE, 24 h), (D) 6 h PEG entrapment (50% w/v PEG, 10% v/v TFE), (E) 24 h PEG entrapment (50% w/v PEG, 10% v/v TFE), and (F) 24 h PEG and PLL-RGD coentrapment (50% w/v PEG, 10% w/v PLL-RGD, 10% v/v TFE).
the PLA component, a phenomenon previously reported for poly(lactic and glycolic acid)-poly(ethylene oxide) copolymers.25 Cell Seeding Studies. The biological relevance of surface PEG presentation by entrapment modification was assessed by studying the adhesion of BAE cells to various PLA materials. The image in Figure 5A illustrates that the cells will readily attach to unmodified PLA. This may occur because of nonspecific interactions or the surface adsorption of adhesion proteins, such as fibronectin and vitronectin, from the serum in the cell media to the PLA interface.26,27 TFE exposure in the absence of PEG results in much faster polymer-solvent interactions, and therefore an estimation of the equivalent treatment time (as determined visually) that creates the same degree of surface swelling was used as a control to account for possible residual solvent effects on cell adhesion. PLA treated in this way was shown to have no inhibitory effect on BAE attachment (Figure 5B). As suggested by the XPS data, the low levels of PEG present following the adsorption process are not sufficient to reduce cell-surface interactions (Figure 5C). However, the entrapment of PEG created a repellent PLA surface upon which cell adhesion was drastically reduced. This was true for both the 6 h and 24 h modifications (Figure 5D,E), suggesting that a surface coverage of 37% PEG is sufficient to create a cellinert surface. Following the successful inhibition of BAE cell adhesion to PLA in serum-containing media conditions by surface entrapment of PEG, the possibility of generating a highly specific polymer interface through coentrapment of cell adhesion sequences was investigated. Cell adhesion to PLA surfaces occurs readily because of nonspecific mech(25) Shard, A. G.; Davies, M. C.; Li, Y. X.; Volland, C.; Kissel, T. Macromolecules 1997, 30, 3051. (26) Burg, K. J. L.; Holder, W. D., Jr.; Culberson, C. R.; Beiler, R. J.; Greene, K. G.; Loebsack, A. B.; Roland, W. D.; Mooney, D. J.; Halberstadt, C. R. J. Biomater. Sci., Polymer Ed. 1999, 10, 147. (27) Cima, L. G.; Ingber, D. E.; Vacanti, J. P.; Langer, R. Biotechnol. Bioeng. 1991, 38, 145.
anisms, and this nonspecificity is further enhanced by the use of serum-containing media. As PEG-entrapped PLA surfaces displayed an inhibition over cell attachment, a reversal of this inhibitory effect by the additional presentation of receptor-specific adhesion ligands at the material surface would signify a highly desirable modification process. The coimmobilization of PLL-RGD with a PEG entrapment shown to prevent cell attachment indeed resulted in the attachment of cells onto the polymer disk (Figure 5F). Thus, the surface engineering strategy described in this paper provides a facile approach to precisely controlling cellular interactions with PLA materials. A quantitative comparison of the actual numbers of BAEs adhered to the various PLA surfaces is shown by the graph in Figure 6. PEG entrapment modification was shown to reduce adhesion by around 95% compared with untreated PLA, whereas neither TFE treatment nor PEG adsorption significantly reduced cell attachment. The additional incorporation of PLL-RGD with PEG-entrapped PLA saw around a 50% increase in the number of cells compared with PEG-modified PLA. Conclusions XPS studies have demonstrated that PEG may be immobilized at PLA surfaces by entrapping the material during the reversible swelling of the starting material. Control over the amount of PEG presented at the surface may be achieved by altering one of a number of process variables, including the solvent/nonsolvent ratio, treatment time, and molecular weight of the modifying polymer. Depth analysis has revealed that entrapped PEG may extend within the bulk of the PLA, but there may be a reduced local PEG concentration at the air-polymer interface possibly because of a reduced entrapment efficiency or surface enrichment effects. The performance of PEG-modified PLA in a cell/serum environment has demonstrated the success of the entrapment strategy in generating a nonadhesive material. These studies have also revealed that entrapment may potentially be used to immobilize multiple modifying species, thus creating a material that can prevent unwanted cell/ protein interactions yet provoke desired responses with particular cell types through the surface presentation of specific adhesion receptor ligands. Acknowledgment. R.Q. thanks the BBSRC for Ph.D. studentship funding. K.M.S. is an EPSRC Advanced Fellow and acknowledges their support for surface engineering studies, including access to the Scienta ESCA300 spectrometer (RUSTI, CLRC Daresbury, U.K.). LA001509A
