Electron-Beam Lithography for Patterning Biomolecules at the Micron

Dec 21, 2011 - This article is part of the Materials for Biological Applications special issue. ... Langmuir 2016 32 (25), 6551-6558 ... D. Maynard. A...
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Electron-Beam Lithography for Patterning Biomolecules at the Micron and Nanometer Scale Christopher M. Kolodziej† and Heather D. Maynard*,† †

Department of Chemistry and Biochemistry and the California NanoSystems Institute, University of California, Los Angeles, 607 Charles E. Young Drive South, Los Angeles, California 90095, United States ABSTRACT: This review summarizes the use of electron beam (e-beam) lithography to pattern biomolecules on surfaces. The focus is on approaches that employ poly(ethylene glycol) (PEG) resists. Overview of the different strategies used, including ablation of self-assembled monolayers and crosslinking of PEG, is provided. Subsequent use of surfaces to immobilize cells for tissue engineering applications is summarized.

KEYWORDS: e-beam lithography, protein patterning, microarrays, nanoarrays, cell adhesion

1.1. INTRODUCTION Electron-beam (e-beam) lithography is a maskless technique for patterning surfaces with nanoscale features that has been recently exploited for patterning biomolecules. In its most conventional form, the substrate is coated with a polymeric thin film or resist, which is exposed to a focused e-beam. The highenergy electrons cause chemical changes in the resist: either cross-linking and reducing solubility (negative resist) or degrading and increasing solubility (positive resist). The substrate is then rinsed with a developer solution to remove the more soluble portion of the resist. E-beam lithography has several advantages over other patterning techniques. E-beam lithography offers the ability to pattern features with lateral dimensions that vary from 101−106 nm with small interfeature spacings. It is also possible to pattern features with arbitrary shape. E-beam lithography can also be used to iteratively pattern features with different chemistries for immobilization of multiple biomolecules on a single substrate, in both two and three dimensions. The technique does suffer from some inherent limitations, notably cost and speed. E-beam writing instruments are expensive and are often installed in cleanroom facilities. It is also a serial technique and thus suffers from slower patterning in comparison to photolithography, stamping, or self-assembly methods capable of writing many features in parallel. E-beam lithography is therefore most amenable to applications for which fewer substrates, high resolutions, and/ or small interfeature spacings are required and for which the ability to prepare substrates with a variety of different patterns is critical. Several recent comprehensive reviews summarize the pros and cons of other patterning techniques, and these will not be covered here.1−6 E-beam lithography has been employed to pattern biomolecules in several ways. It can be used to locally change © 2011 American Chemical Society

the hydrophobicity or functionality of conventional lithographic polymer resists. Biomolecules can then be immobilized on either the patterns or the background via hydrophobic interaction or coupling chemistries, depending on the specific resist used.7,8 It can also be utilized to prepare gold features through a traditional lift off process. These features can then serve as sites for formation of self-assembled monolayers (SAMs) capable of immobilizing biomolecules.9 It can also be employed to ablate SAMs, including silane,10 thiol-on-gold,11 and phosphonic acid-on-aluminum12 SAMs (Figure 1). In such applications, a protein-resistant oligo(ethylene glycol) (OEG)terminated SAM is typically formed and selectively removed to form protein-adhesive regions.13 Likewise, regions of protein monolayers can be degraded for subsequent adsorption of a second protein.14,15 In these cases the monolayer functions as a positive resist. A protein-resistant polymer film can also function as a positive resist in this manner.16 Negative resists can also be patterned by e-beam lithography for immobilization of biomolecules. One such resist is poly(ethylene glycol) or PEG. This method is advantageous in that the features thus formed are composed of PEG and therefore resist protein adsorption.17 This review focuses entirely on patterning proteins on PEG hydrogels produced using e-beam lithography. The first formation of PEG hydrogels via radiation-induced cross-linking was demonstrated by King in 1966 via exposure of dilute aqueous PEG solutions to gamma irradiation.18 Early mechanistic studies of this process identified hydrogen Special Issue: Materials for Biological Applications Received: September 7, 2011 Revised: December 20, 2011 Published: December 21, 2011 774

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analogous peroxide-mediated cross-linking of PEGs, including acetals, olefins, orthoesters, and esters (Scheme 1).21 These radical reactions have also been observed in PEG films under exposure to γ-rays22 and UV irradiation in the presence of a photoinitiator.23,24 E-beam irradiation is also capable of generating radicals in organic materials and was first used by Sofia and Merrill to graft PEG to a surface via radical cross-linking in 1998.25 Krsko and co-workers refined this technique demonstrating the cross-linking of PEG by focused e-beam irradiation, which allows for defined patterns of PEG to be formed on a surface.17 Subsequent research has broadened the scope of e-beam lithography to prepare micro- and nanopatterned PEG hydrogels. To date, the formation by e-beam lithography of 80 nm-wide PEG features with a center-to-center spacing of 320 nm have been reported,26 and 70 nm-wide PEG features with a spacing of 150 nm have been achieved, although certainly the resolution of e-beam lithography is much higher. The features thus formed are generally stable. PEG nanopatterns have been incubated in cell culture media for four days at 37 °C without any observed degradation,27 though nanopatterned lines do appear to broaden when stored under ambient conditions for over a week. This method is also amenable to producing features with a wide range of functional groups for attachment of biomolecules through specific chemistries while maintaining the nonfouling properties. The e-beam patterning of PEGs containing alcohol, amine, aminooxy, alkyne, maleimide, biotin, nitrilotriacetic acid (NTA), and sodium styrene-4-sulfonate has been reported and are discussed in this review. These features

Figure 1. E-beam patterning of self-assembled monolayers (SAMs). Harnett et al. employed e-beam lithography to iteratively pattern 10 × 10 μm squares in an octadecanethiol SAM on a gold substrate. Following each patterning step the ablated regions were backfilled first with cysteamine and then with carboxylic acid coated fluorospheres (a). Rundqvist et al. employed e-beam lithography to deactivate surface-bound fibronectin on a silicon substrate. The patterned substrates were used to culture Swiss 3T3 cells, which adhered preferentially to the unexposed regions and were confined to the shape of the pattern (b). (a) Adapted in part with permission from Harnett et al. Langmuir 2001, 17, 178−182. Copyright 2001 American Chemical Society. (b) Adapted in part with permission from Rundqvist et al. J. Am. Chem. Soc. 2006, 129, 59−67. Copyright 2006 American Chemical Society.

abstraction from the PEG chain by hydroxyl radicals as a key step.19,20 Recent work by Emami and co-workers has further elucidated the many side products produced during the

Scheme 1. Mechanism for Radical Cross-Linking of PEGs Proposed by Emami and Co-workersa

a

Adapted with permission from Emami et al. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 3021−3026. Copyright 2002 Wiley-VCH. 775

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Figure 2. Cross-linking of PEG films as a function of e-beam dose and effect on protein adsorption. In order to form a gel an average of one crosslink per molecule is required (a), thus 200 kDa PEG (circles) has a lower critical dose for gelation than 6.8 kDa PEG (triangles). Swelling ratio of the patterned hydrogels was measured in water and varied with the patterning dose, with a maximum of 16:1 (b). Adsorption of fibronectin on the features was normalized to nonspecific adsorption on the silicon background and varied with swelling ratio (c). Adapted with permission from Krsko et al. Langmuir 2003, 19, 5618−5625. Copyright 2003 American Chemical Society.

Figure 3. Amine-functionalized PEG nanohydrogels patterned by e-beam lithography. Hong and co-workers demonstrated nanoscale e-beam patterning of 5 kDa monoamine-terminated PEG hydrogels. Features with an interpixel spacing as small as 715 nm were resolved by fluorescence microscopy (a). The presence of the amine groups was demonstrated by conjugation of FITC to the patterns (b). Nonspecific conjugation was not observed, and the fluorescence signal could be enhanced by preconjugating BSA to the patterns via EDC coupling, thus increasing the number of amines available for conjugation. Adapted with permission from Hong et al. Langmuir 2004, 20, 11123−11126. Copyright 2004 American Chemical Society.

of e-beam lithography make it an attractive option for the small scale production of tissue engineering substrates and biosensor chips that require functionalized surfaces to attach ligands and biomolecules.

for 2 h, and the adsorbed protein was quantified by immunostaining and fluorescence imaging. The adsorption was found to depend on swelling ratio (Figure 2c). This could be due to chemical changes that can occur at high doses and reduce the hydrophilicity, and thus the protein resistance, of the PEG gels. This dose-dependent protein adsorption was used to prepare continuous PEG pads within which the affinity for fibronectin was controlled on the submicrometer scale to immobilize fibronectin with nanoscale resolution. This work was later extended to immobilize proteins via specific chemistry on individual features with nanoscale dimensions. Hong et al. used e-beam lithography to pattern 5 kDa monoamine-terminated PEG on silicon wafers that had been pretreated with a PEG-silane.28 They achieved arrays of 170 nm-wide features with center-to-center distances of 715 nm. Closer features were attempted but could not be resolved by fluorescence imaging (Figure 3a). These features retained the amine groups, as indicated by binding of FITC (Figure 3b). The amine-containing PEG hydrogels were also able to bind proteins through carbodiimide coupling chemistry and yet still resisted nonspecific protein adsorption. The fluorescence intensity of the stained features required amplification by attachment of bovine serum albumin (BSA), which contains thirty-six surface-accessible amines, and thus provides a greater number of possible conjugation sites for FITC. The scope of functional groups that can be incorporated into these hydrogels via patterning of end-functionalized PEGs was

1.2. DISCUSSION Krsko and co-workers in 2003 reported the first use of e-beam lithography to pattern PEG.17 In this work, they demonstrated the patterning of PEGs with molecular weights of 6.8 and 200 kDa on silicon wafers. They found that the dose required for hydrogel formation varied as a function of molecular weight, with a critical dose for gelation of 6.8 kDa PEG of 70 μC/cm2 and a critical dose for gelation of 200 kDa PEG of 1.5 μC/cm2 (Figure 2a). The doses required for formation of stable gels were higher; 100 μC/cm2 for 6.8 kDa PEG and 3 μC/cm2 for 200 kDa PEG. The difference in dosage was attributed to the requirement for gelation of at least one cross-link per molecule, with longer polymers being more likely to form at least one cross-link at a lower dose. The micropatterned PEG gels were strongly hydroscopic, with swelling ratios as high as 16:1. The swelling ratio was dose-dependent, with decreased swelling at higher doses (Figure 2b). Interestingly, swelling was only observed normal to the silicon surface, which was attributed to constraints imposed by cross-linking of the hydrogels to the silicon substrate. Krsko and co-workers also tested the ability of the micropatterned PEG hydrogels to resist protein adsorption. Surfaces were incubated with 0.1 mg/mL fibronectin at 37 °C 776

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Figure 4. Protein immobilization on patterned 8-arm PEGs terminated with a variety of functional groups. E-beam lithography was employed to cross-link several 8-arm PEGs terminated with biotin (a), maleimide (b), aminooxy (c), and NTA (d) functional groups. These patterns were used to immobilize SAv (a), BSA (b), myoglobin (c), and calmodulin (d) via chemoselective conjugations. Biotin- and maleimide-terminated PEGs could be iteratively patterned and employed to immobilize SAv and BSA from a single solution (e). Immunostaining confirmed the selective binding of the proteins to the desired features (f, g). Nanopatterning was confirmed by AFM imaging (h). Adapted with permission from Christman et al. J. Am. Chem. Soc. 2009, 131, 521−527. Copyright 2009 American Chemical Society.

compared their arrays to traditionally fabricated microarrays on four different commercially available substrates.32 They prepared arrays for capturing ZNF9 by two different methods; one set of arrays was functionalized to contain maleimide groups, which could react with the free cysteine on ZNF9, and the other was functionalized with antiglutathione-S-transferase (anti-GST), which was used to capture a GST-ZNF9 fusion. In both cases, the nanoarrays prepared by e-beam lithography had both the highest signal intensity and the highest signal-tobackground ratio of all arrays tested. Their nanoarrays also had 40-fold greater sensitivity than the traditional microarrays (Figure 5). The authors suggested that the PEG nanohydrogels are more hydrated and thus better able to maintain the conformation of immobilized ZNF9 than the traditional microarrays. They also pointed out that the use of nanopatterned arrays could increase the feature density by a factor of 1000. E-beam patterning of PEG hydrogels has also been applied to the fabrication of high dimensional structures (Figure 6). Brough et al. demonstrated the use of nanopatterned 8-arm amine-terminated PEG as a template for surface-initiated polymerization of actin filaments.26 In this work the aminecontaining hydrogels were biotinylated and used to immobilize SAv. Because SAv has two pairs of binding sites for biotin located on opposite faces of the protein, it can be used as an adapter for the further surface functionalization. In this case, a biotinylated FX45-F-actin complex was bound to the SAvfunctionalized patterns. FX45 was then able to polymerize dissolved G-actin onto the attached F-actin filament in the Z direction. In this way, ∼7.2 μm-long actin filaments were formed from dot patterns as small as 80 nm-wide and also from 100 nm-wide line nanopatterns. Reconfigurable surfaces have also been prepared by e-beam lithography.33 In this work aminooxy-terminated 8-arm PEG was functionalized with a ketone-modified glutathione (GSH), which is a ligand for glutathione-S-transferase (GST). Unlike the biotin-SAv interaction, the binding of GSH to GST is reversible

further expanded by Maynard and co-workers. In 2009, the nanopatterning of biotin-, maleimide-, and aminooxy-terminated 8-arm PEGs and the micropatterning of NTA-terminated 8-arm PEG was reported.29 Streptavidin (SAv), BSA, α-ketoamidemodified myoglobin, and histidine-tagged calmodulin, respectively, were then immobilized on the patterns. The use of 8-arm PEG increased the concentration of reactive end groups, and also effectively introduced some preformed cross-links, depending on the end group. Thus, signal amplification was not required. Iterative patterning of different PEGs to fabricate side-by-side and multilayer structures was also demonstrated. The appropriate proteins could then be immobilized on the features containing the corresponding reactivity: SAv on the biotinylated patterns (Figure 4a), BSA on the maleimidefunctionalized patterns through Michael addition by the free cysteine (Figure 4b), α-ketoamide-modified myoglobin on the aminooxy-functionalized patterns via oxime bond formation (Figure 4c), and histidine-tagged calmodulin on the Ni2+-NTAfunctionalized patterns (Figure 4d). Because the PEG hydrogels resist nonspecific adsorption, multiple proteins could be specifically immobilized on the correct features from a single solution (Figure 4e, f, g, h). This is a critical requirement for the fabrication of protein arrays for diagnostic devices. Nanopatterning of alkyne-terminated 8-arm PEG and binding of azido-ubiquitin to the patterned hydrogels has also been reported.30 This allows for side-by-side and multilayer patterns of different molecules to be achieved using dual Click reactions. Finally, the patterning of a polymethacrylate containing OEG side chains (pOEGMA) terminated in aminooxy groups has been investigated.31 This further increased the effective concentration of reactive end groups compared to polymers with end functional groups only. Arrays of PEG hydrogels patterned by e-beam lithography compare well to commercial protein microarrays in terms of sensitivity. Saaem and co-workers have reported the use of 100 μm diameter clusters of amine-terminated PEG nanohydrogels patterned on glass slides to bind zinc finger 9 (ZNF9) and 777

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Figure 7. Fluorescence images showing the reversibility of GST binding. First, the substrate is incubated with GST (a). Free GSH is then used to dissociate the bound GST (b), 10× longer exposure time than images shown in (a) and (c). The regenerated surface can then be used to bind a second GST protein; GST-bFGF (c). Scale bar = 10 μm. Reproduced by permission of The Royal Society of Chemistry from Kolodziej et al. J. Mater. Chem. 2011, 21, 1457−1461.

Figure 5. Comparison of nanoarrays developed by Saaem and co-workers to commercial microarrays. Anti-GST was immobilized on aminefunctionalized nanopatterned PEG hydrogels and used to capture GSTZNF9. The signal-to-noise ratio was calculated and compared to four commercial microarrays. The white bars show the results for detection of ZNF9 at 50 μg/mL, and the black bars show detection of ZNF9 at 1.25 μg/mL. Reproduced with permission of American Scientific Publishers from Saaem et al. J. Nanosci. Nanotechnol. 2007, 7, 2623−2632.

Figure 8. AFM and fluorescence images of nanopatterned growth factors. (a) Nanoscale patterns of pSS-co-PEGMA are visible in the height image taken with an atomic force microscope in tapping mode. Lines approximately 100 nm in width forming a square, triangle, concentric square, and circle are observed. Fluorescent image of (b) bFGF and (c) VEGF bound to the nanopatterns with antibody staining. Scale bar = 5 μm. Reproduced with permission from Christman, K. L. et al. J. Am. Chem. Soc. 2008, 130, 16585−16591. Copyright 2008 American Chemical Society.

(Figure 7). This weaker attachment allows surface-bound GST to be competitively dissociated by soluble GSH. GST is commonly used as a fusion tag to aid in the purification of expressed proteins; therefore, there are a wide variety of proteins that could be reversibly immobilized on these substrates. Another affinity interaction that has been exploited for the preparation of protein nanopatterns is the binding of growth factors to heparin-mimicking polymers. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been immobilized on nanopatterns of the heparinmimic poly(styrene-4-sulfonate-co-PEG methacrylate) (pSS-coPEGMA) that were fabricated by e-beam lithography (Figure 8).34 This mode of binding mimics the attachment of these growth factors to the natural extracellular matrix (ECM), which is important, because otherwise the proteins can lose bioactivity. Indeed, subsequent work has demonstrated that bFGF is stabilized against denaturation by immobilization on pSS-co-PEGMA nanopatterns.35 The ability to prepare arbitrary patterns offers exciting avenues for controlling cell adhesion for tissue engineering. Krsko and co-workers have reported the use of unmodified hydrogels fabricated by e-beam patterning of 6.8 kDa PEG to control cell

adhesion.36 These hydrogels were 200 nm in diameter and arranged in parallel lines with a spacing of 2.5 μm between each line. Fields of hydrogels with center-to-center distances varying from 1 to 10 μm were also investigated. The background of each substrate was coated with the cell-adhesive protein laminin. They found that both astrocytes and neurites aligned themselves parallel to the PEG line patterns (Figure 9a), with the neurites often forming linear chains of connected cells. The PEG arrays also were able to control cell adhesion by preventing cells from adhering on the patterned features and forcing them to adhere on the adhesive laminin regions. Of particular interest was the ability to direct the outgrowth of neurite axons in a zigzag pattern around the PEG features (Figure 9b).

Figure 6. Confocal images of nanopatterned actin filaments. Actin filaments immobilized on amine-functionalized PEG micropatterns were stained with Alexa Fluor 488 phalloidin. Images were taken at 0 μm (a), 1.8 μm (b), 3.6 μm (c), 5.4 μm (d), and 7.2 μm (e) from the silicon surface. The underlying pattern can be distinguished until 5.4 μm above the surface, above which the image appears uniform. Reproduced from reference 27. 778

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Figure 9. Astrocytes and neurites adhered on e-beam-patterned surfaces prepared by Krsko and co-workers. Neu7 astrocytes preferentially align with the axis of the cell-repellant PEG hydrogels (a). An image of a DRG neurite with its axon grown in a zigzag fashion to avoid the PEG hydrogels (b). Reprinted from Krsko et al. Length-scale mediated adhesion and directed growth of neural cells by surface-patterned poly(ethylene glycol) hydrogels. Biomaterials 2008, 30, 721−729. Copyright 2008, with permission from Elsevier.

on substrates containing only one component. The results indicate that these components modulate cell adhesion in a synergistic fashion. They also suggest that these patterns can be used to control and study cell adhesion.

In addition to fabricating patterns of cell-resistant PEG hydrogels using e-beam lithography, it is also possible to fabricate patterns of PEG hydrogels that contain cell adhesive components. The patterning of a PEG film containing a 1:1 w:w mixture of aminooxy-terminated 8-arm PEG and pSS-coPEGMA has been achieved and the resulting hydrogels used to immobilize both a ketone-functionalized RGD peptide and bFGF.35 RGD is the epitope on a number of ECM proteins that is recognized by integrins, the cell-surface receptors responsible for cell adhesion. These surfaces were shown to adhere endothelial cells via focal adhesions formed at the RGD features. Cells cultured on substrates containing both RGD and bFGF (Figure 10) possessed larger cell areas than cells cultured

1.3. SUMMARY AND OUTLOOK E-beam lithography is a versatile technique for patterning a wide variety of PEGs at varying length scales. To date, the technique has been employed to pattern thin films prepared from linear, multiarm, and graft PEGs containing a variety of different functional groups. Individual feature sizes ranging from tens of nanometers to one millimeter have been achieved, as well as multilayer and multicomponent structures. The hydrogels thus prepared are capable of immobilizing biomolecules containing the appropriate complementary functionality, while resisting nonspecific protein adsorption. E-beam patterning of PEGs has been employed to prepare surfaces for biosensing, which can offer sensitivities and feature densities that are superior to commercially available protein microarrays. It has also been used to prepare surfaces that modulate cell adhesion for tissue engineering applications. Ongoing work in this field will investigate the further application of surfaces patterned by e-beam lithography as substrates for tissue engineering capable of controlling cell processes such as cell migration, differentiation, and proliferation through control over spatial, topographical, and chemical signals. The ability to prepare surfaces that direct adhesion of different cell types to specific locations is also of great interest. Further investigation of e-beam patterned surfaces as diagnostic arrays could lead to greater improvements over current microarray devices. E-beam patterning of PEGs will likely be useful for intricate 3D patterns of multiple chemistries in precise locations to better mimic complex structures found in Nature.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 310-267-5162. Fax: 310-206-4038. E-mail: maynard@ chem.ucla.edu.

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Figure 10. Fluorescence images of HUVEC adhered on ECMmimicking substrate. Actin filaments (red, e) grown from focal adhesions (vinculin, green, b) on patterned substrates (blue, f) are visible in the composite image (a). Focal adhesions are more clearly visible in the single-channel enlarged image corresponding to antivinculin staining (c) and on patterned substrate (d). Scale bar = 20 μm. Reproduced with permission from Kolodziej et al. J. Am. Chem. Soc. 2011, DOI: 10.1021/ja205524x. Copyright 2011 American Chemical Society.

ACKNOWLEDGMENTS The authors are grateful for the National Science Foundation (CAREER CHE-0645793) for funding. ABBREVIATIONS E-beam, electron-beam; SAMs, self-assembled monolayers; OEG, oligo(ethylene glycol); PEG, poly(ethylene glycol); NTA, 779

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(27) Kolodziej, C. M. Fabrication of Protein Nanopatterns for Tissue Engineering and Molecular Sensing Applications. University of California, Los Angeles, Los Angeles, 2011. (28) Hong, Y.; Krsko, P.; Libera, M. Langmuir 2004, 20 (25), 11123−11126. (29) Christman, K. L.; Schopf, E.; Broyer, R. M.; Li, R. C.; Chen, Y.; Maynard, H. D. J. Am. Chem. Soc. 2009, 131 (2), 521−527. (30) Broyer, R. M.; Schopf, E.; Kolodziej, C. M.; Chen, Y.; Maynard, H. D. Soft Matter 2011, 7, 9972−9977. (31) Christman, K. L.; Broyer, R. M.; Schopf, E.; Kolodziej, C. M.; Chen, Y.; Maynard, H. D. Langmuir 2011, 27 (4), 1415−1418. (32) Saaem, I.; Papasotiropoulos, V.; Wang, T.; Soteropoulos, P.; Libera, M. J. Nanosci. Nanotechnol. 2007, 7 (8), 2623−2632. (33) Kolodziej, C. M.; Chang, C.-W.; Maynard, H. D. J. Mater. Chem. 2011, 21 (5), 1457−1461. (34) Christman, K. L.; Vazquez-Dorbatt, V.; Schopf, E.; Kolodziej, C. M.; Li, R. C.; Broyer, R. M.; Chen, Y.; Maynard, H. D. J. Am. Chem. Soc. 2008, 130 (49), 16585−16591. (35) Kolodziej, C. M.; Kim, S. H.; Broyer, R. M.; Saxer, S. S.; Decker, C. G.; Maynard, H. D. J. Am. Chem. Soc. 2011, DOI: 10.1021/ ja205524x. (36) Krsko, P.; McCann, T. E.; Thach, T. T.; Laabs, T. L.; Geller, H. M.; Libera, M. R. Biomaterials 2009, 30 (5), 721−729.

nitrilotriacetic acid; BSA, bovine serum albumin; SAv, streptavidin; pOEGMA, polymethacrylate containing OEG side chains; ZNF9, zinc finger 9; anti-GST, antiglutathione-S-transferase; GSH, ketone-modified glutathione; GST, glutathione-S-transferase; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; pSS-co-PEGMA, poly(styrene-4-sulfonate-co-PEG methacrylate; ECM, extracellular matrix

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REFERENCES

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