Biomacromolecules 2003, 4, 75-79
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Synthesis, Characterization, and Preliminary Biological Study of Poly(3-(tert-butoxycarbonyl)-N-vinyl-2-pyrrolidone) Wei He Department of Chemistry & Institute of Material Science, University of Connecticut, Storrs, Connecticut 06269
Kenneth E. Gonsalves* and John H. Pickett Department of Chemistry & C.C. Cameron Applied Research Center, University of North Carolina, Charlotte, North Carolina 28223
Craig Halberstadt General Surgery Research, Cannon Research Center, Carolinas Medical Center, Charlotte, North Carolina 28232 Received August 27, 2002; Revised Manuscript Received October 22, 2002
Poly(3-(tert-butoxycarbonyl)-N-vinyl-2-pyrrolidone) has been synthesized and characterized by gel permeation chromatography, Fourier transform infrared spectroscopy, NMR spectroscopy, and thermal analysis. The polymer is a chemically amplified photoresist. Arrays of lines with 25 µm width and 25 µm spacing were successfully patterned with this polymer by photolithography. Rat fibroblast cells were seeded on these patterned surfaces as well as the smooth glass surface. Phase contrast microscopy showed that cells on the patterned surfaces were strongly aligned and elongated along the grooves as compared to randomly spreading on the smooth surface. Since controlling cell orientation is critical for the development of advanced forms of tissue repair and cell engineering therapies, for example, peripheral nerve repair, production of tendon and ligament substitutes in vitro, and control of microvascular repair, the described polymer may be useful for applications in tissue reconstruction. 1. Introduction N-Vinyl-2-pyrrolidone (NVP) has been attracting much attention and has been widely investigated for applications in various fields.1-5 Because of its hydrophilic characteristic, polymers of NVP are known to have good biocompatibility with living tissues and extremely low cytoxicity.6 The applications of PNVP can be found in plasma substitutes, soluble drug carriers, and comonomers for UV-curable bioadhesives.7,8 It is known that copolymerization reactions can provide an excellent method for the preparation of macromolecules with specific chemical structures and for the control of properties such as solubility, polarity, and hydrophilic/ hydrophobic balances.9,10 NVP has been copolymerized with various monomers. For example, NVP can be copolymerized with methacrylic acid (MAA) to introduce carboxyl groups that are useful for yielding a wide variety of products. But it is also known that NVP does not copolymerize well because of its low reactivity ratio relative to the comonomers.11 As a result, the copolymer will exhibit pronounced compositional heterogeneity. To avoid this problem, one approach is to prepare homopolymers from functionalized NVP. In the present paper we report the synthesis and characterization of polymers of 3-(tert-butoxycarbonyl)-N-vinyl* Corresponding author. Tel.: +1-704-687-3501. Fax: +1-704-6876106. E-mail address:
[email protected].
2-pyrrolidone (t-BOC-NVP). t-BOC groups are chosen because they can be easily removed either thermally or under classical conditions in acidic medium. Moreover, poly(t-BOC-NVP) can be used as a photoresist material in UV lithography to generate patterns. Rat fibroblast cells were cultured on these patterned poly(t-BOC-NVP) films in a preliminary biocompatibility study. 2. Materials and Methods 2.1. Materials. Diisopropylamine, di-tert-butyl dicarbonate, azobisisobutyronitrile (AIBN), tetrahydrofuran (THF), hexanes, ethyl acetate, petroleum ether, dichloromethane, and propylene glycol methyl ether were obtained from Aldrich. N-Vinyl-2-pyrrolidone, butyllithium, and trifluoroacetic acid were obtained from Acros. Deuterated dimethyl sulfoxide, chloroform, and water were obtained from Cambridge Isotope Laboratories, Inc. Triarylsulfonium hexafluoroantimonate was purchased from Polysciences, Inc. All reagents were used as received unless otherwise noted. 2.2. Monomer Synthesis and Characterization. Monomer 3-(tert-butoxycarbonyl)-N-vinyl-2-pyrrolidone (t-BOCNVP) was synthesized according to the literature12 with modifications. N-Vinyl-2-pyrrolidone (11.12 g, 100 mmol) was added dropwise to a suspension of lithium diisopropylamide (prepared from diisopropylamine (10.12 g, 100 mmol) in anhydrous THF (40 mL) and n-butyllithium (40 mL, 100
10.1021/bm0256505 CCC: $25.00 © 2003 American Chemical Society Published on Web 11/27/2002
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mmol, 2.5 M solution in hexane)) at -78 °C. After the addition was complete, the resulting solution was stirred at this temperature for 2 h. Thereafter, di-tert-butyl dicarbonate (24 g, 100 mmol) was added dropwise, followed by the reaction at -78 °C for 2 h. The solution was allowed to warm to room temperature before saturated NH4Cl solution was added to quench the reaction. The organic layer was separated and washed with deionized water; the aqueous layer was extracted with diethyl ether. The combined organic solution was dried over anhydrous MgSO4 and concentrated with a Bu¨chi rotary evaporator. Purification by column chromatography (silica gel; hexanes/ethyl acetate ) 4/1) gave 10 g of pure 3-(tert-butoxycarbonyl)-1-vinyl-2-pyrrolidone (t-BOC-NVP) as a colorless liquid (47% yield). 1H NMR (DMSO-d6, ppm): δ 6.80-7.00 (dd, 1H), 4.48-4.60 (t, 2H), 3.35-3.60 (m, 3H), 2.10-2.40 (m, 2H), 1.40 (s, 9H). 13C NMR (DMSO-d6, ppm): δ 169.0, 168.5, 128.9, 95.6, 81.1, 49.5, 42.8, 27.5, 21.6. IR (AgCl, cm-1): 1737 (CdO of the ester group), 1704 (CdO, amide), 1638 (CdC). 2.3. Polymer Synthesis. Poly(t-BOC-NVP) was prepared by free radical polymerization in sealed ampules. The following is a representative synthesis. A solution of 2.0 g of t-BOC-NVP in 10 mL of THF (monomer concentration 1.0 mol‚L-1) containing 0.0246 g of AIBN (1.5 × 10-2 mol‚L-1) was heated to 65-75 °C under nitrogen. (Note: AIBN was purified by recrystallization in methanol) After 24 h, the polymer was precipitated in petroleum ether and subsequently redissolved (in THF) and reprecipitated to minimize the presence of residual unreacted monomer. The polymer was dried in vacuo at 40 °C to a constant weight (1.76 g, 88% yield). 2.4. Removal of the t-BOC Groups. The t-BOC protecting groups could be removed by trifluoroacetic acid (TFA). A typical reaction is as follows: The polymer (1 g, 4.74 mmol/t-BOC) was dissolved at room temperature in dichloromethane (10 mL), and TFA (2 mL) was added. A reaction occurred immediately with evolution of gas. After the reaction, the polymer was recovered by solvent evaporation and dried in vacuo. The yield was 0.94 g (94%). 2.5. Polymer Characterization. Gel permeation chromatography (GPC) measurement for molecular weight and molecular weight distribution was carried out on a Waters 2410 chromatography system (eluent THF, narrow molecular weight polystyrene standards, refractive index detection). Fourier transform infrared (FTIR) spectra of the polymers in KBr (spectroscopic grade) were recorded on a Nicolet spectrometer. 1H NMR spectra were recorded with a Varian UNITY 300 spectrometer. Thermal analyses of the polymers were conducted using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) measurements. TGA was performed on a TA instruments TGA 2950 at a heating rate of 20 °C/min in a nitrogen atmosphere. DSC was conducted on a TA instruments DSC 2920. 2.6. Biological Test. 2.6.1. Sample Preparation. Microscope slides (25 × 75 × 1 mm; VWR) were cut into three 25 × 25 mm squares. A 10 wt % quantity of poly(t-BOCNVP) was dissolved in propylene glycol methyl ether. To this solution, triarylsulfonium hexafluoroantimonate, acting as a photoacid generator, was added at an amount of 5 wt
He et al.
% based on the weight of the polymer. The solution was filtrated and spun on glass to form a thin film with a thickness of about 400 nm measured with a Tencor AlphaStep 200 surface profilometer. The clear transparent film was softbaked for 1 min at 120 °C to remove the solvent from the film. The samples were then patterned by exposing to UV light through a chrome mask. After exposure the samples were immediately baked for 1 min on a 120 °C hot plate. In the exposed area, the photoacid generator absorbed UV light and produced acid, which deprotected the polymer by reacting with the t-BOC groups. After deprotection, the polymer became highly hydrophilic. Therefore, the films were dipped in pure water for 60 s to develop the patterns. The features on the mask were lines of 25 µm width and 25 µm spacing. 2.6.2. Cell Culture. All cell culture reagents were obtained from SIGMA unless otherwise noted. Primary Wistar furth rat fibroblast cells (RFB) were isolated from rat dermis. All experimental procedures were done in the AAALACapproved Cannon Research Center vivarium following Institutional Animal Care and Use Committee guidelines. The cells were isolated using a modified collagenease digestion procedure.13 The isolated cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 20% fetal bovine serum (FBS) (heat inactivated), 1.25% L-glutamine (200 nM, GIBCO), 1% fungizone (GIBCO), 1% penicillin (10 000 U/mL)-streptomycin (10 mg/mL), and 2.5% HEPES buffer solution (1 M, GIBCO) at 37 °C in a humidified atmosphere of 92% air and 8% CO2. Cells were grown near confluence in T75 flasks and then subcultured every 4 days by dissociating with trypsinEDTA solution (1×). Glass slides with patterns were sterilized by rinsing with sterile water multiple times. The slides were placed in six-well tissue culture plates purchased from Costar. The plate was precoated with a thin film of 12% poly(hydroxyethyl methacrylate) (Polysciences) to guarantee that cells adhered only to the glass substrates. Cells (1.5 × 105) were seeded in each well. Nonpatterned regular glass slides were used as control. After seeding, fibroblast cells were allowed to stay in contact with the surfaces in an incubator at 37 °C with 8% CO2. The cultures were incubated for 1, 2, and 3 days. At the end of the various incubation periods, the cultures were rinsed with PBS to remove nonattached cells. Cell morphology was observed using an inverted Olympus phase-contrast microscope. 3. Results and Discussion 3.1. Synthesis and Characterization of the Monomer. The use of 3-(tert-butoxycarbonyl)-N-vinyl-2-pyrrolidone (tBOC-NVP) as a DUV photoresist material was first studied by Kim et al.12 Briefly, t-BOC-NVP was prepared by reacting N-vinyl-2-pyrrolidone (NVP) with a strong base (i.e., lithium diisopropylamide (LDA)) in THF at -78 °C to form an enolate, and a t-BOC protecting group was then introduced at the 3-position of NVP. The reagent of choice for introduction of the t-BOC group is di-tert-butyl dicarbonate, a readily available reagent that is both safe and easy to use. FTIR, NMR, elemental analysis, and gas chromatography/ mass spectrometry (GC/MS) were applied to characterize the
Study of a Photoresist Polymer
Figure 1. FTIR spectra of (a) 3-(tert-butoxycarbonyl)-N-vinyl-2pyrrolidone (t-BOC-NVP) and poly(t-BOC-NVP) (b) before deprotection and (c) after deprotection.
monomer. A molecular peak at 211 was detected in the GC/ MS measurement, which is consistent with that calculated from the molecular formula. Elemental analysis of the monomer for C (62.50%), H (8.22%), and N (6.62%) is in good agreement with the calculated values: 62.54% C, 8.11% H, and 6.63% N. In the FTIR spectrum (Figure 1a), a characteristic band of the vinyl group due to CdC stretching was observed at 1638 cm-1. The two peaks overlapping in the 1790-1680 cm-1 region were caused by CdO stretching. The strong absorption at the lower wavenumber (1704 cm-1) is due to CdO stretching of the amide carbonyl group, while the absorption at 1737 cm-1 arises from CdO stretching of the t-BOC carbonyl group. The strong absorption at 1152 cm-1 is caused by the C-C(dO)-O stretching vibration from the ester group. The 1H NMR of the monomer showed a very sharp singlet at 1.4 ppm, which is characteristic for the tert-butyl protons. Protons of the lactam ring showed broad multiplets at 2.12.4 ppm and ∼3.5 ppm, respectively. A triplet at 4.5 ppm for the two methylene protons of the vinyl group, and a doublet-doublet at 6.9 ppm for the methine proton of the vinyl group were also observed. 13C NMR spectrum was recorded to further confirm the chemical structure of the monomer. As expected, nine different carbon peaks were observed corresponding to the nine different carbons in the structure. The peaks at 168.5 and 169.0 ppm can be assigned to the ester carbonyl carbon and amide carbonyl carbon, respectively. 3.2. Synthesis and Characterization of the Polymer. Poly(t-BOC-NVP) was prepared via free radical solution polymerization. The polymer was obtained as a white solid and characterized by GPC, FTIR, 1H NMR, TGA and DSC. The molecular weight of the homopolymer is about 12 500 for Mw with a polydispersity of 1.43. The t-BOC protecting group can be removed either thermally or under classical conditions in acidic medium. The best result is given by trifluoroacetic acid, which removes the t-BOC groups very cleanly. Therefore, trifluoroacetic acid was used in our study for deprotection of poly(t-BOC-NVP). After converting all
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Figure 2. 1H NMR spectra of poly(t-BOC-NVP) (a) before deprotection and (b) after deprotection. Insert is the structure of poly(t-BOC-NVP).
Figure 3. Phase contrast images (20× magnification) of rat fibroblasts on the regular glass slide (a) and on the micropatterned polymeric surface (b) after 1 day of incubation (bar ) 50 µm). The insert displays patterns before cell culturing (4× magnification).
the t-BOC groups to carboxylic groups, the deprotected polymer was characterized with FTIR, 1H NMR, TGA, and DSC. The IR spectrum of the polymer (Figure 1b) shows two peaks at 1726 and 1686 cm-1 for the carbonyl groups, which are characteristic patterns for the t-BOC group and the amide group. The disappearance of the peak at 1638 cm-1 due to
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Figure 4. Phase contrast images (20× magnification) of rat fibroblasts on the regular glass slide (a, c) and on the micropatterned polymeric surface (b, d) after 2 days (a, b) and 3 days (c, d) of incubation (bar ) 50 µm).
vinyl bonds indicated that the polymer was free of residual monomer. After the t-BOC groups were converted to carboxyl groups, a broad O-H stretching absorption is observed in the region of 3600-2500 cm-1 (Figure 1c). The C-C(dO)-O stretching band of the acid group at 1152 cm-1 is much weaker compared to that of the ester group in Figure 1b. The carbonyl stretching bands are also broader because of the intermolecular hydrogen bonding formed between the carboxyl and the amide carbonyl groups. Compared with the ester carbonyl, the CdO stretching of the acid absorbed at a lower frequency. Figure 2 shows the 1H NMR spectra of poly(t-BOC-NVP) before and after deprotection. No peaks are observed between 4.5 and 7.0 ppm in Figure 2a, which further verified the absence of monomers. The major difference between Figure 2a and Figure 2b is the disappearance of the peak at ∼1.4 ppm indicating the removal of all the tert-butyl protons. Since the polymer is very hydrophilic after deprotection, it was insoluble in most organic solvents, thus deuterium oxide (D2O) was used for NMR measurement. The peaks at 1.2 and 4.8 ppm are from the solvent. The thermal behavior of poly(t-BOC-NVP) before and after deprotection was measured by thermogravimetric analysis (TGA) at a heating rate of 20 °C/min in nitrogen atmosphere (results not shown). Poly(t-BOC-NVP) is thermally stable up to 180 °C. When the temperature is higher than 180 °C, rapid deprotection of the tert-butoxy-
carbonyl (t-BOC) group occurs, with evolution of one molecule of carbon dioxide and one molecule of 2-methylpropene per t-BOC group (which amounts to 47% of the total mass of the polymer). After deprotection, the polymer now has become a β-keto acid, and it decarboxylated readily when it was heated to 100 °C, which further confirmed the successful removal of all the t-BOC groups. A glass transition was observed when differential scanning calorimetry was applied to poly(t-BOC-NVP) at a heating rate of 5 °C/min. The glass transition temperature was 124 °C (results not shown), while the values reported in the literature range from 145 to 150 °C, depending on the molecular weight thereof. No obvious glass transition was detected for the deprotected polymer before it reached the decomposition temperature. 3.3. Cell Culture. Poly(t-BOC-NVP) can be used as a photoresist when a photoacid generator (PAG) was applied. The PAG used in our study is triarylsulfonium hexafluoroantimonate, which can produce H+ after absorbing UV light. The acid will then deblock the t-BOC groups resulting in very hydrophilic product which is soluble in water, as shown above. The resist solution was spun on a glass substrate and exposed to UV light through a chrome mask. Lines of 25 µm width and separated by a distance of 25 µm were created. Rat fibroblast cells were seeded on these patterned surfaces at a density of 150 000 cells/sample and cultured for 1, 2, and 3 days. Cells were also cultured on regular glass slides as control.
Study of a Photoresist Polymer
As seen in Figure 3a, after 1 day of culturing, cells had a very flattened appearance on the regular glass slides. They were spindle shaped and randomly distributed over the surface. On the patterned polymer substrate (Figure 3b), it was observed that the cells adhered and spread on the surface, which means the polymer is nontoxic and biocompatible. Moreover, cells were strongly aligned along the engineered grooves (see insert photo). They became bipolar and elongate. Cell alignment along the grooves could be due to several factors. Because of the different chemistry associated with the polymer that formed the grooves, one possible mechanism for selective cell adhesion could be related to preferential protein absorption.14 Primarily, fibronectin and vitronectin that are in the serum may have preferentially coated the surface along the grooves. Hence, the cells adhered to the surface through an integrin/ligand interaction. A second factor could be due to the surface free energy being more suitable for cell adhesion along the groove surface. This phenomenon has been demonstrated by other groups to influence cell behavior on material surfaces.14 A third potential mechanism could be contact guidance. It has been shown that the microtexture of a substrate surface can influence the behavior of the cells growing on such substrates in vitro.15-18 Many cell types, such as fibroblasts, neurons, osteoblasts, and macrophage-like cells, recognize these surface features and react accordingly, probably by reshaping the actin filaments in their surface-probing structures.19 Walboomers et al.20 proposed that the dynamics of actin polymerization could be an explanation of contact guidance. The groove depth in our study was 400 nm. It has been shown that cells such as fibroblasts and endothelias react to depths as shallow as 70 nm.17 The total time of the in vitro experiment was 3 days. As shown in Figure 4, cells were still attached and appeared to be proliferating on both the smooth and the micropatterned surfaces after 2 and 3 days of incubation. On the smooth surfaces (parts a and c of Figure 4), the stellate fibroblast cells were growing randomly, while on the patterned surfaces, the main vector of orientation seemed to be directed parallel to the lines and the shape of the cells were elongated as compared to the control. By the second day, the cells still appeared to be oriented along the grooves. However, cells were able to bridge the gaps and form contacts with cells that were aligned on the parallel grooves. This was evident by the third day in culture and could be due to the short distance of the spaces between the grooves. Our results imply a potential application of using this technique in combination with 3-D constructs to produce an oriented tissue-like structure from fibroblasts, which will have desirable mechanical strength and flexibility similar to that of normal tissue. In general, the ability to control cell orientation is critical for the development of advanced forms of tissue repair and cell engineering therapies, for example, peripheral nerve repair, production of tendon and ligament substitutes in vitro, and the control of microvascular repair.21
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Conclusions A functionalized NVP with incorporation of t-BOC groups at the 3-position was prepared and its homopolymer was synthesized by free radical polymerization. The deblocking t-BOC groups make this polymer applicable to UV photolithography. Lines of 25 µm width separated by 25 µm spacing were generated with such a polymer, and rat fibroblast cells were seeded on such surfaces. Compared to cells growing on smooth glass slides, cells cultured on the patterned surfaces were observed to strongly orient along the lines with elongated shapes. Such orientation was maintained during cell proliferation. Long-term fibroblast cell culturing on the micropatterned surfaces is currently in progress. Protein adsorption on the micropatterned surfaces and its effect on cell attachment, as well as the influence of surface free energy will also be studied soon. Acknowledgment. The authors thank the generous help from Dr. Kristi Harold, MD, and Shirley Coleman for cell culturing work. K.E.G. acknowledges the North Carolina Biotechnology Center for partial support. References and Notes (1) Radic, D.; Gargallo, L. Macromolecules 1997, 30, 817. (2) De Queiroz, A.; Vargas, R. R.; Higa, O. Z.; Ribeiro, R. R.; Vitolo, M. J. Appl. Polym. Sci. 2002, 84, 767. (3) Bajpai, S. K.; Sonkusley, J. J. Appl. Polym. Sci. 2002, 83, 1717. (4) Beitz, T.; Kotz, J.; Wolf, G.; Lleinpeter, E.; Fribery, S. E. J. Colloid Interface Sci. 2001, 240, 581. (5) Basri, M.; Harun, A.; Ahmad, M. B.; Razak, C. A. N.; Salleh, A. B. J. Appl. Polym. Sci. 2001, 82, 1404. (6) Vijayasekaran, S.; Chirila, T. V.; Hong, Y.; Tahija, S. G.; Dalton, P. D.; Constable, I. J.; McAllister, I. L. J. Biomater. Sci., Polym. Ed. 1996, 7, 685. (7) Ranucci, H.; Spagnoli, G.; Sartore, L.; Bignottie, F.; Ferruti, P. Macromol. Chem. Phys. 1995, 196, 763. (8) Kao, F.; Manivannan, G.; Sawan, S. J. Biomed. Mater. Res. 1997, 38, 191. (9) Gallardo, A.; Lemus, A. R.; San Roman, J.; Cifuentes, A.; DiezMasa, J. C. Macromolecules 1999, 32, 610. (10) Brar, A. S.; Kumar, R. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 2225. (11) Brandrup, J., Immergut, E. H., Eds. Polymer Handbook; John Wiley & Sons: New York, 1989. (12) Kim et al. U.S. Patent, 5,750,680, 1998. (13) Doyle, A., Griffiths, J. B., Newell, D. G., Eds. Dissociation of Dermal Fibroblasts in Cell and Tissue Culture: Laboratory Procedures; John Wiley & Sons: New York, 1996. (14) Saltzman, W. M. Cell Interactions with Polymers. In Principles of Tissue Engineering; Lanza, P. P., Langer, R., Vacanti, J., Eds.; Academic Press: New York, 2000. (15) Singhvi, R.; Stephanopoulos, G.; Wang, D. I. C. Biotechnol. Bioeng. 1994, 43, 764. (16) Von Recum, A. F.; Shannon, C. E.; Cannon, E. C.; Long, K. J.; Van Kooten, T. G.; Meyle, J. Tissue Eng. 1996, 2, 241. (17) Curtis, A.; Wilkinson, C. Biomaterials 1997, 18, 1573. (18) Curtis, A.; Wilkinson, C. J. Biomater. Sci. 1998, 9, 1313. (19) Walboomers, X. F.; Croes, H. J. E.; Ginsel, L. A.; Jansen, J. A. Biomaterials 1998, 19, 1861. (20) Walboomers, X. F.; Monaghan, W.; Curtis, A. S. G.; Jansen, J. A. J. Biomed. Mater. Res. 1999, 46, 212. (21) Mudera, V. C.; Pleass, R.; Eastwood, M.; Tarnuzzer, R.; Schultz, G.; Khaw, P.; McGrouther, D. A.; Brown, R. A. Cell Motil. Cytoskeleton 2000, 45, 1.
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