Thermoresponsive Surface-Grafted Poly(N−isopropylacrylamide

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Thermoresponsive Surface-Grafted Poly(N-isopropylacrylamide) Copolymers: Effect of Phase Transitions on Protein and Bacterial Attachment David Cunliffe, Carolina de las Heras Alarco´n, Vanessa Peters, James R. Smith, and Cameron Alexander* School of Pharmacy and Biomedical Sciences, University of Portsmouth, White Swan Road, Portsmouth PO1 2DT, United Kingdom Received August 5, 2002. In Final Form: December 13, 2002 The ability of polymers displaying lower critical solution temperatures (LCSTs) to mediate bioadsorptive processes was assessed. Three carboxyl-terminated polymers P1-3 with LCSTs respectively of 20, 32, and 42 °C were prepared by free-radical polymerization of N-isopropylacrylamide with and without comonomers acrylamide and N-tert-butylacrylamide. The polymers were grafted to amine-functionalized glass substrates, and their surface properties were investigated by contact angle goniometry and atomic force microscopy. Increases in water contact angle of up to 24° were observed between 10 and 37 °C for polymers with LCSTs of 20 and 32 °C, whereas no change was apparent for control amine-functional and the LCST 42 °C polymer surfaces over this temperature range. Variations in topography in water were also apparent from atomic force microscopy (AFM) studies for all the polymer grafts but not the amine surfaces over these temperatures. Adsorption of 3H-labeled bovine serum albumin and cytochrome c also increased to polymer grafts above the LCST, with the greatest change in the amount of attached protein being exhibited by polymer P1 (1.13 pmol‚cm-2 cytochrome c at 10 °C, 3.95 pmol‚cm-2 at 37 °C): adsorption to control surfaces varied by less than 10% in this assay. Incubation of the graft and control substrates with a gram negative and motile bacterium (Salmonella typhimurium) and gram positive, nonmotile species (Bacillus cereus) showed the same overall pattern of attachment as the protein adsorption experiments, with polymers P1 and P2 retaining more bacteria (increases of up to 1350%) at 37 °C than at temperatures below their LCST, while amine-functional and P3 polymer surfaces showed less than 20% changes in the number of attached microorganisms. Further incubations at temperatures below polymer LCST resulted in fewer adsorbed cells at the surfaces showing the reversibility of short-term attachment to these materials. The results show that protein adsorption and short-term bacterial attachment correlate well with observed changes in surface properties as determined by contact angle goniometry and indicate that control of bioadhesion is possible by grafting suitably functionalized polymers capable of temperature-mediated hydrophilichydrophobic switching.

Introduction The fabrication of surfaces that can control bioadhesion is of great theoretical interest and practical significance and presents a continuing challenge for materials scientists.1 In particular, synthetic materials which can bind a biological surface under one set of conditions yet resist attachment under another are desirable for a number of applications in the biomedical sciences.2 These include surface adhesion modifiers,3 selective drug targeting devices,4 and delivery vehicles for therapeutic agents.5,6 The practical value of this class of materials also extends to their use as biochemically triggered actuators or valves,7 as supports for cell culture,8,9 tissue engineering,10 and in * Author to whom correspondence should be addressed. Telephone: +44(0) 23 9284 3598. Fax:: +44(0) 23 9284 3565. E-mail: [email protected]. (1) Kingshott, P.; Griesser, H. J. Curr. Opin. Solid State Mater. Sci. 1999, 4, 403-412. (2) Okano, T.; Kikuchi, A.; Sakurai, Y.; Takei, Y.; Ogata, N. J. Controlled Release 1995, 36 (1-2), 125-133. (3) Koberstein, J. T.; Duch, D. E.; Hu, W.; Lenk, T. J.; Bhatia, R.; Brown, H. R.; Lingelser, J. P.; Gallot, Y. J. Adhes. 1998, 66 (1-4), 229-249. (4) Meyer, D. E.; Shin, B. C.; Kong, G. A.; Dewhirst, M. W.; Chilkoti, A. J. Controlled Release 2001, 74 (1-3), 213-224. (5) Peppas, N. Curr. Opin. Colloid Interface Sci.1997, 2 (5), 531537. (6) Lowman, A.; Morishita, M.; Kajita, M.; Nagai, T.; Peppas, N. J. Pharmaceut. Sci. 1999, 88 (9), 933-937. (7) Li, S. K.; D’Emanuele, A. J. Controlled Release 2001, 75 (1-2), 55-67.

vitro toxicology experiments,11-13 wherein the cells are adhered to the substrate during a particular assay, but can be released at the end of a cycle for analysis or further screening by a simple change in environmental conditions. We have been interested in using environmentally switchable materials, especially thermoresponsive polymers, as probes of biological adhesion mechanisms and as potential mediators of bacterial attachment. Responsive polymers in general have attracted very considerable attention over recent years as “smart” materials14-18 for (8) Aoki, T.; Nagao, Y.; Terada, E.; Sanui, K.; Ogata, N.; Yamada, N.; Sakurai, Y.; Kataoka, K.; Okano, T. J. Biomater. Sci., Polym. Ed. 1995, 7 (7), 539-550. (9) Chen, G. P.; Imanishi, Y.; Ito, Y. J. Biomed. Mater. Res. 1998, 42 (1), 38-44. (10) Yamato, M.; Utsumi, M.; Kushida, A.; Konno, C.; Kikuchi, A.; Okano, T. Tissue Eng. 2001, 7 (4), 473-480. (11) Takei, Y. G.; Aoki, T.; Sanui, K.; Ogata, N.; Sakurai, Y.; Okano, T. Biomaterials 1995, 16 (6 (9)), 667-673. (12) Yamato, M.; Kwon, O. H.; Hirose, M.; Kikuchi, A.; Okano, T. J. Biomed. Mater. Res. 2001, 55 (1), 137-140. (13) Takezawa, T.; Mori, Y.; Yoshizato, K. Biotechnology 1990, 8, 854-856. (14) Saunders, B. R.; Vincent, B. Adv. Colloid Interface Sci. 1999, 80 (1), 1-25. (15) Rajagopalan, S.; Zhu, X. X. Abstr. Pap.-Am. Chem. Soc. 2000, 220, 205. (16) Badiger, M. V.; Lele, A. K.; Bhalerao, V. S.; Varghese, S.; Mashelkar, R. A. J. Chem. Phys. 1998, 109, 1175-1184. (17) Chen, G. H.; Hoffman, A. S. Nature 1995, 373, 49-52. (18) Hoffman, A.; Stayton, P.; Shimoboji, T.; Chen, G.; Ding, Z.; Chilkoti, A.; Long, C.; Miura, M.; Chen, J.; Park, T.; Monji, N.; Cole, C.; Harris, J.; Nakamae, K. Macromol. Symp. 1997, 118, 553-563.

10.1021/la026358l CCC: $25.00 © 2003 American Chemical Society Published on Web 02/19/2003

Thermoresponsive Surface-Grafted Copolymers

a great many applications, including in controlled drug delivery,19-21 in chemical sensing,22 as processing or separation aids,23,24 and even as artificial organs.25 The use of poly(N-isopropylacrylamide) (PNIPAm) copolymers as bacterial adhesion mediators is less well established, but has been generating increasing interest following the first reports in this area from Lopez and co-workers.26-29 In a previous communication,30 we evaluated the ability of surfaces grafted with thermoresponsive PNIPAm to mediate the attachment of the common pathogen Listeria monocytogenes as the polymer underwent a thermally driven switch from a hydrophilic to hydrophobic state. Here we investigate the variation of attachment of model proteins, bovine serum albumin (BSA) and cytochrome c (Cyt c), as well as bacterial pathogens representative of both gram negative and motile species (Salmonella typhimurium (S. typhinurium)) and gram positive, nonmotile (Bacillus cereus (B. cereus)) with phase changes of surface-grafted poly(N-isopropylacrylamide) homopolymer and related copolymers. Specifically we describe the effect of changing temperature on the hydrophilic/ hydrophobic properties of surface-grafted PNIPAm copolymers and demonstrate that this leads to variation in bioadhesion. Experimental Section Materials and Methods. Chemicals. Standard reagents and chemicals were purchased from Fisher Scientific or Aldrich and used as received. D-U-14C)-glucose and 3H-acetic anhydride were obtained from Amersham Life Sciences (Amersham). Cytochrome c (bovine heart) and bovine serum albumin were purchased from Sigma and used as received. Acrylamide, N-tert-butylacrylamide and N-isopropylacrylamide were purchased from Aldrich: the N-alkylacrylamides were recrystallized from hexane before use. Purification of solvents for preparative chemistry was performed by standard methods.31 For contact angle goniometry, double distilled water (surface tension, 72.8 mN m-1 at 20.0 °C), diiodomethane (Aldrich; >99%; surface tension, 50.7 mN m-1 at 20.0 °C) and ethylene glycol (Aldrich; 99.8%; anhydrous; surface tension, 48.0 mN m-1 at 20.0 °C) were used. Preparation of Amine-Functional Glass Surfaces. Silica glass slides (Chance Propper) were cleaned with 5 M sodium hydroxide solution, rinsed in deionized water (10 × 100 mL), dried, immersed in fresh ammonium persulfate/sulfuric acid (1% (19) Kost, J.; Langer, R. Adv. Drug Deliv. Rev. 2001, 46 (1-3), 125148. (20) Neradovic, D.; Hinrichs, W. L. J.; Kettenes-Van Den Bosch, J. J.; Van Nostrum, C. F.; Hennink, W. E. J. Controlled Release 2001, 72 (1-3), 252-253. (21) Inoue, T.; Chen, G. H.; Hoffman, A. S.; Nakamae, K. J. Bioact. Compat. Polym. 1998, 13 (1), 50-64. (22) Chen, J. H.; Yoshida, M.; Maekawa, Y.; Tsubokawa, N. Polymer 2001, 42 (23), 9361-9365. (23) Meyer, D.; Chilkoti, A. Nature Biotechnol. 1999, 17 (11), 11121115. (24) Hoffman, A. S.; Stayton, P. S.; Bulmus, V.; Chen, G.; Chen, J.; Cheung, C.; Chilkoti, A.; Ding, Z.; Dong, L.; Fong, R.; Lackey, C. A.; Long, C. J.; Miura, M.; Morris, J. E.; Murthy, N.; Nabeshima, Y.; Park, T. G.; Press: O. W.; Shimoboji, T.; Shoemaker, S.; Yang, H. J.; Monji, N.; Nowinski, R. C.; Cole, C. A.; Priest, J. H.; Harris, J. M.; Nakamae, K.; Nishino, T.; Miyata, T. J. Biomed. Mater. Res. 2000, 52 (4), 577586. (25) Angelova, N.; Hunkeler, D. Trends Biotechnol. 1999, 17 (10), 409-421. (26) Ista, L. K.; Lopez, G. P. J. Indust. Microbiol. Biotechnol. 1998, 20, 121-125. (27) Ista, L. K.; Perez-Luna, V. H.; Lopez, G. P. Appl. Environ. Microbiol. 1999, 65, 1603-1609. (28) Callow, M. E.; Callow, J. A.; Ista, L. K.; Coleman, S. E.; Nolasco, A. C.; Lopez, G. P. Appl. Environ. Microbiol. 2000, 66 (8), 3249-3254. (29) Ista, L. K.; Mendez, S.; Perez-Luna, V. H.; Lopez, G. P. Langmuir 2001, 17, 2552-2555. (30) Cunliffe, D.; Smart, C. A.; Tsibouklis, J.; Young, S.; Alexander, C.; Vulfson, E. N. Biotechnol. Lett. 2000, 22 (2), 141-145. (31) Perrin, D. D.; Amarego, W. F.; Perrin, D. R. Purification of Laboratory Chemicals; Pergamon: Oxford, U.K., 1980.

Langmuir, Vol. 19, No. 7, 2003 2889 (w/v)) (CAUTION! hazardous oxidizing agents), washed again (10 × 100 mL) in deionized water and ethanol, and then dried at 80 °C for 24 h. The slides were amine functionalized by treatment with (3-aminopropyl)triethoxysilane (APTES) in aqueous acidified methanol solution according to a previously reported method.32 Synthesis of Poly(N-alkylacrylamide) Copolymers. Acrylic monomers (acrylamide, N-isopropylacrylamide, N-tert-butylacrylamide in the required mole fraction, 10.0 g total) were dissolved in propan-2-ol (40 mL) in a thick walled Schlenk tube, and 3-mercaptopropanoic acid (0.344 mmol) and 4,4′-azobis(4cyanovaleric acid) (2.83 mmol) were added. The solution was degassed by freeze-thaw cycles under vacuum at least three times and placed in a thermostated oil bath at 65 °C for 24 h. After cooling to room temperature, the mixture was concentrated under reduced pressure and the residue added to diethyl ether (250 mL). The precipitated polymer was filtered, and the residue was redissolved in tetrahydrofuran (THF) and reprecipitated into diethyl ether (250 mL) three times. The purified polymer was then dried in vacuo at 20 °C overnight. IR and NMR spectra of the purified polymers were consistent with those of the expected products.33 Determination of Cloud Points. Poly(N-alkylacrylamide) copolymers were dissolved in 3-[N-morpholino]propanesulfonic acid (MOPS) buffer (50 mM, pH 7.0) to a concentration of 10 mg‚mL-1. These solutions were first cooled to 5 °C and then heated to 50 °C at 0.5 °C‚min-1 in the thermostated block of the UV spectrometer. The cloud point was taken as the onset of a sharp increase in absorption at 500 nm. Chemical Modification of Surfaces. Amine-functionalized cover slips were placed in MOPS buffer (10 mM, pH 4.75, 10 mL) and cooled to 5 °C. Carboxyl-terminated poly(N-alkylacrylamide) (1.0 g) was added to the buffer solution followed by amounts of 1-ethyl3,3-dimethylaminopropylcarbodiimide hydrochloride (EDC) (3 × 200 mg) every 30 min with gentle stirring. After addition was complete the surfaces were left at 0 °C for a further 72 h and then thoroughly washed with deionized water and dried under a stream of nitrogen. The surfaces were stored in a dry nitrogen atmosphere prior to use. Labeling and Purification of Proteins. BSA and Cyt c were labeled with 3H acetic anhydride as reported previously.34 Protein concentration was determined using the Lowry method for BSA and from a spectrophotometric calibration curve (408 nm) for Cyt c. Adsorption of Proteins to Synthetic Surfaces. To determine protein adsorption to the surfaces, radio-labeled BSA (typically 37.5 µg) or Cyt c (42.5 µg) was added to MOPS (50 mM, pH 7.0, 10 mL) in 15 mL screw capped bottles. Derivatized glass cover slips, prepared and stored as described above, were transferred to the protein solutions. The bottles were incubated, with gentle shaking, for 1 h, rinsed twice in MOPS (50 mM, pH 7.0, 10 mL) and transferred to 5 mL scintillation vials. Growth and Labeling of Microorganisms. Bacterial cultures were maintained on nutrient agar (CM3 Oxoid, containing in g‚L-1: Lab-Lemco powder, 1.0; yeast extract, 2.0; peptone, 5.0; sodium chloride, 5.0; agar, 15.0) at pH 7.4, 4 °C. Stock cultures of Salmonella typhimurium NCTC 12023 and Bacillus cereus (wild strain isolated at the Public Health Laboratory, Portsmouth, U.K.) were grown statically overnight at pH 7.4, 37 °C in nutrient broth (CM1 Oxoid, containing in g‚L-1: Lab-Lemco powder, 1.0; yeast extract, 2.0; peptone, 5.0; sodium chloride, 5.0) to reach stationary phase, and aliquots (100 µL) of each culture in sterile Eppendorf tubes were stored at -70 °C prior to use. For fluorescence microscopy, the bacterial cultures were prestained with ethidium bromide or acridine orange solutions (10 mg‚mL-1, 1 mL), incubated for 3 h, and then centrifuged (5 min, 3000 rpm). The supernatants were removed, and pellets of microorganisms were resuspended in broth (10 mL) by vortex mixing (30 s). To obtain 14C-glucoselabeled bacteria, nutrient broth (6 mL) was first inoculated with (32) Durfor, C. N.; Turner, D. C.; Georger, J. H.; Peek, B. M.; Stenger, D. A. Langmuir 1994, 10, 148-152. (33) Deshmukh, M. V.; Vaidya, A. A.; Kulkarni, M. G.; Rajamohanan, P. R.; Ganapathy, S. Polymer 2000, 41 (22), 7951-7960. (34) Cunliffe, D.; Smart, C. A.; Alexander, C.; Vulfson, E. N. Appl. Environ. Microbiol. 1999, 65, 4995-5002.

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thawed stock culture (50 µL), and an aliquot (100 µL) containing 20 µCi of D-(U-14C)-glucose from a 200 µCi‚mL-1 stock solution was added. The culture was then incubated (37 °C, 24 h) statically. To determine absolute cell numbers, aliquots (200 µL) of unlabeled organisms were enumerated via a serial dilution method. The absolute cell numbers determined in this way were compared with scintillation counts from equivalent aliquots (200 µL) of radio-labeled bacteria. In Vitro Adsorption Studies. Bacterial cultures (107-108 cfu.mL-1), either radio-labeled or stained with ethidium bromide or acridine orange (6 mL) were transferred to centrifuge tubes and centrifuged (8000 rpm, 10 min), washed twice in sterile MOPS 50 mM, pH 7.0, 6 mL), and resuspended in MOPS (6 mL). Aliquots (200 µL) of the suspended bacteria were transferred to sterile MOPS (10 mL) in capped bottles. Functionalized silica glass test surfaces (1 cm2), with and without polymer grafts, were then placed in the capped bottles and incubated, with gentle shaking (40 rpm), for 24 h at 5 or 37 °C. The slides were rinsed twice by immersion in sterile MOPS (10 mL) at the assay temperature and then examined by fluorescence microscopy or transferred to scintillation vials containing Ultima Gold scintillant cocktail (5 mL). All experiments were conducted at least in triplicate. Instrumentation. Contact Angle Goniometry. A Kruss G10 contact angle measuring system equipped with a sealed sample chamber, and automated image analysis system was used. Drops of liquid of known volume (1-4 µL) were applied from a microsyringe to the surface of the test material through a small port at the top of the cell: to avoid cross-contamination of liquids, a dedicated microsyringe was used for each diagnostic liquid. The precision of the angle measurement was (0.5°. Atomic Force Microscopy. Glass surfaces grafted with APTES and/or polymers were rinsed with water (18 MΩ), dried with nitrogen, and mounted on nickel disks (mounting assemblies) prior to atomic force microscopy (AFM) investigation. AFM studies were performed using a TopoMetrix TMX2000 Discoverer scanning probe microscope (ThermoMicroscopes) with a 70 × 70 × 12 µm tripod piezoelectric scanner. Topography measurements were conducted using “V”-shaped silicon nitride cantilevers bearing an integrated standard profile tip (length, 200 µm; nominal spring constant (K), 0.032 N‚m-1; Part. No. 1520-00, ThermoMicroscopes, Santa Clara, CA). Topographic imaging was performed in air and in water using a closed wet cell, modified to allow variable temperature adjustment. Contact mode imaging utilized an applied load and scan rate limited to ca. 1 nN and 3 Hz, respectively, to minimize compression and lateral damage to polymer grafts and underlying surfaces. Infrared Spectroscopy. Infrared spectra were obtained employing a Perkin-Elmer Paragon 1000 FT-IR spectrophotometer, in transmittance mode, at a resolution of 4 cm-1. NMR Spectroscopy. 1H and 13C NMR spectra were recorded on JEOL EX-270 and Eclipse 400 spectrometers at 270 and 399.8 MHz (1H) and 67.6 and 100.5 MHz (13C), respectively. Deuterated solvents used (D2O, CDCl3) were purchased from Spectra-Tech/ Wilmad. All chemical shifts are reported in part per million relative to tetramethylsilane or sodium 3-(trimethylsilyl)-1propanesulfonate. UV Spectroscopy. A Cecil CE1010 spectrophotometer (Cambridge, U.K.) operating at a fixed wavelength of 500 nm was used. Microscopy. A Vickers Epifluorescent microscope, with a broad band mercury lamp fitted with a green excitation filter system, was employed in conventional fluorescence mode. For confocal laser scanning microscopy a Zeiss LSM 510 instrument was used, fitted with Argon and He/Ne lasers operating at excitation wavelengths of 453, 488, and 543 nm, using emission filters of 505-530 and 585-615 nm. Micrographs shown were selected at random from 6 to 10 fields of view recorded per sample and the numbers of cells enumerated over triplicate fields. Scintillation Counting. A Packard Tri-Carb 1900TR liquid scintillation analyzer was used (Packard Instrument Company, Meriden, CT).

Cunliffe et al. Table 1. Characterization Data for Polymers polymer P1 P2 P3

monomer feed composn/(mol %) N-isopropylacrylamide, 80; N-tert-butylacrylamide, 20 N-isopropylacrylamide, 100 N-isopropylacrylamide, 85; acrylamide, 15

molecular cloud point weight (pH 7)/°C 5700

20.0

6500 5400

32.2 42.1

Results and Discussion

of undergoing phase transitions in physiologically relevant conditions of pH and temperature. Accordingly, three carboxyl-terminated polymers were prepared with lower critical solution temperatures (LCSTs) in aqueous buffer at pH 7.0 of 20, 32, and 42 °C: these were respectively poly(N-isopropylacrylamide-co-tert-butylacrylamide) (P1), poly(N-isopropylacrylamide) (P2), and poly(N-isopropylacrylamide-co-acrylamide) (P3). To evaluate the effects of the phase transitions as much as possible in the absence of other variables, chain transfer agents were employed during the polymerizations to ensure the polymers were of similar molecular weight. The measured Mw values were in the range of 5400-6500: other characterization data for the polymers are shown in Table 1. The next stage in our procedure was to prepare surfaces with a high density of functional groups for end-grafting of thermoresponsive and pH-responsive polymers. Silica glass cover slips (ca. 1 cm2) were rigorously cleaned and then oxidized to generate silanol residues prior to reaction with APTES in order to generate the desired functional surfaces. Carboxylic acid terminated polymers P1-3 were grafted to the surfaces by EDC coupling: these conditions were previously employed in our laboratories to give functional densities before grafting of up to 1 amine group/ (34 Å2) and reduction of accessible amine moieties of >87% by derivatization using the EDC coupling procedure.34 Atomic force microscopy in air and under water (Figure 1) demonstrated the transformation of smooth and homogeneous APTES surfaces (Ra ∼ 1 nm) to more disordered coatings following polymer grafting (Ra ∼ 2-4 nm; Table 2). Imaging in aqueous solution using the variable temperature cell allowed the depiction of surface topologies above and below expected polymer solution phase transitions. In all cases, irrespective of temperature, polymer graft surfaces were rough or pitted, with peaks and indentations rather higher (40-60 nm) than those found on APTES substrates (