Integrated Antimicrobial and Nonfouling Hydrogels to Inhibit the

Jun 2, 2010 - Planktonic Bacterial Cells and Keep the Surface Clean ... of antimicrobial agents to inhibit the growth of planktonic bacteria and creat...
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Integrated Antimicrobial and Nonfouling Hydrogels to Inhibit the Growth of Planktonic Bacterial Cells and Keep the Surface Clean Gang Cheng,† Hong Xue,† Guozhu Li,† and Shaoyi Jiang*,† †

Department of Chemical Engineering, University of Washington, Seattle, Washington 98195 Received April 18, 2010. Revised Manuscript Received May 25, 2010

A new strategy integrating antimicrobial and nonfouling/biocompatible properties is presented. A mild antimicrobial agent (salicylate) was incorporated into a carboxybetaine ester hydrogel, poly(N,N-dimethyl-N-(ethylcarbonylmethyl)N-[2-(methacryloyloxy)-ethyl]ammonium salicylate) (pCBMA-1 C2 SA) hydrogel, as its anionic counterion. This new hydrogel provides a sustained release of antimicrobial agents to inhibit the growth of planktonic bacteria and create a nonfouling surface to prevent protein adsorption or bacterial accumulation upon the hydrolysis of carboxybetaine esters into zwitterionic groups. The pCBMA-1 C2 SA hydrogel inhibited the growth of both gram-negative Escherichia coli K12 and gram-positive Staphylococcus epidermidis by 99.9%. This hydrogel holds great potential in applications such as wound dressing and surface coatings for medical devices.

Introduction Microbial adhesion onto biomaterial implants and the subsequent formation of biofilms are major reasons for the failure of implantable biomedical devices, and about 45% of nosocomial infections are caused by biomaterial-associated infections.1 These nosocomial microbial infections associated with implanted biomedical devices such as implantable sensors, catheters, and artificial prosthetics typically lead to the removal of the devices because of the lack of a suitable treatment and increase the duration of hospital stays and hospitalization costs. Currently, there is a constant demand for new materials capable of preventing the colonization of microorganisms onto surfaces of implantable materials. To reduce bacterial attachment and colonization, one method is to coat surfaces with nonfouling materials such as poly(ethylene glycol) (PEG) derivatives or zwitterionic polymers. It was reported that surfaces coated with 2-methacryloyloxyethyl phosphorylcholine (MPC) reduced the attachment of bacteria by 90%.2,3 Recent studies demonstrated that zwitterionic poly(sulfobetaine methacrylate) (pSBMA) and poly(2-carboxy-N,N-dimethyl-N-(20 -(methacryloyloxy)ethyl)-ethanaminium) (pCBMA-2) efficiently reduced the colonization of Pseudomonas aeruginosa and Staphylococcus epidermidis.4,5 These materials are also proven to be highly resistant to protein adsorption from 100% serum and blood plasma.6-8 Although these nonfouling materials can significantly reduce the initial attachment and delay the colonization of microbes on surfaces, they cannot kill or inhibit the growth of pathogenic bacteria cells once bacteria are attached to surfaces. During implantation surgery, there is a great possibility of *Corresponding author. E-mail: [email protected].

(1) Schierholz, J. M.; Beuth, J. J. Hosp. Infect. 2001, 49, 87–93. (2) Lewis, A. L. Colloids Surf., B 2000, 18, 261–275. (3) Hirota, K.; Murakami, K.; Nemoto, K.; Miyake, Y. FEMS Microbiol. Lett. 2005, 248, 37–45. (4) Cheng, G.; Zhang, Z.; Chen, S. F.; Bryers, J. D.; Jiang, S. Y. Biomaterials 2007, 28, 4192–4199. (5) Cheng, G.; Li, G.; Xue, H.; Chen, S.; Bryers, J. D.; Jiang, S. Y. Biomaterials 2009, 30, 5234–5240. (6) Ladd, J.; Zhang, Z.; Chen, S.; Hower, J. C.; Jiang, S. Y. Biomacromolecules 2008, 9, 1357–1361. (7) Yang, W.; Chen, S. F.; Cheng, G.; Vaisocherova, H.; Xue, H.; Li, W.; Zhang, J.; Jiang, S. Y. Langmuir 2008, 24, 9211–9214. (8) Yang, W.; Xue, H.; Li, W.; Zhang, J.; Jiang, S. Y. Langmuir 2009, 25, 11911– 11916.

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introducing pathogenic microbes into the patient, causing the failure of implantation devices. The antimicrobial strategy is another method for preventing bacterial colonization on surfaces. Quaternary ammonium compounds (QACs) are extensively used as antimicrobial agents because of their broad antimicrobial properties.9 These QACs, when covalently linked to material surfaces to make the surfaces permanently microbicidal,10 were efficiently able to kill both bacterial cells and fungal cells.11-16 However, permanent QAC coatings cannot fulfill the requirement of implantable biomaterials for nonfouling and biocompatibility. The inherent drawback of permanent QAC coatings generates a fouling surface and triggers the immune response and chronic inflammation. To overcome the disadvantage of both nonfouling materials and cationic materials, a switchable surface that can catch and kill live bacterial cells and then release dead cells by switching from an antimicrobial cationic surface to a biocompatible nonfouling surface is reported in our previous study.17 Both switchable and permanent cationic surfaces can effectively kill bacterial cells attached to surfaces, but they have limited antimicrobial capacity against the planktonic bacterial cells. The controlled release of antimicrobial agents from surfaces can be used to reduce microbial colonization on surfaces and inhibit the proliferation of planktonic bacteria.18 Typically, antimicrobial agents can be either covalently linked to material surfaces19 or encapsulated (9) Nohr, R. S.; Macdonald, J. G. J. Biomater. Sci., Polym. Ed. 1994, 5, 607–619. (10) Abel, T.; Cohen, J. I.; Engel, R.; Filshtinskaya, M.; Melkonian, A.; Melkonian, K. Carbohydr. Res. 2002, 337, 2495–2499. (11) Lee, S. B.; Koepsel, R. R.; Morley, S. W.; Matyjaszewski, K.; Sun, Y. J.; Russell, A. J. Biomacromolecules 2004, 5, 877–882. (12) Ravikumar, T.; Murata, H.; Koepsel, R. R.; Russell, A. J. Biomacromolecules 2006, 7, 2762–2769. (13) Tiller, J. C.; Liao, C. J.; Lewis, K.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5981–5985. (14) Haldar, J.; An, D. Q.; de Cienfuegos, L. A.; Chen, J. Z.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 17667–17671. (15) Lin, J.; Qiu, S. Y.; Lewis, K.; Klibanov, A. M. Biotechnol. Bioeng. 2003, 83, 168–172. (16) Park, D.; Wang, J.; Klibanov, A. M. Biotechnol. Prog. 2006, 22, 584–589. (17) Cheng, G.; Xue, H.; Zhang, Z.; Chen, S. F.; Jiang, S. Y. Angew. Chem., Int. Ed. 2008, 47, 8831–8834. (18) Hendricks, S. K.; Kwok, C.; Shen, M. C.; Horbett, T. A.; Ratner, B. D.; Bryers, J. D. J. Biomed. Mater. Res. 2000, 50, 160–170. (19) Aumsuwan, N.; Heinhorst, S.; Urban, M. W. Biomacromolecules 2007, 8, 713–718.

Published on Web 06/02/2010

DOI: 10.1021/la101542m

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within materials for temporal release.20 In a previous study, a degradable polymer based on salicylic acid anhydride has been reported to be able to reduce microbial colonization through the controlled release of salicylate that specifically acts against biofilms.21 Hydrogel materials have been broadly used as implantable materials, catheters, and wound dressings because of their high water content and excellent biocompatibility. However, the controlled release of small hydrophilic drugs from hydrophilic surfaces, such as hydrogels, has proven to be challenging because encapsulated drugs are quickly released and depleted through diffusion. The motivation for this study is to develop a multifunctional hydrogel that combines the advantages of both nonfouling and cationic antimicrobial materials while overcoming their respective disadvantages. In this work, an antimicrobial hydrogel is developed on the basis of hydrolyzable cationic compounds N,Ndimethyl-N-(ethylcarbonylmethyl)-N-[2-(methacryloyloxy)ethyl]ammonium salicylate (CBMA-1 C2 SA), with salicylate as its counterion. The release rate of the counterion was controlled through the hydrolysis of the ester group and/or anion exchange. It was observed that this hydrogel inhibited the growth of both gram-negative Escherichia coli K12 and gram-positive Staphylococcus epidermidis by 99.9% after 24 h at 37 °C. After being converted to a nonfouling zwitterionic surface, the pCBMA-1 C2 SA hydrogel surface was highly resistant to nonspecific protein adsorption.

Experimental Section Chemicals. 2-(Dimethylamino)ethyl methacrylate (DMAEMA), tetraethylene glycol dimethacrylate (TEGDMA), ammonium persulfate (APS), sodium metabisulfite, ethyl bromoacetate, phosphate-buffered saline (PBS), sodium salicylate, acetonitrile, ethyl ether, and methanol were purchased from Sigma-Aldrich. (St. Louis, MO). N-Cyclohexyl-3-aminopropanesulfonic acid (CAPS) was purchased from TCI America (Portland, OR). The 2-carboxy-N,N-dimethyl-N-(20 -(methacryloyloxy)ethyl)ethanaminium inner salt (CBMA-2) was synthesized by the reaction of DMAEMA and β-propiolactone using a method published previously.22,23 For the synthesis of N,N-dimethyl-N-(ethylcarbonylmethyl)N-[2- (methacryloyloxy)ethyl]ammonium bromide (CBMA-1 C2), ethyl bromoacetate (16.6 mL, 150 mmol) was added to a solution of 2-(dimethylamino)ethyl metharylate (16.9 mL, 99 mmol) in acetonitrile (100 mL) and stirred at 25 °C for 18 h. The resulting residues were precipitated in ethyl ether, filtered, and subsequently washed twice with ethyl ether. The precipitate was dried in vacuum and analyzed. The yield was 90%. 1H NMR (300 MHz, D2O): δ 7.71-7.67 (m, 1H), 7.35-7.28 (m, 1H), 6.86-6.79 (m, 2H), 5.97 (s, 1H), 5.64 (s, 1H), 4.49 (s, 2H), 4.27 (t, 2H, J = 3.0 Hz), 4.164.09 (t, 2H, J = 3.0 Hz), 3.93-3.90 (t, 2H, J = 3.0 Hz), 3.24 (s, 6H), 1.78 (s, 3H), 1.17 (t, 3H, J = 6.0 Hz). For the synthesis of N,N-dimethyl-N-(ethylcarbonylmethyl)N-[2- (methacryloyloxy)ethyl]ammonium salicylate (CBMA-1 C2 SA), sodium salicylate (1.64 g, 10 mmol) was dissolved in DI water (10 mL) and a sodium salicylate solution was added to a solution of N,N-dimethyl-N-(ethylcarbonylmethyl)-N-[2-(methacryloyloxy)ethyl]ammonium bromide (3.24 g, 10 mmol) in DI water (10 mL). The reaction was stirred at 25 °C for 24 h. The product was extracted with chloroform and dried in vacuum and (20) Zumbuehl, A.; Ferreira, L.; Kuhn, D.; Astashkina, A.; Long, L.; Yeo, Y.; Iaconis, T.; Ghannoum, M.; Fink, G. R.; Langer, R.; Kohane, D. S. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12994–12998. (21) Bryers, J. D.; Jarvis, R. A.; Lebo, J.; Prudencio, A.; Kyriakides, T. R.; Uhrich, K. Biomaterials 2006, 27, 5039–5048. (22) Zhang, Z.; Chen, S. F.; Chang, Y.; Jiang, S. Y. J. Phys. Chem. B 2006, 110, 10799–10804. (23) Zhang, Z.; Chen, S. F.; Jiang, S. Y. Biomacromolecules 2006, 7, 3311–3315.

10426 DOI: 10.1021/la101542m

Scheme 1. Chemical Structures

analyzed. 1H NMR (300 MHz, D2O) (Figure S-1): δ 7.0-7.67 (m, 1H), 7.35-7.28 (m, 1H), 6.86-6.79 (m, 2H), 6.11 (s, 1H), 5.65 (s, 1H), 5.03 (s, 2H), 4.66 (t, 2H, J = 3.0 Hz), 4.42 (t, 2H, J = 3.0 Hz), 4.20 (q, 2H, J = 7.2 Hz), 3.76 (s, 6H), 1.92 (s, 3H), 1.29 (t, 3H, J = 6.0 Hz). Preparation of Chemical Hydrogels. One millimole of CBMA-2 or CBMA-1 C2 SA monomer in 1 mL of a mixed solvent of ethylene glycol/ethanol/H2O (1.5:1:1.5) was mixed with 33 μL of TEGDMA, 8 μL of 40% ammonium persulfate (APS), and 8 μL of 15% sodium metabisulfite (SMS). The reaction was carried out between a pair of glass substrates, separated with a poly(tetrafluoroethylene) (PTFE) spacer with a thickness of 0.76 mm. The reaction mixture was sealed with parafilm, put into a 70 °C oven for 30 min, and left at room temperature for 3 h. The hydrogels were removed from the glass substrates and soaked in deionized water for 24 h at 4 °C, and the water was changed every 3 h. Characterization of Hydrogels. The water content of hydrogels is a basic property of hydrogel materials used for biomedical applications. The wet weight of the hydrogel samples was measured after the removal of excess water from the samples. The dry weight was recorded after the samples had been dried at 65 °C under vacuum for 72 h.

Measurements of Protein Adsorption on Hydrogels by Enzyme-Linked Immunosorbent Assay. pCBMA-2 and pCBMA-1 C2 SA hydrogels in water were transferred to PBS buffer and then punched into 10 mm disks (10 mm biopsy punch, Acuderm Inc., FL). pCBMA-2 and pCBMA-1 C2 SA hydrogel discs were incubated in 10 mM CAPS buffer (pH 10.0) at 37 °C. After 48 h, the samples were placed in PBS (pH 7.4) at room temperature for 2 h. To measure protein adsorption, samples and tissue culture polystyrene (TCPS) were incubated with horseradish peroxidase (HRP)-conjugated antifibrinogen (20 μg/mL buffer) for 0.5 h at room temperature, followed by five washes with PBS. The hydrogel surfaces and TCPS substrates were transferred to individual wells of 24-well plates. o-Phenylenediamine (800 μL of a 1 mg/mL solution) in 0.1 M citrate phosphate pH 5.0 buffer containing 0.03% hydrogen peroxide was added. Enzyme activity was stopped by adding an equal volume of 2 N H2SO4 after 15 min. The absorbance of the supernatant was measured at 492 nm. Bacterial Culture Conditions. E. coli K12 and S. epidermidis were cultured in separate pure cultures overnight at 37 °C on Luria-Bertani medium (LB) (BD, Franklin Lakes, NJ) and trypticase soy broth (TSB) (BD, Franklin Lakes, NJ)24 agar plates. Several colonies were used to inoculate 25 mL of LB (20 g/L) for E. coli K12 and TSB (20 g/L) for S. epidermidis.24 Growth Inhibition Assay. pCBMA-2 and pCBMA-1 C2 SA hydrogels in water were punched into 10 mm disks. The overnight cultures of E. coli K12 or S. epidermidis were washed three times with PBS and diluted in the sterile LB medium to a final (24) Wagner, V. E.; Koberstein, J. T.; Bryers, J. D. Biomaterials 2004, 25, 2247– 2263.

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Figure 1. Hydrolysis was performed in CAPS buffer (pH 10.0) at 37 °C for 48 h. Protein adsorption on all surfaces was normalized to tissue culture polystyrene (TCPS). The results are averaged from three replicates. concentration of 103 cells/mL. A bacterial suspension (1.0 mL) was transferred into each well of a 24-well plate, and each well contained 2 test or control hydrogel disks. The bacterial cultures containing hydrogel disks were incubated at 37 °C for 24 h. After 24 h, both the concentration of live bacteria in solution and the density of accumulated bacteria on hydrogels were measured. To determine the concentration of live bacteria in solution, bacterial cultures were diluted serially in water and spread on LB agar plates for E. coli K12 or TSB agar plates for S. epidermidis. After 18 h at 37 °C, the number of colonies on agar plates was recorded to calculate the concentration of live bacterial cells. To analyze the density of bacteria accumulated on hydrogel surfaces, samples were gently rinsed with water and stained with 1 mL of water containing 20 μM red fluorescent nucleic acid stain propidium iodide and 3.34 μM green fluorescent nucleic acid stain SYTO9 (Invitrogen, Carlsbad, CA). The total number of cells was determined with a CCD-CoolSNAP camera (Roper Scientific, Inc., Trenton, NJ) mounted on Nikon Eclipse 80i with a 100 oil lens through FITC and Texas red filters.

Results and Discussion The percentage of water was measured in the tested hydrogels. Among these two hydrogels, the zwitterionic pCBMA-2 hydrogel (93.71% water content) had a lower swelling ratio than the pCBMA-1 C2 SA hydrogel (96.57% water content). The lower swelling of the zwitterionic pCBMA-2 hydrogel in water is due to its antielectrolyte properties. Increasing solution ionic strength will enhance the swelling of zwitterionic hydrogels. Unlike zwitterionic pCBMA-2, cationic pCBMA-1 C2 SA exhibits polyelectrolyte properties with a higher swelling ratio of pCBMA-1 C2 SA in water. Nonspecific protein adsorption on the pCBMA-1 C2 SA hydrogels surface was measured by ELISA to determine the nonfouling characteristics of the hydrogel (Figure 1). pCBMA1 C2 SA hydrogels were hydrolyzed in N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer (pH 10.0) along with other control hydrogels. Fibrinogen adsorption on pCBMA-1 C2 SA hydrogels before hydrolysis was 180% relative to hydrophobic TCPS because of positively charged quaternary ammonium groups in pCBMA-1 C2 SA hydrogels. After 48 h of hydrolysis, protein adsorption on pCBMA-1 C2 SA hydrogels dropped to 13%, indicating that cationic pCBMA-1 C2 SA hydrogels were hydrolyzed to zwitterionic poly(1-carboxy-N,Ndimethyl-N(2-methacryloyloxyethyl)methanaminium inner salt) (pCBMA-1). Under identical conditions, pCBMA-2 hydrogels still exhibited excellent nonfouling properties with less than 12% protein Langmuir 2010, 26(13), 10425–10428

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Figure 2. Representative photographs of accumulated E. coli K12 on (a) pCBMA-2 and (b) pCBMA-1 C2 SA surfaces and S. epidermidis on (c) pCBMA-2 and (d) pCBMA-1 C2 SA surfaces after culturing at 37 °C for 24 h. These experiments are repeated three times.

adsorption under all conditions. In our previous studies, high protein adsorption was observed on a gold surface coated with pCBMA-1 C2. After hydrolysis by incubating the surface with CAPS buffer at pH 1017 or sodium hydroxide,25 the pCBMA-1 C2 surface switched to an ultra-low-fouling surface. However, protein adsorption on surfaces coated with a permanently positively charged polymer containing a methacrylate polymer backbone was high before and after its incubation with the CAPS buffer at pH 10. The result indicated that hydrolysis occurred on the terminal ester bond instead of the ester bond close to the polymer backbone.17 Wetering et al. also reported that the ester bond in the polymer of methacrylate was quite stable against hydrolysis.26 Results from this study indicate that the obtained zwitterionic hydrogels effectively resist nonspecific protein adsorption,27 which are desirable as surface coatings for implantable medical devices. As shown in Figure 2, the accumulation of S. epidermidis and E. coli K12 on pCBMA-2 and pCBMA-1 C2 SA hydrogel surfaces was observed after 24 h of culturing both strains under static conditions. Although the zwitterionic pCBMA-2 hydrogel cannot inhibit the growth of both E. coli and S. epidermidis in the growth medium, the surface of pCBMA-2 hydrogels showed the low accumulation of bacterial cells because of its excellent nonfouling properties. Because of the inhibited growth of bacteria by pCBMA-1 C2 SA hydrogels and the resulting low-fouling surfaces generated by the partial hydrolysis of the pCBMA-1 C2 SA hydrogel, there are very few attached bacterial cells on the hydrogel surface. The antibacterial activities of pCBMA-1 C2 SA were studied against gram-negative E. coli K12 and gram-positive S. epidermidis. Figure 3 shows that the growth of planktonic E. coli and S. epidermidis was inhibited by the presence of the pCBMA-1 C2 SA hydrogel but not by the presence of pCBMA-2. After 24 h of culturing, the concentrations of E. coli K12 in the growth medium over pCBMA-2 and pCBMA-1 C2 SA hydrogels were 2.36  108 and