Reversible Bacterial Adhesion on Mixed Poly(dimethylaminoethyl

Article pubs.acs.org/Langmuir

Reversible Bacterial Adhesion on Mixed Poly(dimethylaminoethyl methacrylate)/Poly(acrylamidophenyl boronic acid) Brush Surfaces Xinhong Xiong, Zhaoqiang Wu,* Qian Yu, Lulu Xue, Jun Du, and Hong Chen* Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, 199 Ren-ai Road, Suzhou 215123, P. R. China ABSTRACT: A simple and versatile method for the preparation of surfaces to control bacterial adhesion is described. Substrates were first treated with two catechol-based polymerization initiators, one for thermal initiation and one for visible-light photoinitiation. Graft polymerization in sequence of dimethylaminoethyl methacrylate (DMAEMA) and 3-acrylamidebenzene boronic acid (BA) from the surface-bound initiators to form mixed polymer brushes on the substrate was then carried out. The PDMAEMA grafts were thermally initiated and the PBA grafts were visible-light-photoinitiated. Gold, poly(vinyl chloride) (PVC), and poly(dimethylsiloxane) (PDMS) were used as model substrates. X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR), and ellipsometry analysis confirmed the successful grafting of PDMAEMA/PBA mixed brushes. We demonstrated that the resulting surfaces showed chargereversal properties in response to change of pH. The transition in surface charge at a specific pH allowed the surface to be reversibly switched from bacteriaadhesive to bacteria-resistant. At pH 4.5, below the isoelectric points (IEP, pH 5.3) of the mixed brushes, the surfaces are positively charged and the negatively charged Gram-positive S. aureus adheres at high density (2.6 × 106 cells/cm2) due to attractive electrostatic interactions. Subsequently, upon increasing the pH to 9.0 to give negatively charged polymer brush surface, ∼90% of the adherent bacteria are released from the surface, presumably due to repulsive electrostatic interactions. This approach provides a simple method for the preparation of surfaces on which bacterial adhesion can be controlled and is applicable to a wide variety of substrates.

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INTRODUCTION

shown to be an effective strategy for modulating bacterial adhesion.14−16 The work reported herein is based on the design of surfaces that control bacterial adhesion by environmental pH change. Under different pH conditions such surfaces possess different charge states,17−19 and differences in surface charge may influence bacterial interactions because most bacteria are charge-sensitive with isoelectric points (IEP) in the range of 1.5−4.5.20 At pH above the IEP, the bacteria are negatively charged and undergo attractive electrostatic interactions with positively charged surfaces.21−23 In addition, adherent bacteria can be easily released from neutral or negatively charged surfaces by increasing the environmental pH.24,25 For example, Jiang et al. prepared a pH-responsive surface consisting of positively charged polymers based on 2-(acryloyloxy)ethyl trimethylammonium chloride (TMA) and negatively charged polymers based on 2-carboxy ethyl acrylate (CAA) and showed that the transition in surface charge from positive to neutral as a function of pH caused a switch in surface properties from bacteria-adhering to bacteria-releasing.26 Although these materials provide good control of bacterial adhesion, the

The adhesion of bacteria to surfaces is the initial step in the formation of bacterial biofilms.1 In the case of medical devices such as catheters, implants, and artificial organs generally, bacteria can adhere nonspecifically to solid surfaces forming biofilms, which may result in the loss of function of the device and infection of the recipient. Accordingly there is strong interest in surfaces that can prevent or inhibit bacterial adhesion.1−3 In other fields such as biocatalysts and biofuel cells, the adhesion of bacteria to surfaces may be required or desirable.4−6 Thus, control of bacterial adhesion, whether for prevention or promotion, is of great importance in biomedical technology. In general, bacterial behavior such as adhesion and locomotion, is regulated by interactions at interfaces. Such interactions are determined mainly by the physical and chemical properties of material surfaces.7 Therefore, various surfaces have been developed for the control of bacterial interactions by chemical and physical methods.8−16 For example, surfaces have been proposed that can be switched between bacteria-adherent and bacteria-resistant states by environmental stimuli such as enzymes,8 mechanical force,9 pH change,10 humidity change,11 temperature change,12 and electrical charge.13 Control of surface topography has also been © 2015 American Chemical Society

Received: June 1, 2015 Revised: October 13, 2015 Published: October 28, 2015 12054

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Scheme 1. Strategy for Synthesis of PDMAEMA/PBA Brushes by Consecutive Free-Radical- and Visible-Light-Induced Polymerization and Then Bacterial Adhesion and Release in Response to the Change of Environmental pH

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preparation method lacks versatility with limited applicability in terms of substrate. In recent years, dopamine chemistry has been investigated extensively for substrate-independent surface modification.27−31 Along similar lines, versatile methods for surface modification have also been developed in our laboratory using dopamine derivatives.32,33 Herein, we combined these two versatile methods, that is, thermally initiated and visible-light photoinitiated graft polymerization, to create mixed polymer brushes of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) and poly(acrylamidophenylboronic acid) (PBA). We chose thermally initiated graft polymerization to synthesize the first brush component (PDMAEMA) and visible-light photoinitiated graft polymerization for the second brush (PBA). This order was selected because of the two different mechanisms of polymerization. The mechanism of photoinitiated graft polymerization was preferred for the grafting of the second brush component due to the steric hindrance induced by the existing polymer chains.34 The mixed brushes synthesis used here is outlined in Scheme 1. Among the polymer brushes, PDMAEMA is one of the most frequently reported cationic polymers and has been widely used for the conjugation of biomolecules via electrostatic interactions.35,36 At low pH, the tertiary amino groups of PDMAEMA are mostly protonated and the boronic acid groups of PBA are largely neutral, giving the surface a net positive charge. At high pH, the tertiary amino groups are only sparsely protonated while the boronic acid groups bind hydroxyl ions to form negatively charged tetrahedral boronate ions, giving the surface a net negative charge.37,38 Gold was used initially as a model substrate surface and Staphylococcus aureus was used as a model bacterium. To demonstrate the versatility of the approach, poly(vinyl chloride) (PVC) and poly(dimethylsiloxane) (PDMS) were also used as substrates.

EXPERIMENTAL SECTION

Materials. Gold-coated silicon wafers (80 nm Au deposited on a 10 nm chromium adhesion layer) were diced into 0.5 cm × 0.5 cm pieces. PVC films were cast from a 5% (w/v) solution in THF, vacuum-dried, and punched into discs ∼5 mm in diameter and 0.5 mm thick. PDMS films were prepared using the Sylgard silicone elastomer kit from Dow Corning (Midland, MI) and cut into small disks (0.6 cm diameter). Dimanganese decacarbonyl (Mn2(CO)10, 98%), dopamine hydrochloride (DA, 98.5%), 2-bromoisobutyryl bromide (BIBB, 98%), methacryloyl chloride (MAB, 98%), and 3-acrylamidebenzene boric acid (BA, 98%) were purchased from Sigma-Aldrich. SYTO 9 green fluorescent nucleic acid dye was purchased from Invitrogen and used as received. α,α′-Azobis(isobutyronitrile) (AIBN, Aldrich) was recrystallized from methanol and 2-(dimethylamino)ethyl methacrylate (DMAEMA, Aldrich, 98%) was purified by distillation under reduced pressure to remove inhibitors before use. Methanol, acetone, and all other solvents were purchased from the Sinopharm Chemical Reagent (China) and purified before use according to standard methods. Deionized water purified by a Millipore water purification system to give a minimum resistivity of 18.2 MΩ·cm was used in all experiments. Argon gas was of high-purity grade. Gram-positive S. aureus was provided by the China Center for Type Culture Collection. Dopamine methacrylamide (DMA) and 2-bromo-N-(3,4-dihydroxyphenethyl)-2-methylpropanamide (BDAM) were prepared according to previous literature.32,33 Instruments and Measurements. A spectroscopic ellipsometer was used to determine the thickness of the grafted polymers on the gold surface. The chemical composition of the pristine and modified surfaces was determined using an ESCALAB MK II X-ray photoelectron spectrometer (XPS, VG Scientific, England). Fourier transform infrared (FTIR) spectra of the gold surfaces at wavelengths ranging from 400 to 4000 cm−1 over 64 scans were obtained using a Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, U.S.) equipped with a mercury cadmium telluride detector, and the FTIR spectra of the PDMS and PVC were obtained with a horizontal attenuated total reflection (ATR) accessory. Fluorescence images of the bacteria attached to the gold surfaces were obtained by fluorescence microscope (IX71, Olympus, Japan). Surface zeta 12055

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Figure 1. (A) XPS survey spectra of Au, Au-vinyl/Br, Au-PDMAEMA/Br and Au-PDMAEMA/PBA. (B) High-resolution spectrum of the B 1s region of Au-PDMAEMA/PBA.

Table 1. XPS Analysis of Au, Au-vinyl/Br, Au-PDMAEMA/Br, and Au-PDMAEMA/PBA Surfaces XPS atomic concentration (%) sample

[Au]

[C]

Au Au-vinyl/Br Au-PDMAEMA/Bra Au-PDMAEMA/PBAb Au-PDMAEMA/PBAc

62.2 17.6 4.2 0.4 0.3

27.4 62.3 64.0 64.0 67.6

[N]

[O]

[Br]

5.7 6.7 10.4 7.5

10.4 14.0 24.8 21.5 20.7

0.4 0.3 0 0

[B]

thickness (±1 nm)

3.7 3.9

3.5 9.6 19.4 29.6

a

Concentration of PDMAEMA was 1.0 M and the thermal polymerization time was 8 h. bConcentration of BA was 0.1 M. The visible light irradiation time was 30 min. cConcentration of BA was 0.3 M. The visible light irradiation time was 30 min. Bacterial Attachment. Gram-positive S. aureus was used as a model bacterium in this study. Bacteria were first cultured overnight in separate pure cultures at 37 °C with shaking at 190 rmp for 12 h in lysogeny broth (LB) agar plates (10 g/L bacto-tryptone, 5 g/L yeast extract, and 5 g/L sodium chloride, adjusted to pH 7.0 with 1 mmol/L NaOH). After culturing, the optical density of the bacteria-containing broth was measured 1.0 at 600 nm.39 The bacterial cells were collected by low-speed centrifugation, washed three times with sterile, neutral PBS solution, and subsequently diluted with buffers of different pH (solutions of pH 4.5 and 5.3 were obtained from sodium acetate buffer and pH7.4, 9.0 from PBS; all buffers were heat-sterilized at 120 °C for 30 min) until the optical density (OD) as measured at 600 nm reached 0.05. Prior to experiments, the substrates were sterilized with 75% alcohol and then immersed in buffers of different pH. Then, 10 uL of the bacterial suspensions (of different pH) was pipetted into 48-well tissue culture plates and incubated at 37 °C for 30 min. After incubation the materials were washed twice with the corresponding buffers used for preparation to remove nonadherent bacterial cells and subsequently treated with SYTO 9 for morphologic fluorescence imaging of the stained bacteria. The stained bacterial surfaces were observed under the fluorescence microscope. At least three different areas on each substrate were chosen at random for fluorescence imaging as representative of the surface. Bacterial Detachment. First, the S. aureus suspension (pH 4.5) was pipetted onto the surface and treated with SYTO 9 in a similar way as previously described. Then, the bacteria-adhered surface was washed with pH 9.0 buffer and the solution was removed by blotting with filter paper. Finally, fluorescence images of the stained bacteria on the mixed polymer brush surfaces were taken in the fluorescence microscope, and image software (image-Pro Plus v6.0) was used to determine the bacterial surface density.

potentials were determined using a SurPASS Analyzer (Anton Paar, Austria). After filtering the shorter-wave UV light of a 500 W mercury lamp, light of intensity I420nm = 0.2 mW·cm−2 was used to activate the photograft polymerization process. Preparation of Mixed Initiator-Functionalized Surfaces. The mixed vinyl and alkyl bromine functionalized surfaces were prepared from a solution containing DMA and BDAM with a molar ratio of 4:1 in the feed. In brief, dopamine hydrochloride (5 mg, 25 μmol), DMA (22 mg, 100 μmol), and BDAM (15 mg, 50 μmol) were dissolved in a mixed solvent containing 1 mL of methanol and 9 mL of Tris-HCl buffer (pH 8.5). The solid substrates were then immersed in the solution. After incubation for 12 h at room temperature, the substrates were removed, rinsed successively with deionized water and methanol, and dried under a stream of argon. Preparation of PDMAEMA Brushes by Thermal-Initiated Polymerization. Grafting of PDMAEMA on the mixed initiatorfunctionalized surface was performed by thermally initiated free radical polymerization. In brief, DMAEMA (1.57g, 10 mmol) was dissolved in 10 mL of dry methanol and AIBN (16 mmg, 0.1 mmol) was added as initiator. Then the mixed initiator-functionalized surfaces were added to the flask and the solution was degassed by bubbling with argon for 20 min. The polymerization reaction was then carried out at 60 °C under nitrogen for 8 h. The product PDMAEMA-grafted substrates were rinsed with methanol and dried under a stream of argon. Preparation of PBA Brushes by Visible-Light-Photoinitiated Grafting Polymerization. BA (various amounts) and Mn2CO10 (1.95 mg, 0.005 mmol) were placed in a 25 mL conical flask, which contained 2 mL of dry methanol. The PDMAEMA-modified surfaces prepared as above were then placed in the flask, which was capped with a rubber septum. The system was extensively purged with argon before irradiating with visible light for 0.5 h. Following completion of polymerization, the samples were extensively rinsed with methanol to remove all traces of the polymerization solution and subsequently dried in a stream of argon. Moreover, to demonstrate the versatility of the method, mixed polymer brushes of PDMAEMA/PBA were prepared on PVC and PDMS as alternative substrates.

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RESULTS AND DISCUSSION Synthesis of Mixed PDMAEMA/PBA Brushes. The preparation of the PDMAEMA/PBA mixed brushes was carried out in three steps: (1) attachment of mixed initiators to the 12056

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prepared and PDMAEMA grafts were formed by thermal polymerization to confirm that PBA chains were indeed initiated from the alkyl bromine initiator. The PDMAEMAmodified surface was then exposed to the visible light used for BA photopolymerization. No significant change in brush thickness was observed, thus confirming that the BA polymerization was initiated from BDAM. Reflectance FT-IR spectroscopy was used to further confirm the mixed brush synthesis. The PDMAEMA surface showed an intense band at ∼1725 cm−1 (Figure 3) assigned to the carbonyl of the ester group. In contrast, the PBA brush showed a sharp peak at 1660 cm−1 assigned to the CO stretch in the amide group. The mixed PDMAEMA/PBA brush showed both of these characteristic peaks. To test the versatility of this approach using different substrates, we also applied the grafting reactions to PVC and PDMS substrates. Successful formation of mixed PDMAEMA/ PBA brushes was confirmed by the appearance of new peaks at 1725 cm−1 due to the CO stretch in the ester groups of PDMAEMA, and 1660 cm−1 due to the CO stretch in the amide group of PBA (Figure 3B). Although only gold PVC and PDMS substrates were employed for the preparation of the PDMAEMA/PBA mixed brushes in the present work, the initiators of DMA and BDAM, which are dopamine derivatives, have been used in many different substrates for surface modification.32,33,40 We speculate that our approach is suitable for use with a wide range of substrates. Composition of the Mixed Brush. On the basis of the elemental composition of the two monomers, the boron/ nitrogen ratio from XPS was used to estimate the polymer composition of the mixed brushes as follows

substrate, (2) PDMAEMA grafting by thermally initiated radical polymerization, and (3) PBA grafting by visible-lightphotoinitiated polymerization (Scheme 1). In previous work, DMA32 and BDMA33 were successfully attached to solid substrates, respectively. We also found that surface-attached DMA worked well for the thermally initiated radical grafting polymerization of various double-bond monomers, while surface-attached BDAM was appropriate for visible-lightphotoinitiated grafting polymerization using Mn2CO10. In the present work we combined these two surface-initiated radical polymerization techniques to create mixed homopolymer brushes of PDMAEMA and PBA. Initiator immobilization and grafting of mixed polymer brushes on gold were verified by XPS and thickness measurements at each step. As shown in Figure 1A, the unmodified Au surface was composed mainly of gold and carbon with a small amount of oxygen. Successful attachment of the initiators (Au-vinyl/Br) was indicated by a significant increase in carbon concentration (from 27.4 to 62.3%) and a decrease in Au concentration (from 62.2 to 17.6%) as well as by the appearance of N and Br signals (Table 1). After thermally initiated radical grafting polymerization of DMAEMA, a substantial decrease in gold and increase in O and N concentrations were observed, indicating that the PDMAEMA was successfully grafted. Finally, visible-light-induced PBA grafting was confirmed by the appearance of a boron signal and the almost complete disappearance of gold. The thickness of the films modified every step is summarized in Table 1. When the initiators were attached to the gold surface, the thickness was 3.5 nm; then, after surface-initiated grafting PDMAEMA and PBA mixed brushes on Au substrate the thickness came to 9.6 and 19.4 nm, respectively. Namely, the thickness increased after every modification step. To synthesize mixed PDMAEMA/PBA brushes with various PBA chain lengths, we exposed PDMAEMA-grafted surfaces having a constant (∼9.6 nm) dry thickness (Table 1) to photopolymerization conditions at different concentrations of BA for 30 min. As shown in Figure 2, the dry thickness of the grafted PBA layer increased almost linearly as the BA concentration increased, indicating the successful formation of mixed brushes.37,38 A pure DMA initiator layer was first

CB NB‐BA × XPBA = CN NN‐DMAEMA × (1 − XPBA ) + NN‐BA × XPBA

where CB and CN are the atomic concentrations of boron and nitrogen, NB‑BA and NN‑BA are the number of N and B in BA, respectively, NN‑DMAEMA is the number of N in DMAEMA, and XPBA is the mole fraction of PBA in the mixed brush. BA monomer contains one boron atom and one nitrogen atom, whereas DMAEMA contains one nitrogen atom. Thus, for instance, at 0.1 M BA after 30 min of irradiation, CB and CN were 3.67 and 10.42% (Table 1), respectively, corresponding to ∼35 mol % PBA. At 0.3 M BA, CB increased to give XPBA ≈ 51%. Charge-Reversal Properties of the Mixed Brush upon pH Changes. Recognizing that the zeta potential gives an indication of the overall charge of the mixed brushes, the surface of PBA content 35 mol % was chosen to monitor the variation of zeta potential with change in the pH from 3 to 10. As shown in Figure 4, the isoelectric point (IEP) of the mixed brush is at pH 5.3. Below the IEP, the mixed brush is positively charged and showed a zeta potential of +58 mV at pH 3. The tertiary amino groups of PDMAEMA are mostly protonated at this pH and the boronic acid groups of PBA are largely neutral.19,37 With increasing pH, the zeta potential decreased and fell to zero at pH 5.3. As the pH further increased, the zeta potential became negative due to the formation of negatively charged boronate ions.37,38 From these data it is clear that the mixed PDMAEMA/PBA brushes can take on varying charge over a wide range from positive to negative via change of pH. These charge-reversal properties should be useful for the

Figure 2. Film thickness versus concentration of BA for mixed brushes of PDMAEMA and PBA. At time zero we started with a 9.6 nm PDMAEMA brush. For all experiments, the concentration of Mn2(CO)10 was 0.005 M, and the irradiation time was 30 min. Data are mean ± SD (n = 3). 12057

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Figure 3. (A) Reflectance FTIR spectra of the Au-PDMAEMA, Au-PBA, and Au-PDMAEMA/PBA surfaces. (B) Reflectance FTIR spectra of the PVC, PVC-PDMAEMA/PBA, PDMS, and PDMS-PDMAEMA/PBA surfaces.

Figures 5 and 6A. Adhesion on the mixed polymer brush surface showed a strong dependence on pH. At pH 4.5 the brush was positively charged and the cell density was high (2.6 × 106 cells/cm2) due to the attractive electrostatic interactions.21 Under neutral conditions, that is, at the IEP (pH 5.3), the cell density was greatly reduced. Similar behavior has been observed by others.26 Increase in the pH beyond the IEP to give negatively charged polymer brush surfaces led to further decrease in adhesion. The cell density at pH 9.0 was reduced to 9.8 × 104 cells/cm2, presumably due to the repulsive electrostatic interactions between the negatively charged surface and the bacteria.21 In general, the hydrophilicity of surfaces can also affect the bacterial adhesion.42,43 Thus, we investigated the water contact angles of the mixed brush surfaces at different pH. As shown in Figure 6B, the water contact angles did not change significantly from pH 3.0 to 9.0, suggesting that surface charge may be the main determinant of bacterial interactions. These data clearly show that at pH below the IEP of the mixed polymer brush bacterial adhesion is high, and above the IEP adhesion is low. We also investigated the possibility that bacteria, once adherent, could be released from the surface by increasing the pH. As shown in Figure 7, washing at pH 9.0 caused a decrease in cell density to ∼2.3 × 105 cells/cm2, that is, a decrease of ∼90%. In contrast, on the control gold surface, change of pH had no effect on adhesion and release. These results demonstrate that bacterial adhesion and release on this kind

Figure 4. Variation of zeta potential with pH for PDMAEMA/PBA mixed polymer brush.

control of bacterial interactions with the surface in aqueous environment. Bacterial Attachment/Detachment in Response to pH. The effect of surface charge on bacterial interactions was investigated using Gram-positive S. aureus as a model bacterium. Because the isoelectric point of S. aureus is in the range of 2.3 to 2.6.41 The lowest pH testing condition of 4.5 was chosen to ensure a negative surface charge on the bacteria. Data on bacterial adhesion as a function of pH are shown in

Figure 5. Fluorescence images of S. aureus on bare Au and Au-PDMAEMA/PBA surfaces after 30 min of incubation at different pH values. Scale bar = 100 μm. 12058

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Figure 6. (A) Density of S. aureus on bare Au and Au-PDMAEMA/PBA surfaces after 30 min of incubation at different pH. (B) Water contact angles of the Au-PDMAEMA/PBA surfaces at different pH. Data are mean ± standard deviation (n = 3).

Figure 7. Sequential bacteria capture and release. (A) Images of bacteria (scale bar = 100 μm) on the Au and mixed brush surfaces at pH 4.5 and after washing at pH 9.0. (B) Density of S. aureus at pH 4.5 and after washing at pH 9.0. The data are means ± standard error (n = 3).

of surface can be manipulated by varying the ambient pH. The ability to switch from bacteria-adhering to bacteria-releasing should be useful in applications where enrichment, removal, and detection of microorganisms is required.

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CONCLUSIONS

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AUTHOR INFORMATION

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by the National Natural Science Foundation of China (21174098, 21574092) and the National Science Fund for Distinguished Young Scholars (21125418).

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A mixed PDMAEMA/PBA brush-based system that is switchable between bacteria-adhesion and bacteria-detachment states by change of pH has been developed. Immobilization of catechol-based initiators, requiring only a simple mixing procedure, is followed by the formation of a mixed polymer brush by a sequence of thermally initiated radical polymerization and visible-light-photoinitiated polymerization. The methods can be applied to a range of substrates. It is suggested that such mixed polymer brush-based surfaces should be useful in applications where enrichment, removal, and detection of microorganisms is required.

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest. 12059

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DOI: 10.1021/acs.langmuir.5b02002 Langmuir 2015, 31, 12054−12060