Covalently Attached, Silver-Doped Poly(vinyl alcohol) Hydrogel Films

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Covalently Attached, Silver-Doped Poly(vinyl alcohol) Hydrogel Films on Poly(L-lactic acid) Xingjie Zan,† Mikhail Kozlov,‡,§ Thomas J. McCarthy,‡ and Zhaohui Su*,† State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, People’s Republic of China, and Polymer Science and Engineering Department, University of Massachusetts, Amherst, Massachusetts 01003 Received January 15, 2010; Revised Manuscript Received February 27, 2010

Covalently attached, soft poly(vinyl alcohol) (PVA) hydrogel films containing silver particles were prepared on solid biodegradable poly(L-lactic acid) (PLLA) samples by a multistep procedure involving oxygen plasma treatment, UV-initiated graft polymerization, and chemical grafting methods. The modification steps were followed and verified using attenuated total reflection infrared spectroscopy and X-ray photoelectron spectroscopy. 2-Hydroxyethyl methacrylate (HEMA) was graft polymerized from the surface of oxygen plasma-treated PLLA film samples and the alcohol functionality in the grafted polyHEMA chains was oxidized using pyridinium dichromate to obtain an aldehyde-rich surface. PVA was then grafted onto this surface using acid catalysis (acetal formation). The “freeze/ thaw method” was used to form a PVA hydrogel layer that incorporated the covalently grafted PVA chains in the physically cross-linked gel. This composite film (PLLA-PVAgel) was doped with silver ions, which were reduced to silver using NaBH4. Scanning electron microscopy of cross sections of PLLA-PVAgel indicates robust attachment of the PVA hydrogel layer to the PLLA film. PLLA-PVAgel/Ag(0) film samples exhibit both antibacterial and reduced cell adhesion properties due to the antibacterial properties of silver nanoparticles and high water content, respectively. This method provides a route to mechanically sound biodegradable materials with tunable soft material surface properties. Potential applications in tissue engineering and biomedical devices are envisioned.

1. Introduction Poly(L-lactic acid) (PLLA), a biodegradable polymer, has been intensively studied during the past 20 years.1-4 Due to its excellent mechanical strength and suitable biodegradability rates,1-4 PLLA has been widely used in sutures, bone fixation, drug delivery, and temporary matrices or scaffolds in tissue engineering.5,6 PLLA has also been applied as a “new generation” material for biomedical devices, including intravascular stents and in vivo biosensors.7 In these applications, nonspecific protein adsorption to the hydrophobic PLLA surfaces occurs immediately after the device is inserted into the biological environment,8-10 leading to significant bioresponses such as restenosis9 and inflammation.10 An antifouling coating is needed for this type of implanted biomedical device to be functional and stable and various coating materials have been developed for this purpose. Poly(ethylene glycol) (PEG) is effective at interrupting protein adsorption and cell adhesion due to its hydrophilicity, high surface mobility, and low interfacial free energy with water.11 Monolayers of peptides with particular amino acid sequences are found to exhibit protein adsorption resistance in living fluids; these surfaces are comparable to the best proteophobic surfaces known to date (PEG-based surfaces).12 Zwitterioncontaining polymers can also exhibit strong nonspecific protein adsorption resistance and are a viable alternative to PEG-based materials.13-15 Although the origin of resistance to protein adsorption is not yet completely clear, surfaces that are hydrophilic, overall charge-neutral, conformationally flexible, and have hydrogen bond accepting groups have shown the best protein resistance.16 * To whom correspondence should be addressed. Phone: (+86) 43185262854. Fax: (+86) 431-85262126. E-mail: [email protected]. † Chinese Academy of Sciences. ‡ University of Massachusetts. § Current address: Millipore Corporation, 80 Ashby Rd., Bedford, MA 01730.

Hydrogels, three-dimensional cross-linked networks of a water-soluble polymer, are of considerable interest in drug delivery applications, tissue engineering, nanoreactor design, and separation systems.17 An important strength of hydrogel networks is their ability to incorporate abundant amounts of functional materials (solutes, nanoparticles), which can be interactive with the contacting aqueous medium because of their highly swollen state. Due to the high water content and mechanical properties (soft, but strong), nonionic hydrogels can have a strong effect on attenuating the adsorption of proteins and cell adhesion.18,19 Poly(vinyl alcohol) (PVA) hydrogels20,21 were considered as promising candidates for protein and cell adhesion resistance because of their hydrophilic, soft, biocompatible, and neutral nature.19,22 There are several chemical and physical methods to prepare PVA hydrogels;23-25 physical cross-linking (freeze/thaw) due to its simplicity and fact that it does not involve potentially toxic chemical agents has attracted significant attention.26 Hydrogel formation during the freeze/ thaw process is accounted for from a thermodynamic phase separation viewpoint: water crystallization results in interstitial domains of high polymer concentration. Polymer chains in these highly concentrated domains crystallize leading to physical cross-links and gelation of the entire sample. These small PVA crystals do not dissolve during the thawing process. The PVA hydrogel properties can be tuned by doping with functional molecules, choosing PVA samples of particular molecular structure (molecular weight, degree of hydrolysis, tacticity), and varying the number and conditions (temperature and rate) of the freeze-thaw cycles.27,28 Few methods, however, have been developed for attaching hydrogel coatings to hard solid substrates due to the mechanical difficulties in adhering a soft layer to a hard solid surface.29,30 An additional potential problem is that bacteria and microorganisms can grow in hydrogel materials

10.1021/bm100048q  2010 American Chemical Society Published on Web 03/22/2010

Hydrogel Films on Poly(L-lactic acid)

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Scheme 1. Process for Fabricating PLLA-PVAgel/Ag(0) Film

due to their natural biocompatible properties, thus, incorporation of antibacterial agents will be required. Therefore, it is necessary to add antibiotic and antibacterial into the hydrogel. Silver nanoparticles can be introduced into PVA hydrogel by mixing PVA solution with silver ions and then in situ reduction of the silver ions in the hydrogel.31 Although the mechanism of silver’s effects on bacteria is not completely clear, its multilevel antimicrobial mode is well-known and has a very broad antibacterial spectrum even at low concentrations.32,33 In the present work, we report the covalent attachment of PVA hydrogel containing silver nanoparticles to PLLA surfaces by a multistep procedure involving oxygen plasma treatment, UV-initiated graft polymerization, a chemical grafting method and subsequent freeze-thaw hydrogel formation. The PLLA grafted with a PVA hydrogel coating exhibits antibacterial and cell adhesion resistance properties due to the high water content and the antibacterial properties of silver nanoparticles.

2. Experimental Section 2.1. Materials. Poly(L-lactic acid) (PLLA; Mw ) 1.17 × 105) was provided by Prof. Xuesi Chen, and its synthesis has been reported previously.34 2-Hydroxyethyl methacrylate (HEMA), poly(vinyl alcohol) (PVA; Mw ∼ 146000-186000, 99+% hydrolyzed), and pyridinium dichromate (PDC) were purchased from Aldrich. Ether, acetone, sodium borohydride (NaBH4), and sulfuric acid (H2SO4) were all analytical grade and purchased from Beijing Chemical Reagents Company. All these chemicals were used as received except HEMA, which was distilled under vacuum prior to use. Ultrapure water was obtained from a Millipore Simplicity 185 purification unit (18.2 MΩ cm). 2.2. Functionalization of PLLA Surface. The process for functionalization of PLLA with PVA hydrogel/Ag(0) consists of several steps and is illustrated in Scheme 1.

2.2.1. Surface Graft Polymerization with HEMA: PLLA-OH Surface. Compression molded PLLA film with the thickness of ∼100 µm was cut into 1.2 × 1.2 cm2 pieces, rinsed in a mixture of ethanol and water (v/v ) 1:1) for several hours, and then the PLLA film samples were maintained under vacuum for 24 h. These samples were treated with oxygen plasma glow discharge for 10 min in a PDC-M plasma apparatus (Chengdu Mingheng Technologies) at a pressure of approximately 100 Pa and then immersed in aqueous solutions of different concentration of freshly distilled HEMA, which were purged with N2 for 10 min before UV irradiation (1 h under N2). Finally, the films were washed with copious amounts of ultrapure water and soaked overnight at 50 °C to remove residual monomer and then maintained under vacuum for at least 24 h to remove the water. The masses of the films were measured before and after grafting with a PE analytical microbalance, and the graft density (D) was calculated from D ) ∆M/ A, where ∆M is the mass increase after HEMA grafting and A is the surface area of the film. 2.2.2. Aldehyde-Functionalized PLLA Surface: PLLA-CHO Surface. The polyHEMA modified PLLA (PLLA-OH) surface was oxidized using PDC in a round-bottom flask containing freshly distilled ether following a procedure reported in literature.35 Briefly, PLLA-OH film was immersed in an ether solution of PDC (0.5 mol/L), which was stirred for the desired time. After this oxidation reaction, the film was washed with ether and acetone several times (until the film was colorless). The film was maintained in vacuum for at least 24 h to remove solvents. Labeling of the aldehyde groups on the PLLA-CHO film was achieved by immersing the film in 10 mL of ethanol containing 1 mL of freshly distilled aniline for 24 h at room temperature.36 After washing with copious ethanol, the film was dried under reduced pressure for XPS characterization. 2.2.3. Immobilization of PVA onto PLLA Surface: PLLA-PVA surface. The aldehyde-tailored PLLA (PLLA-CHO) film was immersed in a 2 wt % PVA solution with 0.2 M H2SO4 present as a

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catalyst at 37 °C for 2 h for grafting PVA. The PVA-modified PLLA (PLLA-PVA) film was carefully and thoroughly washed with water and maintained wet for next procedure. 2.2.4. Fixation of PVA Hydrogel Containing SilVer Nanoparticles onto PLLA Surface: PLLA-PVAgel/Ag(0). About 0.1 mL of a 10 wt % PVA solution containing 0.5 wt % AgNO3 (PVA/Ag(I)) was cast onto the PLLA-PVA film, and then the film was maintained at -20 °C for at least 12 h before thawing at room temperature. One freeze/thaw cycle was enough to fix the PVA hydrogel onto the PLLA surface. The PLLA-PVAgel/Ag(I) was immersed into freshly prepared 0.010 mol/L sodium borohydride (NaBH4) for 5 min to reduce the silver ions to silver nanoparticles. 2.3. Cell Adhesion Experiments. Hela cells were grown in DMEM medium (Gibco BRL) supplemented with 10% fetal calf serum and 2 mM L-glutamine in a humidified incubator (37 °C, 5% CO2). Exponentially growing cells were used for the experiment. Cells were cultured in a six-well culture plate containing glass, virgin PLLA, or PLLA-PVAgel/Ag(0) films at a concentration of 2.5 × 104 cells/mL. Two days later, cells were viewed using an inverted microscope (TE2000U, Nikon) at 400× magnification. Pictures were taken with a Nikon DXM1200F digital camera. Tests for each specimen were done in triplicate, and at least three random microscope fields were observed for each sample. 2.4. Measurements of Antibacterial Activity. PLLA-PVAgel/Ag(0) was used for antibacterial activity tests. The Kirby-Bauer test was used to evaluate the zone of inhibition (ZOI) around a 1.2 × 1.2 cm2 disk coated with a given antimicrobial film. The sample disks were incubated for 24 h at 37 °C in a gelatinous agar growth medium. This testing was performed according to an established procedure using plates swabbed with solutions of E. coli containing approximately 3 × 106 cell forming units (CFUs) per mL.37 ZOI values for E. coli were based on an average of at least three samples of each composition. 2.5. Characterization. Attenuated Total Reflection Infrared (ATRIR) spectra were obtained on a Bruker Vertex 70 FTIR spectrometer equipped with a DTGS detector using a PIKE ATR accessory with a ZnSe crystal. Surface composition was determined by X-ray photoelectron spectra (XPS) on a ThermoElectron ESCALAB 250 spectrometer equipped with a monochromatic Al X-ray source (1486.6 eV). Spectra were recorded at a 90° takeoff angle with 20 eV pass energy. Scanning electron microscopy (SEM) images were taken on a JEOL JSM 5600LV scanning electron microscope. The surface morphology was observed by atomic force microscopy (AFM; SPA300, Seiko) in the tapping mode. Contact angle analyses were carried out on a KRUSS DSA1 version 1.80 drop shape analyzer with water as the probe liquid. Each contact angle value reported here was an average of at least five measurements.

3. Results and Discussion 3.1. Surface Modification. It is difficult to modify the PLLA surface due to its lack of readily reactive functional groups. Plasma treatment, a harsh, nonselective surface modification method, has been utilized to modify many polymers effectively, and hydrophilic groups, such as hydroxyl, carboxylic acid or carbonyl, and peroxyl or ether, have been introduced to hydrophobic PLLA surfaces by oxygen plasma treatment.38 The introduction of hydrophilic groups to hydrophobic PLLA surfaces can be detected by water contact angle measurements, and the contact angles of the PLLA film treated with plasma for different times are plotted in Figure 1. It can be seen that the water contact angle of the PLLA decreases significantly (from 84 to 38°) after oxygen plasma treatment and reaches a plateau at ∼10 min, indicating that the concentration of the polar groups formed at the surface reaches saturation. All data reported here are from PLLA films that were all exposed to the oxygen plasma for 10 min prior to next surface modification procedure.

Zan et al.

Figure 1. Dependence of the advancing contact angle on the time PLLA treated with the oxygen plasma.

Figure 2. ATR FTIR spectra of pristine PLLA (a) and PLLA-OH prepared at monomer concentration of (b) 1, (c) 5, and (d) 10%.

Next, HEMA was grafted to the plasma-treated films initiated by UV irradiation. Figure 2 shows the ATR FTIR spectra of the film surface after graft polymerization (PLLA-OH) using HEMA at different concentrations. The PLLA film exhibits characteristic absorption bands at 1757, 2997, and 2947 cm-1 attributed to the ester carbonyl stretching and symmetric and antisymmetric stretching vibrations of the C-H, respectively.39 After the grafting, a new broad band at 3000-3700 cm-1 centered at ∼3425 cm-1 characteristic of O-H stretching and a new ester carbonyl stretching at 1726 cm-1 are clearly observed. This is strong evidence that HEMA is grafted onto the PLLA surface. It has been reported that part of the carbonyl groups and the hydroxyl groups in polyHEMA can hydrogen bond, which shifts the carbonyl stretching vibration to lower wavenumbers.39,40 This can be seen by the shoulder at ∼1710 cm-1 that broadens the carbonyl stretching band (Figure 2). The C-H symmetric and antisymmetric stretching vibrations also become stronger due to the higher C-H content in polyHEMA. Furthermore, with increasing monomer concentration, the intensities of the aforementioned characteristic peaks of polyHEMA increase, suggesting that the HEMA layer grafted becomes thicker. This is also reflected in the increase in the peak intensity ratio between the O-H stretching (solely due to polyHEMA) and the C-H stretching (due to polyHEMA and PLLA both), as shown in Table 1. At 10% monomer concentration, the carbonyl stretching of the PLLA at 1757 cm-1 has completely disappeared, indicating the thickness of the grafted layer is at least comparable to the sampling depth of the ATR unit, which is on the order of 1 µm.41 The graft reaction was further assessed by contact angle, which is sensitive to the top several angstroms of the grafted layer, and gravimetry, and the results are listed in Table 1. It can be seen that the polyHEMA surface exhibits higher advancing and lower receding contact angles than virgin PLLA,



Table 1. Change with the Monomer Concentration in Graft Density, Contact Angle, and the IR Absorbance Ratio between O-H Stretching and C-H Stretching Bands (AO-H/AC-H) monomer concentration (%)

θA/θR

graft density (g/m2)

AO-H/AC-H

0 1 5 10

83°/45° 65°/23° 95°/27° 95°/24°

0.108 0.313 0.672

2.14 4.34 4.57

because the hydrophilic functional groups (-OH) in the polyHEMA layer move inward in air and outward in water.42 The wettability, ATR FTIR, and graft density data all exhibit positive dependence on the HEMA monomer concentration, with significant increases at concentrations below 5%, and at higher concentrations, the contact angle and the IR peak ratio both level off because the thickness of the grafted layer is greater than the sampling depth of these two techniques, while the graft density continues to increase with the monomer concentration. These results indicate that monomer concentration can be adjusted to conveniently control the grafting of HEMA onto the PLLA surface and confirm a good coverage of grafted layer on the PLLA surface at 10% monomer concentration, which was used in all subsequent experiments. Various methods for oxidizing alcohols (OH) to aldehyde groups (CHO) are available.43 We chose PDC as the oxidizing agent due to its selectivity: it can oxidize hydroxyls to aldehydes but not to acids.35 Figure 3a shows ATR IR spectra of PLLA-OH before and after oxidation, normalized by the C-H stretching band in the 2900-3000 cm-1 region. It is evident that after oxidation, the intensity of the broad O-H stretching at ∼3425 cm-1 (IOH) characteristic of the hydroxyl in the polyHEMA decreases dramatically, and the CdO stretching band at ∼1730, originally assigned to the ester carbonyl in the polyHEMA, as discussed above, increases greatly due to the contribution of the aldehyde carbonyl stretching band which occurs in the same region, indicative of the successful conversion of the OH in polyHEMA to CHO. More careful inspection shows that the shoulder at ∼1710 cm-1, due to the ester carbonyl hydrogen bonded with the hydroxyl in the polyHEMA, as discussed above, has largely disappeared after the reaction. Apparently a significant portion of the hydrogen bonds was destroyed when the alcohols were converted to aldehydes, which is also consistent with oxidation. The IR bands at ∼3400 (O-H) and ∼1730 cm-1 (CdO) can be used to follow the oxidation process. Figure 3b plots the intensities of these two bands normalized to the C-H stretching band at 2700-3100 cm-1 region as functions of reaction time. It can be seen, as we expected, that over the oxidation time the hydroxyl and the aldehyde contents first decrease and increase, respectively, and then both level off at longer times, indicating the progress of the conversion of the alcohol to the aldehyde. It also shows that not all the alcohols were oxidized in our experimental time scale, in agreement with the XPS analysis (discussed below), but the amount of the aldehyde at the surface was sufficient to graft PVA in the next step. It is known that primary amines (-NH2) readily react with aldehydes to form imines (dN-),36 and this reaction was employed to further verify the presence of aldehyde groups at the surface after the oxidation. After the labeling reaction using aniline, a N1s peak was clearly identified at 398.5 eV in the XPS spectrum due to the dN- groups at the film surface (Figure 4). Therefore, both IR and XPS data confirm the presence of -CHO on the PLLA surface. In the next step, a thin layer of PVA was immobilized on the PLLA-CHO surface via acid-catalyzed acetal formation,

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which has been reported for surface modification.44 As seen in the ATR FTIR spectrum of the PLLA-PVA shown in Figure 3a, the OH stretching band re-emerges, which is consistent with expected chemistry and that PVA is covalently bonded to the film through acetal formation. The surface modification process was also followed by XPS. In the survey spectra of PLLA, PLLA-OH, PLLA-CHO, and PLLA-PVA surfaces, only two peaks, C1s and O1s, are present with different relative intensities. The atomic compositions for these surfaces are listed in Table 2. More information can be obtained from detailed analysis of the C1s region, shown in Figure 5. For these surfaces, three types of carbon are identified at 284.3, 285.9, and 287.9 eV, arising from saturated hydrocarbon (CC-H or CC-C), saturated carbon bound to an oxygen atom (CC-O), and carbon in a carbonyl (CCdO), respectively. It can be seen that there is a significant change in the carbon composition after each modification step. The contents of the three carbon species were obtained as the atomic ratios and compared with the theoretical values calculated based on the atomic composition of the repeat unit of the grafted polymer. It is seen in Table 2 that the experimental data are consistent with the theoretical values, which is in line with our above argument that each step surface modification was successful, even though the conversion of PLLA-OH to PLLA-CHO was not complete in our experimental time scale, as indicated by the slightly lower CCdO and higher CC-O contents. Overall, the XPS results further confirm that the surface modification method utilized here was very effective. 3.2. Fixation of PVA Hydrogel Containing Silver Nanoparticles. After a thin layer of PVA was immobilized on the PLLA-CHO surface, as discussed above, a PVA solution containing silver ions was immediately cast on the surface and a PVA gel layer was formed by the “freeze/thaw” method.27 The silver ions in the hydrogel was reduced by NaBH4 to silver metal in the form of nanoparticles,45 and the colorless PVA gel turned yellow after the reduction due to the presence of the colloidal silver nanoparticles. The reduction of the silver ions to silver nanoparticles in the hydrogel was confirmed by XPS (Figure 6). It can be seen that the two Ag3d peaks at 367.6 and 373.6 eV for Ag+ shift to 368.4 and 374.4 eV, respectively, after the reduction, corresponding to the high binding energies for Ag(0).46 In addition, the N1s signal, observed in the original sample due to the NO3- counterion, has disappeared after the reduction. Figure 7 shows SEM micrographs of the cross section of the PLLA-PVAgel/Ag(0) film. It can be seen that the PVA hydrogel layer is ∼600 nm in thickness (under dry conditions) and that it uniformly covers and attaches firmly to the PLLA substrate. From the magnified micrograph (Figure 7b), no detachment of the PVA layer from the PLLA is visible. This firm attachment ensures the integration of the surface and is important for the cell and antibacterial experiments. As a comparison, PVA solution was cast directly onto the PLLA and PLLA-OH surfaces, respectively, and the hydrogel layer was found to begin detachment in the freezing step and completely detach from the substrate when immersed in water. 3.3. Cell Adhesion Studies. In this work, hela cells were chosen to evaluate the resistance of the PLLA surface to protein and cell adhesion. Figure 8 shows the morphology of hela cells on the surface of PLLA-PVAgel/Ag(0) in comparison to glass and virgin PLLA. It can be seen that on glass the hela cells adhere in abundance and are highly activated with aggregation, pseudopodia, and spread widely, indicating that the glass surface favors hela cells attachment and growth. On the pristine PLLA surface, the cells also attach but show round-shaped morphology,

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Figure 3. (a) ATR FTIR spectra of PLLA-OH before (lower) and after oxidation (middle), and PLLA-PVA (upper). (b) The dependence of the areas of the bands at ∼3400 (AO-H) and ∼1730 cm-1 (ACdO) normalized to that at ∼2950 cm-1 (AC-H) on the oxidation time.

Figure 4. XPS spectra of (a) PLLA-OH and (b) N-labeled PLLA-CHO (inset is the N1s region). Table 2. XPS Surface Atomic Concentrations of Film Samplesa

Figure 6. XPS spectra of (a) PLLA-PVAgel/Ag(I) and (b) PLLA-PVAgel/ The inset is the Ag3d region.

Ag(0).

atomic concentration % (experimental/theoretical) samples

C

O

C1/C2/C3

PLLA PLLA-OH PLLA-CHO PLLA-PVA

60.3/60.0 68.1/66.7 67.2/66.7 67.2/66.7

39.7/40.0 31.9/33.3 32.8/33.3 32.8/33.3

1.00:0.98:1.01/1:1:1 3.04:1.97:1.00/3:2:1 3.00:1.27:1.52/3:1:2 1.00:1.01:0/1:1:0

a Note: C1, C2, and C3 represent the carbon in C-C (284.3 eV), C-O (285.9 eV), and CdO (287.9 eV), respectively.

Figure 7. SEM micrograph of the cross section of a PLLA-PVAgel sample (a), and (b) is a magnified section of (a).

Figure 5. XPS spectra of the core level of C1s of various films surfaces.

in agreement with previous reports.47,48 It is obvious that the PLLA surface prevents the cells from spreading on the surface, demonstrating that the PLLA surface is a poorly adhering substrate but a supportive material to adhesion.47 On the surface of PLLA-PVAgel/Ag(0), however, no hela cell adhesion is observed. Based on the number of adhered hela cells on various

films at the same magnification, the cell adhesion was quantified using the number for a glass substrate as reference (100%). As shown in Figure 8d, the hela attachment on PLLA is 79%, while on it is 0.82%, indicating a high effectiveness in inhibiting hela adhesion. It is known that silver has high toxicity to microorganisms but is almost nontoxic to animal cells.49 For example, human osteoblasts were found to attach and grow well on surfaces containing silver nanoparticles.49,50 Therefore, the high resistance of the PLLA-PVAgel/Ag(0) to hela cell is unlikely due to the embedded silver nanoparticles. The ability of the surface to resist cell adhesion is attributed to its high water content, hydrophilicity (θA ) 10°), and low interfacial tension between the hydrogel surface and the surrounding fluids.19,22 Cells generally prefer to attach to artificial substrates by binding to specific anchoring proteins on the surface of the substrate.47 Hela cell adhesion will not usually occur to a significant extent if no protein has adsorbed onto the surface first. From this point, the PLLA-PVAgel/Ag(0) surface is a promising candidate for resistance to cell and protein adhesion.



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provides a suitable media that facilitates the diffusion of the hydrated silver ions, and the amount of silver loaded can be conveniently controlled via the concentration of the silver salt present in the PVA solution used to prepare the hydrogel. Therefore, the combination of hydrogel with silver nanoparticles gives PLLA-PVAgel/Ag(0) good antibacterial activity.

4. Conclusions

Figure 8. Optical images of adhered hela cells on (a) glass, (b) virgin PLLA, and (c) PLLA-PVAgel/Ag(0); (d) is percent hela adhesion on different substrates relative to that on glass (which served as the control). The scale bar is 100 µm.

Covalent attachment of PVA hydrogel loaded with silver nanoparticles to the surface of PLLA via multiple steps of highly effective modification reactions has been demonstrated. The film shows two properties: antibacterial due to the presence of the silver particles and resistance to protein and cell adhesion due to high water content and the soft nature of the PVA hydrogel. Furthermore, the PVA hydrogel layer can serve as a matrix for incorporation of various functional molecules, which can then be released into the medium due to the properties of the hydrogel, or as a reactor for simple chemistry such as the in situ reduction we demonstrated in this paper. The release rate can be modulated by the freeze-thaw cycle number, freezing temperature, PVA molecular weight, and hydrolysis degree. In addition, the film covalently combines the solid and soft material properties together. This work provides a versatile route to the fabrication of multifunctional films with tunable properties and may have potential applications in tissue engineering and biomedical devices. Acknowledgment. This work was supported by National Natural Science Foundation of China (20423003, 20774097). Z.S. thanks the NSFC Fund for Creative Research Groups (50921062) for support.

References and Notes

Figure9.Antibacterialactivitiesof(a)PLLA-PVAgel and(b)PLLA-PVAgel/ Ag(0).

3.4. Antibacterial Studies. When the Kirby-Bauer technique was used, the antibacterial activity of the PLLA-PVAgel/ gel Ag(0) was assessed in comparison to PLLA-PVA . Figure 9 shows the Kirby-Bauer plates, and the zone of inhibition (ZOI) is clearly observed for the PLLA-PVAgel/Ag(0) surface. The ZOI for PLLA-PVAgel and PLLA-PVAgel/Ag(0) is 0 and 2.94 ( 0.42 mm, respectively, showing significant antibacterial activity of the PLLA-PVAgel/Ag(0), which is apparently due to the silver nanoparticles embedded in the hydrogel. The result is consistent with other reports on the antibacterial activity of silver particles.51 To be active, the silver nanoparticles must first be converted to ionic silver, the effective antimicrobial species, via the oxidation of the zerovalent silver, which limits the concentration of ionic silver available. Therefore, in general, silver nanoparticles exhibit lower bactericidal activity than silver salts. On the other hand, silver in ionic form would diffuse into the media and thus be depleted quickly, while the nanoparticles can serve as a reservoir to supply a steady stream of silver ions for extended periods of time. In addition, the hydrogel matrix

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