Article pubs.acs.org/Biomac
Combined Effects of Ag Nanoparticles and Oxygen Plasma Treatment on PLGA Morphological, Chemical, and Antibacterial Properties Elena Fortunati,† Samantha Mattioli,† Livia Visai,‡,§ Marcello Imbriani,∥,§ Josè Luis G. Fierro,⊥ Josè Maria Kenny,†,# and Ilaria Armentano*,† †
Materials Engineering Center, UdR INSTM, University of Perugia, Str. Pentima 4, 05100 Terni, Italy Department of Molecular Medicine and UdR INSTM, University of Pavia, 27100 Pavia, Italy § Salvatore Maugeri Foundation IRCCS, Via S. Maugeri 4, 27100 Pavia, Italy ∥ Department of Public Health, Experimental Medicine and Forensics, University of Pavia, 27100 Pavia, Italy ⊥ Institute of Catalysis and Petrochemistry, CSIC, Marie Curie 2, Cantoblanco, 28049 Madrid, Spain # Institute of Polymer Science and Technology, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain ‡
ABSTRACT: The purpose of this study is to investigate the combined effects of oxygen plasma treatments and silver nanoparticles (Ag) on PLGA in order to modulate the surface antimicrobial properties through tunable bacteria adhesion mechanisms. PLGA nanocomposite films, produced by solvent casting with 1 wt % and 7 wt % of Ag nanoparticles were investigated. The PLGA and PLGA/Ag nanocomposite surfaces were treated with oxygen plasma. Surface properties of PLGA were investigated by field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), static contact angle (CA), and high resolution X-ray photoelectron spectroscopy (XPS). Antibacterial tests were performed using an Escherichia coli RB (a Gram negative) and Staphylococcus aureus 8325-4 (a Gram positive). The PLGA surface becomes hydrophilic after the oxygen treatment and its roughness increases with the treatment time. The surface treatment and the Ag nanoparticle introduction have a dominant influence on the bacteria adhesion and growth. Oxygen-treated PLGA/Ag systems promote higher reduction of the bacteria viability in comparison to the untreated samples and neat PLGA. The combination of Ag nanoparticles with the oxygen plasma treatment opens new perspectives for the studied biodegradable systems in biomedical applications. to substrates with different surface properties,12 while McAllister et al. found that the irregularities of polymeric surfaces promote bacterial adhesion.13 After the adhesion on the surface of the biomedical device, the bacteria slowly proliferate and form a colony or biofilms that are structurally complex and possess a dynamic architecture.14 The mechanism of adhesion is a two-step process that includes the initial instantaneous and reversible physical phase, which is followed by a time-dependent and irreversible molecular and cellular phase.15,16 Bacterial adhesion takes place due to the initial attraction of the cells to the polymer surface followed by protein adsorption and cell attachment. Bacteria move to the material surface due to physical forces and chemical factors. For these reasons, many approaches to modify the surface of biodegradable polymer supports have been undertaken in order to introduce new surface characteristics to the polymer. Surface treatment techniques, such as plasma
1. INTRODUCTION Multifunctional nanocomposites based on biodegradable polymer matrix and silver (Ag) nanoparticles have attracted great interest in nanobiotechnology due to the silver antimicrobial properties that are retained during polymer degradation.1,2 Silver species can be released in a controlled manner3 and, for this reason, silver containing materials have been extensively used to prevent attack of a broad spectrum of microorganisms in different application fields. Furthermore, physicochemical properties of the Ag nanocomposites surface are known to affect the rate of the initial bacterial adhesion and the subsequent biofilm formation. The design and fabrication of novel nanostructured surfaces, able to interact specifically with biological systems, is a challenging research area to produce new devices exhibiting relevant technological properties, including biosensors,4 tissue engineering,5 and novel antibacterial materials.6 The factors influencing bacteria adhesion to a biomaterial surface include chemical compositions,7,8 surface charge,9 hydrophobicity,10 and surface roughness or physical configuration.11 Bacteria adhere differently © XXXX American Chemical Society
Received: September 28, 2012 Revised: January 11, 2013
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Different treatment times (t; 10, 20, and 30 min) were selected in order to evaluate the effect of the plasma exposition time on the final properties of PLGA nanocomposites. 2.4. Film Characterization. 2.4.1. Morphological Characterization. A morphological analysis of the PLGA and PLGA/Ag film surfaces, before and after the exposure of plasma oxygen treatment, was conducted by means of a field emission scanning electron microscopy (FESEM, Supra 25 Zeiss, Germany). Samples were coated with a thin (about 10 nm) gold layer and analyzed at an accelerating voltage of 2 kV. The surface pore size of the as-prepared nanocomposite films was measured by image analysis (NIS-Elements BR, Nikon, U.K.). Atomic force microscopy (AFM, Easy Scan, Nanosurf, Switzerland) was used to study the topography of the PLGA and PLGA/Ag films before and after plasma treatments. AFM images were recorded in a tapping mode at RT in air, using silicon cantilevers, with a scan rate of 0.5 s/line. The film surface roughness was determined in five random areas per sample, scanning across 30 × 30 μm2 areas. Calculation of the rootmean-square roughness (Sq) was performed using the easy Scan DFM
treatment, ion sputtering, oxidation, and corona discharge, are used to modify the chemical and physical properties of the surface layer without significantly change the bulk material properties.7,17 Most of the literature in this field deals with the plasma process for the modification of the polymer surface chemical composition and properties such as wettability, surface energy, refractive index, hardness, chemical inertness, and biocompatibility18 reporting also the possibility to deposit silver coating layers by means plasma enhanced chemical vapor deposition process7,19 in order to obtain an antibacterial activity. Zimmermann et al. describes the use of plasma process to create effective antibacterial coatings,19 while oxygen plasma treatment influences the surface properties and thereby the bacteria response.7,17,18 In two recent papers,2,20 we demonstrated the antibacterial effect of PLGA/Ag nanocomposite, we underlined also the role of the microstructure nanocomposite surface on the bacteria adhesion properties. Hence, in this paper, for the first time, to the best of our knowledge, we combined the effects of oxygen plasma treatment and silver nanoparticle introduction on PLGA in order to modify the nanocomposite surface at the nanometer level and modulate the surface antimicrobial properties through tunable bacteria adhesion mechanisms. This paper underlined the combined outcomes of plasma surface treatment and nanocomposite approach to design nanostructured silver nanoparticle based PLGA films with modulated surface properties, bacteria adhesion and growth.
software, as Sq =
M−1 ⎛ ⎛ ⎞⎞2 1 Σ Σn − 1⎜z⎜x ; y ⎟⎟ . MN K = 0 l = 0 ⎝ ⎝ k l ⎠⎠
2.4.2. Contact Angle Measurements. Static contact angle (CA) measurements were performed in order to investigate PLGA and PLGA/Ag films wettability before and after the plasma treatment. The contact angles were assessed using the sessile drop method in air by applying a video microscope interfaced to a computer (FTA-1000 Analyzer, U.S.A.) to capture drop images. Sterile water drops of 20 μL (HPLC grade water) were placed on films and the measurements were recorded 10 s after the liquid made contact with the surface. Five independent measurements on five different samples were averaged. 2.4.3. Chemical Characterization. Infrared spectroscopy was carried out in order to characterize the sample surface and to confirm the composition and the treatment effects. Measurements were obtained with attenuated total reflection spectroscopy (ATR) using a JASCO FTIR 615, U.S.A., spectrophotometer with a germanium crystal. Pristine and treated surfaces were analyzed in the range of 400−4000 cm−1, with a 4 cm−1 resolution. Photoelectron spectra (XPS) were obtained with a VG Escalab 200R, U.S.A., spectrometer equipped with a hemispherical electron analyzer and a Mg Kα (hν = 1254.6 eV, 1 eV = 1.6302 × 10−19 J) X-ray source, powered at 120 W. The kinetic energies of photoelectrons were measured using a hemispherical electron analyzer working in the constant pass energy mode at 50 eV. The background pressure in the analysis chamber was kept below 2 × 10−8 mbar during data acquisition. The XPS data signals were taken in increments of 0.1 eV with dwell times of 50 ms. Binding energies were calibrated relative to the C1s peak at 284.9 eV. High resolution spectra envelopes were obtained by curve fitting synthetic peak components using the software XPS peak. The raw data were used with no preliminary smoothing. Symmetric Gaussian− Lorentzian (90G-10L) curves were used to approximate the line shapes of the fitting components. 2.5. In Vitro Bacterial Assays on PLGA and PLGA/Ag Nanocomposites. 2.5.1. Bacterial Strains and Culture Conditions. The microorganisms used in this study were Escherichia coli RB (E. coli RB) and Staphylococcus aureus 8325-4 (S. aureus 8325-4). E. coli RB was an isolate provided by the “Zooprofilattico Institute of Pavia”, Italy, whereas S. aureus 8325-4 was a gift from Timothy J. Foster (Department of Microbiology, Dublin, Ireland). E. coli RB was routinely grown in Luria−Bertani Broth (LB; Difco, Detroit, MI, U.S.A.) and S. aureus 8325-4 in Brian Heart Infusion (BHI; Difco) overnight under aerobic conditions at 37 °C using a shaker incubator (New Brunswick Scientific Co., Edison, NJ, U.S.A.). These cultures, used as source for the experiments, were reduced at a final density of 1 × 1010 cells/mL, as determined by comparing the OD600 of the sample with a standard curve relating OD600 to cell number. 2.5.2. Bacterial Adhesion Assay. To evaluate the adhesion of E. coli or S. aureus cells to each sample of PLGA and PLGA/Ag films, an overnight suspension of both strains were washed with sterile phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH = 7.4) and an aliquot of 200 μL (1 × 106 cells) was
2. MATERIALS AND METHODS 2.1. Materials. Poly(DL-lactide-co-glycolide) (PLGA; inherent viscosity: 0.95−1.20 dL/g, Mw 91600−120000 g/mol) ether terminated with a 50/50 ratio (PLA/PGA) was purchased from Absorbable Polymers Lactel (Durect Corporation, U.K.). Commercial silver nanopowder, P203, with a size distribution ranged from 20 to 80 nm, was supplied by Cima NanoTech (Corporate Headquarters Saint Paul, MN, U.S.A.). A commercially available grade of chloroform (CHCl3), supplied by Sigma Aldrich Chemicals, was used in the sample preparation. 2.2. Fabrication of PLGA/Ag Nanocomposite Films. PLGA nanocomposite films were produced by solvent casting in chloroform and silver nanoparticles were used at 1 wt % and 7 wt % with respect to the polymer matrix. Neat PLGA films were obtained dissolving polymer granules in CHCl3 (10 wt %/v) and using a magnetic stirring at room temperature (RT) to obtain a complete polymer dissolution. PLGA/Ag nanocomposite films were produced by dispersing silver powder in CHCl3 at different percentages by means of sonication treatment for 5 h. The ultrasonic bath was used to disperse the silver nanoparticles in chloroform, to avoid aggregation and Ag cluster presence, to enhance the interaction with the biodegradable matrix. Thereafter, the polymer was added to solutions and the suspensions were magnetically stirred until they were completely dissolved. If silver nanoparticles are well dispersed in the solvent when PLGA polymer is mixed with the silver nanoparticle suspension, the interaction between the nanostructures and the polymer chains is encouraged. The dispersions were cast on a Teflon surface, allowing the solvent to evaporate over 24 h and leaving nanocomposite films of rectangular shape (0.3 mm in thickness).20 Samples were further dried for 48 h in vacuum at RT. Resulting films were designed as PLGA, PLGA/1Ag, and PLGA/7Ag. 2.3. Plasma Treatment. The PLGA and PLGA/Ag films were treated by means of a radiofrequency plasma under oxygen flow, using a Sistec apparatus with Huttinger power supply at 13.56 MHz. The films were placed into the stainless steel chamber that was evacuated for 1 h until a pressure (P) of 9 × 10−3 Torr. The gas flow was maintained at 60 standard cm3/min (sccm). The treatment conditions were P = 6.5 × 10−2 Torr, power supply (RF) = 30 W, and bias voltage (V) = 380 V. The bias voltage is applied to the electrode where the substrate is positioned. B
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Figure 1. FESEM and AFM images of untreated PLGA, PLGA/1Ag, and PLGA/7Ag nanocomposite upper and lower surface. seeded on the sample and incubated for 3 h. Briefly, as previously reported,19 loosely adhering bacteria were removed by gently washing the samples with PBS whereas the bacterial cells tightly adherent to the surfaces were prepared for SEM analysis as indicated. Three samples of each experimental condition were used for Total Viable Count (TVC) estimation. The samples with the adherent bacterial cells were dispersed into 1 mL sterile Ringer solution (Oxoid, Italy) by vortex for 5 min. The cleaned surfaces were stained and observed to ensure complete recovery of cells. Serial dilutions of the bacterial cell suspensions were prepared and 0.1 mL of each dilution was deposited onto BHI or LB agar (Bacto agar, Difco, CA) plates, depending on the bacterial strains. The plates were incubated for 24−48 h at 37 °C and the number of colonies counted. Mean TVC values were calculated for each sample and the percent viability was calculated by setting the bacterial cells grown onto 24 tissue culture plates (TCP) wells equal to 100%. 2.5.3. Scanning Electron Microscopy. Scanning electron microscopy (SEM) was performed on each sample of PLGA and PLGA/Ag films incubated for 3 h at 37 °C with E. coli or S. aureus cells as previously indicated.21 Following incubation, samples were washed once with PBS and then fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 1 h at 4 °C. After additional washing, the samples were incubated using increasing concentrations of ethanol (25, 50, 75, 96%) for 10 min, dried to the critical point using an Emitech K- 850 apparatus, and placed
on a mounting base. Finally, the specimens were coated with gold and examined under SEM at 1000× magnification (model EVO 50 EP; Zeiss-Leica, Cambridge, U.K.). 2.5.4. Antibacterial Activity. To examine the antimicrobial activity of each sample of PLGA and PLGA/Ag films, 200 μL (5 × 103) of an overnight diluted suspension of E. coli or S. aureus cells was added and incubated for 24 h, respectively.22 Wells used as controls were incubated with cell suspension for 24 h. At the end of the incubation time, bacterial suspension was serially diluted and plated on the LB (E. coli) or BHI (S. aureus) agar plates, respectively. The plates were incubated for 24−48 h at 37 °C. The percent viability was calculated by setting the bacterial cells grown onto 24 TCP wells equal to 100%. 2.5.5. Confocal Laser Scanning Microscopy (CLSM). For confocal studies, an aliquot (200 μL) of an overnight diluted suspension of E. coli or S. aureus cells was seeded on sterile PLGA and PLGA/Ag films placed at the bottom of 24-well microplates (Costar) and incubated for 24 h at 37 °C. Following incubation, to determine the viability of bacteria within the different PLGA samples, a BacLight Live/Dead viability kit (Molecular Probes, Eugene, OR, U.S.A.) was used. The kit includes two fluorescent nucleic acid stains: SYTO9, which penetrates both viable and nonviable bacteria, and propidium iodide, which penetrates bacteria with damaged membranes and quenches SYTO9 fluorescence. Dead cells, which take up propidium iodide, fluorescence red, and cells C
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Figure 2. FESEM images of PLGA and PLGA/Ag nanocomposites before and after oxygen plasma treatment at different exposition times.
Figure 3. AFM images of pristine PLGA and PLGA/Ag nanocomposites (first column at left) and oxygen plasma-treated samples at different exposition times (scan size 30 μm A−N and 10 μm C′, G′, and M′). fluorescing green are deemed viable. For assessing viability, 1 μL of the stock solution of each stain was added to 3 mL of PBS and, after being mixed, 500 μL of the solution was dispensed into 24-well microplates containing bacterial suspension, grown on materials, and incubated at 22 °C for 15 min in the dark. Stained bacteria were examined under a Leica CLSM (model TCS SPII; Leica, Heidelberg, Germany) using a 40× oil immersion objective. The excitation and emission wavelengths used for detecting SYTO9 were 488 and 525 nm, respectively. Propidium iodide was excited at 520 nm, and its emission was monitored at 620 nm.23 For each sample, images from three randomly selected positions were acquired. The resulting stacks of images, analyzed using Leica confocal software, were subsequently processed using ImageJ software (Wayne Rasband, National Institutes of Health).
2.6. Statistical Methods. Continuous data were expressed as means and SD. Two-group comparisons were performed by Student’s t test. All analyses were performed using GraphPad Prism 4.0 (Graph Pad Software Inc., San Diego, CA, U.S.A.). Two-tailed p values CO or −COOH (%)
286.9 eV −C−O− (%)
284.8 eV −C−H or −C−C− (%)
532.3 eV
533.8 eV
O/C
PLGA PLGA 10 min PLGA 20 min PLGA 30 min PLGA/1Ag 20 min PLGA/7Ag 20 min
38 41 41 45 7
40 39 34 36 25 45a
22 20 25 19 68 55
44 56 46 51 47 58
56 44 54 49 53 42
0.565 0.859 0.749 1.102 0.269 0.938
The Ag/C ratio of PLGA/7Ag sample is 0.223.
(1500−1340) cm−1 region. The two bands observed at 1452 and 1422 cm−1 correspond to the asymmetric bending of CH3 from
the lactic units and the bending of CH2 from the glycolic units of the polymer. The FT-IR spectra of plasma-treated PLGA films F
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Figure 7. Binding energy of Ag3d5/2 level in PLGA/1Ag and PLGA/7Ag samples and Ag reference (A) and Auger AgMNN lines for PLGA/1Ag, PLGA/ 7Ag, and a metallic Ag reference sample (B).
Figure 8. Bacterial adhesion to PLGA and PLGA/Ag nanocomposite films. E. coli and S. aureus cell adhesion to PLGA and PLGA/7Ag untreated and 30 W, 20 min, oxygen-treated films was determined as CFU/mL after 3 h incubation at 37 °C. Data are expressed as percentage of the ratios between CFU of bacteria adherent to PLGA to CFU of bacteria adherent to 24-well flat-bottom sterile polystyrene microplates. The values represented are the means of the results of each sample performed in duplicate and repeated in three separated experiments. Error bars indicate standard errors of the means. The statistical significance was indicated as follows:*p < 0.05 and **p < 0.01.
were attributed to aliphatic carbon bonds or carbon−hydrogen bonds (−C−H or −C−C−), to −C−O−,28 and to ester groups (>CO or −COOH),28,29 respectively.30 In addition, from the data summarized in Table 1, it is seen that the proportion of these components changed to a great extent for PLGA/1Ag sample, this change is even more drastic for PLGA/7Ag sample. For this latter sample, only two components are observed: a major one at 284.8 eV belonging to C−C bonds and a second one placed at a binding energy in between species (ii) and (iii). It is interesting to be noted that silver is only detected in PLGA/7Ag sample but in no case in its PLGA/1Ag counterpart. The O1s peak of PLGA samples (Figure 6B) was fitted to two components: one placed at 532.3−532.4 eV usually assigned to OC bonds and another at 533.7−533.9 eV often ascribed to O−C bonds.28 Similarly, the Ag-containing samples showed the same two components but appeared somewhat shifted to lower binding energies in PLGA/
demonstrate variation in the lactic unit, with the presence of new peaks at 1460 and 1481 cm−1. The surface chemical composition of PLGA and PLGA nanocomposite films before and after O2 plasma treatment was determined by XPS measurements. To investigate the effect of plasma treatment and silver nanoparticle presence on the chemical properties, XPS investigations were conducted on nanocomposites with the lower (PLGA/1Ag) and higher (PLGA/7Ag) content of silver nanoparticles treated at 30 W for 20 min. The C1s and O1s core-level spectra were recorded for all samples in Figure 6A,B. In addition, in that samples in which silver is expected to be present, the Ag3d core-level spectra were also scanned (Figure 6C). The binding energies of C1s and O1s (and of Ag3d5/2, if present) are compiled in Table 1. The C1s in peaks were fitted to three components at (i) 284.8−284.9, (ii) 286.9−287.0, and (iii) 289.0−289.1 eV, which G
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Figure 9. SEM images of bacteria adherent to PLGA and nanocomposite films. E. coli and S. aureus strains were incubated with the indicated PGLA films for 3 h at 37 °C and then prepared as reported in Materials and Methods; magnification, 1000×.
Figure 10. CLSM images of the antibacterial activity of PLGA and PLGA/Ag nanocomposite films. E. coli and S. aureus strains were incubated for 24 h onto each sample of PLGA films. To determine the viability of bacteria within the PLGA films, bacteria were stained with a BacLight Live/Dead stain, as reported in Materials and Methods; scale bar, 75 μm.
3.3. Effect of Ag Nanoparticles and Plasma Treatment on In Vitro Bacterial Adhesion. Studies on cell attachment performed using two different strains, E. coli RB and S. aureus 8325-4, showed a reduction in bacterial adhesion to all differently treated samples if compared to untreated PLGA systems (Figure 8). In particular, a 15% reduction in bacterial adhesion for both strains on oxygen plasma-treated PLGA matrix was observed if compared to the untreated PLGA. On PLGA/Ag samples the bacterial reduction was dependent on increasing content of Ag nanoparticles, varying from 10% up to 30% reduction in bacterial adhesion (p < 0.05). Finally, the bacterial attachment to oxygen plasma-treated PLGA with increasing concentrations of Ag was reduced up to 60−70%, showing the highest on PLGA/7Ag sample (p < 0.05; Figure 8A,B). To confirm the above indicated quantitative assessment and to envisage the effect of the differently treated PLGA samples on bacterial cell distribution, SEM studies were carried out (Figure 9). Large cellular aggregates were more visible for both S. aureus and E. coli cells either on neat and oxygen plasma-treated PLGA films than on the oxygen-plasma-treated/untreated PLGA/7Ag composites (Figure 9A, B, E, and F). A remarkable reduction in
1Ag sample and much more in its PLGA/7Ag counterpart. The binding energy of Ag3d5/2 level in PLGA/1Ag and PLGA/7Ag samples appears at 367.7 eV and is lower than that found for metallic Ag reference (368.3 eV; Figure 7A). As this difference is not so high, the most intense Auger AgMNN line was also recorded. The Auger AgMNN lines for PLGA/1Ag, PLGA/7Ag and a metallic Ag reference sample are displayed in Figure 7B. The lower value of the Auger AgM4VV line (894.3 eV) of PLGA/ 1Ag and PLGA/7Ag samples with respect to that of reference Ag sample (895.6 eV), together with the corresponding binding energies of the Ag3d5/2 core-level indicate that silver is oxidized (Ag2O) in the surface region of Ag particles.31,32 Atomic O/C fractions were computed from the peak intensity ratios normalized by atomic sensitivity factors (Table 1). The O/ C atomic ratios show a progressive increase with time. Thus, the value of O/C ratio for PLGA 30 min is almost twice the value recorded for PLGA sample. Notwithstanding, this trend is strongly altered for the two silver-containing samples. While the value of the O/C ratio is reduced to about one-half in PLGA/1Ag sample with respect to the original PLGA one, then increased by a factor about four in PLGA/7Ag sample. H
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some differences: the increment was 2−3-folds higher on PLGA/ Ag composites treated with oxygen plasma if compared to untreated PLGA/Ag composites. This difference was highly significant (p < 0.05). To confirm the effect of the different PLGA samples on bacterial viability, cells were studied by CLSM. Bacterial cells of both strains incubated for 24 h with neat PLGA or with oxygen plasma-treated PLGA presented an uniform green fluorescence (live), an indication of a good-medium survivability (Figure 10A, B, E, and F). The results were different on oxygen plasmatreated/untreated PLGA/7Ag nanocomposites: cells of both bacterial strains appeared uniformly red (dead) and quite dispersed (Figure 10C, D, G, and H).
the cell number of both strains was observed when bacterial cells were incubated with PLGA/7Ag composites and even more with oxygen plasma-treated PLGA/7Ag composites (Figure 9C, D, G, and H). Nevertheless, in contrast to the membrane damage observed by CLSM (Figure 10), no apparent alteration in the morphology of cell surfaces could be noted in S. aureus and E. coli cells incubated with the oxygen-treated/untreated PLGA/7Ag nanocomposites. 3.4. Effect of Ag Nanoparticles and Plasma Treatment on In Vitro Antibacterial Activity. In Figure 11, the
4. DISCUSSION In this study, the potential application of plasma-treated PLGA nanocomposites based on silver nanoparticles were explored in order to promote an efficient antimicrobial activity against E. coli and S. aureus. Silver-based nanocomposites, produced by solvent casting, show a specific round-like surface topography that is the result of two combined phenomena during the processing: the chloroform evaporation and the presence of silver nanoparticles that induce the formation of the regular porous surface structure.20,33−35 These structures, in fact, were not observed in the pristine PLGA film. The process of pore formation is controlled by the rapid volatile solvent evaporation. It is clear that the solvent vapor pressure has a critical influence on the pore formation. Moreover, solvent diffusion in polymers and in their nanocomposites plays an important role in the evaporation process as already mentioned. The diffusion coefficients of the solvents and the polymer and solvent solubility parameters as well as the interaction between polymer and solvent are the main parameters affecting the complex mechanism of pore formation. The oxygen treatment is able to induce modifications in surface morphology, roughness, chemistry, and wettability of PLGA and PLGA/Ag nanocomposite films related to the power supply and to the treatment times. PLGA and PLGA nanocomposite surfaces with different roughness from nanoto microscale are obtained after the oxygen treatment. The reduction in contact angle values clearly indicates increased surface hydrophilicity, which may have been caused by the evident superficial roughness and by the introduction of oxygen functional groups on the surface.24,36 Contact angle values are dependent on both surface chemistry and roughness variation due to the specific oxygen plasma treatment selected. The presence of hydroxyl groups on the PLGA surface, as confirmed by XPS and FT-IR, takes a reduction of the contact angle values with the formation of hydrophilic PLGA surface. Moreover, in the case of nanocomposites, the increased contact angle values are correlated with the modification in the surface morphology and with porosity and roughness induced by the oxygen plasma treatment. However, for hydrophilic surfaces (oxygen plasmatreated surface), the effect of roughness is less evident. The surfaces treated for 20 min at 30 W appear to have the most regular topography, and for this reason, it was selected for further chemical and antibacterial characterizations. Our observations demonstrate that these plasma surface treatments combined with nanocomposite approach could readily be used to modulate the physicochemical properties of the PLGA surface. To evaluate the role of surface phenomena and combining function of silver nanoparticles and oxygen plasma treatment on
Figure 11. Antibacterial activity of PLGA and PLGA/Ag nanocomposite films. Surviving fractions of E. coli and S. aureus cells to the indicated PLGA films were determined as CFU/mL after 3 and 24 h incubation times. Data are expressed as percentage of the ratios between CFU of bacteria grown on PLGA films to CFU of bacteria grown in 24well flat-bottom sterile polystyrene plates. The values represented are the means of the results of each sample performed in duplicate and in three separate experiments. Error bars indicate standard errors of the means. The statistical significance was indicated as follows: *p < 0.05 and **p < 0.01.
antibacterial activity exerted by neat PLGA, oxygen plasmatreated PLGA, and oxygen plasma-treated/untreated PLGA/Ag nanocomposites with increasing concentrations of Ag (1 or 7 wt %) on S. aureus and E. coli growth is reported. As expected, the survival of both S. aureus and E. coli cells was particularly high and not significantly different on neat PLGA at 24 h (p > 0.05). The oxygen plasma-treated PLGA samples did not show an elevated antibacterial effect on the growth of both bacterial strains. The antibacterial activity showed by the oxygen plasma untreated/ treated PLGA/Ag nanocomposites either for S. aureus and E. coli was quite similar after 3 h of incubation: the greatest antibacterial activity was exerted by the PLGA sample containing the 7 wt % Ag and treated with plasma oxygen (p > 0.05). Interestingly, after 24 h, the antibacterial effect on both bacterial cells was remarkably enhanced on respect to the 3 h incubation but with I
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bacteria cell wall so that cell contents are lost.37,38 SI binding to proteins may alter the metabolism of bacterial cells and modify membrane permeability and respiration. Both effects lead to death of the bacterial cells. Furthermore, SI can interact with the DNA of bacteria, preventing cell reproduction.37 In a previous study39 was reported that Gram-positive bacteria are generally less susceptible to antibacterial agents containing SI than Gramnegative species. On the contrary, we showed a similar antibacterial activity against either S. aureus or E. coli. Recently, the antibacterial effect against Staphylococcus aureus and Klebsiella pneumonia was shown using PLGA nanofibrous scaffolds having silver nanoparticles of more than 0.5 wt %.40 However, to our knowledge, this is the first work that have combined the nanocomposite approach by using silver nanoparticles and plasma surface modification to develop polymeric surfaces with tunable chemical, physical, and antibacterial properties.
the bacterial activity, antibacterial assays, and bacterial adhesion experiments were carried out. Antibacterial results (Figure 11) indicate that the number of viable colonies is reduced in the presence of silver-based PLGA nanocomposites in comparison to pristine polymer film, and inhibition of bacteria growth on the sample surface is evident in all nanocomposites. Moreover, the antibacterial effect is improved increasing the silver loading. A visible improvement in antibacterial properties of the current oxygen plasma-treated PLGA/Ag nanocomposites was observed because not only the bacterial adhesion for the tested strains was reduced, but also the bacterial growth, was inhibited by the nanocomposites. As reported by our adhesion assays and SEM observations (Figures 8 and 9), the attachment of both bacterial strains to the plasma oxygen-treated PLGA and PLGA/ Ag was reduced but this effect was even more enhanced (up to 50−60%) when the plasma surface treatment was coupled to silver nanoparticles, reaching the highest value with treated PLGA/7Ag. Interestingly both bacterial strains are more observable in the pores of the untreated PLGA/Ag materials (Figure 9C,G). We may argue that the chemical and morphological properties of the PLGA surfaces are different in the pores but it also possible that the diameter of the formed pores is wide enough to allow bacteria to be entrapped. This is confirmed by previous reports showing that bacteria may adhere differently to substrates with different surface properties12 and irregularities.13 Anyhow, we can exclude that the decrement in bacterial adhesion is merely due to the antibacterial activity of Ag. The surface modification of PLGA by using oxygen plasma treatment resulted in a successful decreasing of the initial adhesion of both bacterial strains. As shown by SEM observation, the surface treatment seems to kill the bacteria as they come in contact with the surface reducing the initial adhesion of bacteria to the polymer surface. The oxygen plasma treatment of material surface by itself did not allow to completely prevent bacterial colonization; however, this effect was greatly improved by the addition of Ag nanoparticles at different content. Bacterial adhesion is a very complicated process that is affected by many factors, including material wettability and surface chemical composition. For example, hydrophilic materials are reported to be more resistant to bacterial adhesion than hydrophobic materials.15 Consequently, the marked reduction in bacteria adhesion that characterized the PLGA/7Ag treated system can be attributed to the increased roughness and hydrophilic properties induced by the combination of silver nanoparticles and oxygen flow and confirmed by morphological characterization and wettability studies. The oxygen plasma treatment of PLGA and the addition of Ag nanoparticles both could modify the surface properties of PLGA and therefore alter the bacterial adhesion to the surface. As a consequence of the decrease in cell attachment, the antibacterial activity was enhanced. The oxygen plasma-treated PLGA/Ag showed the best bactericidal effect in comparison to untreated PLGA/Ag or oxygen plasma-treated PLGA matrix for both strains. In particular, it may be possible that the oxygen surface treatment of PLGA could promote a better leaching out of silver nanoparticles or silver ions (SI) from PLGA matrix due to the etching effect of the oxygen plasma treatment on the nanocomposite surface, as previously reported.19,25,26 Hence, the Ag nanoparticles in the nanocomposites are readily available to react with water and to release the SI. Silver ions are known to bind strongly to electron donor groups in biological molecules containing sulfur, oxygen, or nitrogen causing defects in the
5. CONCLUSIONS This work demonstrates that oxygen plasma surface treatment combined with nanocomposite approach could readily reduce bacteria adhesion and growth on silver nanoparticles and PLGA based systems. Of note, this reduction was showed for both type of tested bacterial strains (E. coli and S. aureus). The adopted multistep approach manifests itself as a promising strategy to modulate the topographical and physicochemical surface properties of nanocomposite and, consequently, to regulate the antiadherence properties of biodegradable PLGA-based systems by curbing the adhesion and growth of two important categories of bacteria. The findings of this contribution are expected to be of merit not only from the applied standpoint, but also from the scientific viewpoint, for it lies in the interface of the polymer surface science and biology.
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +390744492914. Fax: +390744492950. E-mail: ilaria.
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from INSTM. L.V. would like to acknowledge financial support by the “Project SAL-45” financed by Regione Lombardia (2010) and by project financed by ALMA MATER TICINENSIS Foundation (2010). We are grateful to P. Vaghi (Centro Grandi Strumenti, University of Pavia) and D. Picenoni (Politecnico di Milano) for their technical assistance in the immunofluorescent and scanning electron microscopic studies, respectively. E.F. is the recipient of the fellowship “L′Oreal Italia per le Donne e la Scienza 2012” for the Project “Progettazione, sviluppo e caratterizzazione di biomateriali nanostrutturati capaci di modulare la risposta e il differenziamento delle cellule staminali”.
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