In Situ Impregnation of Silver Nanoclusters in Microporous Chitosan

Sep 29, 2016 - (16) patented modified acrylate polymer compositions featuring reduced brittleness for manufacturing dental prosthetics. Hussain et al...
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In Situ Impregnation of Silver Nanoclusters in Microporous ChitosanPEG Membranes as an Antibacterial and Drug Delivery Percutaneous Device Sandeep K. Mishra,† Subina Raveendran,† J. M. F. Ferreira,‡ and S. Kannan*,† †

Centre for Nanoscience and Technology, Pondicherry University, Puducherry-605 014, India Department of Materials and Ceramics Engineering, University of Aveiro, CICECO, Aveiro 3810 193, Portugal

‡

S Supporting Information *

ABSTRACT: An in situ synthesis method for preparing silver nanoclusters (AgNCs) embedded in chitosan-polyethylene glycol (CS-PEG) membranes is disclosed. The aim is to develop implantable multifunctional devices for biofilm inhibition and drug release to reduce percutaneous device related complications (PDRCs). A multiple array of characterization techniques confirmed the formation of fluorescent AgNCs with sizes of ∼3 nm uniformly distributed in CS-PEG matrix and their active role in determining the fraction and interconnectivity of the microporous membranes. The presence and increasing contents of AgNCs enhanced the mechanical stability of membranes and decreased their susceptibility to degradation in the presence of lysozyme and H2O2. Moreover, the presence and increasing concentrations of AgNCs hindered biofilm formation against Escherichia coli (Gram negative) and Staphylococcus aureus (Gram positive) and enabled a sustainable release of an anti-inflammatory drug naproxen in vitro until 24 h. The overall results gathered and reported in this work make the AgNCs impregnated CS-PEG membranes highly promising multifunctional devices combining efficient antibacterial activity and biocompatibility with active local drug delivery. including tuning surface topography,8 surface coating9 and antimicrobial modification.10 However, a concrete clinical solution to reduce the percutaneous devices related complications (PDRCs) is yet to be reported. In spite of these open challenges, soft nanocomposite materials offer excellent opportunities for developing micropatterned devices with tunable mechanical, and multifunctional properties (eg., antibacterial, drug release).11 Polymer-based biomaterials in the form of membranes are established for maxillofacial prosthetics,12 repair of hernias,13 neural prosthetics,11,14 and wound dressings.15 Recently, Orlowski et al.16 patented modified acrylate polymer compositions featuring reduced brittleness for manufacturing dental prosthetics. Hussain et al.17

1. INTRODUCTION The indispensability of percutaneous medical devices, such as bone-anchored hearing aids, intravenous catheters, urinary catheters, dental implants, osseo-integrated percutaneous prosthetics, and voice prostheses augmented the infection susceptibility due to penetration through skin as the defense barrier.1−3 Protecting against infections is the utmost priority in the initiation of skin reparative process.4 Implant site infection become severe when pathogens colonize on percutaneous device and thus enhance biofilm formation.5 Through selfproduced polymeric substances, pathogens are protected in biofilm from host defense mechanism and antibiotics.5 The biofilm on the interface of percutaneous device obstructs the integration between devices and skin. Consequently, poor integration of percutaneous devices with skin may cause scar formation, tissue damage, device loosening and hence may result in life-threatening complications.6,7 Attempts were made to improve the integration of percutaneous devices with tissues © XXXX American Chemical Society

Received: July 31, 2016 Revised: September 5, 2016

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mycin, and naproxen were also purchased from Sigma-Aldrich, India. 2.2. Method of Synthesis. CS-PEG membranes with three added molar concentrations of AgNO3, 0.1 mM, 0.5 mM, and 1.0 mM, coded as Ag0.1, Ag0.5, and Ag1.0, respectively, were prepared. For each composition, 500 mg CS was dissolved in 50 mL of 1% aqueous acetic acid in a 250 mL flask and stirred overnight to obtain clear solution. 250 mg PEG dissolved in 100 mL water was subsequently added and the mixed solution was stirred for 6 h at room temperature (RT). The required amount of AgNO3 was then added and the flask was kept refluxing at 90 °C for further 6 h to obtain a golden yellow hydrogel. Membranes were prepared by casting a fixed volume (10 mL) of each hydrogel into Petri dishes (90 mm dia) followed by RT drying for 48 h. Silver nanoparticles were also synthesized by adding 0.2 M NaBH4 solution in CS-PEG solution with 1 mM AgNO3 (AgNPs 1.0).35 2.3. Characterization. 2.3.1. Chemical Characterization. The membranes were characterized by UV−visible spectrophotometry (PerkinElmer Lambda 650 S, USA) in the range of 200−800 nm; photoluminescence spectrophotometer (Horiba Jobin Yvon Fluorolog - FL3−11, USA); and multitechnique Xray photoelectron spectroscopy (XPS, Axis Ultra). FT-IR analysis of membranes and of pure CS, PEG was performed in transmission mode using a FT-IR spectrophotometer (PerkinElmer, USA), and in attenuated total reflection (ATR) mode in the spectral range from 4000 to 400 cm−1. The ATR spectra were obtained using a KRS-5 prism with an incident angle of 45°. Raman spectra were also recorded for membranes to confirm the plasmonic effect of AgNCs using a confocal Raman microscope (Renishaw, UK) at the excitation wavelength of 785 nm. The crystalline state of CS, PEG, and membranes was analyzed using a high resolution powder X-ray diffractometer (XRD) (Rigaku, Ultima IV, Japan) with Cu Kα radiation (λ = 1.5406 Å) produced at 40 kV and 30 mA to scan the diffraction angles (2θ) between 5° and 80°. The thermal stability of membranes was assessed through differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), at a heating rate of 10 °C min−1 in a simultaneous thermal analyzer (TA Instrument Q600 SDT). 2.3.2. Morphological Characterization. A scanning electron microscope (SEM, JEOL Quanta 200, Japan) with an energydispersive X-ray spectrometer (EDX) operating at 15 kV acceleration voltage after being coated with a thin layer of gold, was used to assess the effects of AgNCs content on the surface morphological features. The size and morphology of AgNCs was determined though transmission electron microscopy (FEI Tecnai G2 12 Twin TEM 120 kV, Netherlands). To demonstrate the interconnectivity of pores, 50 μM aqueous solution of fluorescein isothiocyanate (FITC) dye was dropped gently and surface analysis was carried out by fluorescence microscope (Olympus, Japan). 2.3.3. Mechanical Properties. First, all samples were preconditioned at 20 °C and 65% relative humidity for 24 h prior to mechanical testing, then mechanical properties were evaluated with a universal testing machine (Instron, Model 5566, Canton, MA), with a crosshead speed of 10 mm/min and a 200 N static load. The membranes having a thickness of ∼100 μm, were cut into testing samples with 5 cm × 1 cm size for mechanical characterization. The tensile force (N) was recorded up to rupture and plotted as a function of deformation (mm). The percent of elongation at break (deformation divided by initial sample length and multiplying by 100, %); the

patented methods of making bioactive collagen scaffolds for medical prosthetic applications. In recent years, chitosan (CS) has been proposed for several medical applications including wound healing,18,19 drug delivery20 and nonviral gene delivery,21 taking advantages of its salient features such as nontoxicity, biodegradability, biocompatibility, bioadhesive, and absorption enhancing properties.22 However, the fabrication of CS membranes as percutaneous devices faces the demerits of fragility, low mechanical stability and high degradation rates under oxidative environments and in the presence of lysozyme.23 The liability of CS to protonate in water at pH < 6.5 is also considered a strong limitation to explore its antibacterial features at the physiological pH.24 The fragility and poor mechanical stability of CS membranes can be mitigated by cross-linking with synthetic polymers, and polyethylene glycol (PEG) cross-linked CS films have been recently explored in wound healing and tissue engineering applications.25,26 With the outbreaks of infectious antibiotic resistant bacteria, the research focus was deviated to non-antibiotic antimicrobial materials in the form of nanoparticles.25,27 The incorporation of metallic or metal oxides nanoparticles in membranes enhances their antibacterial features at physiological conditions.28 Silver nanocrystals and its compounds exhibit proven potent antimicrobial features against wide range of microbes.29 In this context, the emergence of ligand-protected silver nanoclusters (AgNCs) has received tremendous attraction among researchers. The biological applications of AgNCs are well documented.30 Zhang et al.31 reported synthesis of fluorescent AgNCs in polymer microgels.31 Diez et al.32 recently proposed mechanically strong fluorescent film of AgNCs functionalized cellulose nanofibers that exhibited good antimicrobial efficacy for wound healing applications. The in situ functionalization of clusters by ligands that are either chemically bonded or physically adsorbed prevents clusters from agglomeration and hence, conferring them unique properties.33,34 The present study aims at the in situ synthesis of AgNCs in CS-PEG matrix composite membranes for applications as multifunctional percutaneous devices. The membranes are targeted to combine mechanical stability, biocompatibility, long-term antibacterial activity and sustained drug release. The mechanical stability, oxidative and enzymatic degradation of hydrogel membranes and their ability to prevent biofilm formation were characterized. Naproxen, an anti-inflammatory drug was loaded and in vitro release at physiological condition was carried out to study sustained release. The in vitro drug release, cell viability and cell adhesion behavior support their potential role as occlusive percutaneous devices.

2. MATERIAL AND METHODS 2.1. Materials. Chitosan (Sigma-Aldrich, 80% deacetylation, molecular weight = 190−400 kDa), PEG (MW 2000, Himedia, India), silver(I) nitrate (Sigma-Aldrich), lysozyme (from chicken egg white, Sigma-Aldrich, 100 000 units/mg) and 35% H2O2 (Hi-media, India) were purchased and utilized without further purification. For antibacterial assays, trypton-glucoseyeast extract (TGY) broth and bacteriological grade agar were procured from Hi-media, India. Deionized water was used for all synthesis and mentioned as water throughout. For MTT assay, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), phosphate buffer saline (PBS), penicillin, gentamycin, streptoB

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Langmuir stress at break (MPa) and elastic modulus (MPa) were calculated according to the ASTM D882-91 method (1996). Tensile strength (MPa) was calculated dividing the maximum force by film cross-section (thickness × width). Five different measurements were made for each sample composition. 2.4. In Vitro Tests. 2.4.1. In Vitro Degradation in Lysozyme and H2O2. Degradation studies were performed by incubating pure CS, Ag0.0, Ag0.1, Ag0.5 and Ag1.0 membranes in 10 mL PBS (as control) and in 10 mL PBS solution with added H2O2 or lysozyme under physiological conditions of 37 °C and 7.4 pH, at 50 rpm for up to 30 days. Membranes of 5 cm × 5 cm size were taken and weighed, sequentially neutralized in 1N NaOH, washed with deionized water, sterilized in absolute alcohol, and finally washed in PBS. To mimic in vivo physiological conditions, 20,000 units/mL lysozyme36 and 3.5% H2O237 in PBS solution were taken. The pH changes of the supernatants and the samples’ weight loss were recorded in triplicate assays. At each time point, the samples were removed, dehydrated in ethanol and dried in vacuum desiccator at ambient conditions prior to the determination of weight loss. 2.4.2. In Vitro Silver Release. The Ag release tests were carried out by incubating membranes of 1 g each in 50 mL of PBS (pH = 7.4) at 37.2 °C for 5 days. Aliquots of 0.5 mL each were collected at different time intervals and replaced with an equal amount of fresh PBS. The collected samples were digested with HNO3. Cumulative amounts of Ag released from membranes were quantitatively determined by ICP-AES (Arcos, Spectro, Germany). 2.4.3. Antibacterial Activity and Biofilm Inhibition. The antibacterial efficacy of membranes was assessed according to the ‘modified disc diffusion assay’ and “growth inhibition assay” previously described elsewhere.38 Bacterial cultures of Escherichia coli (Gram negative) and Staphylococcus aureus (Gram positive) were washed in 1× PBS and 1 × 106 CFU/ml were prepared. A suspension of 200 μL was uniformly spread on a trypton-glucose-yeast extract agar (TGYA) plate. All the sample membranes were prepared on paper disks of 6 mm diameter, sterilized in UV radiation for 30 min and then placed on the surface of TGYA plated previously inoculated with bacteria, and incubated overnight at 37 °C. A paper disk (6 mm diameter) soaked with 10 mg of gentamicin was used as a positive control. Finally, zone of inhibition (ZoI) was determined by measuring the diameter of the clearing zone around the membranes. For growth inhibition test, E. coli and S. aureus were cultured in TGY broth in test tubes in the presence of each test membranes of 50 mg for 24 h. At different time points, absorption of culture media at 600 nm was recorded to measure bacterial concentration. A test tube with same amount of TGY broth and bacterial inoculum was used as a positive growth control and mentioned as “control” and all experiments were performed in triplicate. To study biofilm development on the prepared membranes, 10 μL of E. coli culture and 10 μL of S. aureus culture bacteria (2 × 108 CFU/mL) in 980 μL TGY media was spread on hydrogel membrane cast in 30 mm Petri dishes followed by incubation at 37 °C for 48 h. The medium was replaced with fresh media every 24 h. Finally each sample was washed with PBS to eliminate medium and unbound bacteria.39 The biofilm formation on the surface of membranes was observed by optical microscopy after staining with crystal violet followed by safranin. 2.4.4. In Vitro Drug Loading and Release. Hindering inflammation caused by reactive oxygen species is beneficial.40

With this purpose, Naproxen (10 mg/mL) was mixed in the precursor membrane solutions before casting as a model antiinflammatory drug, and analysis of its release was carried out in PBS up to 24 h. A fixed weight of each film (equivalent to 25 mL of drug loaded nanocomposite solution) was immersed in 20 mL of PBS at 37 °C, and supernatant aliquots of 500 μL were taken out at different time points to test the drug release. 500 μL of fresh PBS was added to compensate the loss. The released drug concentrations were measured in triplicate by optical density at 260 nm. The cumulative drug release % was calculated by the following equation: Drug release(%) =

Drug released in supernatant × 100 Loaded drug conc.

2.4.5. Cell Viability Study. Human skin keratinocytes A341 cell lines was procured from the National Centre for Cells Science (NCCS), Pune, India. The cells were maintained and cultured in DMEM with 10% fetal bovine serum and supplemented with penicillin (120 units/mL), and streptomycin (60 mg/mL) at a constant temperature of 37 °C and 5% CO2 with 95% humidity. The membranes of 1 × 1 cm size were treated with ultraviolet light and ethanol for sterilization followed by soaking in DMEM media for 4 h to acclimatize and finally washed with 1× PBS. Then, 50 μL of human skin keratinocytes A341 cell lines (2 × 106 cell/mL) were seeded and incubated for 1 h at 37 °C and 5% CO2 with 95% humidity to allow cell adhesion. Thereafter, 2 mL of DMEM medium was added to each sample followed by incubation for further 24 h. After this period, 200 μL of MTT dye solution in PBS (4 mg/mL) was added to each sample and incubation was extended for further 4 h. Finally, the media was removed and 400 μL of DMSO was added into each Petri dish to solubilize the formazan crystals. The absorbance of the solutions was measured at 570 nm to determine the OD values. All in vitro experiments were repeated in triplicate. The cell viability was calculated as following: Cell viability(%) =

ODtreated × 100 ODcontrol

2.4.6. Preliminary Cell Adhesion and Proliferation Study. As mentioned in Section 2.4.5, the membranes of 1 × 1 cm size were sterilized and acclimatized followed by washing with PBS. Then, on both CS and Ag 1.0 membranes, 2 × 106 cell/mL human skin keratinocytes A341 cells were seeded and incubated for 1 h at 37 °C and 5% CO2 with 95% humidity to allow cell adhesion. Thereafter, 2 mL of DMEM medium was added to each sample followed by incubation for further 24 h. The membranes with adhered cells were washed thrice with warm Dulbecco’s phosphate buffered saline to remove nonadherent cells and then fixed with 4% paraformaldehyde for 20 min. Further, cells were washed with PBS and stained with DAPI (4′,6-diamidino-2-phenylindole) fluorescent dye. The number of adherent cells on each surface was calculated by counting the number of stained nuclei using a fluorescence microscope (OLYMPUS, JAPAN) at six different fields.

3. RESULTS AND DISCUSSION 3.1. Formation of Silver Nanoclusters. The synthesis of AgNCs in CS-PEG matrix was accomplished by the mild in situ reduction of silver ions created upon refluxing AgNO3 in the mixed CS-PEG aqueous solution at 90 °C for 6 h, confirmed by a clear golden yellow color of the resulting colloidal system. In C

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Langmuir Scheme 1. Formation Mechanism of AgNCs in CS-PEG Solution

Figure 1. (a) Absorbance and (b) fluorescence emission (λex = 490) spectra of silver ion, silver nanoparticles (AgNPs) and silver nanoclusters (AgNCs) equivalent to 1 mM silver atoms. (c) XRD reflections of AgNCs, (d) TEM micrograph of AgNCs1.0 with particle distribution (inset) (e) HR-TEM image of an individual AgNCs representing crystal fringes; (f) Selected area electron diffraction (SAED) pattern of the AgNCs reflecting two distinct rings, which can be attributed to two main diffracting planes, the {111} and the {200} planes of Ag. Photographs of AgNCs1.0 film under bright light (g) and illuminated by a UV lamp of 365 nm (h). EDS image revealing uniform distribution of silver on AgNCs1.0 surface (i).

3.2. Characterization of in Situ Synthesized AgNCs. The absorption and fluorescent emission spectra of AgNCs protected by CS-PEG membranes are compared to those of dispersed Ag nanoparticles (AgNPs) and silver ions in Figure 1a,b, respectively. While AgNPs display single peak at 410 nm, AgNCs exhibit a broad absorption band with transition peaks at 432 and 540 nm. The absorption peak at 540 nm is characteristic of ultrasmall AgNCs capped with CS-PEG. The 432 nm absorption peak is ascribed to the surface plasmon resonance (SPR) of aggregated larger AgNCs in CS-PEG matrix.45

the absence of PEG, reduction was very slow and no substantial color change was observed while varying pH from 3 to 7 at 90 °C up to 8 h. However, in the presence of PEG, reduction was faster and at pH 7.0 ± 0.3, CS-PEG matrix color changed to golden yellow within 6 h. Scheme 1 shows the possible mechanism of AgNCs synthesis in CS-PEG solution. The pH of the mixed solution was adjusted to 7.0 ± 0.3 by adding of CH3COOH and NaOH. The Ag+ ions initially coordinated with the nonprotonated electron donor amine groups of CS are subsequently stabilized as AgNCs during reduction process that occurs on heating.41−44 D

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Figure 2. Fitted XPS spectra of AgNCs 1.0 (a) full width (b) Ag 3d (c) N 1s (d) C 1s (e) O 1s.

The digital images of the AgNCs 1.0 film before (Figure 1g) and after irradiation with UV light (365 nm) (Figure 1h) offer visual evidence of Ag0 nanoclusters formation. The uniform distribution of silver in film was confirmed with EDX elemental mapping (Figure 1i). X-ray photoelectron spectroscopy (XPS) was performed to determine the oxidation states of Ag after reduction and its capping ability with CS (Figure 2).The wide XPS scan spectra of AgNCs 1.0 depicts carbon (C 1s), oxygen (O 1s), nitrogen (N 1s) and silver (Ag 3d). The analysis confirms that the mild reducing environment created within the mixed CS-PEG aqueous solution led to the in situ formation of Ag0. Reported binding energies for the Ag 3d5/2 peak range from 368.1 to 369.0 eV for neutral Ag, from 367.3 to 367.6 for Ag+, and from 367.8 to 368.0 eV for Ag2+.27,48 The XPS spectra exhibit Ag 3d5/2 and 3d3/2 peaks at 369.3 and 375.2 eV, respectively, thus indicating the presence of Ag in uncharged state.27,48,49 The C 1s spectrum of AgNCs was deconvoluted with three binding energies at 286.1, 287.4, and 289.4 eV. The 287.4 and 289.4 eV peaks are assigned to amide (N−CO) and carbonyl (−C O) groups respectively, in good agreement with literature reports.50,51 The binding energy assigned to C−O groups of

The AgNCs solution excited at 490 nm exhibits a broad emission band with a maximum at 710 nm, while the AgNPs and Ag(ion) excitated at same wavelength did not display any significant emission. This enables inferring the successful synthesis of AgNCs in CS-PEG matrix.45,46 The fluorescent emission of metal nanoclusters may either arise from the metal core or from the protective ligands.47 The absence of fluorescent emission in AgNPs and Ag(ion) having the same ligand shell confirms that fluorescence of AgNCs is mostly governed by the metallic core. The powder XRD pattern of AgNCs 1.0 (Figure 1c) shows an excellent coincidence with the standard Joint Committee on Powder Diffraction Standards (JCPDS) Card No. 65-2871 for Ag. The TEM images (Figures 1d,e) reveal the nanometre-size of AgNCs. Their average diameter was found to be 3.5 ± 2 nm by applying Gaussian fit (Figure 1d inset). HR-TEM image reflect crystal fringes of 0.23 nm d-spacing that correspond to the 111 lattice planes of FCC-Ag. The selected area electron diffraction (SAED) pattern of AgNCs reflects two distinct rings, which can be attributed to two main diffracting planes, the 111 and the 200 planes of Ag (Figure 1f). These results are in reasonable agreement with the observation obtained from XRD reflections. E

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Langmuir CS, including the primary alcohol group (−CH2−OH), reported at 285.9 eV,27,48 appears slightly shifted to a higher value (286.1 eV). The O1s spectrum exhibits a predominant binding energy at 533.7 eV. The N s1 binding energy was deconvoluted in two peaks at 400.6 and 402.0 eV, corresponding to amine and acetamide groups, respectively.48,51 The upper shift of the peak to 400.6 eV from its original value (399.7 eV) indicates the interference of other more electronegative atoms like oxygen and associations of nitrogen with the AgNCs.48 3.3. Physicochemical Analysis. The inter- and intramolecular interactions between polymers and how they are affected by the presence of AgNCs were assessed by FTIR and Raman spectroscopy. The FTIR spectra of CS, PEG, and of AgNCs impregnated membranes are displayed in Figure 3a. The characteristic bands of CS at 1656 cm−1 [(CO−NHR), amide−I group], at 1590 cm−1 [N−H stretching of the secondary amide (amide−II)], and at 1256 cm−1 [amide−III], appear in both CS−PEG (Ag 0.0) and AgNCs impregnated

CS−PEG membranes (Ag0.1, Ag0.5 and Ag1.0), but at lower wavenumbers respectively of 1636, 1552, and 1252 cm−1. These significant down shifts reflect the strong intramolecular interactions between CS and PEG polymeric chains.52 The presence of AgNCs induced significant changes in the stretching vibrations of amide bands of CS at 1656 and 1590 cm−1. The 1424 cm−1 band assigned to the bending vibration of −OH in both CS and PEG was shifted to 1405 cm−1 in the case of AgNCs incorporated membranes. The two peaks at 1070 and 1020 cm−1 are respectively assigned to the secondary (C6− OH) and primary (C2−OH) vibrations of alcohol groups of CS. The intensity of this bifurcation is stronger for composite membranes, suggesting the occurrence of long-range intermolecular/interfibrilar cross-links between CS and PEG chains.20 Moreover, the broad band centered at 3493 cm−1, attributed to the stretching vibrations of −OH and −NH2 groups in CS and PEG, indicates a prominent shift to 3317 cm−1 with diminishing intensity for membranes. These changes imply a reduction in intrachain interactions in CS and an increase in long chain order arrangements of CS and PEG.20,53 Hence, the FTIR spectra provide evidence about the effective coordination of −OH and −NH2 groups of CS molecules with PEG and AgNCs. The surface enhanced Raman spectra (SERS) of membranes were recorded to assess the effects of AgNCs contents. Figure 3b shows that the characteristic peaks of CS due to C−H vibrations of β-anomeric residues at 898 cm−1 and the stretching vibration of C−H in pyranose ring at 2930 cm−1 were unaffected, demonstrating the absence of any structural loss in CS chain. This is essential for biological applications.54 The intensities of the 1086, 1262, and 1378 cm−1 peaks that corresponds to C−O deformation of C6−OH are enhanced with increasing concentrations of AgNCs from Ag0.1 to Ag1.0. The intensity of the 1042 cm−1 peak (−C−OH vibration of C2−OH) also increases with increasing concentrations of AgNCs, proving the interactions between AgNCs and CS chain.38,54 The 1460 cm−1 band that corresponds to the −C−N stretching of −NH2 is observed in a diminishing trend with increasing AgNCs contents, substantiating the polar interaction of AgNCs with amine group of CS. The outcome from the Raman analysis confirms strong interactions between CS and PEG, and also dipolar interactions of AgNCs with polymer chains. The FT-IR spectra revealed intense disruption of intra- and intermolecular hydrogen bonding of CS after interaction with PEG and AgNCs. Such disruptions are expected to change the crystalline state of CS. All the samples were subjected to XRD analysis in order to explore any crystalline change in membranes. As shown in Figure 3c, CS exhibits characteristic reflections at 11° and 20° 2θ. The CS−PEG blend shows broad diffraction patterns characteristic of both CS and PEG reflections. The AgNCs impregnated samples also indicated broad diffraction patterns with suppressed intensity. These findings are in accordance with the results obtained from FT-IR analysis. Figure 4a,b present the DSC and TGA curves, respectively, for CS, PEG, CS−PEG blend (Ag 0.0), and AgNCs impregnated membranes. PEG did not undergo significant changes until 300 °C, and its degradation occurred in a single thermal event centered at ∼410 °C. CS exhibited two stages of weight loss, the first (endothermic) peak between ∼60−160 °C is due to moisture removal (∼18%), and a second one between ∼280−480 °C corresponds to CS decomposition (∼60%). The

Figure 3. (a) FT-IR spectra of pure chitosan (CS), polyethylene glycol (PEG) and membranes; (b) confocal Raman spectra showing enhanced Raman shifts of CS due to Surface Plasmon Resonance effect of silver clusters; (c) XRD pattern of CS, PEG, and membranes. F

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Figure 4. Thermal analysis (a) DSC and (b) TGA of CS, PEG, and AgNCs impregnated CS−PEG membranes.

composite membranes underwent three thermal degradation stages (Table S1). The first endothermic peak due to water loss and two exothermic peaks related to the degradation of CS and PEG components, respectively. From the significant variations in DSC peak position and area, one can infer that composite membranes differ from either CS or PEG individuals in water holding capacity. This is not surprising as hydration properties of polymers depend on both primary and supramolecular structures.55 Further, the presence of polar AgNCs is likely to enhance the water absorption capacity of hydrogel blends. For the CS−PEG blends, the exothermic peak at ∼287 °C shows noticeable down shifts with increasing contents of AgNCs, a symptom that thermal stability and crystallinity of CS were gradually reduced. Marginal down shifts can also be noticed in the exothermic peak due to PEG degradation at ∼407 °C, again pointing out a certain decrease of thermal stability of the PEG component in the blends. The increasing residual weight of membranes clearly reflects the concentration of AgNCs in samples. 3.4. Surface Morphology. The SEM and fluorescent microscopy images shown in Figure 5 reveal microporous hydrogel membranes with uniformly distributed AgNCs. The images of the Ag0.0 reveal a puffy blunt surface with few visible micropores. The gradual incorporation of AgNCs led to membranes with increasing porosity and uniformity as the result of clustering Ag+ ions around polymer chains, providing the possibility of ionotropic gelation. The interconnectivity of porous AgNCs impregnated membranes was confirmed by fluorescent dye sensitized surface analysis as indicated in Figure 5 (images b, d, f, and h). Figure S1 reveals the blunt solid surface of CS film. Figure S2 displays the elemental mappings of C, O, N and Ag in Ag1.0, which clearly indicate the uniform distribution of silver throughout the film. 3.5. Mechanical Evaluation. The relevant mechanical properties of porous CS−PEG membranes without and with increasing AgNCs contents are compared with those of the CS in Figure 6. The elongation at break (Eb) and maximum breaking force (Fmax) have shown slight but significant increases with increasing concentrations of AgNCs, confirming their positive contribution to the mechanical stability of membranes (Figure 6a). The breaking force (Fmax) was also gradually enhanced the values measured for Ag0.1, Ag0.5, and Ag1.0 being respectively 20%, 70%, and 105% higher, in comparison to that of CS. This strengthening effect of AgNCs is likely due to the ionotropic gelation role of Ag+ ions. The Eb of Ag1.0 was found ∼30% higher than for CS film. The Young’s modulus shows an exponential increasing trend with increasing AgNCs

Figure 5. SEM (Scale = 10 μm) and fluorescent microscope (Scale = 40 μm) images of Ag 0.0 (a, b), Ag 0.1 (c, d), Ag 0.5 (e, f), and Ag 1.0 (g, h).

contents, while tensile strength (TS) and stress at break (SB) were also somewhat improved but in a less steady way as shown in Figure 6b. The ample plasticizing role of PEG is evident from the ∼70 times increase in SB for Ag 0.0 in comparison to CS. The enhanced mechanical stability lend by the AgNCs to the microporous membranes based on chitosan is of prime significance for their intended applications. 3.6. In Vitro Degradation of Membranes in Lysozyme and H2O2. Biodegradable materials are generally desired for percutaneous scaffolds. CS is susceptible of undergoing in vivo degradation by enzymes like lysozyme as well as oxidative degradation by H2O2, since these molecules are secreted by macrophages during inflammatory reactions.37,56 Thus, the in vitro degradation was assessed by incubating the membranes in PBS in the absence (control) and in the presence of lysozyme or H2O2 at 37 °C for 30 days and changes in pH and weight loss were recorded as a function of time (Figure 7). The control G

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Figure 6. Mechanical properties of blank chitosan and CS-PEG membranes with different AgNCs contents: (a) Maximum breaking force (N) and % of elongation at break; (b) tensile strength (MPa), stress at break (MPa), and Young’s modulus (MPa).

Figure 7. Degradation kinetics of films: (a) weight loss in lysozyme; (b) pH change in lysozyme; (c) weight loss in 3.5% H2O2; (d) pH change in 3.5% H2O2.

part per billion (ppb) is essential to elicit effective antimicrobial activity.57 The cumulative silver release profile as a function of time is displayed in Figure 8. After an initial fast release from all samples within the first few hours, likely due to the most peripheral and loosely bound Ag, the process slows down,

samples underwent negligible changes in pH and weight loss over the entire period (data not presented). CS underwent the maximum weight loss in comparison to CS−PEG and AgNCs impregnated membranes, which showed increased stability against lysozyme with increasing AgNCs contents. The preservation of weight in Ag0.5 and Ag1.0 is attributed to the enhanced ionotropic interactions of AgNCs with polymeric chains and the consequent deficiency of active sites to interact with lysozyme. The oxidative degradation by H2O2 showed expected linear kinetics corresponding to random decomposition of the polysaccharide chain, with the least weight loss being observed for Ag1.0. The oxidative degradation and consequent weight loss was accompanied by a proportional decrease in pH caused by the degradation products as observed in Figure 7b,d. 3.7. In Vitro Release Profile of Silver from Membranes. The antimicrobial activity of commercially available silver based bandages is attributed to a suitable silver release profile. An initial rapid release followed by a subsequent sustainable prolonged release of silver at a minimum concentration of 0.1

Figure 8. Ag release kinetics from membranes with AgNCs. Error bars represent standard deviations of triplicate experiments. H

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Figure 9. Inhibition zone against: (a) E. coli; (b) S. aureus; growth inhibition curve in TGY broth against: (c) E. coli; (d) S. aureus. Biofilm development on the surface of films: (e) CS; (f) Ag0.0; (g) Ag0.1; (h) Ag0.5; (i) Ag1.0. White scale bars represent 400 μm.

against the growth of both the bacteria, while the AgNCs impregnated membranes are likely to inhibit infection by the investigated microbes. Considering the sustainable slow Ag+ release, it is suggested that AgNCs impregnated membranes might also exert antimicrobial activity by the direct surface contact effect. To test this hypothesis, the effect of AgNCs on the biofilm formation onto the membranes and bacteria destruction were analyzed by crystal violet staining method (Figure 9e−i).58 The results confirmed the absence of any bacterial survival on the surface of Ag1.0. The surface of CS membrane was almost completely covered with a biofilm. The biofilm coverage of AgNCs impregnated membranes was inversely proportional to AgNCs concentration. 3.9. Drug Loading and Release. The in vitro drug release behavior of naproxen from CS, Ag0.0 and Ag1.0 membranes was investigated. The results plotted in Figure 10 disclose a burst release from CS within 2 h, attributed to its excessive swelling. Sustained naproxen release was observed from both composite membranes up to 24 h, but the process was slower for Ag1.0. In all time points, the slow drug release rate of the Ag1.0 membrane makes it promising for potential applications as a drug delivery device. 3.10. Cell Viability Study. The results of cell viability and proliferation displayed in Figure 11a confirm the absence of any cytotoxic effects from CS−PEG and AgNCs embedded membranes in comparison to the control membrane of pristine CS. The AgNCs demonstrated that the complete absence of cytotoxicity is evident for the AgNCs impregnated membranes. The AgNP-containing ones also reveal insignificant cytotoxicity levels. Oppositely, significant cytotoxicity was observed for the Ag (ion) membrane with a reduction of cell viability up to 56%.

being mediated by the swelling and associated structure opening of membranes. The profiles of Ag0.1 and Ag0.5 tended to attain plateaux after about 24 and 60 h, respectively; however, the sample with the highest silver content (Ag1.0) continued displaying a steady and sustainable release up to 120 h. 3.8. Antibacterial Study. The sustained silver release profiles obtained from ICP-AES (Figure 8) are expected to confer antibacterial ability to the membranes. This property was assessed through ‘modified disc diffusion assay’ (Figure 9a,b) and “growth inhibition assay” (Figure 9c,d), tested on Gram negative (E. coli) and Gram positive bacteria (S. aureus). CS membranes showed significantly less antibacterial activity as no zone of inhibition (ZoI) (>6 mm) is observed in disc diffusion method. This negligence could be attributed to the nonprotonated state of CS after neutralization in NaOH.38 AgNCs incorporated membranes exhibited antibacterial activity against both tested bacteria, with increasing ZoI as a function of AgNCs concentration. The highest AgNCs contained sample Ag1.0 showed 633 mm2 and 755 mm2 inhibition area against S. aureus and E. coli, respectively, which is comparably equivalent to the inhibition efficiency of 7−8 mg gentamycin (GM), as 10 mg GM has exposed 765 mm2 and 1017 mm2 inhibition area against S. aureus and E. coli, respectively. Moreover, the inhibition area of Ag0.1 was found 40−90% higher than that of CS, but as time passed the inhibition area encroached, due to ineffective concentration of AgNCs. To confirm the extent of antibacterial activity, growth inhibition tests were performed and showed as growth inhibition curves for E. coli and S. aureus (Figure 9c,d). The Ag1.0 demonstrated the absence of any microbial growth. CS showed no efficient inhibitory activity I

DOI: 10.1021/acs.langmuir.6b02844 Langmuir XXXX, XXX, XXX−XXX

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Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b02844. Table S1: TGA-DSC data of CS-PEG membranes; Figure S1: Optical image of CS film; Figure S2: Elemental mapping of C, O, N and Cu in Ag1.0 (PDF)

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Figure 10. In vitro release kinetics of model drug “naproxen” in PBS at 7.4 pH.

AUTHOR INFORMATION

Corresponding Author

*Address: Centre for Nanoscience and Technology, Pondicherry University, Puducherry-605014, India. E-mail: para_ [email protected]; Phone: 0091-413-2654973.

DAPI-stained fluorescent imaging analysis of cells adhered onto the surface of CS (Figure 11b) and Ag1.0 (Figure 11c) membranes, reveals that enhanced cellular adhesion and proliferation took place for the AgNCs impregnated membrane. The number of cells adhered to this membrane was found to be ∼1.8 times higher than those adhered to the CS one. The enhanced adhesion and proliferation of keratinocytes onto the surface of the Ag1.0 membrane (Figure 11c) confirms its biocompatibility. The overall in vitro results confirm that the AgNCs impregnated membranes are the most promising percutaneous devices among all the tested candidate materials.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial assistance received from Department of Science and Technology, DST [Reference: SB/FT/CS-101/2012(SR)], India is acknowledged. The facilities availed from Central Instrumentation Facility (CIF) of Pondicherry University is also acknowledged. The characterization facilities availed from IIT Bombay and IISc Bangalore under INUP which is sponsored by DeitY, MCIT, Government of India is gratefully acknowledged. The support of CICECO-Aveiro Institute of Materials (ref. UID/CTM/50011/2013), funded by FEDER funds through the Operational Programme Competitiveness Factors (COMPETE 2020) and the Portuguese Foundation for Science and Technology (FCT) is acknowledged.

4. CONCLUSION A novel method of in situ synthesis of AgNCs embedded in a CS-PEG matrix blend is disclosed. The reduced state of Ag and its capping with chitosan were confirmed by XPS and the size of AgNCs formed was ∼3 nm. The porosity of membranes could be varied through alterations in the AgNCs contents. The mechanical stability and antibacterial features of the membranes were also enhanced with increasing contents of AgNCs. The sustainable release of Ag from AgNCs-containing membranes restricts biofilm formation. Moreover, the AgNCs-containing membranes exhibit slow enzymatic and oxidative degradation during lysozyme and H2O2 tests. The silver contents investigated in the present study were enough to inhibit biofilm formation and to stimulate cell viability and adhesion, combined with sustained slow release of drug. The microporous surface and superior cell attachment ability of AgNCscontaining membranes are expected to provide comprehensive integration of percutaneous multifunctional devices with decreased infection risk.

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REFERENCES

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Figure 11. (a) Cell viability of human skin keratinocytes against different films; microscopic images of cell adhesion on (b) CS film and (c) Ag1.0 film. J

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