In Vitro Biocompatibility and Antimicrobial Activity of Poly(Îµ

Article pubs.acs.org/Biomac

In Vitro Biocompatibility and Antimicrobial Activity of Poly(εcaprolactone)/Montmorillonite Nanocomposites T. Corrales,*,† I. Larraza,† F. Catalina,† T. Portolés,‡ C. Ramírez-Santillán,‡ M. Matesanz,‡ and C. Abrusci§ †

Polymer Photochemistry Group, Instituto de Ciencia y Tecnología de Polímeros, C.S.I.C. Juan de la Cierva 3, 28006 Madrid, Spain Biochemistry and Molecular Biology I, Faculty of Chemistry, Universidad Complutense de Madrid, Ciudad Universitaria s/n, UCM, 28040 Madrid, Spain § Departamento de Biología Molecular, Facultad de Ciencias, Universidad Autónoma de Madrid-UAM, Cantoblanco, 28049 Madrid, Spain ‡

S Supporting Information *

ABSTRACT: A triblock copolymer based on poly(ε-caprolactone) (PCL) and 2-(N,N-diethylamino)ethyl methacrylate (DEAEMA)/ 2-(methyl-7-nitrobenzofurazan)amino ethyl acrylate (NBD-NAcri), was synthesized via atom transfer radical polymerization (ATRP). The corresponding chlorohydrated copolymer, named as PCL-bDEAEMA, was prepared and anchored via cationic exchange on montmorillonite (MMT) surface. (PCL)/layered silicate nanocomposites were prepared through melt intercalation, and XRD and TEM analysis showed an exfoliated/intercalated morphology for organomodified clay. The surface characterization of the nanocomposites was undertaken by using contact angle and AFM. An increase in the contact angle was observed in the PCL/ MMT(PCL‑b‑DEAEMA) nanocomposites with respect to PCL. The AFM analysis showed that the surface of the nanocomposites became rougher with respect to the PCL when MMTk10 or MMT(PCL‑b‑DEAEMA) was incorporated, and the value increased with the clay content. The antimicrobial activity of the nanocomposites against B. subtilis and P. putida was tested. It is remarkable that the biodegradation of PCL/MMT(PCL‑b‑DEAEMA) nanocomposites, monitored by the production of carbon dioxide and by chemiluminescence emission, was inhibited or retarded with respect to the PCL and PCL/1-MMTk10. It would indicate that nature of organomodifier in the clay play an important role in B. subtilis and P. putida adhesion processes. Biocompatibility studies demonstrate that both PCL and PCL/MMT materials allow the culture of murine L929 fibroblasts on its surface with high viability, very low apoptosis, and without plasma membrane damage, making these materials very adequate for tissue engineering.

1. INTRODUCTION In the last decades, the design of materials with antimicrobial properties have raised great interest, since infections by pathogenic microorganisms are of great concern in many biomedical applications, especially in tissue engineering, surgery equipments, and hygienic products.1 The infection is attributed to initial attachment of a viable bacterial population to the surface of a material and subsequent formation of biofilm.2−4 Then a promising approach to limiting microbial colonization will be prevention of bacterial adhesion to material prior to colonization, which depends on factors such as surface hydrophobicity/hydrophilicity, roughness, charge, and functional groups. In this sense, polymers have been used as antimicrobial materials in many areas, and several strategies are proposed:5−8 polymers with intrinsic antimicrobial properties, systems comprising mixtures of polymers, and other antimicrobial materials, such organic and inorganic additives, or by regulation of mechanical stiffness of substrata material. Three main methods may be used to incorporate inorganic additives to polymers, including in situ polymerization of monomer/clay intercalates, solution-intercalation, and melt© 2012 American Chemical Society

compounding. A key factor in the nanocomposite preparation is to compatibilize the matrix and the filler, which will affect the nanostructure (intercalated/exfoliated) and, consequently, the factors controlling the interface polymer/nanoclay, contributing to the design of systems for valued-added applications. Because most polymers are organophilic, organo-modified layered silicates are used to obtain better affinity between the filler and matrix. One of the more common modification methods is the introduction of an ammonium or phosphonium salt, bearing a suitable organic functionality, inside the interlayer space through a cation exchange reaction.9 The organic salt substitutes the metal ions present inside the clay mineral galleries, enhances the interlayer between the platelets and, for instance, facilites the intercalation of polymer within galleries and is also responsible for changes in the surface properties of the clay. Received: October 2, 2012 Revised: November 12, 2012 Published: November 15, 2012 4247

dx.doi.org/10.1021/bm301537g | Biomacromolecules 2012, 13, 4247−4256

Biomacromolecules

Article

Figure 1. Structure of the chlorohydrated triblock copolymer, named as PCL-b-DEAEMA, based on 2-(N,N-ethylamino)ethyl methacrylate/2(methyl-7-nitrobenzofurazan)amino ethyl acrylate and ε-caprolactone.

substance is converted to an inorganic product such as water and carbon dioxide) by indirect impedance technique, and chemiluminescence emission of biodegraded film samples. Both techniques26,27 were previously employed and proved their suitability to study polymer biodegradation. The biocompatibility of materials was analyzed after the culture of L929 cells on PCL nanocomposites.

The efficiency against microorganisms of polymer nanocomposites prepared by inclusion of antimicrobial-modified inorganic systems, such as organoclay montmorillonites modified with cationic surfactants, has been investigated.5,10,11 Clay minerals have attracted great interest due to their nontoxic, environmentally friendly characteristics and easy preparation by intercalation with antimicrobial organic modifiers.12 The studies showed that the interaction between the cells and the organoclay surfaces may be the responsibility of the antimicrobial activity, and the intercalation of positively charged polymers on the clay contribute to avoid the migration of the biocidal cationic surfactant, enhancing their antimicrobial properties. Nowadays, there are very few reports in the open literature concerning the biocompatibility and applicability of antimicrobial nanocomposites as implantable biomaterials.13−15 PCL is regarded as a soft and hard tissue compatible bioresorbable material, and it has been considered as a potential substrate for wide applications, such as tissue-engineered skin,16 drug delivery systems,17 and scaffolds for supporting fibroblast and osteoblast growth.18,19 PCL substrates and nanocomposite materials have been reported as a matrix for cell growth20,21 and sustained release of silver or benzoate derivatives as antimicrobial agents.22,23 The aim of this work is the development of new approaches for clay modification in order to improve interactions between polymer and organoclays to further prepare nanocomposites with antimicrobial properties adequate for tissue engineering. The study investigates the influence of organoclay on the biocompatibility and antimicrobial activity of nanocomposites based on poly(ε-caprolactone). For that purpose, the modification of the montmorillonite clay by a chlorohydrated block copolymer (PCL-b-DEAEMA), based on PCL and 2(N,N-diethylamino)ethyl methacrylate and synthesized by atom transfer radical polymerization (ATRP), and its characterization are presented. Quaternary ammonium compounds are widely used as cationic disinfectants or biocidal agents to prevent the growth of microorganism.24,25 The protonated amino groups of block copolymer that do not interact electrostatically could be effective against colonization of bacteria and may play an important role on safe materials for fighting biofilm. The morphology of the PCL nanocomposites along with the thermal, mechanical, and surface properties of the materials was characterized. Biodegradation of PCL nanocomposites was studied using Bacillus subtilis and Pseudomonas putida bacteria, which are two bacterial strains most commonly associated with implant infections. Two different techniques were employed to evaluate biodegradation of PCL nanocomposites, measurement of the carbon dioxide production or mineralization (process where an organic

2. MATERIALS AND METHODS 2.1. Materials. Poly(caprolactone) diol (Mn = 2000 g/mol), 2bromoisobutyryl bromide, 2,2′-bipyridine, and 2-(N,N-diethylamino)ethyl methacrylate (DEAEMA) were purchased from Aldrich and used as received. Triethylamine (TEA) was purchased from Fischer. Copper chloride (CuCl) was purchased from Aldrich and was purified according to the method of Keller and Wycoff.28 Toluene (BDH, 98%) was degassed by bubbling with nitrogen for 30 min, and the water used in all experiments was Milli-Q grade. The fluorescent probe 2-(methyl-7-nitrobenzofurazan)amino ethyl acrylate (NBD-NAcri) was synthesized according to the procedure described.29 Polycaprolactone (Mn = 80000 g/mol) was purchased from Aldrich. The clay employed was montmorillonite k10 (MMT-k10; surface area of 220− 240 m2/g, C.E.C. = 92 meq/100 g) purchased from Aldrich. 2.2. Block Copolymer Synthesis. Synthesis of Macroinitiator. The bromide-ended PCL macroinitiator was synthesized according to the procedure described.30 Synthesis of Triblock Copolymer. The ratio between the concentrations of initiator, catalyst, and ligand was ([I]/[C]/[L] = 1:2:4.2). The ratio of monomer concentration to initiator was 70:1. A typical polymerization procedure is detailed below. PCL based macroinitiator (2 g, 0.88 mmol) was placed in a Schlenk tube and dissolved in deoxygenated toluene (60 mL). 2-(N,N-Diethylamino) ethyl methacrylate (9.2 mL, 46 mmol) and the fluorescent probe (NBD-NAcri, 0.257g, 0.88 mmol) were added to the solution. Then, the tube was sealed with a rubber septum, and the mixture was degassed via three freeze−pump−thaw cycles. CuCl (0.174 g, 1.76 mmol) was added to the frozen mixture and it was deoxygenated by three vacuum−N2 cycles. The reaction mixture, under an atmosphere of nitrogen, was placed in an oil bath at 80 °C. Once the solution reached the desired reaction temperature, 2,2′-bypyridine (0.58 g, 3.7 mmol; t = 0) was added. Samples were taken periodically throughout the reaction in order to follow the polymerization by 1H NMR in CDCl3. Termination reaction occurred after 5 h by exposing the reaction solution to air, leading to aerial oxidation of the catalyst. Catalyst residues were removed by filtering through an activated basic alumina column. The volatiles were removed by rotary evaporation and under high vacuum at ambient temperature, yielding an orange polymer. 1 H NMR (CDCl3, 400 MHz) δ (ppm): 0.89 [m, 64 N-(CH2CH3)2-DEAEMA, 384H], 1.38 [m, 16 O2C (CH2)2CH2 (CH2)2O-PCL, 32H], 1.65 [m, 16 O2C-CH2-CH2-CH2-CH2-CH2-O-PCL, 64H], 1.85 [s, 4 CH3‑PCL, 12H], 1.95 [m, 64 CH3-C-DEAEMA, 192H], 2.31 [t, 16 O2C-CH2-(CH2)4O-PCL, 32H], 2.57 [m, 64 N-(CH2-CH3)2‑DEAEMA, 256H], 3.65 [m, 2 O-CH2-CH2-OCO-PCL, 4H], 4.05 [m, 16 O2C(CH2)4-CH2-O-PCL, 32H], 4.20 [t, 2 O-CH2-CH2-O-CO-PCL, 4H]. 4248


Biomacromolecules

Article

tip, and a nominal spring constant of 20−80 N m−1. A resonance frequency of the cantilever typically at 275 kHz was chosen for the tapping mode oscillation. Moderate tapping forces were used by setting the set-point ratio between 0.6 and 0.7. The AFM images were obtained with a maximum scan range of 20 × 20 μm2. Root-meansquare (rms) roughness measurements were determined using standard Digital Instruments software. The tensile strength tests were carried out using a MTS QTEST Elite tensile tester equipped with a 1 kN load cell and interfaced to a computer for data collection. Sample sizes of 10 × 3 mm were tested at a constant rate of 5 mm/ min. The data of corresponding values are the average of five independent measurements. Contact angle was measured using a CAM200 KSV contact angle meter. The wetting liquids used for contact angles measurements were demineralized distilled water and diiodomethane, as Owens and Wendt suggested.31 All measurements were made at room temperature. Considering advancing contact angles for different liquids, the surface tension was calculated by the method described in the literature.32 In Vitro Biocompatibility Studies. Initially, PCL and PCL/MMT films were cut in circular pieces, sterilized by 1 h UV irradiation on each side, and then submerged in Modified Eagle's Medium (DMEM, Sigma Chemical Company, St. Louis, MO, U.S.A.) with penicillin (800 μg/mL, BioWhittaker Europe, Belgium) and streptomycin (800 μg/ mL, BioWhittaker Europe, Belgium) for 24 h at 37 °C. Cell Adhesion and Proliferation Studies. Murine L929 fibroblasts were seeded on PCL and PCL/MMT films, previously introduced into six-well culture plates, at a density of 105 cells/mL in Dulbecco's Modified Eagle's Medium (DMEM, Sigma Chemical Company, St. Louis, MO, U.S.A.) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, BRL), 1 mM L-glutamine (BioWhittaker Europe, Belgium), penicillin (200 μg/mL, BioWhittaker Europe, Belgium), and streptomycin (200 μg/mL, BioWhittaker Europe, Belgium), under a 5% CO2 atmosphere and at 37 °C for different times. The culture medium was renewed every two days. Tissue culture polystyrene (TCP) was used as control surface. To evaluate the cell adhesion and proliferation on the substrates after 1, 4, and 6 days culture, the medium was aspirated and the attached cells were harvested using 0.25% trypsin-EDTA solution and counted with a Neubauer hemocytometer. The nonattached cells were previously discarded by washing the cultures with PBS. Lactate Dehydrogenase (LDH) Measurement. Lactate dehydrogenase activity was measured in the culture medium by an enzymatic ́ method at 340 nm (BIOAnalitica, S.L.) using a Beckman DU 640 UV−visible spectrophotometer. The medium was collected after every culture time, centrifuged 2 min at 12000g at 4 °C, and the enzymatic assay was performed in the supernatant. Flow Cytometry Studies. Cells cultured for 1, 4, and 6 days on the PCL and PCL/MMT films were washed twice with PBS and incubated at 37 °C with trypsin-EDTA solution for cell detachment. After 5−10 min, the reaction was stopped with culture medium; cells were centrifuged at 310 rpm for 10 min and resuspended in fresh medium. After the incubation with the different probes, as it is described below, the conditions for the data acquisition and analysis were established using negative and positive controls with the CellQuest Program of Becton Dickinson and these conditions were maintained during all the experiments. Each experiment was carried out three times, and single representative experiments are displayed. For statistical significance, at least 10000 cells were analyzed in each sample and the mean of the fluorescence emitted by these single cells was used. Cell Cycle Analysis and SubG1 Fraction as Indicative of Apoptosis. Cell suspensions were incubated with Hoechst 33258 (PolySciences, Inc., Warrington, PA, U.S.A.) (Hoechst 5 μg/mL, ethanol 30% vol/vol, and BSA 1% wt/vol in PBS), used as a nucleic acid stain, for 30 min at room temperature in darkness. The fluorescence of Hoechst was excited at 350 nm and the emitted fluorescence was measured at 450 nm in a LSR Becton Dickinson flow cytometer. The cell percentage in each cycle phase, G0/G1, S, and G2/ M, was calculated with the CellQuest Program of Becton Dickinson and the SubG1 fraction was used as indicative of apoptosis.

Calculations of conversion were performed by 1H NMR with comparison of the relative integration of the signals at 5.6 and 6.2 ppm assigned to the protons of monomeric double bond and that at 4.20 ppm for ethylene protons of PDEAEMA. The theoretical molecular weight of copolymer (Mn,th = 11100 g/mol) was calculated by using the following equation:30

M̅ n,th = (([monomer]0 × M w,monomer × conversion) /[macroinitiator]0 ) + M̅ n,macroinitiator Protonation of Triblock Copolymer with HCl. Polymer (7.1 g) was dissolved in 200 mL of water and 80 mL of 0.5 M HCl (aq) under stirring. The solution was frozen in liquid nitrogen and lyophilized to give 8.3 g of the corresponding chlorohydrated copolymer, Figure 1. IR (KBr) ν (cm−1): 2316−2745 (+HN-(CH2-CH3)2‑DEAEMA). The weight percentage of fluorescent probe in the copolymer was determined in dichloromethane by UV−vis spectroscopy, using the molar absorption coefficient of the corresponding model compound, NBD-NAcri, (ε = 1.56 × 10−4 L mol−1 cm−1 at λmax = 467 nm). The probe content determined was 1.7 w/w. 2.3. Clay Modification and Films Preparation. 2.3.1. Clay Modification. To obtain monocationic montmorillonite, MMT-k10 was treated according to the procedure described.7 The chlorohydrated block copolymer was dissolved in methanol and added in 2:1 mass, to a MMT suspension in water (0.5 g/L), and stirred overnight at room temperature. Then, the suspension was filtered under reduced pressure and the obtained organomodified clay MMT(PCL‑b‑DEAEMA) was dried under vacuum at 40 °C. 2.3.2. Preparation of Nanocomposites. Neat polymer and composites were prepared by melt processing in a Haake MiniLab mixer extruder with two counter-rotating screws. First, PCL/clay masterbatch at a clay percentage of 20% was prepared. The processing temperature was set at 90 °C, and the rotating speed of the rotor and the mixing time were, respectively, 50 rpm and 10 min. Then, nanocomposites with 1, 2, 4, 6, and 10% of nature or organomodified nanoclay were prepared at 90 °C for 10 min and screw speed at 80 rpm. Polymer films were made by compression molding of a fixed amount of blended powder (1 g) in a Collin-200 press under the same temperature (100 °C) and pressure cycle (2 min at 0 bar, and 1 min at 200 bar). Also, the cooling rate from 100 °C until room temperature was controlled and maintained constant. Under such conditions, circular polymer films of 10 cm diameter and 200 μm thickness were obtained. The samples will be referred as PCL/x-MMTk10 and PCL/ x-MMT(PCL‑b‑DEAEMA) for nature and organomodified clay, respectively, and being x the content of clay in the nanocomposites. 2.4. Characterization Techniques and Methods. 1H NMR spectra were recorded in CDCl3 solution on a Bruker AM-400 instrument at 400 MHz. UV absorption spectra were recorded by means of a Lambda 35 UV−vis spectrometer (Perkin-Elmer). FTIR spectra were obtained using a Perkin Elmer BX-FTIR spectrometer. Diffraction X-ray patterns (XRD) were obtained using an X Bruker D8 Advance diffractometer with Cu Kα radiation. The scanning speed and the step size were 0.5°/min and 0.02°, respectively. All the experiments were carried out with 2θ varying from 1 to 30°. The dspacing between the silicate layers of the clay was calculated using the Bragǵs equation. Thermal gravimetric analyses (TGA) were performed using a TGA Q500-0885 (TA Instruments). The heating rate for the dynamic conditions was fixed at 5 °C min−1, and the nitrogen flow was maintained constant at 60 mL min−1. Differential scanning calorimetry (DSC) was performed on a Mettler DSC-821e calorimeter over the temperature range −100 to 130 °C. The measurements were made at a heating rate of 10 °C min−1 under an inert atmosphere of nitrogen and the instrument was calibrated with an indium standard (Tm = 429 K, Hm = 25.75 J/g). Transmision electron microscopy (TEM) analyses were carried out on a Zeiss 910 microscopy operating at 100 KV. Tapping-mode atomic force microscopy (TM-AFM) measurements were conducted in air with a Nanoscope IV system (Digital Instruments) with a triangular microfabricated cantilever with a length of 115−135 μm, 1−10 Ohm−cm phosphorus (n) doped Si pyramidal 4249


Biomacromolecules

Article

Cell Size and Complexity. The light scattering properties of cells were examined by flow cytometry in a FACScalibur Becton Dickinson flow cytometer, measuring forward angle (FSC) and side angle (SSC) light scatters as indicators of cell size and complexity, respectively. Cell Viability. Cell viability was evaluated by exclusion of propidium iodide (PI; 0.005% wt/vol in PBS, Sigma-Aldrich Corporation, St. Louis, MO, USA). PI was added to the cell suspensions in order to stain the DNA of dead cells. The fluorescence of PI was excited by a 15 mW laser tuned to 488 nm and the emitted fluorescence was measured with a 530/30 band-pass filter in a FACScalibur Becton Dickinson flow cytometer. Statistics. Data are expressed as mean + standard deviations of a representative of three experiments carried out in triplicate. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 19 software. Statistical comparisons were made by analysis of variance (ANOVA). Scheffé test was used for posthoc evaluations of differences among groups. In all statistical evaluations, p < 0.05 was considered as statistically significant. Bioassay Procedure and Indirect Impedance Technique. Strains used in this study were B. subtilis (DSM-No1088) and P. putida (DSM-No50194) both strains obtained from the German collection of microorganisms and cell cultures (DSMZ GmbH, Brauschweig, Germany). Aerobic biodegradation of PCL film samples by bacteria were conducted at 30 °C in bioreactors of 7 mL of capacity, filled with 1.5 mL of bacteria suspension in Minimal Growth Medium (MGM) of 2.5 × 107 cells/mL of concentration, prepared as detailed before.33 The composition of MGM used in this work contained NH4H2PO4 8 g, K2HPO4 2 g, MgSO4·7H2O 0.5 g, NaSO4 0.5 g, ZnCl2·2H2O 8 mg, MnSO4·7H2O 8 mg, FeSO4·7H2O 10 mg, CaCl2 0.05 g, distilled water 1000 mL. In this way, a suspension of each bacterium was prepared. After that, two discs of film samples of 4 mm in diameter (3 mg) were added to the medium. These containers were introduced in disposable cylindrical cells of 20 mL charged with 1.5 mL of 2 g·L−1 KOH aqueous solution and provided by four stainless steel electrodes to measure impedance on a Bac-Trac 4300 (SY-LAB Geräte GmbH, Neupurkerdorf, Austria). The experimental device and procedure were described before.26 The device monitors the relative change in the initial impedance value of KOH solution, which is converted in concentration of carbon dioxide by a calibration curve of impedance variation versus concentration of CO 2 . The percentage of biodegradation of the sample can be calculated taking into account the theoretical amount of carbon dioxide ([CO2]theor.) of the sample, % biodegradation = [CO2]t · 100/[CO2]theor.. Chemiluminescence Analysis. The formation of hydroperoxide species on the samples surfaces during the bacterial degradation was evaluated by chemiluminescence emission analysis in a CL400 ChemiLume Analyzer from Atlas Electric Device Co. The samples were held in a small aluminum pan (20 mm diameter) in the sample cell under a continuous flow of dry nitrogen (60 mL/min). The cells are temperature-controlled and were heated from 25 to 250 °C, at a heating rate of 2 °C/min. To ensure reproducibility of CL signals, the dimensions of the specimens were kept constant: discs of 4 mm diameter. The films were covered by a lens focusing the emitted light from the sample to the water-cooled photon counting photomultiplier, which was previously calibrated using a radioactive standard provided by Atlas. The light emitted was measured as a function of temperature.

Figure 2. XRD spectra of clays MMTk10, MMT(PCL‑b‑DEAEMA), and nanocomposites PCL/1-MMTk10, PCL/6-MMTk10, PCL/1MMT(PCL‑b‑DEAEMA), PCL/6-MMT(PCL‑b‑DEAEMA).

of the polymeric ammonium salt. It has been described that surface energy of uncoated layered silicates is large, and the forces keeping the layers together is very strong. The surface tension of clay decreases with the organophilization, the low surface energy leads to weaker forces between layers and facilitates the exfoliation. 34 For the organomodified MMT(PCL‑b‑DEAEMA) clay, a significantly broadened reflection was observed with respect to MMTk10, which reflected an intercalated structure less spatially ordered. That fact has been previously described for MMTk10 modified with hyperbranched polymers,7 and it was explained in terms of the structure of organomodifier and the presence of several charged groups that could interact electrostatically with different but neighboring platelets surfaces.35 The thermogravimetric analysis (TGA) of pure clay and clay intercalated with the PCL-b-DEAEMA copolymer was undertaken in nitrogen. As expected, MMTk10 showed a high thermal stability, and a total weight loss of 9% was observed when heating due to the water physically adsorbed in the montmorillonite clay. For the intercalated clay, MMT(PCL‑b‑DEAEMA), when heating up to 800 °C, there is a weight loss above 100 °C, which indicates the presence of the triblock copolymer on clay platelets, because the decomposition and combustion occurs at the same temperature range and the overall degradation behavior is quite similar as that of pure copolymer. At temperatures above 100 °C the influence of water content is eliminated and the weight loss corresponding to the intercalated organomodifier takes place. Then, the content of the organic component in the modified clay was 6%, and it has been determined as the weight loss from 100 to 800 °C. 3.2. Morphology and Properties of Nanocomposites. The XRD patterns of nanocomposites containing 1 and 6% of pure and modified clays are presented in Figure 2. The d001 peak of the clay in the PCL/MMT(PCL‑b‑DEAEMA) nanocomposites nearly disappeared and a slight increase in the interlayer spacing to 2.1 nm was observed, indicating the formation of intercalated/exfoliated structure in the nanocomposites. The clay treatment lead to a widening of the clay interlamellar spacing and, together with the organophilic character of the treated clay, allows an easy penetration of the polymer into the lamellar structure inducing intercalation and eventually exfoliation. However, the behavior with the

3. RESULTS AND DISCUSSION 3.1. Clay Modification. The organomodified nanoclay studied in this work was characterized using XRD and TGA to confirm the intercalation of the triblock copolymer to the MMTk10 clay. The X-ray diffraction patterns for the unmodified MMTk10 showed a primary silicate (001) reflection at 8.9°, which corresponds to a d-spacing of 1 nm, Figure 2. Upon the incorporation of the PCL-b-DEAEMA, an increase in the interlayer spacing to 1.8 nm is observed for the organomodified MMT sample when compared to the unmodified clay, which indicates the successful intercalation 4250


Biomacromolecules

Article

Figure 3. TEM images of nanocomposites PCL/1-MMT(PCL‑b‑DEAEMA) and PCL/6-MMT(PCL‑b‑DEAEMA).

Table 1. Static Contac Angle and Surface Free Energy for PCL, PCL/MMTk10, and PCL/MMTPCL‑b‑PDEAEMA Nanocomposites surface free energy (mN·m−1)

static contact angle (deg) sample name PCL PCL/1MMTk10 PCL/2MMTk10 PCL/4MMTk10 PCL/6MMTk10 PCL/10MMTk10 PCL/1MMTPCL‑b‑PDEAEMA PCL/2MMTPCL‑b‑PDEAEMA PCL/4MMTPCL‑b‑PDEAEMA PCL/6MMTPCL‑b‑PDEAEMA PCL/10MMTPCL‑b‑PDEAEMA

sater 79.3 73.3 74.6 78.1 75.4 69.3 81.2 82.9 85.2 88.4 84.6

± ± ± ± ± ± ± ± ± ± ±

0.7 0.4 1.3 1.3 1.4 3.1 1.1 2.6 4.0 0.9 0.7

diiodomethane

γsd

γsp

γs

± ± ± ± ± ± ± ± ± ± ±

38.8 35.3 37.2 37.2 34.3 35.4 33.7 35.1 40.8 36.2 37.5

3.9 7.2 6.1 4.7 6.6 9.1 4.3 3.4 1.8 1.7 2.5

42.7 42.5 43.3 41.9 40.9 44.5 38.0 38.5 42.6 37.9 40.0

35.5 39.9 36.8 38.2 42.6 38.5 45.5 43.6 34.0 43.6 39.9

3.4 2.1 3.9 1.1 1.5 2.8 1.8 2.7 4.7 1.1 2.1

leads to increased surface hydrophilicity, and γps increased with increasing MMTk10 concentration. The dispersive component of surface energy, γds , was observed to be less sensitive to the clay. Otherwise, concerning the organomodification of clay and the incorporation in PCL, contact angle measurements in water showed the increase of the contact angle values with the clay content, in the PCL/MMT(PCL‑b‑DEAEMA) nanocomposites with respect to PCL, and the surface energy and the polar component decreased with increased MMT(PCL‑b‑DEAEMA) content. That effect would be related to the influence of others factors, such as roughness on the contact angle, which has been determined by several methods and related to the adhesion and friction.38,39 TM-AFM images of films are displayed in Figure 4. The brighter areas in phase image correspond to the hard domains where clay is included and the interaction between tip and sample is dominated by repulsive force.40 The phase images exhibited reasonably the morphology of nanocomposites that provided good contrast between the polymer and polymer/clay regions. The size, dispersity, and spacing of the bright domains vary within the sample. As it is evident from the images provided a clear change on the appearance of the domains was observed when natural and organomodified clay were incorporated. In the case of PCL/MMT(PCL‑b‑DEAEMA) nanocomposites, images revealed that the clay was dispersed uniformly and randomly at 1 and 6% in the polymer matrix during mixing in the molten state. Otherwise, PCL/MMTk10 revealed more aggregated structures in partial areas of the AFM phase image.

addition of the untreated hydrophilic clay is quite different, a significant primary peak at 8.9° is observed with small secondary peak around 6.4°, indicating only an intercalated morphology for the PCL/MMTk10 nanocomposites. Because XRD analysis gives the macroscopic conformation of a sample, to further investigate the clay dispersion in the polymer matrix, TEM analysis was performed, as shown in Figure 3. The TEM image for PCL/MMT(PCL‑b‑DEAEMA) nanocomposites reveals that the clay was dispersed uniformly and randomly in the polymer matrix during mixing in the molten state, and exfoliated into thin tactoids which contain few clay layers. This result further supports the XRD analysis, the organomodification makes the layer distance enough for formation of exfoliated morphology. The surface characterization of the nanocomposites was undertaken by using contact angle and AFM. The static contact angle was measured in water and diiodomethane, and the surface energy of the samples was determined by using the two probe-liquids approach of Owens and Wendt.31 The contact angles values and the surface free energy (γs) and its dispersive (γds ) and polar (γps ) components, are summarized in Table 1. Two of the critical surface properties for materials are wetting (hydrophilicity) and real area of contact. Hydrophobicity is characterized by the contact angle of the surface, which depends on factors, such as roughness or method employed for preparing the surface. The incorporation of MMTk10 caused a decrease on the contact angle values, which is on agreement with the results obtained by other authors.36,37 It would be related to the hydrophilic nature of clay incorporated, which 4251


Biomacromolecules

Article

Figure 4. AFM phase images of PCL, PCL/1-MMTk10, PCL/6-MMTk10, PCL/1-MMT(PCL‑b‑DEAEMA), and PCL/6-MMT(PCL‑b‑DEAEMA).

Figure 5. AFM height images and microroughness of PCL, PCL/1-MMTk10, PCL/6-MMTk10, PCL/1-MMT(PCL‑b‑DEAEMA), and PCL/6MMT(PCL‑b‑DEAEMA).

The microroughness of the films was measured by AFM height images within 5 × 5 μm2, Figure 5. The surface of the nanocomposites became rougher with respect to the PCL when MMTk10 was incorporated, and the microroughness value increased with the clay content from 26.7 nm for PCL, to 60.2 and 70.0 nm for PCL/1-MMTk10 and PCL/6-MMTk10, respectively. Also, a higher enhancement was observed for PCL/MMT(PCL‑b‑DEAEMA) nanocomposites (76 and 98.7 nm for PCL/1-MMT(PCL‑b‑DEAEMA) and PCL/6-MMT(PCL‑b‑DEAEMA),

respectively). This behavior corresponds to the morphology of the samples previously described. The obtained results correlate with the model describing roughness with hydrophobicity, a rough surface has larger solid−liquid interface area and it is responsible for the increase of the contact angle for a hydrophobic surface. Differential scanning calorimetry was utilized to investigate the thermal properties of nanocomposites. Despite the variations in morphology, there are no significant differences 4252


Biomacromolecules

Article

between nanocomposites in terms of thermal properties. The addition of organoclay was shown to have no effect on the glass transition temperature (Tg), and the values obtained for nanocomposites were approximately the same as the pure polymer film (−63 °C). The percentage of crystallinity was affected as the amount of clay loading increases. The crystallinity of PCL was 36%, whereas 1 and 2% MMT(PCL‑b‑DEAEMA) additions increased this content up to 41%, and at a higher silicate content of 10%, a decrease to 31% was found. This behavior can be explained by the assumption that, at a lower content, organomodified clay acts as nucleating agent for the crystallization of the PCL. By increasing the clay content, other factors hindering the crystallization process should be considered, such as the confinement of the intercalated polymer chains within the clay galleries, which restrict polymer chain motions, as a result from strong interactions between the clay layers and the polymer chains.41 That latter effect was predominant in PCL/MMTk10 nanocomposites as consequence of their intercalated morphology, and the crystallinity decreased with respect to the neat polymer to 29 and 25% with incorporation of 1 and 10% clay, respectively. The influence of content and organic modification of clay on the mechanical properties of PCL nanocomposites has been studied by tensile testing, as reported in Table 2. In general, the

two principal factors, the degree of crystallinity and the degree of dispersion of the clay, which affected the interfacial area derived from the exfoliation.43 3.3. Cell Growth. The potential application of the PCL/ MMT nanocomposites in biomedical devices has been evaluated. For that purpose, different biocompatibility parameters such as adhesion, proliferation, and LDH release were analyzed in murine L929 fibroblasts cultured on circular membranes of these biomaterials and compared to those obtained for polycaprolactone. These cells are recommended as a reference cell line for the cytotoxicity testing of biopolymers. Figure 6 shows the number of adhered cells on each film

Table 2. Mechanical Properties of PCL, PCL/MMTk10, and PCL/MMTPCL‑b‑PDEAEMA Nanocomposites

Figure 6. Cell number of L929 fibroblasts on PCL and PCL/MMT nanocomposites films evaluated during the proliferation assay.

sample PCL PCL/1MMTk10 PCL/2MMTk10 PCL/4MMTk10 PCL/6MMTk10 PCL/10MMTk10 PCL/ 1MMTPCL‑b‑PDEAEMA PCL/ 2MMTPCL‑b‑PDEAEMA PCL/ 4MMTPCL‑b‑PDEAEMA PCL/ 6MMTPCL‑b‑PDEAEMA PCL/ 10MMTPCL‑b‑PDEAEMA

Young's modulus (MPa) 283 252 232 256 236 299 284

± ± ± ± ± ± ±

13 25 14 13 18 15 7

tensile strength (MPa)

elongation at break (%)

± ± ± ± ± ± ±

811 ± 103 1107 ± 19 1031 ± 102 713 ± 53 684 ± 98 541 ± 59 880 ± 49

31 46 41 31 29 22 37

9 2 5 6 5 3 6

295 ± 12

35 ± 4

807 ± 66

302 ± 11

29 ± 5

727 ± 61

394 ± 20

24 ± 4

413 ± 21

433 ± 3

27 ± 2

590 ± 18

surface after 1, 4, and 6 days culture. PCL and PCL/MMT materials allowed a similar adhesion and proliferation of fibroblasts on its surface, independently on the roughness of the samples and the nature of clay incorporated. In general, the cell number was lower than on the control surface, as it was previously found.19 However, it is important to take into account that this control surface is the tissue culture plastic (polystyrene) commercially available and specifically treated to ensure the optimal cell adhesion and proliferation and viability. High values (92−99%) of cell viability, Figure 7, and very low apoptosis levels (0.1−0.8%) measured through the SubG1

interaction between silicate and polymer decreases proportionally with surface tension, although it would be compensated by a large extent of exfoliation, which drives to a large contact area and the improvement in mechanical properties. The Young's modulus of PCL/MMTk10 was basically independent of the clay content and similar as the neat polymer (283 MPa). In contrast, organomodified clay exhibited reinforcing action. Similar to studies reported by other authors,42 the Young's Modulus of PCL/MMT(PCL‑b‑DEAEMA) nanocomposites was significantly increased as a function of the clay content, from 284 to 433 MPa for filler contents of 1−10%, respectively. The addition up to 2% of clay in the polycaprolactone also increased slightly the tensile strength and the elongation at break compared to the neat polymer, and with a further increase in clay loading, a moderate decrease of tensile strength and ductility was observed. This behavior would be attributed to

Figure 7. Cell viability on PCL and PCL/MMT nanocomposites films evaluated as a function of the cell culture time.

fraction, Figure 8, were obtained in cells after culture on PCL and PCL/MMT films. The analysis of LDH levels at different times in the culture medium, Figure 9, did not reveal significant changes with respect to control cells cultured on tissue culture plastic, demonstrating the plasma membrane integrity of the 4253


Biomacromolecules

Article

and PCL/6-MMT(PCL‑b‑DEAEMA), was monitored by the production of carbon dioxide using an indirect impedance technique. The production of carbon dioxide during the bacteria metabolism was then correlated to the degree of biodegradation in a period of six weeks, and the profiles are shown in Figure 10. Both bacterial strains were able to biodegrade pure PCL, being Bacillus subtilis more efficient than Pseudomonas putida. Biodegradation occurred practically from the start of the bioassay and reached values of 25 and 13% after 42 days. In the case of B. subtilis, PCL/1-MMTk10 showed a clear inhibition regarding the unfilled PCL, although in the case of P. putida, the biodegradation profile was very similar as the one for PCL. Despite the variations in the contact angle and roughness on the PCL/MMT(PCL‑b‑DEAEMA) nanocomposites, it is remarkable that the biodegradation of those samples was inhibited or retarded with respect to the PCL and PCL/1-MMTk10. It has been reported that surface stiffness, hydrophobic termination, and high roughness properties of surfaces can regulate adhesion of bacteria.44 A linear behavior between surface hydrophobicity and the number of adherent cells has been observed.45 Several reports showed that the extent of microbial colonization was higher as the surface roughness increased.46 The obtained results would indicate that the nature of the organomodifier in the clay plays an important role in B. subtilis and P. putida adhesion processes, and the high efficiency against microorganism may be related to the presence of block copolymer with a high density of ammonium groups attached to the clay. These results are in agreement with the antimicrobial effect of quaternary ammonium functionality reported by other authors.47 The activity of the quaternary ammonium compounds has been proposed to involve a general perturbation of lipid bilayer membranes that constitute the bacterial cytoplasmic membrane of Gram negative bacteria.25 Interaction between the positively charged ammonium groups with the anionic molecules at the cell surface would change the permeability of the cell membrane of the microorganism, and the leakage of intercellular components take place. Prior to this work, chemiluminescence (CL) emission measurements have been used to evaluate the degree of oxidation produced by microorganisms in polymer materials, such as gelatin, cellulose triacetate, and polyethylene.26,48,49 The chemiluminescence in polymers is due to the light emission that accompanies the thermal decomposition of the thermooxidative degradation products (hydroperoxides), which are formed by exposure to various factors such as heat, oxygen, UV light, humidity, and microorganism, during processing or

Figure 8. Sub G1 (%) on PCL and PCL/MMT nanocomposites films evaluated as a function of the cell culture time.

Figure 9. LDH concentration at different times in the medium of L929 fibroblasts cultured on PCL and PCL/MMT nanocomposites films.

L929 fibroblasts grown on these biomaterials. When cell size and complexity were analyzed by flow cytometry, no changes were observed (data not shown). All these results demonstrate the biocompatibility of PCL and PCL/MMT films, which allow the culture of murine L929 fibroblasts on its surface with high viability, very low apoptosis, and without plasma membrane damage. 3.4. Antimicrobial Activity. To analyze the antimicrobial activity onto the nanocomposites materials, the biodegradation of the samples was carried out in the presence of Bacillus subtilis and Pseudomonas putida, which are two bacterial strains most commonly associated with implant infections. The biodegradation of the PCL, PCL/1-MMTk10, PCL/1-MMT(PCL‑b‑DEAEMA),

Figure 10. Biodegradation profiles in a period of 42 days in the presence of Bacillus subtilis and Pseudomonas putida. 4254


Biomacromolecules

Article

in-service life of the material under ambient conditions. This bimolecular reaction promotes ketone products to its lowest triplet state and the radiative deactivation gives chemiluminescence emission in the visible region.50 CL has been very useful in studying the long-term degradation of polycaprolactone films in biologically related fluids,51 in the presence or absence of fibroblasts.52 The characterization of the degraded substrates allowed to explain transitory oxidative stress phenomena described in the fibroblasts and its relation with cell adhesion processes. The measurements of CL in a temperature dynamic experiment under an inert gas flow such as nitrogen provides valuable information about the oxidation state of the studied material. For the material prepared without montmorillonite, the emission of CL was detected from temperatures above 65 °C that correspond to the melting temperature of the material, where the efficiency of the quantum yield of chemiluminescence emission was enhanced by the increase in mobility of the hydroperoxides, which favor the disproportion reaction responsible for the emission. The nanocomposites that contained MMTk10 and MMT(PCL‑b‑DEAEMA) presented an improved thermal stability regarding the unloaded material, as it can be seen in Figure 11a. In general, the CL intensity decreases and the onset is delayed to higher temperature with the content of clay, and that effect is more pronounced for the organomodified clay. The chemiluminescence emission of the materials after 42 days of bacterial exposure was collected and the results are shown in Figure 11b,c. In general, a higher chemiluminescence emission and lower onset temperature were observed for all samples after biodegradation with respect to the initial samples, and that effect was more evidenced for PCL and in presence of Bacillus subtilis compared to Pseudomonas putida, Figure 11b,c. In the case of the materials exposed to B. subtilis, the PCL presented the lowest onset temperature of chemiluminescence emission, as well as the highest intensity, regarding the loaded materials. The material PCL/1-MMTk10 showed an onset of temperature slightly shifted to a higher temperature with respect to PCL. This is in agreement with the biodegradation results and with the beneficial effect that the clay produces in the stability of the polymer matrix. The materials containing organomodified MMT showed lower chemiluminescence emission. The results obtained confirmed those obtained by the capture of carbon dioxide produced during the bacterial mineralization. The presence of quaternary ammonium cations inhibited the bacterial biodegradation and therefore decreased the oxidation species and the chemiluminescence emission. Otherwise, others factors such as the influence of the crystallinity content may be considered. As the biodegradation starts in the amorphous region, it is reasonable to expect the rate of biodegradation to be more sluggish for those samples with higher crystallinity. In PCL/ MMT(PCL‑b‑DEAEMA) nanocomposites, the antimicrobial quaternary ammonium cations would be located close to the interphase intercalated polymer chains within the clay galleries, where the crystallization process would be more hindered.

Figure 11. Chemiluminiscence emission in a range of temperatures under a nitrogen flow of the initial (a) and biodegraded samples in the presence of B. subtilis (b) and P. putida (c).

prepared through melt intercalation, and XRD and TEM analysis showed an exfoliated/intercalated morphology for organomodified clay. The Young's modulus of PCL/MMTk10 was basically independent of the clay content and similar as the neat polymer. Otherwise, PCL/MMT(PCL‑b‑DEAEMA) nanocomposites exhibited a marked increase of Young's modulus as a function of the clay content, ascribed to the high degree of dispersion of clay. The contact angle measurements in water demonstrated that the incorporation of MMTk10 caused a decrease on the contact angle values, related to the hydrophilic nature of clay. Otherwise, increase in the contact angle happens in the PCL/ MMT(PCL‑b‑DEAEMA) nanocomposites with respect to PCL, and the surface energy and the polar component decreased with increased organomodified clay content. The AFM analysis showed that the surface of the nanocomposites became rougher with respect to the PCL when MMTk10 or MMT(PCL‑b‑DEAEMA) was incorporated, and the value increased with the clay content. The results obtained correlate with the model describing roughness with contact angle, a rough surface has larger solid−

4. CONCLUSIONS A triblock copolymer based on PCL and PDEAEMA synthesized via atom transfer radical polymerization (ATRP). The corresponding chlorohydrated copolymer, PCL-b-DEAEMA, was prepared and anchored via cationic exchange on MMTk10 clay surface. PCL/layered silicate nanocomposites with 1 to 10% content of MMTk10 or MMT(PCL‑b‑DEAEMA) were 4255


Biomacromolecules

Article

(14) Hsu, S.; Wang, M.; Lin, J. Appl. Clay Sci. 2012, 56, 53. (15) Madhavan, R. V.; Rosemary, M. J.; Nandkumar, M. A.; Krishnan, K. V.; Krishnan, L. K. Tissue Eng. 2011, 17, 439. (16) Kee, W. N.; Hutmacher, D. W.; Schantz, J. T.; Chin, S. N.; Too, H. P.; Thiam, C. L.; Toan, T. P.; Swee, H. T. Tissue Eng. 2001, 7, 441. (17) Zhong, Z. K.; Sun, X. Z. Polymer 2001, 42, 6961. (18) Hutmacher, D. W.; Schantz, T.; Zein, I.; Ng, K. W.; Teoh, S. H.; Tan, K. C. J. Biomed. Mater. Res. 2001, 55, 203. (19) Serrano, M. C.; Pagani, R.; Vallet-Regi, M.; Peña, J.; Rámila, A.; Izquierdo, I.; Portolés, M. T. Biomaterials 2004, 25, 5603. (20) Gümüsḑ erelioglu, M.; Dalkiranoglu, S.; Aydin, R. S. T.; Ç akmak, S. J. Biomed. Mater. Res., Part A 2011, 98, 461. (21) Kim, S. Y. J. Appl. Polym. Sci. 2011, 121, 1921. (22) Bugatti, V.; Gorrasi, G.; Montanari, F.; Nocchetti, M.; Tammaro, L.; Vittoria, V. Appl. Clay Sci. 2011, 52, 34. (23) Olgun, U.; Tunç, K.; Ö zaslan, V. Polym. Adv. Technol. 2011, 22, 232. (24) Hong, S. I.; Rhim, J.W. J. Nanosci. Nanotechnol. 2008, 8, 5818. (25) Gilbert, P.; Moore, L. E. J. Appl. Microbiol. 2005, 99, 703. (26) Abrusci, C.; Marquina, D.; Del Amo, A.; Catalina, F. Int. Biodeterior. Biodegrad. 2007, 185, 188. (27) Abrusci, C.; Marquina, D.; Santos, A.; Del Amo, A.; Corrales, T.; Catalina, F. J. Photochem. Photobiol., A 2007, 185, 188. (28) Keller, R.; Wycoff, H. D. Inorg. Synth. 1947, 2, 1. (29) Alonso, A.; Catalina, F.; Salvador, E. F.; Peinado, C. Macromol. Chem. Phys. 2001, 202, 2293. (30) San Miguel, V.; Limer, A. J.; Haddleton, D. M.; Catalina, F.; Peinado, C. Eur. Polym. J. 2008, 44, 3853. (31) Owens, D. K.; Wendt, R. C. J. Appl. Polym. Sci. 1969, 13, 1741. (32) Fowkes, F. M. Ind. Eng. Chem. 1964, 56, 40. (33) Abrusci, C.; Marquina, A.; Del Amo, A.; Corrales, T.; Catalina, F. Polym. Deg. Stab. 2004, 86, 283. (34) Kádár, F.; Százdi, L.; Fekete, E.; Pukánszky, B. Langmuir 2006, 22, 7848. (35) Fan, X.; Xia, C.; Advincula, R. C. Langmuir 2003, 19, 4381. (36) Zhou, Q.; Pramoda, K. P.; Lee, J. M.; Wang, K.; Loo, L. S. J. Colloid Interface Sci. 2011, 355, 222. (37) Anadão, P.; Sato, L. F.; Wiebeck, H.; Valenzuela-Díaz, F. R. Appl. Clay Sci. 2010, 48, 127. (38) Wenzel, R. N. Ind. Eng. Chem. 1936, 28, 988. (39) Burton, Z.; Bhushan, B. Nano Lett. 2005, 5, 1607. (40) Peinado, C.; Corrales, T.; Catalina, F.; Pedrón, S.; Ruiz Santa Quiteria, V.; Parellada, M. D.; Barrio, J. A.; Olmos, D.; GonzálezBenito, J. Polym. Degrad. Stab. 2010, 95, 975. (41) Chen, B.; Evans, J. R. G. Macromolecules 2006, 39, 747. (42) Gorrasi, G.; Tortora, M.; Vittoria, V.; Pollet, E.; Lepoittevin, B.; Alexandre, M. Polymer 2003, 44, 2271. (43) Pavlidou, S.; Papaspyrides, C. D. Prog. Polym. Sci. 2008, 33, 1119. (44) Lichter, J. A.; Todd, M.; Delgadillo, M.; Nishikawa, T.; Rubner, M.; Van Vliet, K. J. Biomacromolecules 2008, 9, 1571. (45) Oliveira, R.; Azeredo, J.; Texeira, P.; Fonseca, A. P. In: Gilbert, P, Allison, D, Branding, M, Verran, J, Walker, J, Eds. Biofilm Community Interactions: Chances or Necessity; BioLine: Cardiff, 2001; p 11. (46) Texeira, P.; Oliveira, R. J. Adhes. Sci. Technol. 1999, 13, 1287. (47) Hong, S. I.; Rhim, J. W. J. Nanosci. Nanotechnol. 2008, 8, 5818. (48) Abrusci, C.; Marquina, D.; Santos, A.; Del Amo, A.; Corrales, T.; Catalina, F. Int. Biodeterior. Biodegrad. 2009, 63, 759. (49) Abrusci, C.; Pablos, J. L.; Marín, I.; Espí, E.; Corrales, T.; Catalina, F. J. Appl. Polym. Sci. 2012, 126, 1664. (50) Billingham, N. C.; Then, E. T. H.; Gijman, P. H. Polym. Degrad. Stab. 1991, 34, 263. (51) Peña, J.; Corrales, T.; Izquierdo-Barba, I.; Doadrio, A. L.; ValletRegí, M. Polym. Degrad. Stab. 2006, 91, 1424. (52) Peña, J.; Corrales, T.; Izquierdo-Barba, I.; Serrano, M. C.; Portolés, M. T.; Pagani, R.; Vallet-Regí, M. J. Biomed. Mater. Res. 2006, 76A, 788.

liquid interface area and it is responsible for the increase of the contact angle for a hydrophobic surface. The novel nanocomposites were tested for antimicrobial activity against B. subtilis and P. putida. The biodegradation was monitored by the production of carbon dioxide using an indirect impedance technique, and by chemiluminescence emission. It is remarkable that the biodegradation of PCL/ MMT(PCL‑b‑DEAEMA) nanocomposites was inhibited or retarded with respect to the PCL and PCL/1-MMTk10. It would indicate that nature of organomodifier in the clay play an important role in B. subtilis and P. putida adhesion processes, and the high efficiency against microorganism may be related to the presence of block copolymer with a high density of ammonium groups attached to the clay. The cell growth on the sample surfaces was studied, revealing that the presence of MMTk10 or MMT(PCL‑b‑DEAEMA) has no significant effect on the viability of L929 fibroblasts, which show neither plasma membrane damage nor relevant apoptosis induction, making these materials very adequate for tissue engineering.

■

ASSOCIATED CONTENT

* Supporting Information S

TGA thermograms of MMTk10, MMT(PCL‑b‑DEAEMA), and triblock copolymer PCl-b-DEAEMA. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors would like to thanks to the MICINN and Comunidad de Madrid (Spain) for financial support (MAT2009-09671 and S2009/MAT-1472). C.A. also thanks to Ramon y Cajal Program.

■

REFERENCES

(1) Muñoz-Bonilla, A.; Fernández-García, M. Prog. Polym. Sci. 2012, 37, 281. (2) Lorite, G. S.; Rodrigues, C. M.; de Souza, A. A.; Kranz, C.; Mizaikoff, B.; Cotta, M. A. J. Colloid Interface Sci. 2011, 359, 289. (3) Katsikogianni, M.; Missirlis, Y. F. Eur. Cells Mater. 2004, 8, 37. (4) Rijnaarts, H. H. M.; Norbe, W.; Bouwer, E. J.; Lyklema, J.; Zehnder, A. J. B. Colloids Surf., B 1995, 14, 179. (5) Meng, N.; Zhou, N.; Zhang, S.; Shen. J. Appl. Clay Sci. 2009, 42, 667. (6) Galya, T.; Sedlarik, V.; Kuritka, I.; Novotny, R.; Sedlarikova, J.; Saha, P. J. Appl. Polym. Sci. 2008, 110, 3178. (7) Larraza, I.; Peinado, C.; Abrusci, C.; Catalina, F.; Corrales, T. J. Photochem. Photobiol., A 2011, 224, 46. (8) Lichter, J. A.; Todd, M.; Delgadillo, M.; Nishikawa, T.; Rubner, M. F.; Van Vliet, K. J. Biomacromolecules 2008, 9, 1571. (9) Giannelis, E. P. Adv. Mater. 1996, 8, 29. (10) Nigmatullin, R.; Gao, F.; Konovalova, V. J. Mater. Sci. 2008, 43, 5728. (11) Huskic, M.; Zagar, E.; Zigon, M.; Brnardic, I.; Macan, J.; Ivankovic, M. Appl. Clay Sci. 2009, 43, 420. (12) Wu, T.; Xie, A.; Tan, S.; Cai, X. Colloids Surf., B 2011, 86, 232. (13) Behlau, I.; Mukherjee, K.; Todani, A.; Tisdale, A. S.; Cade, F.; Wang, L.; Leonard, E. M.; Zakka, F. R.; Gilmore, M. S.; Jakobiec, F. A.; Dohlman, C. H.; Klibanov, A. M. Biomaterials 2011, 32, 8783. 4256


In Vitro Biocompatibility and Antimicrobial Activity of Poly(Îµ

Recommend Documents