Polyethylene Glycol-Modified Graphene

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Poly(propylene fumarate)/Polyethylene Glycol-Modified Graphene Oxide Nanocomposites for Tissue Engineering Ana M. Díez-Pascual*,† and Angel L. Díez-Vicente‡ †

Analytical Chemistry, Physical Chemistry and Chemical Engineering Department, Faculty of Biology, Environmental Sciences and Chemistry, Alcalá University, E-28871 Alcalá de Henares, Madrid, Spain ‡ Airbus Operations S. L., John Lennon s/n, 28906 Getafe, Madrid, Spain S Supporting Information *

ABSTRACT: Poly(propylene fumarate) (PPF)-based nanocomposites incorporating different amounts of polyethylene glycolfunctionalized graphene oxide (PEG-GO) have been prepared via sonication and thermal curing, and their surface morphology, structure, thermal stability, hydrophilicity, water absorption, biodegradation, cytotoxicity, mechanical, viscoelastic and antibacterial properties have been investigated. SEM and TEM images corroborated that the noncovalent functionalization with PEG caused the exfoliation of GO into thinner flakes. IR spectra suggested the presence of strong hydrogen-bonding interactions between the nanocomposite components. A gradual rise in the level of hydrophilicity, water uptake, biodegradation rate, surface roughness, protein absorption capability and thermal stability was found upon increasing GO concentration in the composites. Tensile tests revealed improved stiffness, strength and toughness for the composites compared to unfilled PPF, ascribed to a homogeneous GO dispersion within the matrix along with a strong PPF/PEG-GO interfacial adhesion via polar and hydrogen bonding interactions. Further, the nanocomposites retained enough stiffness and strength under a biological state to provide effective support for bone tissue formation. The antibacterial activity was investigated against Gram-positive Staphylococcus aureus and Staphylococcus epidermidis as well as Gram-negative Pseudomonas aeruginosa and Escherichia coli microorganisms, and it rose sharply upon increasing GO concentration; systematically, the biocide effect was stronger versus Gram-positive bacteria. Cell viability data demonstrated that PPF/PEG-GO composites do not induce toxicity over human dermal fibroblasts. These novel materials show great potential to be applied in the bone tissue engineering field. KEYWORDS: poly(propylene fumarate), graphene oxide, polyethylene glycol, nanocomposites, mechanical properties, tissue engineering

1. INTRODUCTION Biodegradable synthetic polymers present numerous advantages over other materials for the development of scaffolds for tissue engineering, including the capacity to tailor mechanical performance and degradation kinetics to fit a variety of applications. Further, they can be fabricated into diverse shapes with desired pore morphologies and allow the easy incorporation of different chemical groups, facts that favor the growth of tissues.1 A wide number of biodegradable polymers belong to the polyester family. The most frequently employed synthetic biopolymers for 3D scaffolds in tissue engineering are saturated poly(α-hydroxy esters), such as poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA) and poly(ε-caprolactone) (PCL), which display outstanding © XXXX American Chemical Society

mechanical properties and biological affinity. Nevertheless, their brittleness, poor barrier properties and deficiency of functional groups in the backbone have interfered with modulation of their morphology, degradation rate and mechanical performance.2 Recently, polyesters based on fumaric acid, a component of the Krebs cycle, have involved a lot of attention for medical applications owed to their good biocompatibility and biodegradability.3 Among them, the most widely investigated is poly(propylene fumarate) (PPF), a linear copolyester that holds two ester bonds and one unsaturated Received: May 11, 2016 Accepted: June 27, 2016

A

DOI: 10.1021/acsami.6b05635 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 1. Schematic Representation of the Synthesis Procedure of PPF

and biological tissues, it is a promising material for medical applications such as tissue engineering, molecular imaging, drug/gene delivery and antibacterial materials.22,23 Further, because of π−π* transitions, a little energy is necessary for electron movement, a crucial property for biosensor and bioimaging applications.22 GO also has a very high surface area, hence has great potential for the fabrication of microelectrical devices, batteries, supercapacitors and solar cells.24 Besides, it can simply be mixed with several polymers and improve the properties of the resulting composites including mechanical, conductivity, bioactivity and so forth.25−27 To attain high property improvements, GO has to be homogeneously distributed within the matrix.25 However, the large GO surface area and strong van der Waals forces among the flakes can lead to strong aggregation in the composite. To achieve stable GO dispersions and tailor the microstructure of the nanocomposites, noncovalent or covalent functionalization with polymers might be required.28 The noncovalent functionalization, which is based on van der Waals forces, hydrogen bonding, electrostatic or π−π stacking interactions, offers successful ways to tailor the properties and solubility of the sheets without altering their chemical structure. The covalent functionalization relies on the reaction between the functional groups on the GO surface, namely epoxides, hydroxyls and carbonyls on their basal planes and carboxyls on the edges, and specific functional groups on the polymer.18 Compared with noncovalent functionalization, the covalent approach provides a lot of possibilities owed to the rich surface chemistry of GO. However, the covalent attachment of GO to polymer chains frequently generates defects on the sheets that have detrimental effects on the conductivity and mechanical properties.28 Polyethylene glycol (PEG), a biocompatible and biodegradable polymer widely used for the development of tissue engineering scaffolds,1 is a promising compound to functionalize graphene and its derivatives for medical applications. Previous studies have assessed the biological activity and cytotoxicity of G and GO covalently modified with PEG and its derivatives,29−31 which show great potential as drug carries. However, scarce works have dealt with PEG-functionalized graphene (or GO) via noncovalent chemistry.32 The present article deals with the preparation and characterization of PPFbased nanocomposites reinforced with GO noncovalently functionalized with PEG. Different GO loadings have been incorporated in the copolyester matrix, and their morphology, structure, thermal stability, water uptake, protein absorption capability, cytotoxicity, biodegradability, antibacterial, viscoe-

carbon−carbon double bond as repeating units (Scheme 1). The double bonds allow PPF to be cross-linked into a polymeric network via thermal treatment or using a photoinitiator.4 Upon hydrolysis of its ester linkages, PPF forms biocompatible degradation products, mainly fumaric acid and propylene glycol.5 The main disadvantages of PPF are that at room temperature is a liquid, which makes the managing difficult, and its poor mechanical strength owed to bendy C− O−C regions in the polymer backbone, which limits its use for bone tissue engineering. With the aim to enhance mechanical properties and spread its potential applications, novel approaches are pursued such as the incorporation of fillers6−10 or polymer blending.11,12 PPF can be combined with other polymers to increase its degree of hydrophilicity, which is an important property when considering medical applications. More hydrophilic surfaces generally result in enhanced cell attachment, spreading and proliferation.13 On the other hand, composites of PPF with ceramics such as tricalcium phosphate,6 calcium sulfate7 or hydroxyapatite (HA)14 have been reported to be suitable for bone replacement, owed to the outstanding biocompatibility, osteoconductivity and ability to promote cellular functions of bioceramics.15 Further, preceding studies have evaluated the usefulness of fullerenes and single-walled carbon nanotubes (SWCNTs) as reinforcing agents of crosslinked PPF.9,10 Fullerenes slightly improved the mechanical properties of the polymer whereas SWCNTs lead to moderate improvements. Nevertheless, the results of those works give insight into two other approaches that can be used to enhance further the mechanical behavior of PPF/filler nanocomposites: covalent and noncovalent functionalization of the nanofillers to improve their distribution within the matrix and diminution of the filler aspect ratio. Graphene oxide (GO), a one atom thick hexagonal lattice of sp2 bonded carbon atoms prepared by the powerful oxidation of graphite, has recently attracted a lot of attention as nanostructured material.16 GO is an oxidized form of graphene, and is highly hydrophilic because incorporates numerous oxygencontaining functional groups, therefore yielding stable dispersions in water and other solvents.17 GO can be synthesized via four basic ways:18,19 Staudenmaier, Hofmann, Brodie and Hummers, although numerous variations of these approaches have been reported.20,21 This layered two-dimensional nanomaterial possesses outstanding strength, unique optical, physical, mechanical, and electronic properties. Owed to its good aqueous processability, fluorescence quenching ability, surface functionalization capability, high antibacterial activity, versatility, biocompatibility, and capacity to interact with cells B


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ACS Applied Materials & Interfaces Scheme 2. Representation of the Synthesis Method of the Nanocomposites

A schematic representation of the synthesis procedure of the nanocomposites is shown in Scheme 2. To improve the stability and dispersibility of GO sheets, PEG (10 mg) was mixed with a GO solution in chloroform (0.1 mg/mL) at room temperature. Subsequently, the solution was ultrasonicated in a bath for 15 min followed by probe sonication for 5 min at 240 W. The GO dispersion in PEG was stable for more than 3 months. Separately, PPF and NVP (1:1 mass ratio) were mixed in chloroform and then a certain amount of the GO dispersion was added. The nanocomposite mixture was bath sonicated for another 15 min and the chloroform was completely removed using a rotary evaporator. Subsequently, BP (free radical initiator) was dissolved in DEF and added to the nanocomposite mixture to initiate polymerization. The mixture was finally poured into Teflon molds and cured under vacuum at 80 °C for 24 h. Five nanocomposites were prepared with GO weight percentages of 0.1, 0.5, 1.0, 2.0 and 3.0 wt %. To apply the approach proposed in this work for the development of biocomposites, a nanocomposite with 3.0 wt % GO (designated by an asterisk) was prepared following a similar fabrication process in which PEG was simply mixed with an aqueous GO solution at room temperature followed by ultrasonication under identical conditions. Subsequently, PPF and NVP were mixed in the absence of chloroform and then the GO dispersion was added. The results obtained from the different characterizations of this bionanocomposite are collected in Tables S1 and S2 (see Supporting Information). Characterization Techniques. The surface morphology was investigated with a SU8000 Hitachi scanning electron microscope (SEM).33 A contact profilometer was used to estimate the average surface roughness of the samples. A Philips Tecnai 20 FEG (LaB6 filament) electron microscope fitted with an EDAX detector was used to acquire the transmission electron microscopy (TEM) images. The FT-IR spectral scans were recorded at room temperature with a PerkinElmer Spectrum One spectrometer in the wavenumber range 4000−600 cm−1. A Bruker D8 Advance diffractometer equipped with a copper X-ray source was used to record the X-ray diffractograms (XRD) of the samples in the angular range (2θ values) between 2 and 40°. The thermal stability was investigated using a TA Instruments Q50 thermobalance.33 Thermogravimetric analysis (TGA) experiments

lastic and mechanical properties have been investigated in detail throughout this work.

2. EXPERIMENTAL SECTION Materials. Natural graphite was obtained from Bay Carbon, Inc. (Michigan, USA) and used for synthesizing graphene oxide (GO). Diethyl fumarate (DEF) was provided by Acros Organics. H2SO4, KMnO4, P2O5, K2S2O8, H2O2 (30 wt % in water), hydroquinone, Nvinylpyrrolidone (NVP), ZnCl2, benzoyl peroxide (BP), dichloromethane, propylene glycol (PG), sodium sulfate, chloroform and polyethylene glycol (PEG, Mw = 4000 g/mol) were purchased from Sigma-Aldrich and used as received. Synthesis of PPF. PPF was synthesized via a two-step reaction of DEF and PG (Scheme 1) adapted from Kasper et al.3 In brief, both reagents in a molar ratio of 1:3 were mixed in a three-neck roundbottomed flask in the presence of ZnCl2 as acatalyst and hydroquinone as a cross-linking inhibitor, which were added in a 0.01:0.003:1 molar ratio to DEF, respectively. The temperature was then increased to 130 °C and the reaction was allowed to proceed for 10 h under an inert atmosphere and mechanical stirring. This stage lead to bis(hydroxypropyl) fumarate (BHPF) intermediate and ethanol. In the second stage, the intermediate was heated to 150 °C and the reaction was carried out with mechanical stirring and reduced pressure for 8 h. The polymer was allowed to cool, washed thoroughly with dichloromethane, hydrochloric acid and distilled water, and finally dried under vacuum at room temperature. Nanocomposite Preparation. GO was synthesized using a modified Hummers’ method.18,20 In short, graphite powder, H2SO4, K2S2O8 and P2O5 were heated at 80 °C for 6 h. Upon cooling, deionized water was added to the mixture and was maintained overnight with stirring. The resulting product was then filtered, dried under air and further oxidized by addition of H2SO4, KMnO4 and water in an ice−water bath. Subsequent to dilution with water, excess KMnO4 was decomposed by addition of 30 wt % H2O2 aqueous solution and then 5 wt % HCl aqueous solution. The product was filtered again and finally washed with deionized water several times. GO powder was obtained after freeze-drying the GO aqueous solution under reduced pressure. C


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ACS Applied Materials & Interfaces

Figure 1. Representative SEM images at different magnifications of GO (a), PEG-modified GO (b) and PPF/PEG-modified GO nanocomposite with 3.0 wt % GO loading (c, d, e and f). were carried out from 100 to 650 °C at a rate of 10 °C/min under an inert atmosphere. Water uptake was determined by immersing the samples in a simulated body fluid (SBF)34 at 37 °C for 14 days. It was calculated as (W − W0)/W0 × 100, where W0 and W are the weight of the sample before and after immersion in SFB, respectively. Static contact angle measurements at 25 °C were carried out with a Krüss G10 goniometer.34 Nonenzymatic hydrolytic degradation was evaluated by means of submerging the samples in phosphate buffered saline (PBS) at pH 7.4 and 37 °C for 7 weeks.34 The amount of adsorbed protein was determined by placing the nanocomposites for 4 h in a culture medium containing 10% fetal bovine serum at 37 °C. Subsequently, they were transferred into well plates and washed three times with PBS. 400 μL of 1% sodium dodecyl sulfate (SDS) solution were then added to each well and allowed to react for 1 h at room temperature. The SDS solution was collected in a plastic flask and new SDS solution was put into the wells for another 1 h. The protein content in the solutions was determined using a microplate reader with a Micro BCA protein assay kit (ThemoFisher Scientific). Tensile tests were performed with a 4204 mechanical tester at 23 ± 2 °C and 50 ± 5% RH following the UNE-EN ISO 527-1 standard.35 Experiments were also carried out on samples immersed in SBF at 37 °C for 6 weeks. Dynamic mechanical analysis (DMA) tests were carried out on a Mettler DMA861 dynamic mechanical analyzer.35 The antibacterial activity of the nanocomposites was tested against Gram-positive Staphylococcus aureus (S. aureus, ATCC 12600) and Staphylococcus epidermidis (S. epidermidis, ATCC 12228) as well as Gram-negative Escherichia coli (E. coli, ATCC 25922) and Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853), as described elsewhere.35 It was calculated as log(viable cell countcontrol/viable cell countcomposite),

where a beaker with bacteria and without sample was taken as control. Cell viability of the samples was evaluated by the Alamar blue assay (AbDSerotec) using normal human dermal fibroblasts (NHDF), as reported previously;35 the relative cell viability (%) is expressed as a percentage relative to the untreated control cells.

3. RESULTS AND DISCUSSION Surface Morphology. SEM analysis was performed to gain insight into the state of dispersion of PEG-modified GO within PPF matrix and to estimate the average flake thickness, and representative images at different magnifications of GO, PEGmodified GO and the nanocomposite with 3.0 wt % GO are compared in Figure 1. The raw GO powder is composed of agglomerated stacked nanosheets (Figure 1a); because of its high specific surface area, GO flakes are prone to form agglomerates via π−π interactions, van der Waals forces and Hbonding. Upon noncovalent functionalization with PEG, disaggregation into thinner layers (ca. 50 nm size, Figure 1b) takes place, which suggests that the polymer chains are intercalated between the GO sheets in the nanocomposite.28 The hydrogen bonding interactions between the oxygencontaining functional groups of GO sheets and the hydroxyl moieties of PEG would exceed the van der Waals forces and π−π stacking interactions that maintain the graphene sheets together, leading to exfoliation of the nanomaterial during the ultrasonication process, as previously reported for other hydrophilic polymers like poly(ethylene oxide) (PEO), polyvinylpyrrolidone (PVP) and poly(vinyl alcohol) (PVA).36 D


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Table 1. Mean Roughness Ra, Water Uptake, Weight Loss in PBS, Glass Transition Temperature Tg, Initial Degradation Temperature Ti, Temperature of 10% Weight Loss T10 and Temperature of Maximum Rate of Weight Loss Tmax material PPF PPF/PEG-GO PPF/PEG-GO PPF/PEG-GO PPF/PEG-GO PPF/PEG-GO

(0.1) (0.5) (1.0) (2.0) (3.0)

Ra (μm)

water uptake (%)

weight loss (%)

Tg (°C)

Ti (°C)

T10 (°C)

Tmax (°C)

1.30 1.49 1.88 2.06 1.94 2.37

2.9 4.3 5.1 7.2 8.4 8.8

3.5 3.7 4.0 4.4 4.9 5.6

22.8 22.6 27.1 30.5 32.0 35.3

285.2 283.0 284.8 297.0 303.2 310.9

309.0 311.3 317.5 328.9 332.0 345.2

352.2 364.8 370.3 377.2 386.4 395.7

previously reported for epoxy composites filled with graphene nanoplatelets when the nanofiller concentration was increased up to 0.5 wt %.37 The increased surface roughness of the nanocomposites compared to PPF could be highly advantage for cell proliferation and differentiation. Overall, SEM and TEM observations demonstrate that the ultrasonication step was indeed effective to disrupt the aggregation of GO nanosheets, thus increasing the surface area available for interacting with the polymer matrix, consequently resulting in a satisfactory dispersion of PEG-modified GO within PPF. FT-IR Study. The FTIR spectra of GO, PEG-GO, neat PPF and the nanocomposites with 0.5 and 3.0 wt % GO loading are shown in Figure 2. GO contains epoxide and hydroxyl

It should be noted that the GO sheets are not completely exfoliated, given that no individual flakes can be visualized, but remain gathered in small bundles. These results are in agreement with those found by Park et al.32 for GO noncovalently functionalized with NH2−PEG−NH2, where the original size of GO was significantly reduced after sonication in the presence of the polymer derivative, albeit only partial debundling was attained. Nonetheless, the delamination of GO obtained herein upon ultrasonication with PEG is critical to attain effective nanofiller−matrix load transfer, hence to improve the performance of the composites. Regarding the composite with 3.0 wt % GO, a random and homogeneous dispersion of the GO flakes within PPF matrix can be observed (Figure 1c−f). The flakes show a wide size distribution, with thickness in the range of 7−45 nm and a mean value of 18 nm. Further, the flakes appear to be bended, showing a high degree of flexibility. Similar morphology was found for the rest of nanocomposites, without the occurrence of clusters or agglomerates. Further information about the morphology of PEG and PEG-GO was obtained from TEM micrographs. Figure S1 (Supporting Information) presents representative TEM images of GO at different magnifications, revealing stacked nanosheets with a crumpled and rippled structure. In contrast, in the micrographs of PEG-GO (Figure S3), the PEG chains appear intercalated between the GO sheets (dark features), resulting in an intrinsically unstacked structure. No voids or discontinuities are detected between the GO nanosheets and the polymer, indicating good compatibility between the two phases. The histograms of the thickness and width distribution obtained from 100 flakes of GO and PEG-GO are shown in Figures S2 and S4, respectively. As can be observed from these statistics, the GO flake thickness distribution is unimodal in the range of 20−100 nm, with a weighted mean value of 66 nm, whereas the GO width shows a broad trimodal distribution between 0.2 and 1.2 μm, with local maximums at 0.25, 0.55, and 0.95 μm, and a weighted average of 0.63 μm. Regarding PEG-GO, the polydispersity is reduced; both the flake thickness and width distributions are unimodal, showing weighted mean values of 55 nm and 0.54 μm, respectively. These results confirm that the ultrasonication process in the presence of the polymer induces the debundling of the GO sheets, resulting in thinner and narrower flakes. On the other hand, surface roughness is an important factor modulating cell adhesion and proliferation, because adhesion strength generally improves with increasing surface roughness. Therefore, the mean surface roughness (Ra) of neat PPF and the nanocomposites was determined, and the results are summarized in Table 1. As can be observed, Ra increases gradually on increasing GO loading: PPF shows the smoothest surface (Ra ≈ 1.3 μm) whereas PPF/PEG-GO (3.0 wt %) is the roughest (Ra ≈ 2.4 μm). A strong increase in Ra has been

Figure 2. IR spectra of GO, PEG-GO, PPF and PPF/PEG-GO nanocomposites with 0.5 and 3.0 wt % GO loading.

functional groups on either side of its basal plane and carboxyl moieties at the edge sites. The peak at ∼3520 cm −1 corresponds to the OH stretching vibrations and that at about 1740 cm−1 is typical of the CO stretching of the carboxylic groups. The band at 1628 cm−1 can be assigned to the aromatic CC stretching, that at ∼1400 cm−1 corresponds to the OH deformation and those at approximately 1280 and 1060 cm−1 are related to epoxy CO stretching vibrations.38,39 The spectrum of PEG-modified GO is similar to that of GO, with some additional bands characteristic of PEG polymer such as the CH stretching in the range of 2900−2980 cm−1 and the C−O−C stretching vibrations between 1270 and 1050 cm−1. Compared with the spectrum of GO, a widening and downshift of the peak that corresponds to the hydroxyl stretching is detected, centered at ∼3486 cm−1. Analogously, a shift of the CO stretching toward lower wavenumber is detected, and these phenomena are indicative of H-bonding interactions between the OH moieties of PEG and the carboxylic and hydroxyl groups of GO. Similar shifts have been reported for PEG/GO composite papers.40 E


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small increase in the interlayer spacing of GO due to the presence of the PPF chains. Further, the (001) plane of PEG is slightly visible in the diffractogram of the composite with 3.0 wt % GO. Thermal Stability. The thermal stability of GO, PEG-GO, neat PPF and the different nanocomposites was investigated under a nitrogen atmosphere, and typical thermograms are compared in Figure 4. GO experiences about 15% weight loss

The spectrum of PPF shows the CH stretching bands in the range of 2900−3010 cm−1, the CO stretching of the ester groups at 1730 cm−1 and the CC stretching of alkenes at around 1650 cm−1.41 The CH scissoring and methyl rocking appear at 1460 and 1382 cm−1, respectively, and the CH outof-plane bending at ∼750 cm−1; further, the bands at about 1060, 1140 and 1270 cm−1 are ascribed to COC stretching vibrations.11 Regarding the composites, the spectrum is quite similar to that of PPF, with some bands arising from PEG-GO like the OH stretching which is broader and occurs at lower wavenumber compared to the neat polymer, ascribed to H-bonding interactions between the carbonyl of the ester group of PPF and the OH moieties of PEG-GO. Thus, the interactions between molecules containing OH groups and GO result in a broadening of the OH stretching band and induce a moderate red shift.38 A similar downshift can be observed for the CO stretching band, which occurs at about 7 and 17 cm−1 lower than that of neat PPF in the nanocomposites with 0.5 and 3.0 wt % GO, respectively, yet another sign of the intense polymer−nanofiller interactions. XRD Measurements. X-ray diffractograms were also recorded to investigate potential changes in the crystalline structure. As known, raw graphite presents a sharp diffraction peak at 2θ = 26.5°, corresponding to a d-spacing of 0.336 nm.42 As can be observed in the XRD of GO (Figure 3), the

Figure 4. TGA thermograms under nitrogen atmosphere of GO, PEG, PEG-GO, PPF and the nanocomposites with different GO loading.

below 200 °C as a result of the evaporation of adsorbed water, and another 20% mass loss between 200 and 250 °C ascribed to the decomposition of oxygen-functional groups at the GO surface such as epoxide, hydroxyl, carboxylic acid, etc.35 The steady mass loss above 250 °C is attributed to the additional elimination of functional groups.39 The thermogram of PEGGO displays several decomposition stages: two below 250 °C, similar to those found in GO, and the third in the range of 390−480 °C, attributed to the degradation of PEG chains adsorbed onto the GO surface.40 The thermal stability of PEGGO is higher than that of GO, ascribed to the presence of the polymeric chains on GO that interact via strong inter- and intramolecular interactions.26 On the other hand, neat PPF exhibits a single degradation step that starts (Ti) at about 285 °C and exhibits the maximum rate of weight loss (Tmax) at about 352 °C (Table 1). A one-step decomposition process can also be observed for the composites, while shifted to higher temperatures, corroborating the outstanding thermal stabilization effect induced by the presence of PEG-GO. As shown in Table 1, Tmax gradually rises upon increasing GO concentration, by up to 43 °C for the composite with 3.0 wt % loading. An analogous tendency is found for Ti and the temperature of 10% weight loss (T10) that exhibit maximum increments of 25 and 36 °C, respectively. The PEG-GO randomly and well distributed inside the matrix could act as a barrier and a thermal protective material to isolate the polymeric chains from the flame and retard the diffusion of decomposition products from the interior of the matrix to the gas phase via formation of a tortuous pathway. A similar effect of thermal stability enhancement has been reported for polypropylene (PP)/ chemically reduced graphene oxide (rGO)46 and poly(ethylene) (PE)/functionalized GO nanocomposites,47 ascribed to the free radical transfer between the matrix and graphene nanosheets and the barrier effect of rGO or GO. Further, the strong PPF−PEG and PPF-GO H-bonding and polar interactions would restrict main chain rotational movement, thus decreasing the amplitude of the molecules moving

Figure 3. X-ray diffraction patterns of GO, PEG-GO, PPF and PPF/ PEG-GO nanocomposites with 0.5 and 3.0 wt % GO content.

characteristic (002) peak of graphite at 26.5° vanished upon oxidation, whereas another peak appears at 11.4° corresponding to the (001) reflection of GO.43 The d-spacing in GO is 0.776 nm, about 2.3-fold larger than that of graphite. The rise in the d-spacing is attributed to the presence of epoxide and hydroxyl groups intercalated between the GO layers and a change of hybridization of the carbon atoms from sp2 to sp3.44 Regarding PEG-GO, the (001) reflection is broader, less intense and appears at 2θ = 7.3°, which matches with a d-spacing of 1.211 nm. This left shift can be attributed to the intercalation of PEG chains in the interlayer spacing of GO, and has been previously described for other PEG/GO composites.45 In addition, two diffraction peaks at 2θ = 19.3° and 23.4° due to the diffraction of the (120) and (001) planes of PEG monoclinic unit cell can be observed. Regarding the diffractogram of PPF, a single wide reflection in the range of 2θ = 8−28° can be visualized, which corroborates its amorphous nature. This broad peak can also be observed in the diffractogram of the nanocomposites, albeit shows reduced intensity, together with that corresponding to the (001) reflection of GO; this peak occurs slightly shifted toward lower 2θ compared to that of PEG-GO, suggesting a F


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composites, indicating higher level of hydrophilicity (wettability), ascribed to the large number of surface oxygencontaining groups of GO combined with the hydroxyl moieties of PEG that can form H-bonds with the water molecules. The developed nanocomposites display θ values in the range of 43− 61°, hence are postulated to be appropriate for cell attachment and proliferation. With regard to the water uptake (Table 1), PPF presents a low value of about ∼3%, which rises gradually on increasing PEG-GO loading, consistent with its highly hydrophilic nature, and the composite with the highest nanofiller concentration displays a water absorption close to 9%. On the whole, the composites display reasonably good water resistance, which is advantageous in order to preserve their dimensional stability after contacting with fluid environments. The biodegradability is another key factor for the choice of materials for tissue engineering. In general, a high biodegradation rate is desired, and the ideal in vivo degradation rate would be similar to that of tissue formation. Consequently, an in vitro degradation investigation in PBS at 37 °C was performed, and the percentage mass loss for the diverse samples is collected in Table 1. Neat PPF shows a relatively small weight loss of about 3.5% after 7 weeks, attributed to the hydrolytic degradation of the ester bonds, leading to fumaric acid and propylene glycol.3 An increase in weight loss is found for the nanocomposites, similarly to the trend observed for the water absorption, albeit the percentage of increase is significantly smaller. The polar hydrophilic groups on the GO surface accelerate the hydrolysis, hence the weight loss increases with increasing nanofiller loading. A faster hydrolysis has also been observed for other polyester biocomposites such as PLLA/GO.49 This increased degradation rate would be

under the temperature influence, thereby resulting in improved thermal stability. Contact Angle, Water Uptake, Biodegradability and Protein Absorption. With a view to use the developed nanocomposites for medical applications, it is important to assess their degree of hydrophilicity, which can be evaluated via water contact angle (θ) measurements. Low θ values indicate the formation of a more hydrophilic surface and are a sign of cell attachment preference compared to more hydrophobic surfaces. Indeed, most favorable θ values for protein adsorption and cell adhesion have been reported to be in the range of 40− 70°.48 Figure 5 compares the contact angle values for the

Figure 5. Water contact angle θ (solid bars) and protein adsorption (dashed bars) for PPF/PEG-GO composites with different GO contents.

different samples. PPF can be regarded as a hydrophilic material, because it presents a θ value lower than 90°. A clear drop in θ is observed on increasing PEG-GO content in the

Figure 6. Tensile properties of PPF/PEG-GO composites with various GO loadings at 23 °C and 50% RH and at 37 °C in SBF medium: (a) Young’s modulus; (b) tensile strength; (c) elongation at break; (d) toughness. G


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Often materials for tissue engineering stay in the body for long periods of time, and their mechanical performance should be preserved until the new tissue can assume the mechanical function; consequently, it is crucial to assess the mechanical characteristics of the developed nanocomposites in a biological state such as in a SBF medium at 37 °C, and the data obtained under such conditions are also displayed in Figure 6. The analysis of the results attained under dry environment and after subjecting to SBF corroborates that the stiffness and strength of every sample drops upon testing in aqueous conditions, even though the degree of the fall rises on increasing GO content. This trend is likely associated with the increase in the degree of hydrophilicity, biodegradation rate and water uptake found on increasing nanofiller loading (Table 1), hence higher plasticization effect of absorbed water, which diffuses through the samples causing them to swell, leading to a diminution in the degree of cross-linking among PPF chains, thus a fall in stiffness and strength. Accordingly, neat PPF, which does not contain readily reactable groups, displays the smallest reduction in E and σy (about 40 and 25%, respectively), whereas the nanocomposite with the highest GO loading, with larger amount of oxygen-containing groups, shows around 56 and 38% reduction, respectively, compared to dry conditions. The loss in properties found herein is smaller than that reported for poly(propylene fumarate-co-caprolactone diol) reinforced with HA,52 which could be related to the higher level of hydrophilicity of HA compared to GO. Interestingly, the elongation at break of neat PPF and the composites with low GO loading decreases in SBF compared to dry conditions, whereas εb of the composites with 2.0 and 3.0 wt % GO loading rises. This behavior could be attributed to the competition of two opposite factors: on the one hand, the hydrolytic degradation provokes the breakage of the ester linkages, therefore reducing the polymer chains,53 which would be reflected in lower εb values. On the other hand, the presence of residual moisture would act as a plasticizer, increasing the ductility of the polymer. It seems that at low GO concentrations, the former factor predominates, whereas at higher loadings the plasticizing effect prevails, and the overall result is an increase in εb. However, the toughness of the samples is systematically lower under SBF medium, hinting that in the composites with higher nanofiller loadings the loss in strength overweighs the rise in ductility. Nonetheless, the differences between dry and aqueous conditions diminish on increasing GO loading, and the variation in the composite with the highest loading is lower than 15%. On the whole, the diminution (in percentage) in mechanical properties after immersion in SBF environment seems to be straightforwardly connected to the level of hydrophilicity, water absorption and biodegradation of the nanocomposites. Analogous behavior was found for various chitosan/polyester blends, where the drop in mechanical performance was stronger for the samples that displayed higher water uptake.54 Further, experimental results reveal that the developed composites can retain sufficient mechanical strength in the biological state for providing effective support for new tissue development. Dynamic Mechanical Analysis. The viscoelastic properties of the composites were analyzed by DMA, which gives information on the stiffness of the materials, and can be useful to find their glass transition temperature (Tg) and to differentiate transitions arising from other molecular motions. The temperature dependence of the storage modulus (E′) and loss factor (tan δ) for the different samples is depicted in

beneficial from a practical viewpoint because it would enable a faster elimination of the material from the body. On the other hand, the capability to adsorb proteins from the culture medium can also influence cell adhesion.1 Therefore, the protein absorption ability of the nanocomposites was studied and the data obtained are displayed in Figure 5. A noticeable rise in this property is found on increasing GO content, leading to around 3-fold increase at 3.0 wt % loading. This enhanced capability for adsorbing protein might be induced by the rougher surface of this composite (Table 1), as explained previously for poly(caprolactone fumarate) (PCLF)/ PPF blends.11 Thus, increased roughness combined with higher hydrophilicity is postulated to enhance protein adsorption, as reported for PE/graphene composites,50 and consequently favor cell attachment and proliferation. Tensile Properties. The intrinsic mechanical properties of composites are crucial for tissue engineering applications. For instance, for bone tissue engineering, materials need to have a moderately high Young’s modulus in order to resist the load at the position of the wound. Even so, a good balance between flexibility and strength is generally desired. In this regard, the static mechanical properties of PPF/PEG-GO nanocomposites were investigated via tensile experiments, and their Young’s modulus (E), tensile strength (σy), elongation at break (εb) and toughness (T) at 23 °C and 50% RH are compared in Figure 6. Neat PPF exhibits an E value of about 1 GPa, consistent with the results reported previously.9 The addition of PEG-GO causes a significant increase in the biopolymer modulus, leading to about 3-fold enhancement at 3.0 wt % GO loading. The outstanding modulus improvement observed in these nanocomposites indicates de high reinforcing efficiency of PEG-GO, likely arising from the combination of a random and homogeneous nanofiller dispersion, a strong PPF−PEG-GO adhesion via hydrogen bonding and polar interactions combined with the high modulus of GO (∼210 GPa).51 An analogous trend is found for σy, which rises gradually on increasing GO concentration, leading to approximately 2.2-fold increase at the highest loading. Interestingly, the reinforcement effect found here is better than that reported for comparable loadings of other nanofillers like SWCNTs, multiwalled carbon nanotubes (MWCNTs) or fullerenes.9,10 The noncovalent functionalization of GO would result in an improved dispersion within the matrix and more effective stress-transfer, yielding higher reinforcement effect. Focusing on the ductility of the samples (Figure 6c), PPF presents a low elongation at break value (∼4.3%), which decreases at high GO contents, the decrement being ∼43% for the composite with the highest loading. This reduced plasticity is likely related to the presence of matrix−nanofiller H-bonding interactions that noticeably hamper the ductile flow of the polymer segments, thus resulting in smaller εb values. Regarding the toughness, estimated by the area under the stress−strain curve (Figure 6d), a rise is detected with increasing GO loading, showing a maximum in the range of 0.5−1.0 wt % and then decreases slightly. The hydrogen bonding interactions between GO and the biopolymer prevent nanofiller aggregation, which frequently causes slippage between nanofillers or acts as a source of stress concentration sites or crack initiators under applied loads, which is reflected in improved toughness. This enhancement is very significant because PPF is a brittle material, and a high toughness is of crucial importance for bone tissue engineering applications. H


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ACS Applied Materials & Interfaces Figures 7a,b, respectively, and their Tg values are summarized in Table 1. Neat PPF shows a constant although gentle drop in E′

which is only partly visible in Figure 7b, might be related to intrasegmental cooperative motions.58 With regard to Tg data for the different nanocomposites (Table 1), a gradual rise is found on increasing GO loading, indicating limited motion of the PPF chains in the presence of the nanofiller. Thus, Tg increases by ∼13 °C for the composite with the highest GO loading compared to that of neat PPF, ascribed to strong PEGGO−matrix interfacial interactions that hamper rotational motion within the chains. A slight increase in Tg has also been observed for other GO-reinforced polyester composites,56 attributed to the limiting effect of the molecular movement of polyester by GO. Figure 7b also reveals that the maximum value of tan δ decreases upon rising GO loading, also indicative of reduced chain mobility. Besides, high tan δ values suggest deficiencies in the elasticity of a system. Hence, the reduced tan δ for the nanocomposites suggests a faster recovery rate when the stress is removed compared to neat PPF. Moreover, a widening of tan δ is observed on increasing GO content. Thus, the peak width at half height rises from 16 °C for neat PPF to 23 °C for the nanocomposites with 1.0 and 2.0 wt % GO, which has also been related to a bigger interface.59 Accordingly, an increase in GO concentration should lead to enhanced GO−matrix interactions via hydrogen bonding and larger interfacial area. Overall, DMA data corroborate that the developed nanocomposites exhibit better rigidity than that of the neat biopolymer in a wide temperature range. Antibacterial Properties. Microbial contamination of materials is a severe and widespread problem in surgery because it frequently causes infections that can provoke implant loosening, arthrodeses or amputations. The development of the microbial colonization depends on many factors such as the material chemistry, the physical properties of the surface, the implanted anatomical site, the extent of surgical invasion, the application time and the location. Numerous bacteria and fungi are involved in material infections, although most belong to endogenous bacteria like Gram-negative E. coli and P. aeruginosa or the commensal skin community such as Grampositive S. aureus and S. epidermidis.60 Hence, the antibacterial activity of the developed nanocomposites was assessed using the aforementioned microorganisms, and the results are displayed in Figure 8. Neat PPF does not show any antimicrobial action. In all cases, the antibacterial action rises sharply upon increasing GO concentration, and the best antibacterial activity is found for the nanocomposite with 3.0 wt % loading. This behavior can be explained considering the very

Figure 7. DMA measurements for PPF/PEG-GO composites with various GO loadings: (a) Storage modulus E′ and (b) tan δ as a function of temperature.

with increasing temperature, less prominent than that reported for other synthetic polyesters such as PLLA53 although comparable to that described for natural polyesters like poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).55 At temperatures below Tg, a noteworthy enhancement in E′ can be observed on increasing GO concentration, which corroborates the outstanding reinforcing effect of these 2D nanosheets. Indeed, E′ indicates the ability of a material to accumulate mechanical energy; the higher E′, the stiffer the material is. For instance, at −100 °C, more than 2-fold E′ increment is found for the composite with 3.0 wt % GO. This improvement should be related to the strong nanofiller−matrix interfacial adhesion due to hydrogen bonding interactions between the oxygen containing groups of GO and the −OH moieties of PEG with the CO and −O− groups of PPF, as mentioned previously. These results are in contrast to those reported for a thermoset polyester resin with styrene,56 where E′ decreased for GO loadings ≥0.01 wt % due to nanofiller agglomeration and weak adhesion with the matrix. Interestingly, the enhancement in modulus is even larger at temperatures higher than the Tg (more than 4-fold rise at 100 °C for the nanocomposite with 3.0 wt % loading compared to that of neat PPF). This suggests that the stiffening effect is more significant above the Tg of the matrix, in agreement with the results reported for other polyester-based composites.55,57 This behavior has been ascribed to the formation of a percolated system of filler held together by hydrogen bonding at higher temperatures. Figure 7b displays the evolution of tan δ (damping capacity) vs temperature for the different samples. The position of tan δ peak indicates the Tg. Neat PPF displays three relaxation peaks: the one at low temperature (about −100 °C) has been recognized as a subglass β transition process, like that found in other polyesters, assigned to the reorientation of the ester groups;54 that at about 22 °C, named α relaxation, corresponds to the glass transition and the one in the range of 80−130 °C,

Figure 8. Antibacterial activity of PPF and PPF/PEG-GO composites against S. aureus, S. epidermidis, P. aeruginosa and E. coli. I


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demonstrate that neat PPF does not exhibit any toxicity to NHDF, with cell viability values >97% after 24 and 72 h of incubation. This behavior is in agreement with preceding investigations that confirmed the exceptional biocompatibility of PPF with diverse cell types, and that it behaves similarly to known noncytotoxic polymers like PE.66 Composites with GO ≤ 2.0 wt % are also nontoxic, displaying cell viability values in the range of 90−96% after 24 h of incubation, and these somewhat increase after 72 h of incubation. Nonetheless, cell viability drops slightly, by about 11% compared to neat PPF, when NHDF cells are incubated with the nanocomposite with 3.0 wt % GO, albeit it can still be regarded as nontoxic material (cell viability ≥ 85%). The smaller viability observed at 24 h compared to values attained after 72 h of incubation might be associated with the interaction between GO and the cells.34 Afterward, the residual viable cells would begin to proliferate thereby leading to increased viability. A number of groups have recently explored the in vitro cytotoxic effects of graphene and its derivatives, and contradictory results have been reported.31,64,67−70 Some of them suggested that, analogous to other carbon nanomaterials, they can induce cell damage via different mechanisms including plasma membrane disruption,67 impairment of mitochondrial activity68 as well as induction of oxidative stress and DNA damage64 that might lead to apoptotic and/or necrotic cell death. Wang et al.69 proved that GO induced important cytotoxicity to NHDF at concentrations above 50 mg/L, whereas Hu et al.70 stated that this nanomaterial only slightly reduced cell proliferation rates without causing apoptosis or cell death at exposure concentrations ≥85 mg/L. Conversely, upon coating with a biocompatible polymer (e.g., PEGylation) GO exhibited negligible toxicity toward many cell lines even at high concentrations (up to 100 mg/L).31 There is a general agreement on the fact that the toxicity of GO is dose- and time-dependent, and that it does not exert toxicity when applied at low concentrations. More importantly, polymer coating can alter GO uptake mechanism, rendering it less toxic and more biocompatible. Thus, cell viability data suggest that the noncovalent functionalization of GO with PEG, a nontoxic and biocompatible polymer, reduces its cytotoxicity toward human cells, likely related to the presence of the biopolymer physically attached to the GO surface and the improved GO dispersion, facts that are postulated to reduce the nanomaterial toxicity. On the whole, experimental data indicate that PPF/ PEG-GO nanocomposites have good in vitro cytocompatibility and hence are prospective candidates for medical applications.

large GO surface area that enables a large bacteria−nanofiller interfacial contact area. Systematically, the biocide effect is stronger versus Gram-positive cells, reaching a value close to 2.5 for the composite with the highest nanofiller loading. Thus, for this type of bacteria, efficient antibacterial activity is already attained at 1.0 wt % GO content, whereas in the case of Gramnegative ones, the bacterial inactivation is only effective for the composites with the highest GO concentration. On the other hand, hardly difference is observed between the activity toward S. aureus and S. epidermidis, and the same occurs for the inactivation of E. coli and P. aeruginosa, indicating that the different toxicity is mainly related to the different characteristics of the cell wall of Gram-negative and Gram-positive bacteria.61 In addition, the Gram-negative bacteria has an external membrane incorporating lipopolysaccharides that shields the peptidoglycan layer. Besides, the discrepancies might be associated with their different form and size.35 The antibacterial activity of graphene-based materials is well recognized, albeit the factors responsible for the biocide action of GO nanosheets are not understood yet. Several mechanisms have been proposed including cell membrane harm induced by direct contact of the bacteria with the very sharp edges of the GO nanowalls,62 trapping of microorganism within the graphene nanosheets63 and oxidative stress due to the generation of reactive oxygen species (ROS).64 In particular, GO nanosheets can generate hydroxyl radicals that attack the carbonyl groups of the peptide linkages of the bacterial cell wall and damage the cellular components such as lipids, proteins and DNA, thus leading to the destruction of the bacteria. Several studies have provided evidence supporting the ROS induced toxicity of graphene and GO nanosheets on cellular systems.64,65 Therefore, the Gram-negative bacteria with an outer membrane are found to be more resistant to the membrane injure provoked by GO nanosheets than the Grampositive microorganisms missing the external membrane. Overall, experimental results disclose the high capability of the composites with high GO loading to control the growth of human pathogenic bacteria. Cytotoxicity. From an application viewpoint, it is crucial to assess the toxicity of the developed composites. Normal human dermal fibroblasts (NHDF) were selected to evaluate the cytotoxicity of neat PPF and the different composites, because these represent one of the most common tissues that PPF/ PEG-GO composites could interact with as bone tissue scaffolds, and the results are shown in Figure 9. The results

4. CONCLUSIONS PPF/PEG-GO nanocomposites have been successfully prepared via sonication and thermal curing, and their morphology, structure, thermal stability, water absorption, hydrophilicity, hydrolytic degradation, protein absorption capability, mechanical performance, cytotoxicity viscoelastic and antibacterial properties have been evaluated. The noncovalent functionalization of GO with PEG reduced its aggregation tendency and cytotoxicity without impairing its outstanding properties. X-ray diffractograms indicated the presence of PEG chains in the interlayer spacing of GO. SEM images showed a random and homogeneous distribution of PEG-GO within the copolyester matrix, and IR spectra suggested the existence of strong Hbonding interactions between the carbonyl of the ester group of PPF and the OH moieties of PEG-GO. The nanofiller acted as a barrier and a thermal protective material and significantly

Figure 9. Cytotoxicity of PPF and PPF/PEG-GO composites against NHDF for incubation periods of 24 h (solid lines) and 72 h (dashed lines). J


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phate) Composites: Preparation, Characterization and In Vitro Degradation. Acta Biomater. 2009, 5, 628−635. (8) Mistry, A. S.; Pham, Q. P.; Schouten, C.; Yeh, T.; Christenson, E. M.; Mikos, A. G.; Jansen, J. A. In Vivo Bone Biocompatibility and Degradation of Porous Fumarate-based Polymer/Alumoxane Nanocomposites for Bone Tissue Engineering. J. Biomed. Mater. Res., Part A 2010, 92, 451−462. (9) Shi, X.; Hudson, J. L.; Spicer, P. P.; Tour, J. M.; Krishnamoorti, R.; Mikos, A. G. Rheological Behaviour and Mechanical Characterization of Injectable Poly(propylene Fumarate)/Single-Walled Carbon Nanotube Composites for Bone Tissue Engineering. Nanotechnology 2005, 16, S531−538. (10) Lalwani, G.; Henslee, A. M.; Farshid, B.; Parmar, B.; Lin, L.; Qin, Y.-X.; Kasper, F. K.; Mikos, A. G.; Sitharaman, B. Tungsten Disulfide Nanotubes Reinforced Biodegradable Polymers for Bone Tissue Engineering. Acta Biomater. 2013, 9, 8365−8373. (11) Wang, S.; Kempen, D. H.; Simha, N. K.; Lewis, J. L.; Windebank, A. J.; Yaszemski, M. J.; Lu, L. Photo-crosslinked Hybrid Polymer Networks Consisting of Poly(propylene fumarate) (PPF) and Poly(caprolactonefumarate) (PCLF): Controlled Physical Properties and Regulated Bone and Nerve Cell Responses. Biomacromolecules 2008, 9, 1229−1241. (12) Wang, K.; Cai, L.; Hao, F.; Xu, X.; Cui, M.; Wang, S. Distinct Cell Responses to Substrates Consisting of Poly(ε-caprolactone) and Poly(propylene fumarate) in the Presence or Absence of Cross-links. Biomacromolecules 2010, 11, 2748−2759. (13) Hollinge, J. O. An Introduction to Biomaterials, 2nd ed., CRC Press: Boca Raton, FL, 2011. (14) Hakimimehr, D.; Liu, D.-M.; Troczynski, T. In-situ Preparation of Poly(propylene Fumarate)-Hydroxyapatite Composite. Biomaterials 2005, 26, 7297−7303. (15) Mohandes, F.; Salavati-Niasari, M.; Fathi, M.; Fereshteh, Z. Hydroxyapatite Nanocrystals: Simple Preparation, Characterization and Formation Mechanism. Mater. Sci. Eng., C 2014, 45, 29−36. (16) Dreyer, D. R.; Park, S.; Bielawski, C. W.; Ruoff, R. S. The Chemistry of Graphene Oxide. Chem. Soc. Rev. 2010, 39, 228−240. (17) Tavakoli, F.; Salavati-Niasari, M.; Badiei, A.; Mohandes, F. Green Synthesis and Characterization of Graphene Nanosheets. Mater. Res. Bull. 2015, 63, 51−57. (18) Hummers, W. S.; Offeman, R. E. Preparation of Graphitic Oxide. J. Am. Chem. Soc. 1958, 80, 1339−1339. (19) Dreyer, D. R.; Park, S.; Bielawski, C. W.; Ruoff, R. S. The Chemistry of Graphene Oxide. Chem. Soc. Rev. 2010, 39, 228−240. (20) Kovtyukhova, N. I.; Ollivier, P. J.; Martin, B. R.; Mallouk, T. E.; Chizhik, S. A.; Buzaneva, E. V.; Gorchinskiy, A. D. Layer-by-Layer Assembly of Ultrathin Composite Films from Micron-Sized Graphite Oxide Sheets and Polycations. Chem. Mater. 1999, 11, 771−778. (21) Poh, H. L.; Šaněk, F.; Ambrosi, A.; Zhao, G.; Sofer, Z.; Pumera, M. Graphenes Prepared by Staudenmaier, Hofmann and Hummers Methods with Consequent Thermal Exfoliation Exhibit Very Different Electrochemical Properties. Nanoscale 2012, 4, 3515−3522. (22) Nanda, S. S.; Papaefthymiou, G. C.; Yi, D. K. Functionalization of Graphene Oxide and its Biomedical Applications. Crit. Rev. Solid State Mater. Sci. 2015, 40, 291−315. (23) Zhang, Y.; Nayak, T. R.; Hong, H.; Cai, W. Graphene: a Versatile Nanoplatform for Biomedical Applications. Nanoscale 2012, 4, 3833−3842. (24) Lightcap, I.; Kamat, P. V. Graphitic Design: Prospects of Graphene-Based Nanocomposites for Solar Energy Conversion, Storage, and Sensing. Acc. Chem. Res. 2013, 46, 2235−2243. (25) Díez-Pascual, A. M.; Gómez-Fatou, M. A.; Ania, F.; Flores, A. Nanoindentation in Polymer Nanocomposites. Prog. Mater. Sci. 2015, 67, 1−94. (26) Mohandes, F.; Salavati-Niasari, M. In Vitro Comparative Study of Pure Hydroxyapatite Nanorods and Novel Polyethylene Glycol/ Graphene Oxide/Hydroxyapatite Nanocomposite. J. Nanopart. Res. 2014, 16, 2604. (27) Mohandes, F.; Salavati-Niasari, M. Freeze-drying Synthesis, Characterization and In Vitro Bioactivity of Chitosan/Graphene

increased the thermal stability of the matrix. The water uptake, level of hydrophilicity, weight loss in PBS and protein absorption capability rose with increasing GO content in the nanocomposites, which is beneficial for cell adhesion and proliferation. Further, the Young’s modulus, tensile strength and toughness calculated from the stress−strain curves raised on increasing GO loading. More importantly, the nanocomposites maintained acceptable stiffness and strength under physiological conditions. The antibacterial action was investigated toward human pathogen bacteria: Gram-positive S. aureus and S. epidermidis as well as Gram-negative P. aeruginosa and E. coli. The biocide action was enhanced upon increasing GO loading, and was systematically stronger against Grampositive microorganisms. Cell viability experiments corroborated that the developed nanocomposites do not induce toxic effects over NHDF. The results obtained herein suggest the great potential of these new nanocomposites for medical applications, mainly in the bone tissue engineering field.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b05635. TEM images of GO and PEG-modified GO, histograms showing the size distributions of GO and PEG-GO flakes, comparison of data of PPF and nanocomposites with 3.0 wt % GO prepared using either chloroform or water as solvent (PDF).

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AUTHOR INFORMATION

Corresponding Author

́ *E-mail: [email protected] (A. M. Diez-Pascual). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS ́ Dr. Ana Diez-Pascual acknowledges the Ministerio de ́ Economia y Competitividad (MINECO) for a “Ramón y Cajal” Senior Research Fellowship cofinanced by the EU.

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REFERENCES

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Polyethylene Glycol-Modified Graphene

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