Antibacterial zinc oxide hybrid with gelatin coating

Accepted Manuscript Antibacterial zinc oxide hybrid with gelatin coating

Jun Lin, Jianxun Ding, Yanfeng Dai, Xiaolei Wang, Junchao Wei, Yiwang Chen PII: DOI: Reference:

S0928-4931(17)32311-1 doi: 10.1016/j.msec.2017.08.009 MSC 8214

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

19 June 2017 29 July 2017 2 August 2017

Please cite this article as: Jun Lin, Jianxun Ding, Yanfeng Dai, Xiaolei Wang, Junchao Wei, Yiwang Chen , Antibacterial zinc oxide hybrid with gelatin coating, Materials Science & Engineering C (2017), doi: 10.1016/j.msec.2017.08.009

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ACCEPTED MANUSCRIPT Antibacterial zinc oxide hybrid with gelatin coating Jun Lin a, Jianxun Ding b,*,, Yanfeng Dai a, Xiaolei Wang a,c, Junchao Wei a,* Yiwang Chena a

College of Chemistry, Nanchang University, Nanchang 330031, P. R. China Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China c Institue of Translational Medicine, Nanchang University, Nanchang 330031, P. R. China

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* Corresponding authors. E-mail addresses: [email protected] (J. Wei), [email protected] (J. Ding).

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ABSTRACT ZnO has been widely investigated as important biomaterials and antibacterial materials. However, the aggregation of nanoparticles and its potential toxicity may hinder its final application. Herein, biocompatible gelatin chains were grafted on the surface of ZnO via mussel inspired method to prevent the aggregation of the ZnO nanoparticles. The in vitro test showed that the gelatin can greatly improve the biocompatibility of ZnO, while the antibacterial properties of ZnO against both E. Coli and S. Aureus were maintained. Keywords: Zinc oxide; Gelatin; Biocompatibility; Antibacteria; Surface Modification

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1. Introduction Various bacterial infections have been serious problems in clinics, and thus more and more antibiotics or antibacterial peptides have been explored [1]. However, the multi-drug resistance limits the progress of conventional antibacterial agent [2]. Inorganic nanoparticles, due to their excellent properties, have shown much applications in optics, electronics, energy storage, controlled delivery and many other biomedical fields [3-7]. Ag [8], CuO [9], TiO2 [10], ZnO [11], carbon dots [12] and many other inorganic nanoparticles with antibacterial properties have aroused much attention [13]. ZnO has been widely applied in optical devices [14], sensors [15], imaging [16, 17], drug delivery [17, 18] and antibacterial fields [19]. Furthermore, ZnO can also works as functional nanofillers in polymer composites. However, its application has been hindered by limited dispersibility and surface modification techniques [20]. Up to now, many methods, such as ring opening polymerization [21], atom transfer radical polymerization [20, 22] have been used to tune its surface properties and improve its optical, dispersion and biocompatible properties. Various polymer chains have been connected to the surface of ZnO surface via “grafting to” or “grafting from”, for example, Poly(N-isopropylamide) was grafted on the surface of ZnO to realize its thermal responsive properties and can works as a smart drug carrier [23], poly(methyl methacrylate) was tethered on the surface of ZnO to tune the composites’ optical and dielectric properties [22], Poly(lactide) was graft on the surface of ZnO to tune its dispersion and interaction with polymer matrix and thus to improve its mechanical properties [24]. Although much progress has been achieved to graft polymer chains on the surface 1

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of ZnO, much more simple or green modification method is still needed. Furthermore, whether the surface grafted polymer brush may have some effect on its biocompatibility or antibacterial properties should be addressed. Inspired by the adhesion proteins of mussel, Messersmith group proposed a universal surface modification method by dip-coating of objects in the aqueous solution of dopamine [25]. Due to the self-polymerization of dopamine, polydopamine can be formed on various surface, furthermore, the polydopamine can react with various molecules. This green method has been widely used to graft different molecules on various substrates and realize the functionalization of different materials. In this work, gelatin, a biocompatible polymer, was grafted on the surface of ZnO to form a new hybrid ZnO@Gelatin via mussel inspired method (scheme 1). Firstly, ZnO nanoparticles were treated with dopamine to form a layer of polydopamine on its surface, and then gelatin was grafted via the reaction between gelatin and polydopamine. The optical and in vitro test showed that the surface modification by Gelatin may increase the biocompatibility of ZnO, in addition, the new hybrid also show good antibacterial properties, which have similar antibacterial ability as ZnO, showing its potential application as a biocompatible antibacterial agent.

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Scheme 1. Synthesis route of Zinc Oxide with Gelatin coating 2. Experimental section 2.1. Materials Zinc acetate dihydrate (Zn(CH3COO)2·2H2O) was purchased from Sigma Aldrich. KOH was purchased from Tianjin Damao Chemical Reagent Factory. Methanol and ethanol absolute were purchased from XiLong Sciences Ltd.. Dopamine, Tetrabutylammonium bromide and tris(hydroxymethyl)aminomethane(Tris) were obtained from Energy Chemical Reagent Co. Ltd.. Gelatin is purchased from Sinopharm Chemical Reagent Co. Ltd.. Nutrient broth, nutrient agar, agar-agar used for antimicrobial studies were purchased from Hangzhou basebio Biotechnology Co., Ltd.. PBS Phosphate buffer are purchased from Beijing Solarbio Technology Co., Ltd.. All chemicals are used without further purification. 2.2. Synthesis of ZnO NPs ZnO NPs were synthesized by the modified sol-gel route [26]. Briefly, 2.75 g of Zn(CH3COO)2·2H2O and 4.02 g of tetrabutylammonium bromide were added into 150 mL of ethanol. The mixture was refluxed for 1 h, and then 5mL of KOH (1.7 M) ethanol solution was added. 12 hours later, the white precipitates were collected by centrifugation, and washed several 2

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times with methanol. 2.3. Preparation of gelatin-functionalized ZnO (ZnO@Gelatin) ZnO NPs was dispersed in 10 mM Tris-buffer solution (pH=8.5), and then Dopamine (2 mg/mL) was added into suspensions under stirring. After 2 hours, polydopamine coated ZnO (ZnO@PDA) were collected by centrifugation, and then washed with demonized water for several times. ZnO@Gelatin was prepared via the reaction between Gelatin and ZnO@PDA. Briefly, gelatin was added into the ZnO@PDA suspensions and stirred at 35℃ for 24 hours. The final product ZnO@Gelatin were obtained by centrifugation and washed with water. 2.4. Characterizations Fourier-transform infrared (FT-IR) spectra were recorded on a Fourier-transform infrared spectrophotometer (Shimadzu IR Prestige-21, Nakagyo-ku, Japan) using potassium bromide (KBr) disks. X-ray photoelectron spectroscopy (XPS) was measured on a X-ray photoelectron spectroscopy (Thermo-VG; ESCALAB 250) with an Al Kα radiation X-ray source. Morphologies and microstructures of different samples were investigated by transmission electron microscopy (TEM, Tecnai G20, FEI, USA). The crystalline structure was measured by X-ray diffraction (XRD, Mini Flex 600, Japan). Thermal gravimetric analyses (TGA) were carried out under N2 atmosphere from room temperature to 800 °C at a heating rate of 10 °C/min with a thermogravimetric analyzer (Netzsch STA409PC, Selb, Germany). The UV-Vis spectra and photoluminescence spectra were measured by UV-Vis spectrophotometer (Lambda 750s, PerkinElmer, USA) and Fluorescence spectrometer (Shimadzu, RF-5301 PC, Japan), respectively. The zeta potential and particle size were measured by a High Sensitive Zeta potential and particle size analyzer (Brookhaven, 90Plus PALS, USA). 2.5. Cytotoxicity tests MTT assay was used to test the cytotoxicity of different samples against 3T3 cells by adapting a previously described method [27]. Briefly, Different amounts of ZnO, ZnO@PDA and ZnO@Gelatin were dispersed in cell culture medium (DMEM) and the concentration of each samples were set to 0.3125, 0.625, 1.25, 2.5, 5.0, 10.0 mg/mL, respectively. After 24 h, the solid samples were removed from the culture medium, which were used for cell culture. 3T3 cell were suspended in DMEM supplemented with 10% (v/v) FBS at a density of 104 cells/mL, and 100 μL of cell suspension solution was pipetted into 96-well plates. After incubation at 37℃ under 5% CO2 atmosphere for 24 h, the medium was replaced by the previously treated DMEM culture medium. After 24 hours’ incubation, the optical density (O.D.) was read on a multi-well microplate reader at 630 nm. The cytotoxicity for each sample was tested in triplicates. Cells cultured with fresh DMEM were used as Control. 2.6. Antibacterial assays The antibacterial activities were studied by the bacterial growth kinetic analysis according to reference’s method [28]. Briefly, in an aseptic condition, 80 μL of respective bacteria culture was diluted to 8 mL using nutrient broth, followed by growth till mid log phase at 37 °C. And then, 100 μL of bacterial mother cultures were added to 3 mL of nanoparticles suspensions at different concentrations (16, 25, 50, 100, 250 and 500 μg/ml), and then cultured at 37 °C, after a required time, the optical density (O.D.) at 600 nm of each sample were measured by UV-Vis spectrophotometer. Culture without samples was taken as positive control. Each test was measured in triplicates. 3

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3. Results and discussion 3.1. Graft of gelatin onto surface of ZnO When ZnO nanoparticles were dispersed in dopamine solution, due to the self-polymerization of dopamine, a thin layer of poly(dopamine) was coated on the surface of ZnO, and then, the gelatin was grafted on the surface of ZnO via the reaction between gelatin and polydopamine. This process can be verified by the XPS spectra of different samples. As shown in Fig. 1, characteristic peaks at 1021 and 1044 eV are ascribed to the characteristic peaks of Zn2p, while the peak at 530 eV is attributed to the signal O1s. Most peaks are nearly the same with the spectra of ZnO, ZnO@PDA and ZnO@Gelatin. When PDA was coated on the surface of ZnO, a minor peak around 399 eV, derived from the N1s signal, was observed (the inset in Fig. 1), and this demonstrated the existence of PDA layers. The quantitative element content in ZnO@PDA was 40.6% (Zn), 2.3% (N) and 57.07% (O). When gelatin was grafted on the surface of ZnO, the N content will increased a lot (Fig. 1), and the quantitative element content were 13.1% (Zn), N 28.4% (N) and 58.5% (O), respectively.

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Fig. 1. XPS spectra of ZnO, ZnO@PDA, and ZnO@Gelatin

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The coating amount of PDA and gelatin on the surface of ZnO was measured by thermal gravimetric analysis (TGA). As shown in Fig. 2, the pristine ZnO displays a weight loss of about 9.3% when heated from 25 to 600 °C. The weight loss can be classified into two steps. The first is below 120 °C, which is due to the evaporation of absorbed water and solvent. While the second step is above 120 °C, and the weight loss in this step is 6.3%, which is mainly derived from the decomposition of the surface organic content (tetrabutylammonium bromide). In the period between 120 and 600 °C, The weight loss of ZnO@PDA and ZnO@Gelatin was 7.4% and 23.4%, respectively, which are derived from the decomposition of PDA and Gelatin. Compared with the weight loss of ZnO, the coating amount of PDA and gelatin was about 1.1 and 16.0%, respectively. These results further demonstrated the successful preparation of ZnO@PDA and ZnO@Gelatin hybrid materials.

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Fig. 2. TGA curves of ZnO, ZnO@PDA, and ZnO@Gelatin The X-ray diffraction (XRD) patterns of different samples are shown in Fig. 3. The typical crystalline face (100), (002), (101), (102), (110), (103) and (112) appeared in the XRD patterns of ZnO, showing the representative wurtzite nanostructures as observed elsewhere [29]. All these peaks remain the sample in the XRD patterns of ZnO@PDA and ZnO@Gelatin, which are consistent with the pristine ZnO, implying that the hexagonal wurtzite structure of ZnO is well-maintained after the coating process.

Fig. 3. XRD patterns of ZnO, ZnO@PDA, and ZnO@PDA@Gelatin The morphologies of different samples were shown in Fig. 4. The ZnO particles show sphere structure, however, the nanoparticles aggregate heavily (Fig. 4A). When PDA was coated on the surface of ZnO, the nanoparticles size did not change too much, however, due to the PDA layer contains many hydroxyl or amino groups, and thus there may be strong hydrogen bonds between ZnO@PDA, which makes the nanoparticles connect one by one (Fig. 4B). When gelatin was grafted on the surface of ZnO nanoparticles, the strong hydrogen bonds still exist, however, the gelatin chains can stretch in to the solvent, the interactions between the solvent and gelatin chains 5

ACCEPTED MANUSCRIPT decrease the hydrogen bonds and make the distance of nanoparticles become longer, so it can prevent the aggregation of nanoparticles, and also reduce the strong interactions between particles, and thus the particles can disperse well (Fig. 4c). This result can demonstrated that it is a good method to prevent the aggregation of ZnO nanoparticles by grafting gelatin chains on its surface, which may have potential applications to prepare ZnO based polymer composites by realizing the homogeneous dispersion of ZnO nanoparticles. A

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Fig. 4. TEM images of ZnO, ZnO@PDA, and ZnO@Gelatin

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When the surface composition of nanoparticles was changed, the physical properties may also change. The zeta potential of ZnO was positive 41.8 (mV), after coating with PDA layers, the surface zeta potential changed to negative (-22.7 mV), this implying the surface composition of ZnO have been changed. When gelatin was grafted on the surface of ZnO, the zeta potential of ZnO@Gelatin was -27.8 mV. In addition, the particle sizes of ZnO, ZnO@PDA and ZnO@Gelatin was about 65, 177 and 331nm, respectively. These results can also demonstrate that the surface grafting reaction was successful. As shown in Fig. 5, the characteristic absorption peaks of ZnO, ZnO@PDA, and ZnO@Gelatin are at 346, 348, and 354 nm, respectively. The observed red-shift of UV absorption spectra may results from the increase of particle sizes, and showed decrease in the band gap [30]. Furthermore, the photoluminescence spectra were performed to explore the photoconductivity characteristics of NPs using a laser source with an excitation wavelength at 325 nm (Fig. 5B). As expected, a primary peak is observed at 373 nm and a broad PL band is centered at 539 nm in the wavelength ranging from 400 nm to 700 nm. The dominant peak belongs to the recombination of free excitons through an exciton–exciton collision process [31]. The broad PL band is attributed to the oxygen vacancies and Zn interstitials [32,33]. On the contrary, upon coating PDA on the surface of ZnO, the luminescent peaks ranging from 400 to 700nm were quenched, and the reasons may be that, during the coating process, some dopamine was oxided to quinine structure. The PDA layer may works as electron acceptor, and thus when the ZnO@PDA was excited with UV light, the released electron will interact with PDA layer, resulting in the quenching of PL spectra. This result is a little like the published schemes of ZnO as a probe to measure the content of dopamine [34].

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Fig. 5. UV-Vis spectra (A) and PL spectra (B) of ZnO, ZnO@PDA, and ZnO@Gelatin

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An efficient surface modification method should improve certain properties of nanomaterials without decrease its bulk properties. Gelatin is an important biopolymer, and has been widely used in tissue engineering and drug delivery. ZnO is also an excellent candidate in biomedical field. Although its low toxicity makes it possible to use, there were still some reports about its toxicity or surface defect induced toxicity. When gelatin was grafted on the surface, the biocompatibility of the hybrid should be better than the pure ZnO. Fig. 6 shows the cytotoxicity test about ZnO, ZnO@PDA and ZnO@Gelatin against 3T3cells. The cell viability of ZnO was about 82%, event at higher concentration, the cell viability did not change too much, showing its good biocompatibility. The cell viability of ZnO@PDA was a little lower than that of ZnO nanoparticle, however, their results did not show much evident difference, which can demonstrated that PDA layer did not bring too much cytotoxicity. When gelatin was grafted, the cell viability of ZnO@Gelatin increased to more than 95%, much higher than that of ZnO, implying that this kind of materials is good candidate biomaterials. Furthermore, it may be concluded that this method is able to increase the biocompatibility of nanoparticles via grating biocompatible Gelatin on their surface.

Fig. 6. MTT assay of ZnO, ZnO@PDA and Zno@Gelatin against 3T3 Cells The antibacterial properties of ZnO, ZnO@PDA and ZnO@Gelatin against gram positive and negative bacterials cells were measured by the growth kinetic curves. As shown in Fig. 7, all the samples show evident render effect on both kind of bacterial cells, and showing a dose-dependent tendency. ZnO@Gelatin and ZnO shows similar results toward different cells. As for the E.coli, the inhibition results are not very good. When the concentration is 500 ug/mL, the particles can 7

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show killing effect. While for the S. Aureus cells, the ZnO and ZnO@Gelatin can show evident hindering effect at a lower concentration (100 ug/ML). These results demonstrated that the surface gelatin did not change the antibacterial properties of ZnO.

Fig. 7. E.Coli bacterial growth curves at different concentrations of ZnO (A), ZnO@PDA (B) and ZnO@Gelatin (C). S.Aureaus growth curves at different concentrations of ZnO (D), ZnO@PDA (E) and ZnO@Gelatin (F) 4. Conclusion An efficient mussel inspired method was proposed to graft Gelatin on the surface of ZnO nanoparticles. The UV-Vis and photoluminencent results showed that this method could quench the Luminescent of ZnO ranging from 400-700nm. The Gelatin layer can greatly improve the biocompatibility of ZnO nanomaterials, meantime, the surface modification process does not affect the antibacterial properties of ZnO. All these results demonstrated that Gelatin modifed ZnO showed excellent biocompatibility, and have much potential application as biocompatible 8

ACCEPTED MANUSCRIPT antibacterial materials. The surface modification may greatly promote ZnO’s application in clinic by reducing its cytotoxicity. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Nos. 51663017, 51463013, 51673190, and 51673187)

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ACCEPTED MANUSCRIPT Highlights * Biocompatible gelatin was successfully grafted on the surface of ZnO nanoparticles and thus prevent the aggregation of ZnO. * The prepared ZnO@Gelatin showed low cytotoxicity and can be used as

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biocompatible antibacterial agent.

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