Materials Science and Engineering C 55 (2015) 155–165

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Bioinspired synthesis of polydopamine/Ag nanocomposite particles with antibacterial activities Chengjiao Wu a,1, Guoxing Zhang b,1, Tian Xia b,1, Zhenni Li a, Kai Zhao a, Ziwei Deng a,⁎, Dingzong Guo b,⁎, Bo Peng c,⁎ a b c

School of Materials Science and Engineering, Shaanxi Normal University, Xi'an 710062, China College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, United Kingdom

a r t i c l e

i n f o

Article history: Received 25 December 2014 Received in revised form 31 March 2015 Accepted 8 May 2015 Available online 9 May 2015 Keywords: Polydopamine Silver Nanocomposite Particles Antibacterial activity

a b s t r a c t Mussel-inspired chemistry (polydopamine) offers great opportunities to develop inexpensive and efficient process for many types of materials with complex shapes and functions in a mild and friendly environment. This paper describes a facile, yet green approach to synthesize polydopamine/silver (PDA/Ag) nanocomposite particles with a combination use of polydopamine chemistry and electroless metallization of Ag. In this approach, monodisperse spherical polydopamine particles are first synthesized by the oxidation and self-polymerization of dopamine (monomer) in an alkaline water–ethanol solution at room temperature, which are served as the active templates for secondary reactions due to the abundant catechol and amine groups on the surface. Subsequently, the silver precursor-[Ag(NH3)2]+ ions introduced are easily absorbed onto the surface of the PDA particles, and are immediately in situ reduced to metallic Ag nanoparticles with the help of these active catechol and amine groups. During the preparation, no additional reductants, toxic reagents and intricate instruments are needed. These as-synthesized PDA/Ag nanocomposite particles are ideal candidates for antibacterial application because they do not show significant cytotoxicity against HEK293T human embryonic kidney cells in the in vitro cytotoxicity assay, whereas demonstrate enhanced antibacterial abilities against Escherichia coli (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria) in the antibacterial assays. Owing to their excellent cytocompatibilities and antibacterial activities, these PDA/Ag nanocomposite particles can be considered as the promising antibacterial materials for future biomedical applications. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Human health has been greatly threatened by some serious public health risks in recent years, e.g., the outbreaks of the infectious diseases caused by various pathogenic bacteria and the risks of new resistant strains of the bacteria to the current antibiotics. Hence, it is an urgent demand for the pharmaceutical and medical researchers to develop new and effective antibacterial agents against the increasing hygiene demands in the public health care [1–4]. Silver (Ag) has been widely employed as an antibacterial material since ancient times to fight against infections and control spoilage [1, 4]. The past few years have been witness to an unprecedented revolution in particle synthetic methodology, specifically, nanotechnology, because nanoparticles possess unexpected characteristics which cannot be realized by the bulk materials alone. Ag nanoparticles as one of the ⁎ Corresponding authors. E-mail addresses: [email protected] (Z. Deng), [email protected] (D. Guo), [email protected] (B. Peng). 1 These three authors contributed equally to this work.

http://dx.doi.org/10.1016/j.msec.2015.05.032 0928-4931/© 2015 Elsevier B.V. All rights reserved.

most effective antimicrobial agents have been mostly investigated owing to its strong bactericidal activities and broad inhibitory biocidal spectra for many different types of microorganisms including bacteria [1], fungi and virus [5,6],and their relatively low toxicities to humans and other animals [7,8]. Although Ag nanoparticles have been proven as the well-known antibacterial agents in the medical fields to treat infections, burns and chronic wounds, their practical applications are often hampered by the problems of the ease of aggregation, which will inevitably weaken or lose their antibacterial activities [9]. In principle, the antibacterial activities of the nano-structured materials are greatly affected by their nano-scaled sizes. Therefore, the aggregation of the Ag nanoparticles will cause their antibacterial activities to be diminished or even totally lost [10]. To solve this problem, extensive efforts have been spent on the immobilization of the Ag nanoparticles into/onto various matrices so as to enhance their stability and antibacterial activity. So far, a long list of materials, such as silica [11], polymers [12], TiO2 [13], iron oxides [14], multi-walled carbon nanotubes [15] and carbonaceous nanomaterials [16], have been employed as the matrices for supporting Ag nanoparticles. In spite of the success of some methods in which the stability and antibacterial activity of the

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silver nanoparticles could be maintained, it is still worth pointing out that the complicated synthetic strategies and accompanying with the usage of some certain reagents including stabilizers, surfactants and reducing agents are usually required during the preparation [17,18]. In addition, some of the preparation processes are not only resulting in much time and high cost consumption in the practical applications, but also lead to a toxicity for the environment or biological hazards due to the usage of non-biocompatible and hazardous reductants [19–21]. Therefore, a straightforward and environmentally friendly synthesis of silver-based nanocomposite materials with desirable biocompatibility and excellent antibacterial activity is highly expected. Mussels, which are underwater fouling organisms, are able to achieve long-lasting adhesion in a wet environment and attach to any natural or synthetic substrates based on the proteins secreted in the byssus [22]. Inspired by mussel-adhesion phenomena in nature, dopamine, a biomolecule which contains amply catechol and amine groups can self-polymerize and spontaneously form a thin, surface-adherent polydopamine (PDA) coating on various material surfaces. PDA shows the similar characteristics to the adhesive foot protein, Mefp-5 (Mytilus edulis foot protein 5) secreted by mussels [23,24]. More importantly, thanks to the rich amount of active catechol and amine groups, these surface-sticky polydopamine coatings (PDA) were able to serve as the reductants, binding reagents and universal platforms for diverse secondary reactions (e.g., electroless metallization, inorganic mineralization, grafting of various organic molecules/polymers), which will, ultimately, lead to a variety of composite materials with promising functional features [23]. Very recently, the reducing ability of the polydopamine with the metal ions (e.g., silver ions) and its robust adhesion ability have been jointly used for the in situ synthesis of Ag nanoparticles onto diverse material substrates [25–28]. In spite of their success, it is worth pointing out that the substrate materials need to be prepared prior to the coating of PDA, which means that the extra efforts on the substrate materials are required. Therefore, skipping the preparation of substrate materials would, in principle, largely increase the efficiency and avoid the problems that may be encountered during the substrate preparation and/or the subsequent PDA coating step. Additionally, similar to the adhesive foot protein, Mefp-5, PDA has also been demonstrating a good biocompatibility and relatively low toxicity against the human cells [29,30]. The presence of the substrate materials may influence the excellent biocompatibility of the PDA, or cause the problems during the decoration of the Ag nanoparticles, or weaken the unique antibacterial activities of the Ag nanoparticles. Therefore, the template-free synthesis of the PDA/Ag composite particles is highly desired. Herein, we present a straightforward, mild and green approach to prepare polydopamine/silver (PDA/Ag) nanocomposite particles. In this approach, monodisperse submicron-sized polydopamine particles were first synthesized through the oxidation and self-polymerization from its monomer (dopamine) in an alkaline water–ethanol environment at room temperature. Neither core material selection nor core preparation is needed. In light of the abundant surface catechol and amine groups, the surface of polydopamine particles are the active sites for the in situ reduction of the silver precursor [Ag(NH3)2]+ ions, and the formed Ag nanoparticles can be fixed there. This approach shows four highlighted unique characteristics: (1) The whole procedure is easy and carried out in a mild environment, and no intricate instruments and special reagents are needed. In addition, the preparation process is green from the reaction chemicals to products. No toxic or environmentally unfriendly chemicals are involved; (2) Monodisperse submicron-sized PDA particles are straightforwardly synthesized through the self-polymerization of monomer dopamine in a water–ethanol solution at room temperature. Extra template preparation is not necessary, which is not only saving time but also lowering the experimental cost; (3) Benefiting from the abundant active catechol and amine groups on the PDA particle surface, the in situ binding and

reduction of the silver precursor-[Ag(NH3)2]+ ions takes place. Additional functionalization of the template particles and reductants are not needed during this so-called polydopamine-assisted electroless Ag metallization. It is also worth pointing out that these PDA particles can be served as a universal platform for decoration of nanoparticles which is not only restricted in Ag nanoparticles; and (4) The coverage of the Ag nanoparticles can be easily tuned via the adjustment of the concentration of silver precursor in the reaction media. The obtained system is secondary-nucleation-free. In addition, the submicron sized PDA/Ag particles are stable during the antibacterial assays, and can be easily collected after their usage by centrifugation, which is ideal for the recyclable use. Additionally, to demonstrate their potential biomedical applications, the biocompatibility and antibacterial activities of these PDA/Ag nanocomposite particles have also been studied in this work. They exhibit a reasonably good cytocompatibility with human cells and an excellent antibacterial performance against Escherichia coli (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria). 2. Experimental section 2.1. Materials Silver nitrate (AgNO3, ≥ 99.8%), aqueous ammonia (25–28 wt.% aqueous solution) and absolute ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (China) and used without further purification. 3-Hydroxytyramine hydrochloride (Dopamine hydrochloride) was purchased from Sigma-Aldrich and used as received. Ultrapure water (resistivity N 17 MΩ cm−1) from a GZY-P10 water system was used throughout the experiments. 2.2. Preparation of monodisperse polydopamine particles In general, uniform polydopamine (PDA) particles were prepared through the oxidation and self-polymerization of their monomer (dopamine) in an alkaline water–ethanol environment at room temperature [31], as illustrated in Scheme 1a. In detail, first, all of ethanol (40 mL), deionized water (90 mL) and ammonia aqueous solution (NH4OH, 0.6 mL, 25–28%) were mixed with a mild stirring at room temperature for 30 min. Subsequently, a dopamine hydrochloride aqueous solution (50 mg/mL) prepared by dissolving dopamine hydrochloride (0.5 g) in water (10 mL) was injected into the alkaline water–ethanol mixture. The color of this mixture immediately turned to pale brownish and further gradually became too dark brownish. This reaction was allowed to proceed for 30 h before completion. The monodisperse PDA particles were obtained by centrifugation and rinse with water three times. The obtained particles were dried 24 h in vacuum at room temperature for the further use. 2.3. Preparation of polydopamine/Ag nanocomposite particles As illustrated in Scheme 1b, a series of transparent [Ag(NH3)2]+ ion aqueous solutions (1.18 × 10−2 mol/L–1.96 × 10−2 mol/L) were freshly prepared by feeding aqueous ammonia into AgNO3 solution. Subsequently, 0.1 g of PDA particle power was added into 300 mL of freshly prepared [Ag(NH3)2]+ ion aqueous solution. The system was kept magnetically stirring at a speed of 500 rpm in an ice-water bath. After 1 h, the reaction was believed to be complete, and PDA/Ag nanocomposite particles were obtained. These PDA/Ag nanocomposite particles were collected by centrifugation and washing with deionized water several times, and then, they were dried in vacuum at room temperature for 24 h. In the end, this PDA/Ag nanocomposite particle powder was collected and stored in vials for further characterization and biological applications.

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Scheme 1. Schematic diagram illustrating the formation of the PDA/Ag nanocomposite particles.

2.4. Cell culture HEK293T human embryonic kidney cells (kindly provided by Professor Shaobo Xiao, Huazhong Agricultural University, China) were routinely cultured in the Dulbecco's Modified Eagle Medium (Gibco) supplemented with a 10% fetal bovine serum (Gibco), penicillin–streptomycin solution (100 U/mL, 100 μg/mL, Beyotime) at 37 °C in a humidified atmosphere containing 5% CO2. These HEK293T human embryonic kidney cells were seeded at a density of 1 × 104 cells/well in the 96-welled plates in a 100 μL Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C for 24 h under a humidified 5% CO2-contained atmosphere and grew up overnight prior to further studies. 2.5. In vitro cytotoxicity and cell viability evaluation The in vitro cytotoxicities of the PDA particles and PDA/Ag nanocomposite particles were assessed by using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan) assay with HEK293T human embryonic kidney cells. Specifically, the HEK293T human embryonic kidney cells were plated at a density of 1 × 104 cells/well in 96-well plates and cultured under a humidified atmosphere consisting of 5% CO2 at 37 °C for 24 h prior to use. Then, these HEK293T human embryonic kidney cells were incubated in the standard growth medium in the presence of different concentrations (2, 4, 8, 12, 16 and 32 μg/mL) of PDA particles and PDA/Ag nanocomposite particles for another 24 h. Meanwhile, the controls only comprising of culture medium were also prepared. Subsequently, 10 μL of CCK-8 dye aqueous solution was added to each cell cultures, and the systems were maintained at 37 °C for another 4 h. The absorbance was measured with the single wavelength spectrophotometry at 450 nm with a microplate reader (Bio-Rad 680). The experimental results were averaged from nine measurements, and the relative cell viability (%) was determined by comparing the absorbance at 450 nm with the controls in which cell culture mediums were contained. 2.6. Bacterial culture E. coli (ATCC 25922) and S. aureus (ATCC 29213) were selected as the model Gram-negative and Gram-positive bacteria, respectively. For the bacterial culture, two bacterial suspensions were prepared by firstly taking a single colony from the stock bacterial culture with a loop, and then inoculating in 5 mL sterile nutrient broth medium. After 12 h incubation in a constant temperature vibrator at 37 °C with a speed of 200 rpm, both bacterial species at the exponential growth phase were harvested.

control groups. Pure PDA particles (50 μg/mL in both of E. coli and S. aurous samples) were also set as the control groups for both bacterial strains. After shaking for 14 h in a temperature-constant vibrator the turbidities of the bacterial suspensions and two control groups were characterized to estimate the antibacterial activities of the PDA/Ag nanocomposite particles. For the kinetic test, the E. coli and S. aurous suspensions inoculated in the LB liquid medium containing PDA/Ag nanocomposite particles at divers contents (5, 10, 15, 20, 30, 40 μg/mL for E. coli and 5, 10, 20, 40, 60 μg/mL for S. aureus, respectively) were cultivated for 14 h in the temperature-constant (37 °C) vibrator at a speed of 200 rpm. The bacterial inhibition growth curves were expressed by the optical density (O.D.) of the nutrient broth in both mediums measured at a wavelength of 600 nm from 0 to 14 h with an interval of 1 h. Similarly, the bacterial suspensions in which only LB liquid medium, and pure PDA particles and LB liquid medium (40 μg/mL for E. coli and 60 μg/mL for S. aureus) were selected as the control groups. To better expose the performance of all groups in the kinetic tests, the characterizations have been carried out for three times. To further examine the antibacterial properties of the PDA and PDA/ Ag particles, the systems were stained with the live/dead Bacterial Viability Kit according to the following protocol (GENMED). In detail: 2 μL of DAPI (4,6-diamidino-2-phenylindole) and PI (propidium iodide) were mixed before adding the buffer to 100 μL to obtain a 2× staining solution. The same volume of the fluorescently dyed mixture was added into the bacterial suspensions, which were fixed in advance with an appropriate volume of fixative. Then, the samples were mixed thoroughly and incubated in dark at room temperature for 15 min. To observe the dyed samples with a Fluorescence Microscope (Olympus IX71), 10 μL of the stained bacterial suspension was taken and dropped to a glass slide with a square cover slip. 2.8. Characterization TEM observation. Transmission electron microscopy (TEM, JEOL JEM2100, Japan) was used to observe the morphologies of the PDA particles and PDA/Ag nanocomposite particles. All samples were diluted with ethanol and ultrasonicated at 25 °C for 10 min, and then dried onto the carbon-coated copper grids prior to examination. The average diameters of the particles were obtained based on the measurement of more than 100 particles in the TEM images. The polydispersity index (PDI) was calculated as follows: Dn ¼

2.7. Antibacterial assays against E. coli and S. aureus To evaluate the antibacterial activities of the PDA/Ag nanocomposite particles against E. coli and S. aureus, Luria–Bertani (LB) liquid medium turbidity assays were chosen. In detail, 150 μL of bacterial suspension were inoculated in 15 mL of LB liquid medium supplemented with PDA/Ag nanocomposite particles (10 and 50 μg/mL for E. coli and S. aurous, respectively) at various concentrations. The bacterial suspensions only contained LB liquid medium were selected as the negative

Dw ¼

k X

k X N i Di = Ni

i¼1

i¼1

k X

k X Ni D4i = Ni D3i

i¼1

i¼1

PDI ¼ Dw =Dn

ð1Þ

ð2Þ ð3Þ

where Dn represents the number-average diameter of the particles, Dw the weight-average diameter of the particles, and Ni the number of the particles having diameter Di.

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SEM observation. The surface morphologies of the PDA particles and PDA/Ag nanocomposite particles were further characterized by the scanning electron microscopy (SEM, Hitachi S-4800, Hitachi, Ltd. Japan). All the dispersions were diluted with ethanol and dried on the silica wafers at room temperature prepared for SEM observation. EDX analysis. An energy-dispersive X-ray spectroscopy (EDX) attached to the scanning electron microscopy (SEM, Hitachi S-4800, Hitachi, Ltd. Japan) was used to examine the surface compositions of the PDA particles and PDA/Ag nanocomposite particles. X-ray photoelectron spectroscopy. X-ray photoelectron spectroscopy (XPS) measurement was carried out on an AXIS Ultra X-ray photoelectron spectrometer (Kratos Analytical Ltd. U.K.) equipped with a monochromatized Al Kα X-ray source (1486.6 eV). All binding energies were calibrated by using the containment carbon (C1s = 284.6 eV). X-ray diffraction. PDA/Ag powders were deposited on a glass substrate and examined by a DX-2700 X-ray diffractometer equipped with a Cu tube and a diffracted beam curved graphite monochromator operating at 40 kV and 30 mA. X-ray diffraction spectra were obtained by scanning the samples with a rate of 0.02° (2θ) per second in the range of 20° and 90° (2θ). TG analysis. The thermogravimetric analysis (TGA) of dried PDA particles and PDA/Ag nanocomposite particles was performed on the SDT Q600 (TA Instruments, U.S.A.). All powder samples were heated

from 30 to 800 °C with a rate of 10 °C/min under a nitrogen atmosphere with a flow rate of 50 mL/min. 3. Results and discussion 3.1. Preparation and characterization of polydopamine particles The detailed preparation process of monodisperse polydopamine (PDA) particles has been illustrated in Scheme 1a. In this process, dopamine self-polymerizes and forms monodisperse PDA particles via the oxidation in the alkaline water–ethanol solution at room temperature [31]. As shown in Fig. 1a and c, the typical transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of the obtained PDA particles are monodisperse at a large scale in both spherical shape and size, which is quantified by the measurement of the mean diameter of 586 nm and the polydispersity index (PDI) of 1.0148 (by averaging more than 100 particles in the TEM micrographs). Therefore, these PDA particles offer a fine uniformity for their serving as the monodisperse templates. Moreover, PDA particles ‘inherit’ ample amount of catechol and amine groups from their starting materialdopamine, which allows them as an active platform for diverse secondary reactions [23,24]. In addition, PDA particles behave similar to those of naturally occurring melanin, which confirms an excellent

Fig. 1. Transmission electron microscopy (TEM) images of (a) PDA particles and (b) PDA/Ag nanocomposite particles. Scanning electron microscopy (SEM) images and the corresponding energy dispersive X-ray (EDX) spectra of (c, e) PDA particles and (d, f) PDA/Ag nanocomposite particles (PDA particles: 0.1 g; [Ag(NH3)2]+ ions: 1.96 × 10−2 mol/L in aqueous solution; time: 1 h; temperature: ice-water bath).

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biocompatibility of the PDA [30,32]. In all, the as-synthesized PDA particles are an ideal biocompatible substrate for the convenient synthesis of a rich variety of nanocomposite materials with promising features. 3.2. Preparation and characterization of polydopamine/Ag nanocomposite particles As mentioned, PDA particles can be used as the active templates for the synthesis of nanocomposite particles due to their surfaces containing abundant catechol and amine groups which exhibit extraordinary active nature, such as the good binding and reducing abilities for metal ions [25,33]. Here, by making full use of these advantageous features, PDA/Ag nanocomposite particles have been successfully fabricated though a so-called polydopamine-assisted electroless Ag metallization [28], as illustrated in Scheme 1b. With feeding a fresh [Ag(NH3)2]+ ion aqueous solution into the as-synthesized PDA particle aqueous dispersion, silver ions are simultaneously bonded onto the oppositely charged surface of the PDA and immediately in situ reduced to metallic Ag nanoparticles with the help of the reductive nature of these active catechol and amine groups [28]. During this process, no additional reductants, toxic reagents or intricate instruments are needed, revealing the easy and eco-friendly inherences of this approach. The in situ formed Ag nanoparticles are tightly bonded onto the PDA template particles even the sonication cannot break the link between each other. Moreover, the other key advantage of preparing PDA/Ag nanocomposite particles is to protect the Ag nanoparticles from aggregation, in other words, enhances the stability of the Ag nanoparticles. As expected, the obtained PDA/Ag nanocomposite particles were able to disperse into aqueous media from their dry state, even later on, dispersing in a complicated biological environment, no visible aggregation has been observed. Fig. 1b and d displays the TEM and SEM images of the as-synthesized PDA/Ag nanocomposite particles. In comparison with their starters PDA

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particles (Fig. 1a and c), PDA/Ag nanocomposite particles with relative rough surfaces are observed. The high contrast between PDA particles and Ag nanoparticles is a strong evidence which confirms the successful decoration of the Ag nanoparticles on the PDA template particles. Moreover, EDX spectra were also employed to analyze element variation in this system. As shown in Fig. 1e and f, it is observed that new peaks ascribed to Ag element emerge after the decoration. Combining with previous TEM and SEM observation, a result in which PDA/Ag nanoparticles has been successfully prepared can be concluded. It is noted that the Si element signals in a high intensity originated from the silica wafer. In addition, XPS spectroscopy was employed to further analyze the surface chemical composition of the PDA particles and PDA/Ag nanocomposite particles. Before coating with Ag nanoparticles, the wide scanning XPS spectrum of the template PDA particles reveals that the main strong carbon (C1s) and oxygen (O1s) signal peaks accompanying with the low intensities of the nitrogen (N1s) signal peak are present as illustrated in Fig. 2a. The occurrence of oxygen (O1s) and nitrogen (N1s) signal peaks with low intensities confirms the presence of the catechol and amine groups in the template PDA particles. In contrast, the surface chemical information of the PDA/Ag nanocomposite particles examined by XPS is shown in Fig. 2b. Comparing with the XPS spectrum of the template PDA particles in Fig. 2a, except C1s, N1s, O1s, the new signal peaks including Ag3d, Ag3p (Ag3p5/2, Ag3p3/2) and Ag3s emerge in the XPS spectrum of the PDA/Ag nanocomposite particles in Fig. 2b. The strong Ag signal peaks at a binding energy of around 370.0 eV certify the emergence of the silver element in the PDA/Ag nanocomposite particles. A close inspection on Ag3d peaks in Fig. 2b, two individual peaks occur, as shown in Fig. 2c. These two peaks at 367.5 eV and 373.5 eV with a spin-orbit separation of 6.0 eV correspond to the binding energies of Ag3d5/2 and Ag3d3/2, respectively. These two characteristic peaks (Ag3d5/2 (367.5 eV) and Ag3d3/2 (373.5 eV)) present in the XPS spectrum of the PDA/Ag nanocomposite particles

Fig. 2. X-ray photoelectron spectroscopy (XPS) scans of (a) PDA particles; (b) PDA/Ag nanocomposite particles; (c) Ag3d core-level spectrum of the PDA/Ag nanocomposite particles (PDA particles: 0.1 g; [Ag(NH3)2]+ ions: 1.96 × 10−2 mol/L in aqueous solution; time: 1 h; temperature: ice-water bath).

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are the other valid evidence that proves the existence of the Ag0 species [11,28]. It further reveals the fact that PDA enables to in situ reduce Ag ions to Ag nanoparticles at room temperature, neither additional reductants nor the complicated treatment are needed. 3.3. Effect of the concentrations of [Ag(NH3)2]+ ions In this polydopamine-assisted electroless Ag metallization strategy, the concentration of the silver precursor-[Ag(NH3)2]+ ions is playing a vital role in the morphological control of the target particles. Fig. 3 illustrates the SEM and TEM observation of the PDA/Ag nanocomposite particles prepared by using different concentrations of [Ag(NH3)2]+ ions. As just discussed, the silver precursor-[Ag(NH3)2]+ ions are bonded and in situ reduced to Ag nanoparticles at the surface of the PDA template particles with the help of the adequate catechol and amine groups. Indeed, Ag nanoparticles are only present at the surface of the PDA template, no free Ag nanoparticles were observed as shown in Fig. 3. When the concentration of the [Ag(NH3)2]+ ions added into the PDA dispersion was low, for example, 1.18 × 10−2 mol/L, Ag nanoparticles sparsely cover the surface of the template particles (see Fig. 3a and b). The polydisperse Ag nanoparticles attached to the PDA particles in Fig. 3c reveals that the mergence between Ag nanoparticles may take place during the reduction. As the concentration of the [Ag(NH3)2]+ ions increased to 1.57 × 10−2 mol/L the coverage of the Ag nanoparticles on the PDA particles rose, as shown in Fig. 3d and e. The mean size of the Ag nanoparticles increases whereas their shapes become more irregular (see Fig. 3f). While increasing further the concentration of the [Ag(NH3)2]+ ions to 1.96 × 10−2 mol/L, some Ag nanoparticles become big enough to touch each other, forming an incomplete Ag nanoshell forms (see Fig. 3h–i). It is worth pointing out that the absence of free

Ag nanoparticles confirms the excellent absorption ability of the PDA with nanoparticles, in other words, the strong interaction between PDA and Ag nanoparticle guarantees to against the formation of the Ag nanoparticles in the solution phase. We believe that with increasing further the concentration of Ag precursor or rationally modifying the strategy (e.g., continual feed of Ag precursor) a complete Ag shell can be obtained. In addition, X-ray diffraction (XRD) was employed to determine the crystal structure and grain size of the Ag nanoparticles. As shown in Fig. 4a, there are broad peaks at 2θ angles of 20° and 30°, which are attributed to the PDA template particles [34]. After decoration with Ag nanoparticles new peaks appear at 2θ angles of 37.9°, 44.1°, 64.3°, 77.2° and 81.4°, which correspond to the reflections of (111), (200), (220), (311) and (222) crystalline planes of the fcc structured Ag (JCPDS No. 04-0783), respectively. Varying the concentration of the Ag precursor would not alter the location of the characteristic peaks of the Ag, but it influences the size of crystal grain. As shown in Fig. 4b–d, the XRD patterns become sharper as more Ag precursor was added (from 1.18 × 10−2 to 1.96 × 10−2 mol/L). The strongest characteristic reflection (111) of Ag can be used to estimate the average crystallite size of the Ag nanoparticles according to the Debye–Scherrer equation, by which the average sizes of the Ag nanoparticles were calculated. In good agreement with previous TEM and SEM observation in Fig. 3, the average size of Ag nanoparticles increases from 52 to 83 nm with the concentration of [Ag(NH3)2]+ ions increasing from 1.18 × 10−2 to 1.96 × 10−2 mol/L, correspondingly. Thermogravimetric analysis (TGA) was performed to examine the thermal stability of the PDA particles and PDA/Ag nanocomposite particles. As illustrated in Fig. 5a, pristine PDA particles start to lose their weight at about 100 °C. And, in the end, 53.68 wt.% of the particles has left over at 800 °C. The weight loss below 300 °C is ascribed to the

Fig. 3. SEM (a, d, g) and TEM (b, c, e, f, h, i) images of PDA/Ag nanocomposite particles prepared with various concentrations of [Ag(NH3)2]+ ions: (a, b, c) 1.18 × 10−2 mol/L; (d, e, f) 1.57 × 10−2 mol/L; (g, h, i) 1.96 × 10−2 mol/L. The high magnification TEM images in (c), (f) and (i) show one typical PDA/Ag nanocomposite particle in each case (PDA particles: 0.1 g; time: 1 h; temperature: ice-water bath).

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Fig. 4. X-ray diffraction (XRD) patterns of (a) PDA particles and (b, c, d) PDA/Ag nanocomposite particles prepared by using various concentrations of [Ag(NH3)2]+ ions: (b) 1.18 × 10−2 mol/L, (c) 1.57 × 10−2 mol/L and (d) 1.96 × 10−2 mol/L (PDA particles: 0.1 g; time: 1 h; temperature: ice-water bath).

solvent and unreacted monomer. Starting from 300 °C the weight loss of pure PDA particles accelerates, suggesting a quick decomposition of the PDA particles. The situation becomes different when the PDA particles are covered with Ag nanoparticles. As shown in Fig. 5b–d, the residues of the PDA/Ag nanocomposite particles are much higher than that of the PDA particles. The rate of the weight loss of the PDA/Ag nanocomposite particles seems keeping constant, revealing an improved thermal stability when the PDA particles have been ‘protected’ with Ag nanoparticles. As discussed previously, the surface coverage of the Ag nanoparticles on the PDA particles increases with the incremental content of the [Ag(NH3)2]+ ions. The more Ag nanoparticles are loaded, the more residues are left over after sintering. The final residue weight ratios of the composite particles are 76.15% (b), 77.22% (c) and 79.69% (d), respectively (see Fig. 5b–d).

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cytocompatibility is the one of the primary concerns in the use of these silver-based nanomaterials as the antimicrobial agent in vivo for the clinical therapeutics [7]. The PDA/Ag nanocomposite particles prepared at the [Ag(NH3)2]+ ions concentration of 1.96 × 10−2 mol/L, who load the most amount of the Ag nanoparticles in our experiment, are picked up as an extreme to uncover the cytotoxicity in a series of in vivo tests. First, this sample with the HEK293T human embryonic kidney cells were incubated with the final concentrations ranging from 2 to 32 μg/mL, and were used to treat with cells on incubation at 37 °C for 24 h. As a comparison, the same treatment was also applied to the pure PDA particles as well. Subsequently, the in vitro cytotoxicities of the PDA particles and PDA/Ag nanocomposite particles were evaluated referenced to the pure HEK293T human embryonic kidney cells using the Cell Counting Kit-8 (CCK-8) assay. As shown in Fig. 6, in general, both the PDA particles and PDA/Ag nanocomposite particles do not show the significant cytotoxicities against the HEK293T human embryonic kidney cells. Specifically, up to 93.72% of HEK293T human embryonic kidney cells are alive even if a concentration as high as 32 μg/mL of the PDA particles were incubated with the cells. This result confirms that polydopamine particles have a good biocompatibility with the human cells. While more than 84% of the HEK293T human embryonic kidney cells are viable in the comparison sample where PDA/ Ag nanocomposite particles are present. Therefore, the introduction of the Ag nanoparticles onto the surface of the PDA particles having a minor effect on the cytocompatibility can be concluded. The reasonably good cytocompatibility of the PDA/Ag nanocomposite particles performed paves the possible avenues towards the in vivo biomedical application of these particles for diagnostics and therapeutics.

3.4. In vitro cytotoxicity analysis Before practically employing PDA/Ag nanocomposite particles as the antibacterial materials for biomedical applications, their cytocompatibilities should be evaluated at first. Basically,

Fig. 5. Thermogravimetric analysis (TGA) curves of (a) PDA particles and (b, c, d) PDA/Ag nanocomposite particles prepared in the presence of [Ag(NH3)2]+ ions at various concentrations: (b) 1.18 × 10−2 mol/L, (c) 1.57 × 10−2 mol/L and (d) 1.96 × 10−2 mol/L (PDA particles: 0.1 g; time: 1 h; temperature: ice-water bath).

Fig. 6. In vitro cytotoxicity (CCK-8 assay) of (a) PDA particles and (b) PDA/Ag nanocomposite particles against HEK293T human embryonic kidney cells after incubation for 24 h at 37 °C.

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3.5. Antibacterial property Concerning the prospective applications of these as-synthesized PDA/Ag nanocomposite particles as the antibacterial materials, two bacterial species E. coli (Gram-negative bacteria) and S. aureus (Grampositive bacteria) were selected to practically evaluate the antibacterial properties of the PDA/Ag nanocomposite particles in the current work. In detail, Luria–Bertani (LB) liquid medium turbidity assays were employed to initially evaluate the antibacterial properties of the PDA/ Ag nanocomposite particles against E. coli and S. aureus, respectively. In these antibacterial assays, PDA/Ag nanocomposite particles prepared at a [Ag(NH3)2]+ ion concentration of 1.96 × 10−2 mol/L were chosen as the typical antibacterial agent for the investigation. A series of bacteria (E. coli and S. aureus) solutions were prepared in the LB liquid media for 14 h in the absence/presence of the as synthesized particles (PDA particles and PDA/Ag nanocomposite particles), among which only two control bacterial suspensions in the absence of PDA or PDA/Ag particles became turbid (see Fig. 7a and c, the two control groups), suggesting that bacteria (both E. coli and S. aureus) were rapidly proliferated in the LB environment. Moreover, the addition of the PDA particles (50 μg/mL) did not effectively inhibit the growth of the

bacterial (see Fig. 7a and c, the two PDA groups), since the mixtures in which PDA particles were added became turbid after 14 h incubation of the bacteria in the LB liquid medium. For the PDA/Ag groups, the media containing a high concentration of the PDA/Ag nanocomposite particles (for example, 50 μg/mL) remained pellucid, which indicated that few bacteria proliferated. While the mixtures with a low concentration of the PDA/Ag nanocomposite particles (e.g., 10 μg/mL) became a little turbid after the incubation for 14 h, indicating a small amount of the bacteria having proliferated (see Fig. 7a and c, the PDA/Ag groups). In general, PDA/Ag nanocomposite particles can effectively inhibit the bacterial growth of E. coli and S. aureus at the certain concentrations. Their antibacterial activities are believed mainly originating from the Ag nanoparticles deposited on the surfaces. Nevertheless, the pure PDA particles did not show an antibacterial activity against E. coli and S. aureus. We further quantitatively evaluated the antibacterial properties of the PDA/Ag nanocomposite particles by studying their bacteria growth kinetics in the LB liquid media. In this process, the bacteria (E. coli and S. aurous) suspensions were first inoculated in the LB liquid medium in the presence of various concentrations of the PDA/Ag nanocomposite particles. Subsequently, the bacterial proliferation was monitored by

Fig. 7. LB liquid medium turbidity assays (a, c) and bacterial growth curves (b, d) are employed to evaluate the antibacterial activities of the PDA/Ag nanocomposite particles against Escherichia coli (a, b) and Staphylococcus aureus (c, d). (a, c) The bacteria in LB liquid medium containing 10 and 50 μg/mL of the PDA/Ag nanocomposite particles and 50 μg/mL of the PDA particles are studied, respectively. The LB liquid medium is used in the control experiments. Photographs are taken at the times when the samples having been incubated for 0 h and 14 h, respectively. (b, d) Different concentrations of the PDA/Ag nanocomposite particles (5–40 μg/mL for E. coli and 5–60 μg/mL for S. aureus) and pure PDA particles (40 μg/mL for E. coli and 60 μg/mL for S. aureus) are added to the culture of E. coli and S. aureus. LB liquid medium is used in the control experiments. The growth of the bacteria was measured by judgment of the O.D. at a wavelength of 600 nm. The initial addition of the PDA/Ag nanocomposite particles to the LB bacterium suspension was regarded as the starting point.

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measuring the optical density at 600 nm (O.D. 600) based on the turbidity of the cell suspensions within 14 h. The growth curves of the bacteria showed a typical dose-dependent antibacterial effect of the PDA/Ag nanocomposite particles (see Fig. 7b and d). Compared with the growth curves of the control groups, the bacterial growth in (E. coli and S. aureus) suspensions where the low concentrations (e.g., 5 μg/mL and 10 μg/mL) of the PDA/Ag nanocomposite particles were present were slightly retarded, which are in consistence with those observed in the Luria–Bertani (LB) liquid medium turbidity assays. As the higher concentration of the PDA/Ag nanocomposite particles were dispersed in the bacteria suspensions, the smaller the bacterial growth rates can be achieved. As shown in Fig. 7b and d, when the concentrations of the PDA/Ag nanocomposite particles were increased to 40 μg/mL for E. coli and 60 μg/mL for S. aureus, respectively, the growths of the bacteria were completely inhibited. On the other hand, the antibacterial activities of the PDA/Ag nanocomposite particles against E. coli and S. aureus have also been compared statistically. According to the statistical analysis, the PDA particles did not effectively inhibit the growth of the bacteria (E. coli and S. aureus), because there was no significant difference from the PDA particles groups in comparison with the control groups. While, for the PDA/Ag nanocomposite particles groups, when a low concentration (e.g., 5 μg/mL) of the PDA/Ag nanocomposite particles were used in the bacteria suspensions, after 4 h incubation, they did show a minor inhibition against the bacterial growth, in which the difference was significant (P b 0.05) in comparison with that of the control groups. Furthermore, as the high concentrations (e.g., 10 μg/mL–60 μg/mL) of the PDA/Ag nanocomposite particles were used, the effectively inhibition against the bacterial growth after 2 h incubation were shown, in which the significant difference (P b 0.05) was exhibited when was compared with the control groups. With the increase of the incubation time, the antibacterial activities of the PDA/ Ag nanocomposite particles groups were also varying. Therefore, two facts can be summarized from these results; one is the presence of the Ag nanoparticles is the essential element of the antibacterial feature of the composite particles, since pure PDA does not retard the growth of the bacteria at all; the other is the antibacterial activity of the nanocomposite particles is mainly determined by the concentration of the Ag nanoparticles introduced, the more PDA/Ag nanocomposite particles are employed, the better antibacterial effects against E. coli and S. aureus are shown. It is shown that E. coli (Gram-negative bacteria) is more sensitive to the PDA/Ag nanocomposite particles than S. aureus (Gram-positive bacteria), which may be attributed to the fact that the bacterial membranes of the Gram-positive bacteria are thicker and more stable than those of the Gram-negative bacteria [35]. Moreover, the Grampositive bacteria are generally less susceptible than the Gram-negative species against the Ag based antibacterial agents [36,37]. In addition, the template PDA particles were also set as the other control groups for both bacterial strains, by which we can further confirm which components in PDA/Ag nanocomposite particles play the key antibacterial role. As shown, the bacteria (E. coli and S. aureus) suspensions containing PDA particles have the close growth rates with that of the control groups. Therefore, only pure PDA cannot inhibit both strains of the bacterial growth. This result, again, confirms that the antibacterial activities of the PDA/Ag nanocomposite particles are mainly from the Ag nanoparticles attached on the surfaces of the PDA spheres. In addition, the antibacterial activities of PDA/Ag nanocomposite particles were also compared with the commercial antibiotic, kanamycin. According to our previous results, the minimum inhibition concentrations (MIC) of kanamycin against E. coli and S. aureus were at 20 μg/mL and 10 μg/mL, respectively [28]. While, the MIC of PDA/Ag nanocomposite particles have been evaluated at 40 μg/mL for E. coli and 60 μg/mL for S. aureus, respectively (see Fig. 7b and d). Therefore, we can conclude that the as-synthesized PDA/Ag nanocomposite particles with the lower antibacterial efficiencies against both E. coli and S. aureus in comparison with those of kanamycin. However, kanamycin

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as an aminoglycoside bacteriocidal antibiotic has excellent antibacterial properties, but its instability and side effects (such as tinnitus or loss of hearing, toxicity to kidneys) often limit its application. On the contrary, these PDA/Ag nanocomposite particles are stable during the antibacterial assays, and they exhibited a reasonably good cytocompatibility with human cells and an excellent antibacterial performance against E. coli and S. aureus. On the other hand, we choose the live/dead bacterial cell fluorescence stain assays to further confirm the effect of the PDA/Ag nanocomposite particles on the bacterial viabilities. The bacterial cells of both strains were first incubated for 14 h in the LB liquid medium where 15 μg/mL of PDA/Ag nanocomposite particles were added. In contrast, the bacterial cells solely containing the LB liquid media were considered as the control groups. Subsequently, all the bacterial cells were harvested for staining. Blue fluorescent dye was used to stain both live and dead cells, while red fluorescent dye only stained dead cells. As Fig. 8a, b, e and f has shown, the bacterial cells in both strains where the PDA/Ag nanocomposite particles were absent can keep a high survivability. On the contrary, the majority of the bacterial cells with a strong red fluorescence can be observed in the strains cultured in the presence of the PDA/Ag nanocomposite particles (see Fig. 8c, d, g, and h). The poor survivability is a strong evident which directly exhibits that the PDA/ Ag nanocomposite particles are excellent antibacterial candidates. Although all results have demonstrated that the introduction of the Ag nanoparticles is the key with which PDA/Ag nanocomposite particles show a promising antibacterial activities against E. coli (Gram-negative bacteria) and S. aureus (Gram-positive bacteria), the antibacterial activities are still not fully understood. On the basis of our antibacterial results and the knowledge from literatures [9,36,38–41], a possible mechanism can be explained as follows: The silver-based materials can interact with the sulfur-contained proteins in the cell membrane of the bacteria, causing the structural changes or functional damage to the bacterial cell membrane, and finally, result in the death of the bacteria [38,39]. In addition, the released Ag ions from silver-based materials can interact with the disulfide or sulfhydryl groups of the enzymes, causing the structural changes and further destroying the metabolic process of the bacterial cells, all of which can inactivate the microorganism cells and lead to cell death in the end [42,43]. 4. Conclusions In summary, we describe a facile, mild and green approach to fabricate PDA/Ag nanocomposite particles by combining musselinspired PDA chemistry and electroless metallization of Ag. In this polydopamine-assisted electroless Ag metallization procedure, no additional reductants, toxic reagents and intricate instruments are needed. The formation of the PDA/Ag nanocomposite particles has been fully investigated with a rich variety of characterizations (TEM, SEM, EDX and XPS). Moreover, through adjusting the concentration of the silver precursor-[Ag(NH3)2]+ ions, the size of the nanoparticle and the surface coverage of the Ag nanoparticles on the PDA particles can be easily tuned. XRD results combining with the TEM and SEM observation confirmed the formation of the single-crystal domain of the Ag nanoparticles, and determined the exact size of the Ag nanoparticles as well. These PDA/Ag nanocomposite particles showed a fine cytocompatibility against the HEK293T human embryonic kidney cells, whereas the preliminary antibacterial assays indicated that the PDA/ Ag nanocomposite particles exhibited the extraordinary antibacterial activities against E. coli (Gram-negative bacteria) and S. aureus (Grampositive bacteria). Regarding these advantages, these PDA/Ag nanocomposite particles produced in this environmentally friendly process is an ideal antibacterial materials for various biomedical applications. In addition, this approach presents a versatile paradigm for the fabrication of many types of materials with complex shapes [44, 45]. On the basis of this technique, many kinds of composite particles coated with various materials including metal, metal oxides,

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Fig. 8. Fluorescent images of antibacterial activities of PDA/Ag nanocomposite particles against Escherichia coli (a–d) and Staphylococcus aureus (e–h). (a and b, e and f) Control groups for E. coli and S. aureus, respectively; (c and d, g and h) PDA/Ag nanocomposite particle treated groups for E. coli and S. aureus, respectively. E. coli and S. aureus strains were incubated for 14 h in the LB medium containing 15 μg/mL of the PDA/Ag nanocomposite particles. The red fluorescent dye stained only dead cells, whereas the blue fluorescent dye stained both live and dead cells. The scale bar is 50 μm for E. coli and 10 μm for S. aureus, respectively.

ceramics and polymers could be achieved [23] and these composite particles could have a great potential for biomedical and environmental applications. Acknowledgments The financial support from the National Key Technology R&D Program of China (No. 2012BAD12B03), the National Natural Science Foundation of China (Nos. 51203087, 51473089), the Natural Science Basic Research Plan in Shaanxi Province of China (No. 2013JQ2006), the Fundamental Research Funds for the Central Universities (Nos. GK201503039, GK201501002, GK201101003, GK201301002) and the

Scientific Research Foundation for the Returned Overseas Chinese Scholars State Education Ministry is appreciated. We also thank Miss. Ying Cong (Department of Materials Science and the Advanced Coatings Research Center of China Educational Ministry, Fudan University, China) for the help with the schematic diagram drawn in this work. References [1] V.K. Sharma, R.A. Yngard, Y. Lin, Adv. Colloid Interface Sci. 145 (2009) 83–96. [2] M. Rai, A. Yadav, A. Gade, Biotechnol. Adv. 27 (2009) 76–83. [3] C.N. Lok, C.M. Ho, R. Chen, Q.Y. He, W.Y. Yu, H. Sun, P.K.H. Tam, J.F. Chiu, C.M. Che, J. Biol. Inorg. Chem. 12 (2007) 527–534. [4] C. Marambio-Jones, E.M.V. Hoek, J. Nanopart. Res. 12 (2010) 1531–1551.

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