Materials Science and Engineering C 58 (2016) 1160–1169

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Antibacterial nanocarriers of resveratrol with gold and silver nanoparticles Sohyun Park a, Song-Hyun Cha b, Inyoung Cho c, Soomin Park b, Yohan Park a, Seonho Cho b, Youmie Park a,b,⁎ a b c

College of Pharmacy, Inje University, 197 Inje-ro Gimhae, Gyeongnam 621-749, Republic of Korea National Creative Research Initiatives (NCRI) Center for Isogeometric Optimal Design, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-744, Republic of Korea School of Civil, Environmental and Architecture Engineering, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 136-701, Republic of Korea

a r t i c l e

i n f o

Article history: Received 6 April 2015 Received in revised form 13 August 2015 Accepted 16 September 2015 Available online 18 September 2015 Keywords: Resveratrol Nanocarriers Gold nanoparticles Silver nanoparticles Antibacterial activity

a b s t r a c t This study focused on the preparation of resveratrol nanocarrier systems and the evaluation of their in vitro antibacterial activities. Gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) for resveratrol nanocarrier systems were synthesized using green synthetic routes. During the synthesis steps, resveratrol was utilized as a reducing agent to chemically reduce gold and silver ions to AuNPs and AgNPs. This system provides green and eco-friendly synthesis routes that do not involve additional chemical reducing agents. Resveratrol nanocarriers with AuNPs (Res-AuNPs) and AgNPs (Res-AgNPs) were observed to be spherical and to exhibit characteristic surface plasmon resonance at 547 nm and at 412–417 nm, respectively. The mean size of the nanoparticles ranged from 8.32 to 21.84 nm, as determined by high-resolution transmission electron microscopy. The face-centered cubic structure of the Res-AuNPs was confirmed by high-resolution X-ray diffraction. Fouriertransform infrared spectra indicated that the hydroxyl groups and C_C in the aromatic ring of resveratrol were involved in the reduction reaction. Res-AuNPs retained excellent colloidal stability during ultracentrifugation and re-dispersion, suggesting that resveratrol also played a role as a capping agent. Zeta potentials of ResAuNPs and Res-AgNPs were in the range of − 20.58 to − 48.54 mV. Generally, against Gram-positive and Gram-negative bacteria, the Res-AuNPs and Res-AgNPs exhibited greater antibacterial activity compared to that of resveratrol alone. Among the tested strains, the highest antibacterial activity of the Res-AuNPs was observed against Streptococcus pneumoniae. The addition of sodium dodecyl sulfate during the synthesis of ResAgNPs slightly increased their antibacterial activity. These results suggest that the newly developed resveratrol nanocarrier systems with metallic nanoparticles show potential for application as nano-antibacterial agents with enhanced activities. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Resveratrol (3,5,4′-trihydroxystilbene) is a polyphenol and phytoalexin present in various plants and plant products, including wine, berries, grapes, pistachio, peanuts and cocoa powder [1,2]. The resveratrol concentration in red wine generally ranges from 1 to 14 mg/mL [1]. As observed in the “French paradox”, the consumption of red wine, especially resveratrol, prevents heart disease [1]. In nature, resveratrol occurs as trans and cis isomers; the trans-isomer is known as the biologically active form. The biological activities of resveratrol include cardioprotection, neuroprotection, anti-oxidant, anti-inflammatory, anti-carcinogenic and anti-aging activity [1,2]. However, the poor water solubility, poor bioavailability, and rapid metabolism of resveratrol, as well as its transformation from the trans to cis isomer, make in vivo delivery and subsequent exploitation of its health benefits ⁎ Corresponding author at: College of Pharmacy, Inje University, 197 Inje-ro, Gimhae, Gyeongnam 621-749, Republic of Korea. E-mail address: [email protected] (Y. Park).

http://dx.doi.org/10.1016/j.msec.2015.09.068 0928-4931/© 2015 Elsevier B.V. All rights reserved.

difficult. Thus, nano- and micro-encapsulation techniques have been used to increase the water solubility, bioavailability and photostability of trans-resveratrol against light-induced degradation [1–3]. The most common carrier systems for resveratrol are polymeric nanoparticles, liposomes, emulsions, micelles, solid lipid nanoparticles, cyclodextrin complexes, protein complexes, yeast cell micro-encapsulation and pectinate beads [1–4]. Many research articles have reported the loading of resveratrol onto polymeric nanoparticles and the subsequent applications of the loaded nanoparticles [5–11]. To protect resveratrol from light-induced degradation, chitosan-alginate-coated poly(D,L-lactide-co-glycolide) nanoparticles (spherical-shaped, 135–580 nm) were prepared by a nanoprecipitation method [5]. trans-Resveratrol was loaded onto chitosan nanoparticles (spherical-shaped, 319 nm) modified with biotin and avidin, and these nanoparticles were capable of targeting hepatic carcinoma [6]. Resveratrol-loaded polymeric micelles (spherical-shaped, less than 100 nm) were prepared with polycaprolactone and polyethylene glycol [7]. These micelles demonstrated the feasibility of protecting cells from Aβ toxicity. Resveratrol was also loaded onto poly(ε-caprolactone) and poly(D,L-lactic-co-glycolic acid)-poly(ethylene glycol) blend (spherical-

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shaped, 150 nm) for prostate cancer treatment [8]. In another system, resveratrol was loaded onto poly(N-vinylpyrrolidone)-b-poly(εcaprolactone) polymer, thereby generating spherical shapes with an average size of 89 nm [9]. The neuroprotective effects of resveratrol were enhanced in rat cortical cell culture with these polymeric nanoparticles [9]. A conjugate of transferrin-modified polyethylene glycol-poly lactic acidresveratrol (150 nm) was effective in glioma therapy [10]. The polyethylene glycol-poly lactic acid-resveratrol conjugate suppressed tumor growth and glucose metabolism [11]. However, only several reports have reported using metallic nanoparticles for the loading of resveratrol [12,13]. Mohanty and co-workers reported the preparation of resveratrol-stabilized gold nanoparticles (AuNPs), which enabled the surface loading of doxorubicin [12]. They concluded that resveratrolstabilized AuNPs were suitable as a doxorubicin carrier. Bacterial AuNPs prepared using the culture supernatant of the Delftia sp. strain KCM-006 were conjugated to resveratrol [13]. This system was 65% more effective as an anticancer agent than resveratrol alone on a human lung cancer cell line (A549). Microbial resistance to conventional antibiotics poses many problems related to public health. Specifically, the appearance of multidrug-resistant (MDR) bacteria has increased the need for novel antibacterial agents. With the development of nanotechnology, nanoparticles have garnered interest as a therapeutic tool serving as antibacterial agents [14,15]. Among nanoparticles, metallic nanoparticles, such as AuNPs and silver nanoparticles (AgNPs), are gaining attention as excellent antimicrobial platforms [14–19]. The use of AuNPs and AgNPs as antibacterial agents provides advantages over conventional antibiotics because AuNPs and AgNPs employ multifaceted antibacterial mechanisms and are therefore not likely to grow bacterial resistance [14]. The synthetic steps for producing AuNPs and AgNPs commonly involve chemical reducing agents to induce the reduction of Au ions and Ag ions. However, this strategy poses toxicity-related problems due to the use of noxious chemicals, which further decrease the biocompatibility of the resulting nanoparticles. Thus, green (eco-friendly or environmentally friendly) reducing agents have been used for the synthesis of AuNPs and AgNPs [20–24]. Diverse natural products, such as primary and secondary metabolites of plants [20,25–28], play a role as green reducing agents. In this study, resveratrol was utilized as a green reducing agent to develop novel nanocarriers of resveratrol by converting gold ions to AuNPs (hereafter referred to as Res-AuNPs) and silver ions to AgNPs (referred to hereafter as Res-AgNPs). The novel Res-AuNPs and ResAgNPs were characterized using UV–visible spectrophotometry, inductively coupled plasma mass spectrometry (ICP-MS), Fourier-transform infrared spectroscopy (FT-IR), high-resolution transmission electron microscopy (HR-TEM), atomic force microscopy (AFM) and highresolution X-ray diffraction (HR-XRD). Finally, the in vitro antibacterial activity of the nanoparticles was evaluated against twenty-two strains of Gram-positive and Gram-negative bacteria using minimum inhibitory concentration (MIC) methods. 2. Materials and methods 2.1. Materials Gold(III) chloride trihydrate (HAuCl4·3H2O) and silver nitrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Resveratrol (trans form) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Unless otherwise stated, all reagents were used as received.

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AFM were performed to obtain information related to the nanoparticle size and shape. For the acquisition of HR-TEM images, the sample solution was loaded onto a carbon-coated copper grid (carbon type-B, 300 mesh copper grids, Ted Pella, Inc., Redding, CA, USA), and the sample-loaded grid was subsequently air-dried or dried in an oven at 37 °C. HR-TEM images were acquired with a JEM-3010 microscope operated at 300 kV (JEOL, Ltd., Tokyo, Japan). AFM images were obtained with a Dimension® Icon® system (Bruker Nano, Santa Barbara, CA, USA) operated in tapping mode with an RTESP probe (premium highresolution silicon probe with tapping mode, Bruker Nano, Inc.). The nanoparticle solution was loaded onto a mica substrate (grade V-1, 25 mm × 25 mm length, 0.15 mm thick, SPI Supplies Division, Structure Probe, Inc., West Chester, PA, USA), and the sample-loaded mica was air-dried or dried in an oven at 37 °C. HR-XRD analysis provides information regarding crystalline nature of nanoparticles. A Bruker D8 Discover high-resolution X-ray diffractometer (Bruker, Karlsruhe, Germany) was used to obtain powder HR-XRD patterns; the instrument was equipped with a Cu-Kα radiation source (λ = 0.154056 nm), and the samples were scanned over the range of 20°–90° (2θ scale). The functional groups of resveratrol that were involved in nanoparticle synthesis were characterized by FT-IR. FT-IR spectra were acquired with a Varian 640-IR using the KBr pellet method (Agilent Technologies, Santa Clara, CA, USA). ICP-MS was performed to determine the synthetic yield with an ELAN 6100 (Perkin-Elmer SCIEX, Waltham, MA, USA). A Brookhaven 90Plus was used to obtain zeta potential values at 25 °C (Brookhaven Instruments Co., Holtsville, NY, USA). Each sample was measured ten times and averaged to get mean values. 2.3. Synthesis of Res-AuNPs and Res-AgNPs as nanocarriers of resveratrol Stock solutions of gold(III) chloride trihydrate and silver nitrate were prepared in deionized water, and resveratrol was dissolved in 2% DMSO. For the preparation of Res-AuNPs in a volume of 1 mL, various concentrations of gold(III) chloride trihydrate and resveratrol were placed in an 80 °C oven. The reaction time was varied from 2 h to 24 h. UV–visible spectra were recorded under each set of reaction conditions. The optimal conditions for the synthesis of Res-AuNPs were determined to be a gold(III) chloride trihydrate final concentration of 0.6 mM and a resveratrol final concentration of 0.5 mM in a 1 mL volume. The mixture was incubated in an 80 °C oven for 2 h. There was another preparation method of Res-AuNPs; gold(III) chloride trihydrate (final concentration of 0.6 mM) in a glass vial was heated on a hot plate (set at 80 °C) under stirring. To this solution, a resveratrol solution (in 2% DMSO, final concentration of 0.5 mM) was added to make a final volume of 1 mL. Then, the mixture was further incubated in an 80 °C oven for 2 h. Two types of Res-AgNPs were prepared by the addition of SDS (ResAgNPs-SDS) or NaOH (Res-AgNPs-NaOH). In a volume of 1 mL, various concentrations of silver nitrate and resveratrol were placed in an 80 °C oven for 2 h. The optimal conditions for the synthesis of Res-AgNPs were determined to be a silver nitrate final concentration of 0.05 mM and a resveratrol final concentration of 0.7 mM. For Res-AgNPs-SDS, the final concentrations of SDS that were tested were 0.1, 1.0 and 2.0 mM; the concentration of 2.0 mM was selected for further investigation. Therefore, the optimum reaction conditions for the synthesis of Res-AgNPs-SDS were 0.05 mM silver nitrate, 0.7 mM resveratrol, and 2.0 mM SDS in a 1-mL volume in an 80 °C oven for 2 h. For ResAgNPs-NaOH, the concentration of NaOH was varied from 0.1 mM to 14.0 mM. The optimum reaction conditions for the synthesis of ResAgNPs-NaOH were 0.05 mM silver nitrate, 0.7 mM resveratrol, and 6.0 mM NaOH in a 1-mL volume in an 80 °C oven for 2 h.

2.2. Instruments 2.4. Measurement of in vitro antibacterial activity UV–visible spectra were acquired with a Shimadzu UV-2600 spectrophotometer to follow the reaction progress (Shimadzu Corporation, Kyoto, Japan). Ultracentrifugation was performed using an Eppendorf 5424R centrifuge (Eppendorf AG, Hamburg, Germany). HR-TEM and

As shown in Table 2, twenty-two strains of Gram-positive and Gramnegative bacteria were screened to evaluate the in vitro antibacterial activity of Res-AuNPs, Res-AgNPs-SDS, Res-AgNPs-NaOH and the

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Fig. 2. UV–visible spectra of Res-AuNPs. (a) “Before incubation” is a mixture of resveratrol and gold(III) chloride trihydrate under optimal reaction conditions. (b) The supernatant of the Res-AuNPs after ultracentrifugation. (c) Res-AuNP pellets re-dispersed in deionized water after ultracentrifugation. (d) Synthesized Res-AuNPs “after incubation.”

resveratrol standard according to our previously reported method [29]. The strain names and their CCARM numbers are listed in Table 2. The MIC values are expressed in terms of the resveratrol concentration in each case. Norfloxacin was used as a standard antibiotic. To increase the solubility of resveratrol in deionized water, 2% DMSO was added. Thus, the solvent itself (2% v/v DMSO in deionized water) was also screened as a negative control to determine the effect of this solvent system. The results showed that the solvent did not affect the antibacterial activity (data not shown). 3. Results and discussion 3.1. UV–visible spectra, synthetic yields and zeta potentials of Res-AuNPs and Res-AgNPs

Fig. 1. UV–visible spectra of (a) Res-AuNPs, (b) Res-AgNPs-SDS and (c) Res-AgNPs-NaOH. The bold and dotted lines in the spectra indicate “before incubation” and “after incubation,” respectively. The inset shows the color of the solution. The bottles on the left and right contain samples “before incubation” and “after incubation,” respectively.

UV–visible spectrophotometry is a convenient method for monitoring the progress of a reaction or for observing the surface plasmon resonance (SPR) of metallic nanoparticles. The optimum reaction conditions in the case of Res-AuNPs were gold(III) chloride trihydrate with a final concentration of 0.6 mM and resveratrol with a final concentration of 0.5 mM in a 1-mL volume. The mixture was incubated at 80 °C in an oven for 2 h. As shown in Fig. 1a, the SPR of Res-AuNPs prepared under the optimum reaction conditions appeared at 547 nm. Before incubation, the color of the solution was pale-yellow; it became a dark-wine color after incubation (Fig. 1a, inset, bottle on the right). When the incubation time was increased to 4 h, 6 h, 8 h, 10 h, 12 h, 16 h, 20 h, and 24 h, the color of the solution became a pale-wine color and a precipitate was finally formed. Thus, the optimum reaction time was established as 2 h. This result indicated that resveratrol acted as a reducing agent to produce Res-AuNPs from Au ions. In the case of Res-AgNPs, two different methods were employed, depending on the addition of SDS (ResAgNPs-SDS) or NaOH (Res-AgNPs-NaOH). During the synthesis of AgNPs, researchers commonly add surfactants or polymers to increase the colloidal stability and enhance the antibacterial activities of AgNPs [30]. In the present work, three types of surfactants were tested, including myristyltrimethylammonium bromide (cationic surfactant), sodium dodecyl sulfate (SDS, anionic surfactant) and Tween 80 (nonionic surfactant). Among these surfactants, SDS was selected to produce ResAgNPs-SDS (Fig. 1b). The observation of SPR at 419 nm suggested that Res-AgNPs-SDS were successfully synthesized, resulting in a yellowcolored solution (Fig. 1b, inset, bottle on the right). Second, the pH of the solution was varied by the addition of HCl or NaOH during the synthesis. When HCl (final concentration of 1.0–14.0 mM) was added, no

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Fig. 3. (a, b and e) HR-TEM images and a size histogram of Res-AuNPs and (c, d and f) HR-TEM images and a size histogram of Res-AuNPs subjected to ultracentrifugation and re-dispersion processes.

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Fig. 4. AFM images of (a and b) Res-AuNPs and (c and d) Res-AuNPs subjected to ultracentrifugation and re-dispersion processes: (a) 3D height image (1.0 μm × 1.0 μm), (b) 3D phase image (1.0 μm × 1.0 μm), (c) 3D height image (2.5 μm × 2.5 μm), and (d) 3D amplitude error image (2.5 μm × 2.5 μm).

reaction progress was observed. Upon the addition of NaOH (final concentration of 6.0 mM), the pH of the solution was 10.62 and Res-AgNPsNaOH were synthesized. The brown color of Res-AgNPs-NaOH appeared, along with SPR at 412 nm, after incubation (Fig. 1c, inset, bottle on the right). Before oven incubation, the mixture of resveratrol and gold(III) chloride trihydrate under optimal reaction conditions did not exhibit any SPR associated with the Res-AuNPs (Fig. 2a). As previously mentioned the absorbance at 547 nm appeared after oven incubation (80 °C, 2 h) and suggested the successful synthesis of Res-AuNPs (Fig. 2d). To evaluate the colloidal stability of the particles, the solution of Res-AuNPs was subjected to ultracentrifugation and re-dispersion in deionized water. Ultracentrifugation (21,130 × g, 30 min) was conducted to separate the Res-AuNPs as pellets at the bottom of the tube. The SPR of the Res-AuNPs at 547 nm completely disappeared in the supernatant after ultracentrifugation, indicating that the Res-AuNPs could be recovered from the pellet (Fig. 2b). The pellet (Res-AuNPs) was then reTable 1 Zeta potentials of Res-AuNPs, Res-AgNPs-SDS and Res-AgNPs-NaOH. Nanoparticles

Res-AuNPs

Res-AgNPs-SDS

Res-AgNPs-NaOH

Zeta potential (mV)

−20.58 ± 1.21

−48.54 ± 3.11

−37.49 ± 1.65

dispersed in deionized water, and the SPR at 547 nm was recovered (Fig. 2c). As indicated by the UV–visible spectra shown in Fig. 2, the Res-AuNPs maintained good colloidal stability during the ultracentrifugation and re-dispersion processes, suggesting that resveratrol also played a role as a capping agent. We next acquired HR-TEM and AFM images to observe the shape, size and 2D/3D-topography of the ResAuNPs before and after the ultracentrifugation and re-dispersion processes; the results are discussed in the next section (Figs. 3 and 4). The synthetic yield of the Res-AuNPs was estimated to be 99.89% by ICP-MS. The yield was high, indicating that the current reaction conditions were optimized. The yield was calculated according to the following equation: Yield (%) = 100 − [(Au concentration in the supernatant after ultracentrifugation) / (total Au concentration used for Res-AuNP synthesis)] × 100. As shown in Table 1, zeta potentials of Res-AuNPs, Res-AgNPs-SDS and Res-AgNPs-NaOH were measured as − 20.58 ± 1.21 mV, − 48.54 ± 3.11 mV and − 37.49 ± 1.65 mV, respectively (n = 10). These results clearly indicated that resveratrol was bound or complexed onto the surface of nanoparticles, generating electrostatically stable nanoparticles. The zeta potential of Res-AuNPs was −20.58 ± 1.21 mV, which is well matched with the value (− 21.2 mV) of ResAuNPs from the previous report by Mohanty and co-workers [12]. They prepared Res-AuNPs by mixing both resveratrol solution (in NaOH) and chloroauric acid solution under stirring [12]. Possibly due

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Fig. 5. (a, b and e) HR-TEM images and a size histogram of Res-AgNPs-SDS and (c, d and f) HR-TEM images and a size histogram of Res-AgNPs-NaOH.

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Fig. 6. AFM images of (a and b) Res-AgNPs-SDS, and (c and d) Res-AgNPs-NaOH: (a) 3D height image (1.0 μm × 1.0 μm), (b) 3D phase image (1.0 μm × 1.0 μm), (c) 3D height image (1.0 μm × 1.0 μm), and (d) 3D amplitude error image (1.0 μm × 1.0 μm).

to the negative charges of SDS, the Res-AgNPs-SDS exerted the highest negative values of zeta potential (−48.54 ± 3.11 mV). 3.2. HR-TEM and AFM images of Res-AuNPs As shown in Fig. 3a and b, spherical Res-AuNPs were observed. On the basis of the HR-TEM images, 118 nanoparticles were randomly

selected; the corresponding size histogram is presented in Fig. 3e, showing a mean particle size of 14.60 ± 2.97 nm. The dispersion state was quite excellent, without the appearance of aggregation of nanoparticles. After the processes of ultracentrifugation and redispersion discussed in the previous section, this good dispersion state was maintained, as confirmed by the HR-TEM images (Fig. 3c and d). The particle size did not substantially change after the process. The size histogram of the re-dispersed Res-AuNPs is shown in Fig. 3f, showing a mean particle size of 11.99 ± 1.84 nm from 132 discrete nanoparticles. AFM is a powerful tool for the visualization of nanoparticles, and AFM images provide information concerning particle size, 2D/3D shape and nanotopography. Primarily spherical Res-AuNPs were observed in the AFM images (Fig. 4a–b); this observation was corroborated by the HR-TEM images. The ultracentrifugation and re-dispersion processes did not change the shape of the ResAuNPs (Fig. 4c–d). However the interparticle distance of the ResAuNPs shown in Fig. 4c–d was slightly shorter than that of the particles shown in Fig. 4a–b. 3.3. HR-TEM and AFM images of Res-AgNPs

Fig. 7. HR-XRD analysis of Res-AuNPs.

Similar to the Res-AuNPs, primarily spherical Res-AgNPs-SDS and Res-AgNPs-NaOH were observed in the HR-TEM images (Fig. 5a–d). The mean particle sizes of the Res-AgNPs-SDS and Res-AgNPsNaOH were measured at 17.9 ± 3.94 nm (23 nanoparticles) and 11.5 ± 3.18 nm (19 nanoparticles), respectively, from the HR-TEM images (Fig. 5e–f). With respect to the mean particle size, the

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Fig. 8. FT-IR spectra of (a) the resveratrol standard, (b) Res-AuNPs, (c) Res-AgNPs-SDS, and (d) Res-AgNPs-NaOH.

addition of NaOH produced smaller particles compared to those formed by the addition of SDS. The AFM images further confirmed the spherical shape of the Res-AgNPs-SDS and Res-AgNPs-NaOH (Fig. 6a–d).

3.4. HR-XRD analysis The crystalline nature of the Res-AuNPs was confirmed by HR-XRD analysis, as shown in Fig. 7. The strong diffraction peaks at 38.18°,

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Table 2 MIC values of Res-AuNPs, Res-AgNPs-SDS, Res-AgNPs-NaOH, resveratrol standard and norfloxacin. Norfloxacin was used as a standard antibiotic. The MIC values were measured and are expressed in terms of the resveratrol concentration. Strains

Staphylococcus aureus SG511 Staphylococcus aureus 285 Staphylococcus aureus 503 Streptococcus pyogenes 308A Streptococcus pyogenes T 12 A Streptococcus pyogenes 77 A Pseudomonas aeruginosa 9027 Pseudomonas aeruginosa 1592E Pseudomonas aeruginosa 1771 Pseudomonas aeruginosa 1771 M Escherichia coli 078 Escherichia coli TEM Escherichia coli 1507 Escherichia coli DC 0 Escherichia coli DC 2 Salmonella typhimurium Klebsiella oxytoca 1082E Klebsiella aerogenes 1522E Enterobacter cloacae P99 Enterobacter cloacae 1321E Escherichia coli Streptococcus pneumoniae

CCARM no.

0201 0204 0205 0206 0207 0208 0219 0223 0224 0225 0230 0235 0236 0237 0238 0240 0248 0249 0252 0253 0010 0031

MIC values (μg/mL) Res-AuNPs

ResAgNPsSDS

ResAgNPsNaOH

Resveratrol standard

Norfloxacin

N57 N57 N57 N57 N57 N57 N57 N57 N57 N57 N57 N57 N57 N57 N57 N57 N57 N57 N57 N57 N57 14.25

40 40 40 80 80 80 N80 N80 N80 80 N80 N80 N80 N80 40 80 40 N80 N80 N80 N80 40

N80 N80 N80 80 N80 N80 N80 N80 80 80 80 80 N80 80 80 N80 N80 N80 80 80 80 N80

114 N114 N114 57 114 N114 N114 N114 N114 N114 N114 N114 N114 N114 114 114 N114 N114 N114 N114 N114 28.5

b0.25 1 1 8 1 4 1 0.5 0.5 0.25 b0.06 0.12 b0.06 1 0.25 b0.06 b0.06 0.12 b0.06 b0.06 b0.06 2

44.42°, 64.62°, 77.52°, and 81.52° correspond to the (111), (200), (220), (311) and (222) planes of the Au crystal, respectively. The Scherrer equation (D = 0.89 λ/W·cosθ) was employed to roughly estimate the particle size from the (111) peak, where D is the particle size, W is the full-width at half-maximum of the (111) peak in radians, θ is the diffraction angle of the (111) peak, and λ is the wavelength of the Cu-Kα radiation (0.154056 nm). The rough size estimated from the equation was 33.26 nm, which was slightly larger than that measured from the HRTEM images (14.60 ± 2.97 nm). 3.5. FT-IR spectra FT-IR is routinely performed to obtain information about the functional groups involved in synthesis steps. As shown in Fig. 8, the stretching vibration of the –OH functional groups of the resveratrol standard appeared at 3234 cm−1; this peak was shifted to 3406 cm−1 upon the synthesis of Res-AuNPs (Fig. 8a–b). This result suggested that the –OH functional groups of resveratrol were involved in the reduction reaction or were complexed onto the surface of Res-AuNPs. The C_C ring stretching vibration of the aromatic hydrocarbon (arene) of the resveratrol standard appeared at 1585 cm−1 (Fig. 8a). This peak was shifted to 1593 cm−1 (Res-AgNPs-SDS) and 1613 cm−1 (Res-AgNPs-NaOH) (Fig. 8c–d). In addition, the stretching vibration of the –OH functional groups was shifted to 3733/3538 cm−1 (ResAgNPs-SDS, Fig. 8c) and 3393 cm−1 (Res-AgNPs-NaOH, Fig. 8d). These results indicate that the C_C in the aromatic ring and the –OH functional groups were involved in the synthesis of Res-AgNPs-SDS and ResAgNPs-NaOH. 3.6. Evaluation of in vitro antibacterial activity Among the twenty-two strains, Streptococcus pneumoniae was the most susceptible strain (Table 2). The antibacterial activity (two-fold increase) of the Res-AuNPs (MIC 14.25 μg/mL) against S. pneumoniae was observed to be enhanced compared with that of the resveratrol standard (MIC 28.5 μg/mL). However, the Res-AgNPs-SDS (MIC 40 μg/mL) and Res-AgNPs-NaOH (MIC N 80 μg/mL) did not improve the antibacterial activity against S. pneumoniae. On the basis of the MIC values, the Res-AuNPs are the most effective antibacterial agent

against S. pneumoniae as nanocarriers of resveratrol. When considered other strains except S. pneumoniae, the nanocarriers of Res-AuNPs, Res-AgNPs-SDS, and Res-AgNPs-NaOH increased the antibacterial activity compared with that of the resveratrol standard. In comparing the two types of Res-AgNPs, the Res-AgNPs-SDS were observed to exert slightly higher (two-fold increase) antibacterial activity than the ResAgNPs-NaOH against Staphylococcus aureus (SG511, 285 and 503), Escherichia coli DC 2, Klebsiella oxytoca 1082E and S. pneumoniae. As previously mentioned, nanoparticles utilize multifaceted mechanisms to exert their antimicrobial activity; therefore, they are not likely to develop microbial resistance [14]. Among metallic nanoparticles, AuNPs and AgNPs are considered the most promising antibacterial agents. The antibacterial mechanisms of these nanoparticles have not been completely elucidated; however, several hypotheses have been proposed to explain their mechanisms of action [17]: (i) the binding of AgNPs with disulfide or sulfhydryl groups of essential enzymes disrupts the metabolic processes of bacteria; (ii) metallic nanoparticles release ions that damage bacterial membranes; and (iii) metallic nanoparticles generate reactive oxygen species that cause the inhibition of DNA replication, induce protein denaturation and disrupt the cell wall. In this study, interestingly, the resveratrol standard exhibited at least a four-fold increase in antibacterial activity against S. pneumoniae (28.5 μg/mL) compared to its activities against other Gram-positive and Gram-negative strains (114 or N114 μg/mL). The Res-AuNP nanocarriers (14.25 μg/mL) further enhanced the antibacterial activity against S. pneumoniae. Bibbis et al. reported that spherical silver polyvinyl pyrrolidone nanoparticles with a diameter of 20 nm exhibited a greater bactericidal effect against S. pneumoniae than did Au nanospheres and titanium dioxide [31]. To the best of our knowledge, no studies have used Au nanocarriers of resveratrol (Res-AuNPs) as efficient antibacterial agents against S. pneumoniae. 4. Conclusions Owing to the development of nanotechnology, the delivery of active components using nanocarriers has received considerable attention. Among metallic nanoparticles, AuNPs and AgNPs are the most promising antibacterial nanocarriers because of their unique chemical and physical properties, which differ from the properties of their bulk

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counterparts. In this study, resveratrol nanocarriers (Res-AuNPs and Res-AgNPs) were developed, and their properties were characterized using microscopic and spectroscopic methods. Spherical nanocarriers with a particle size of 8.32–21.84 nm were synthesized. Resveratrol was observed to play a dual role as a green reducing agent for nanocarrier synthesis and as an antibacterial agent. The novel metallic nanocarriers of resveratrol were also observed to enhance antibacterial activity against Gram-positive and Gram-negative bacteria compared to the activity of resveratrol alone. These results indicate that the combination of metallic nanoparticles with resveratrol improves the antibacterial activity of resveratrol. Specifically, Res-AuNPs are the most effective antibacterial agent against S. pneumoniae. Additionally, Res-AuNPs maintained good colloidal stability when submitted to ultracentrifugation and re-dispersion processes, revealing their potential as novel antibacterial nanocarriers for the treatment of S. pneumoniae infections. We concluded that the green synthetic process elaborated herein for the antibacterial nanocarriers of AuNPs and AgNPs using plant primary and secondary metabolites is simple and cost-effective; thus, these nanocarriers can substitute for conventional antibiotics as alternatives or as adjuvants. Conflict of interest The authors declare no conflicts of interest in this work. Acknowledgments This work was supported by the Korea Foundation for the Advancement of Science & Creativity (KOFAC) and funded by the Korean Government (MOE). This study was partially supported by the National Research Foundation of Korea (NRF) through a grant funded by the Korean Government, by the Ministry of Education (NRF-2012R1A1A2042224) and by the Ministry of Science, ICT & Future Planning (NRF-2010-18282). References [1] A.R. Neves, M. Lúcio, J.L. Lima, S. Reis, Resveratrol in medicinal chemistry: a critical review of its pharmacokinetics, drug-delivery, and membrane interactions, Curr. Med. Chem. 19 (2012) 1663–1681. [2] M.A. Augustin, L. Sanguansri, T. Lockett, Nano- and micro-encapsulated systems for enhancing the delivery of resveratrol, Ann. N. Y. Acad. Sci. 1290 (2013) 107–112. [3] S. Wang, R. Su, S. Nie, M. Sun, W.D. Zhang, N. Moustaid-Moussa, application of nanotechnology in improving bioavailability and bioactivity of diet-derived phytochemicals, J. Nutr. Biochem. 25 (2014) 363–376. [4] N. Summerlin, E. Soo, S. Thakur, Z. Qu, S. Jambhrunkar, A. Popat, Resveratrol nanoformulations: challenges and opportunities, Int. J. Pharm. 479 (2015) 282–290. [5] V. Sanna, A.M. Roggio, S. Siliani, M. Piccinini, S. Marceddu, A. Mariani, M. Sechi, Development of novel cationic chitosan- and anionic alginate-coated poly(D,L-lactideco-glycolide) nanoparticles for controlled release and light protection of resveratrol, Int. J. Nanomedicine 7 (2012) 5501–5516. [6] L. Bu, L.C. Gan, X.Q. Guo, F.Z. Chen, Q. Song, Qi-Zhao, X.J. Gou, S.X. Hou, Q. Yao, Transresveratrol loaded chitosan nanoparticles modified with biotin and avidin to target hepatic carcinoma, Int. J. Pharm. 452 (2013) 355–362. [7] X. Lu, C. Ji, H. Xu, X. Li, H. Ding, M. Ye, Z. Zhu, D. Ding, X. Jiang, X. Ding, X. Guo, Resveratrol-loaded polymeric micelles protect cells from Aβ-induced oxidative stress, Int. J. Pharm. 375 (2009) 89–96.

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Antibacterial nanocarriers of resveratrol with gold and silver nanoparticles.

This study focused on the preparation of resveratrol nanocarrier systems and the evaluation of their in vitro antibacterial activities. Gold nanoparti...
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