Preparation of mono-dispersed silver nanoparticles assisted by chitosan-g-poly(ɛ-caprolactone) micelles and their antimicrobial application

Applied Surface Science 301 (2014) 273–279

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Preparation of mono-dispersed silver nanoparticles assisted by chitosan-g-poly(␧-caprolactone) micelles and their antimicrobial application Chunhua Gu a , Huan Zhang b , Meidong Lang a,∗ a Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237, China b State Key Laboratory of Bioreactor Engineering, New World Biotechnology Institute, East China University of Science and Technology, Shanghai 200237, China

a r t i c l e

i n f o

Article history: Received 29 July 2013 Received in revised form 7 February 2014 Accepted 12 February 2014 Available online 20 February 2014 Keywords: Chitosan Chemical coupling Micelled Silver nanoparticles Antimicrobial activity

a b s t r a c t Amphiphilic chitosan-graft-poly(␧-caprolactone) (CS-g-PCLs) copolymers were synthesized by a homogeneous coupling method and characterized by 1 H NMR, FTIR and ninhydrin assay. The graft copolymers were subsequently self-assembled into micelles, which were measured by DLS and TEM. The particle size of the micelles decreased as the segment grafting fraction was increased. Thereafter, silver nanoparticles were prepared in the presence of chitosan-based micelles under UV irradiation. The molar ratio and radiation time of silver to micelles were optimized with process monitored via UV–vis spectrophotometer. DLS and TEM were used to illustrate the particle structure and size while XRD patterns were applied to characterize the crystal structures of polymer-assisted silver nanoparticles. Films impregnated with silver nanoparticles were conducted with results of strong antimicrobial activities against Escherichia coli and Staphylococcus aureus as model Gram-negative and positive bacteria. © 2014 Elsevier B.V. All rights reserved.

Introduction Recently nanoparticles have been widely studied since the nanotechnology endows traditional materials, especially noble metals (e.g. Au, Ag, Pt and Pd), with novel functions and unique properties [1,2]. Owing to the small size effect and high surface area, metal nanoparticles exhibit special optical, thermal, mechanical and magnetic properties. Therefore, great efforts have been made to take the new properties for further applications, including catalysis, optical sensor and data storage [3–5]. However, these novel characteristics are closely related to their size and shape. In order to produce stable nanoparticles, many chemical and physical methods have been developed. Generally, there are two main approaches to prepare metal nanoparticles [6]. One is the physical route such as laser ablation, evaporation/condensation and irradiation with visible light or ultraviolet, etc. [7]. The other is the chemical route which utilizes reducing agent (e.g. citrate, sodium

∗ Corresponding author. Tel.: +86 21 64253916; fax: +86 21 64253916. E-mail address: [email protected] (M. Lang). http://dx.doi.org/10.1016/j.apsusc.2014.02.059 0169-4332/© 2014 Elsevier B.V. All rights reserved.

borohydride and ascorbate, etc.) to favor the formation of small metal clusters or aggregates [8]. Among several noble metal nanoparticles, silver nanoparticles (silver NPs) have attracted special attention because of its superior antibacterial characteristics as compared to bulk silver [9]. Besides, silver NPs have also found applications in fields of catalyst, optical data storage, electroluminescent displays, medical diagnosis and biomedical imaging [10]. However, the high surface energy caused by small size is unfavorable to the stability of silver NPs [11]. In order to prevent this issue, several approaches have been proposed and could be summarized into two general categories, electrostatic stabilization and steric stabilization. The former method is to make use of anionic species (e.g. halides, carboxylates) to coordinate with NPs via the formation of electrical multilayer, while steric stabilization is achieved by the presence of bulky materials to prevent the diffusion of nanoparticles [12]. In recent years, polymer-assisted fabrication has become a promising way to enhance or control spatical distribution of NPs. For example, Tarasankar et al. apply the TX-100 (poly(oxyethylene)isooctyl phenyl ether) micelles as template/capping agents to control the size and shape of gold NPs [13]. Xia et al. exploit poly(vinyl pyrrolidone) as stabilizer to generate silver nanowires by a solution-phase method [14]. Moreover,

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different polymer matrixes have been widely explored as stabilizer or scaffold for immobilization, such as dendrimers and block copolymer micelles [15,16]. Chitosan is an eco-friendly natural material, second only to cellulose in annual production. Due to its unique properties (e.g. biocompatibility, biodegradability, non-toxicity and antibacterial), it has been extensively explored in various fields including cosmetics, food, medicine and tissue engineering [17]. Importantly, the amine groups on the backbone of chitosan could favor the stabilization of silver NPs [18]. However, the strong inter- and intra-molecular hydrogen bonding make it undissolved in most common solvents, which brings the difficulty of preparing silver NPs. To overcome this issue, graft modification is adopted [19]. For example, Hu and his co-workers [20] prepared a series of chitosan-g-poly(L-lactide) (CS-g-PLLA) and chitosan-g-poly(Dlactide) (CS-g-PDLA) copolymers via “graft onto” method, which were able to dissolve in water and various common organic solvents (e.g. dichloromethane, tetrahydrofuran and dimethylformamide). Huang and Duan applied chitosan-based graft copolymers as drug delivery systems by self-assembling into core–shell micelles or as scaffolds by spinning into nanofibers [21,22]. However, few researches have been focused on its stabilization with metal NPs. In this study, CS-g-PCL was synthesized by grafting poly(␧caprolactone) onto phthaloyl-protected chitosan, followed by deprotection of phthaloyl groups to regenerate free amino groups. Then the chemical modified polymers were self-assembled into micelles with poly(␧-caprolactone) segment as core and chitosan as shell. Thereafter, the obtained micelles were used as stabilizers to prepare silver nanoparticles triggered by UV light. This method required no reducing agent or any manipulative skill, and it is reproducible. Materials and methods Materials Chitosan (CS) powder (125 mPa s) was purchased from Zhejiang Aoxing Biotechnology Co. Ltd. (China) and treated in a 40 wt% NaOH solution for 2 h at 100 ◦ C twice. The degree of deacetylation (DD) was 98% and the viscosity-average molecular weight was 1.03 × 105 Da according to the reference [23]. ␧-Caprolactone (␧-CL, Aldrich), benzyl alcohol and N,N-dimethylformide (DMF) were dried over CaH2 and distilled under a reduced pressure. Phthalic anhydride was dried under vacuum after recrystallizing from chloroform, and toluene were distilled under nitrogen after refluxing over a benzophenone-Na complex. Stannous octoate (Sn(Oct)2 ) was purchased from Aldrich and used as received. 4-Dimethylaminopyridine (DMAP) and 1-hydroxybenzotrizole (HOBt) were purchased from Aladdin Reagent (Shanghai) Co. Ltd. All other reagents were purchased from Shanghai Chemical Reagents Co. (Shanghai, China) and used without further purification. Preparation of carboxyl-terminated poly(␧-caprolactone) (PCL-COOH) and phthaloylchitosan (PHCS) Monohydroxy-terminated poly(␧-caprolactone) (PCL–OH) was synthesized by ring-opening polymerization of ␧-CL using benzyl alcohol as the initiator and Sn(Oct)2 as the catalyst in bulk at 120 ◦ C for 24 h. Then PCL–OH polymer (1 mmol) was conducted with succinic anhydride (1.2 mmol) in dry toluene (20 mL) in the presence of DMAP (1.2 mmol) at room temperature. After 48 h reaction, the solution was precipitated in hexane. The crude polymer was redissolved in dichloromethane, following the washing process of HCl

aqueous solution (10% in v/v) and saturated NaCl solution for three times, respectively. Extracted by aqueous solution, the organic layer was reprecipitated in hexane and dried in vacuum. To prepare phthaloyal-protected chitosan (PHCS), chitosan (5 mmol) was reacted with phthalic anhydride (14 mmol) in DMF (20 mL) under nitrogen at 120 ◦ C for 8 h to form pale tan solution. After the reaction, the mixture was cooled to room temperature, poured into ice water and then filtered to obtain crude product. Thereafter, the obtained PHCS was washed with methanol and vacuum-dried to a constant weight at 40 ◦ C. Preparation of chitosan-g-poly(␧-caprolactone) (CS-g-PCL) polymers CS-g-PCL was synthesized via a two-step reaction pathway. PHCS and PCL-COOH with different ratios were dissolved in dried DMF containing HOBt in an ice water bath, followed by EDC (HOBt and EDC, 2 mol equivalent to the PCL-COOH). After 30 min, the reaction was warmed up to room temperature and continued for 24 h under stirring. The product was obtained by extracting in a Soxhlet apparatus with acetone for 24 h to remove the homopolymer. Deprotection of the graft polymer was conducted in DMF containing hydrazine monohydrate at 100 ◦ C under nitrogen for 2 h. Then the reaction mixture was cooled to room temperature to precipitate. The collected precipitate was washed with water and methanol for purification and freezing-dried to obtain the powdery graft polymer. Preparation of silver NPs in micelles from the graft polymers The aqueous micelle solution was prepared by the dialysis method [24]. Generally, 10 mg of graft polymer was first dissolved in 2 mL DMSO and then added into 20 mL acetic acid aqueous solution (pH 3.5) under stirring for 2 h. The solution was dialyzed against ultrapure water with a regular change for 3 days. The micelles were finally obtained with a concentration of 0.5 mg/mL. The preparation of silver NPs was conducted using a high pressure mercury lamp (500 w, 365 nm). First, AgNO3 /CS-g-PCL29 complex with fixed molar ratio (1/1) was treated for different time scales (0–25 min). Then irradiation time was settled, various amounts of AgNO3 were mixed CS-g-PCL29 micelles under 365 nm UV irradiation for 20 min. Finally, AgNO3 /CS-g-PCL29 complex (1/1) was exposed under sunlight with substitution of UV light. Characterization FTIR spectra were recorded in KBr pellets by a Nicolet 5700 spectrometer using an absorbance mode in the range between 4000 and 400 cm−1 . 1 H NMR spectra were recorded on a Bruker Avance 500 spectrometer with D2 O/CF3 COOH (95/5, v/v) for deprotected graft polymers. Elemental analysis was carried out with a Vario ELIII. The amount of primary amines after deprotecting was determined by ninhydrin assay using glycine as a standard calibration curve according to the literature [25]. UV–vis spectra were recorded during the preparation of silver NPs in the range between 300 and 550 nm on a SPECTRA max PLUS384 spectrophotometer. The DLS spectra were recorded by a Malver Instruments Nano-ZS Nanosizer with helium neon ( = 633 nm) as a laser source and scattering angle as 173◦ . The samples were filtered through a 0.45 ␮m cellulose membrane filter before measurements. The TEM observation was performed on a JEM 1400 transmission electron microscope at the accelerating voltage of 75 kV. All samples were prepared by dropping on the copper grids and then dried up at room temperature. The XRD measurements were conducted on a Bruker AXS D8 powder diffraction

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bone since the degree of deacetylation was determined as 98%. After chemical coupling and deprotection, four peaks appeared at 1.24, 1.42, 2.40 and 3.30 ppm, which belonged to characteristic proton signals of PCL. Therefore, it confirmed that PCL segments were successfully coupled onto CS in this study. The grafting degree (GD) of PCL segments was then calculated to examine the controllability of chemical coupling. It represented the graft level of PCL macromolecules per 100 CS units. The value was calculated by the following equation. GDPCL =

Fig. 1. The chemical coupling route of CS-g-PCL graft polymer.

and scanned from 10◦ to 80◦ with a rate of 0.1◦ /s by using Cu K˛ radiation. Antimicrobial activity study

I2.40 114 × × 100 I3.20 2000

where 114 was the molecular weight of CL unit and 2000 was the average molecular weight of PCL. The subscript of samples (Table 1) represented the grafting degree of PCL segments calculated by feed ratios of PCL/PHCS. In this study, five graft copolymers were prepared. The GD values determined by 1 H NMR were in accordance with theoretical ones except for the last sample. This exception was attributed to the increasing amount of PCL, which hindered its further coupling because of the steric effect. The results demonstrated that the graft-onto reaction was controllable when weight ratios of PHCS to PCL were less than 1:3. In addition, amounts of free amino groups after deprotection were evaluated by ninhydrin assay at 570 nm via a UV–vis spectrophotometer. The obtained values were

The antimicrobial activity of silver NPs was investigated against Escherichia coli as the model Gram-negative bacteria and Staphylococcus aureus as the model Gram-positive bacteria by disc diffusion method. Plain films were cut into a disc shape with 2 cm diameter, sterilized by 75% ethanol and UV lamp. The inhibition zone was monitored after incubation on agar plate for 24 h at 37 ◦ C. Results and discussion Synthesis and characterization of CS-g-PCL The graft copolymers were synthesized via protected/deprotected method to maintain free primary amino groups in the backbone of polysaccharide (Fig. 1). First, Chitosan was reacted with phthalic anhydride to protect free amino groups. In addition, the protection could make the product soluble in DMF, which was convenient for the following “graft onto” reaction. The PCL segment was subsequently coupled with phthaloylchitosan via the OH/COOH groups. Finally, polymers with free amino groups were obtained after treatment with hydrazine monohydrate as reducing agent. Fig. 2 presented the FTIR spectra of the polymer from each step. After alkali treatment, CS showed two characteristic peaks at 890 and 1147 cm−1 , assigned to ether groups on saccharide structures and another peak at 1605 cm−1 , assigned to the amino group. The broad band at 3200–3700 cm−1 was attributed to stretching vibration of the hydroxyl and amino groups [26]. After reacting with excess phthalic anhydride, there emerged the twin absorptions at 1712 and 1777 cm−1 , assigned to imide group. Another characteristic peak at 731 cm−1 was attributed to the benzene from the protected group [27]. The other two spectra confirmed that coupling with PCL was successful owing to the emergence of 1724 cm−1 and enhancement of 2800–3000 cm−1 . They were attributed to ester carbonyl stretching band (C=O) and methylene vibration absorption peaks (C–H), respectively. 1 H NMR spectra was also used to verify the chemical structures (Fig. 3). The peaks at 3.4–3.9 and 3.2 were assigned to H3,4,5,6 and H2 of glucosamine repeat units in CS. Besides, a weak signal at 1.99 ppm was observed in the spectra. It was ascribed to the methyl groups of N-acetyl residues which remained in CS back-

(1)

Fig. 2. FTIR spectra of (a) CS, (b) PHCS, (c) PHCS-g-PCL and (d) CS-g-PCL.

Fig. 3.

1

H NMR spectra of (a) CS-g-PCL in D2 O/CF3 COOD (95/5, v/v) and (b) CS.

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Table 1 Grafting degree of PCL and amount of primary amine in CS-g-PCLs. Sample

Weight feed ratio of PHCS/PCL

GDa of CS-g-PCL

Yield (%)

Theoretical values of primary amines (mmol/g)

Experimental values of primary aminesb (mmol/g)

CS-g-PCL5 CS-g-PCL7 CS-g-PCL15 CS-g-PCL29 CS-g-PCL44

3:1 2:1 1:1 1:2 1:3

4.4 7.3 14.6 29.2 29.8

87 84 75 72 44

3.83 3.33 2.17 1.35 1.31

3.67 ± 0.08 3.25 ± 0.04 2.04 ± 0.04 1.26 ± 0.05 1.20 ± 0.05

a b

Determined by 1 H NMR, GD means the number of grafting branches per 100 CS units. Determined by ninhydrin assay.

ticle distribution index (PDI) became a constant value when GD of PCL reached up to 15. Based on above results, micelles prepared from CS-g-PCL29 graft copolymers were used to stabilize silver NPs. As well acknowledged, chemical reduction was the most frequent approach for preparation of silver NPs. But the usage of chemical reductants or organic agents became the major disadvantage from the green chemistry perspectives. Therefore, an eco-friendly method was adopted in this study with assistance of polysaccharide-based polymer micelles [28]. As an important member of polysaccharide family, chitosan was modified by PCL with reservation of free amino groups on the surface of micelles, which was able to chelate the heavy metal ions [29]. In another way, these amino groups provided a suitable environment to coordinate with silver ions. The hydrated electrons (eaq − ) produced during irradiation in solution reduced some silver ions (Ag+ ) to neutral silver atoms (Ag0 ). The process was written as follows [30]: Fig. 4. Schematic formation of CS-g-PCL micelle and silver nanoparticles.

in good agreement with theoretical ones, which indicated that the deprotection was complete. Characterization of CS-g-PCL micelles and polymer-assisted Ag NPs To prepare silver nanoparticles, CS-g-PCLs were first selfassembled into micelles via the dialysis method. As illustrated in Fig. 4, the hydrophobic chain (PCL) formed the core and the hydrophilic chain (CS) formed the shell. Besides, free amino groups on CS backbone extended to the surface of amphiphilic micelles as an important site for the following preparation of silver nanoparticles. The mean size and zeta potential of nanoparticles were measured by DLS. It was found that the particle size enlarged from 43.7 to 131.3 nm with increase of GD (Fig. 5). In addition, the par-

Fig. 5. DG course of changes in the particle size and PDI.

hv

•

•

+ n · H2 O−→e− aq + H3 O + H + OH + H2 + H2 O2 + · · · reduction

0 Ag+ + e− aq −→ Ag

(2) (3)

But these neutral silver atoms would further react with excess ions in the solution to form higher order silver ion clusters (Agn + ). After reduction, neutral silver clusters and nanoparticles formed. The coalescence processes to form dimer, timer and higher order silver ion clusters were shortly described as follows: Ag0 + Ag+ → Ag+ 2

(4)

Ag0 + Ag+ → Ag+ 0 3

(5)

(n − 3)Ag0 + Ag+ → Ag+ n 3

(6)

Ag+ n

(7)

+ ne− aq

→ Agn

The final formed silver NPs could be well dispersed in the solution as micelles facilitated the reduction of silver ions and promoted their growth selectively on the surface of micelles. A visual and direct illustration could refer to Fig. 4. The micelles were used to prevent the silver clusters from agglomeration and to stabilize the silver NPs [30]. In this study, preparation of polymer-assisted silver NPs was investigated in two aspects. They were the selection of a proper irradiation time and an optimal micelle/silver ions molar ratio. First, irradiation time (5–25 min) was optimized by means of UV–vis measurement. From Fig. 6, it was viewed that a broad absorption peak gradually red-shifted from 400 to 420 nm and the intensity was strengthened as time went by. Accordingly, the color of the polymer-assisted silver NPs changed from transparent to dark red as shown in the inset of Fig. 6. These could be attributed to the growth of silver nanoparticles triggered by UV irradiation. Then these silver clusters redispersed and formed smaller nanoparticles when they were adsorbed onto the surface of graft polymers. So the color made a change as time went by. Second, different molar ratios of graft polymer and silver ions were investigated. The amount of

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Fig. 6. Time-dependent curves of UV–vis spectra and photographs in preparation of silver NPs for (a) 5 min, (b) 10 min, (c) 15 min, (d) 20 min and (e) 25 min.

AgNO3 increased from 0 to 7.5 mg with the same amount of CSg-PCL29 micelles. Besides, the AgNO3 solution without polymer micelles was added as a comparison to investigate the influence of polymer micelles. All samples exhibited broad absorption peaks at 420 nm except for the pure silver NPs at 422 nm (Fig. 7). When the silver ions increased, the intensity strengthened and the color changed, as well. For pure silver NPs, agglomeration occurred after UV radiation for 20 min and black particles were clearly observed in Fig. 7f. But no agglomeration was found in polymer-assisted sil-

Fig. 7. Ratio-dependent curves of UV–vis spectra and photographs in preparation of silver NPs with molar ratios of AgNO3 /CS-g-PCL29 for (a) 1/0.1, (b) 1/0.2, (c) 1/0.5, (d) 1/1.0, (e) 1/1.5 and (f) 1/0 for 20 min.

ver NPs. So it was concluded that CS-g-PCL polymer micelles could helped to stabilize silver NPs, avoiding its agglomeration. Fig. 8 showed the change of particle sizes before and after radiation measured by DLS and TEM. Pure micelles were uniformly distributed with the size of 129 ± 2 nm (Fig. 8a). When AgNO3 solution was added into pure micelles and irradiated by UV light, silver NPs were uniformly distributed in spherical structures with an

Fig. 8. Size distribution histogram and TEM images of (a–b) CS-g-PCL29 micelles and (c–d) silver NPs with CS-g-PCL29 micelles irradiated by UV of 365 nm for 20 min.

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ally strengthened with the increase of silver ions/polymer micelles. The XRD patterns confirmed the preparation of polymer-assisted silver NPs and its stability. The antimicrobial activities were conducted to study its efficiency against the model Gram-positive and negative bacteria (E. coli and S. aureus) as a film additive. Acknowledgments This research was supported by the Fundamental Research Funds for the Central Universities (WD0913008), The National Natural Science Foundation of China (21274039), Basic Research Key Program Project of Commission of Science and Technology of Shanghai (12JC1403000, 12JC1403100) and “Shu Guang” Project of Shanghai Municipal Education Commission. Appendix A. Supplementary data Fig. 9. The XRD pattern of (a) pure CS-g-PCL29 micelles and (b) silver NPs prepared from CS-g-PCL29 .

average diameter of 20 ± 1 nm as shown in Fig. 8c. At the same time, a size division was detected by DLS measurement. This was consistent with theoretical micelles-assisted silver NPs as smaller size belonged to silver NPs and larger one belonged to micelles (Fig. 8c). The X-ray diffraction (XRD) was used to examine the formation of silver NPs and the crystal structure irradiated by UV light. It showed six sharp peaks at 22.4◦ , 24.0◦ , 38.1◦ , 44.3◦ , 66.4◦ and 78.0◦ for the metal NPs prepared from CS-g-PCL29 micelles (Fig. 9). The former two peaks were corresponding to the characteristic peaks for PCL segments of the graft copolymers. The latter four peaks were assigned as the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of the face centered cubic structure of silver NPs [17]. Thus, it could be concluded that silver nanoparticles were successfully prepared by CS-g-PCL graft polymers with well dispersion and stabilization for the first time. Antimicrobial activity of polymer-assisted Ag NPs Emerged with development of nanotechnology, silver NPs have been widely studied in fields of biofouling and biomedical devices. Considering the structure of CS-g-PCL graft copolymers, PCL was selected as the matrix because it was easier to form film and did not show any antimicrobial activity alone. Then polymer-assisted silver NPs were further added into PCL films and two bacteria, E. coli and S. aureus, were selected as the model Gram-negative and Gram-positive bacteria. By means of disc diffusion method, growth inhibition rings were observed in both polymer-assisted silver NPs complex films after incubation for 24 h. They were measured as 6 and 7 mm. But no inhibition zone was found for the control group (see Supporting information). So it could be concluded that polymer-assisted silver NPs showed good antimicrobial activity and was able to be applied as an additive of other films. Conclusions To summarize, CS-g-PCLs were synthesized via chemical coupling by means of protection/deprotection to obtain graft polymers with free amino groups. Thereafter, amphiphilic polymers with different grafting degree of PCL segments were self-assembled into micelles with uniform core–shell structures. The mean size of polymer micelles decreased with the increase of PCL. Silver nanoparticles were prepared in the presence of polymer micelles. Different time scales and silver ions/polymer ratios were investigated to obtain an optimal condition irradiated by 365 nm UV irradiation with 20 min. The intensity of UV absorbance was gradu-

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