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May 8, 2017 - of Medical Sciences & Peking Union Medical College, Tianjin 300192, P. R. China. ‡. State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of ... Institute of Polymer Chemistry, Collaborative Innovation Center of Chemical ...... P.; Tajik, H.; Abubakar, S.; Zandi, K. A Review on Antibacterial,.

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Silver-Decorated Polymeric Micelles Combined with Curcumin for Enhanced Antibacterial Activity Fan Huang,† Yang Gao,† Yumin Zhang,† Tangjian Cheng,‡ Hanlin Ou,‡ Lijun Yang,† Jinjian Liu,*,† Linqi Shi,*,‡ and Jianfeng Liu*,† †

Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192, P. R. China ‡ State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, P. R. China S Supporting Information *

ABSTRACT: Because of the mounting prevalence of complicated infections induced by multidrug-resistant bacteria, it is imperative to develop innovative and efficient antibacterial agents. In this work, we design a novel polymeric micelle for simultaneous decorating of silver nanoparticles and encapsulating of curcumin as a combination strategy to improve the antibacterial efficiency. In the constructed combination system, silver nanoparticles were decorated in the micellar shell because of the in situ reduction of silver ions, which were absorbed by the poly(aspartic acid) (PAsp) chains in the shell. Meanwhile, natural curcumin was encapsulated into the poly(εcaprolactone) (PCL) core of the micelle through hydrophobic interaction. This strategy could prevent aggregation of silver nanoparticles and improve the water solubility of curcumin at the same time, which showed enhanced antibacterial activity toward Gram-negative P.aeruginosa and Gram-positive S.aureus compared with sliver-decorated micelle and curcumin-loaded micelle alone, due to the cooperative antibacterial effects of the silver nanoparticles and curcumin. Furthermore, the achieved combinational micelles had good biocompatibility and low hemolytic activity. Thus, our study provides a new pathway in the rational design of combination strategy for efficiently preventing the ubiquitous bacterial infections. KEYWORDS: silver nanoparticle, polymeric micelle, curcumin, antibacterial, combination therapy

1. INTRODUCTION

antibacterial agents because of their broad-spectrum antibacterial activity, limited bacterial resistance, and relatively low toxicity toward mammalian cells.24−28 Silver nanoparticles can increase the membrane permeability of bacteria, penetrate into the bacterial cytoplasm, denature the bacterial proteins, and interfere with DNA replication, leading to the bacterial death.29−31 Thus, significant efforts have been made to achieve synergistic antibacterial activities against Gram-negative and Gram-positive bacteria by combining silver nanoparticles with conventional antibiotics, including ampicillin, Penicillin G, erythromycin, kanamycin, vancomycin, etc.32−36 Nevertheless, these methods might associate with the concerns of the aggregation of silver nanoparticles, high cost, and adverse effects of antibiotics.30,37 Hence, it is very appealing and promising to develop a silver nanoparticle-based combination therapy using friendly and inexpensive materials as the other

In recent years, the rapid emergence of multi-drug-resistant microorganisms has become a growing global issue, which was identified as one of the top three threats to human health by the World Health Organization.1−3 Thus, exploring new antibacterial agents with favorable safety and strong antibacterial activity that will not engender bacterial resistance are urgently needed. To this end, a variety of antibacterial materials such as antimicrobial peptides,4−6 cationic polymers,7,8 carbonbased nanomaterials,9−12 and polymeric13−18 and inorganic nanoparticles19−21 have been widely studied to improve the antibacterial performance. However, for many complicated bacterial infections, monotherapy is increasingly no longer adequate.22 For example, physicians often use two or more antimicrobial drugs to increase the therapeutic effect for Mycobacterium tuberculosis patients.23 Therefore, the demands of developing new and efficient combination therapy are becoming crucial for the treatment of bacterial infections. Silver nanoparticles, which are extensively used in consumer products, have been considered as one of the most excellent © 2017 American Chemical Society

Received: March 8, 2017 Accepted: May 8, 2017 Published: May 8, 2017 16880

DOI: 10.1021/acsami.7b03347 ACS Appl. Mater. Interfaces 2017, 9, 16880−16889

Research Article

ACS Applied Materials & Interfaces

Scheme 1. Schematic Illustration of the Formation of Silver-Decorated Polymeric Micelles Encapsulating Curcumin Simultaneously for Enhanced Antibacterial Activity

caprolactone)-block-poly(aspartic acid) (PCL-b-PAsp) in the aqueous solution, resulting in the micelles with hydrophobic PCL core and negatively charged PAsp shell. By micelle templating, the Ag+ ions could be easily absorbed by the PAsp segment due to the electrostatic interaction and in situ generated silver nanoparticles once addition of reducing agent. After silver-decorated micelles formation, curcumin was loaded into the hydrophobic core of the micelle and the new combination system consisting of silver nanoparticles and curcumin was thus achieved. The silver nanoparticles embedded in the micelle shell could damage the structure of bacterial membrane and the curcumin encapsulated in the micelle core could be rapidly released once interacting with the bacteria, because of the degradation of PCL by bacterial lipases, which were abundant in microorganism.51−53 Therefore, this polymeric micelle concurrently decorating silver nanoparticles and encapsulating curcumin would be a promising combination therapy for enhancing antibacterial activity.

component and simultaneously resisting silver nanoparticle aggregation. There are numerous research studies showing that utilizing a proper support material, including silica nanoparticles,38,39 graphene oxide,9,40 carbon nanotubes,41 and polymer structures,42−44 was an attractive strategy to effectively load silver nanoparticles and prevent their unfavorable aggregation which could sharply reduce the antibacterial efficiency. Among a diverse selection of templates, polymeric micelles selfassembled from amphiphilic block copolymers were a commendable candidate for growing silver nanoparticles and stabilizing them, because of their versatile compositions and unique advantages in biomedical applications.45 Moreover, polymeric micelles also offer the opportunities to simultaneously deliver different therapeutic agents.46 Curcumin, a natural polyphenolic compound, can be extracted from the rhizomes of the herb Curcuma longa. It is a highly potent, nontoxic and cheaper drug that possesses a wide range of biological activities, such as anticancer, anti-HIV, antioxidant, anti-inflammatory and antimicrobial properties.47 Recently, it has been shown that curcumin could exert synergistic effect against various bacteria when combined with other agents, including lactoferrin, N-acetylcysteine and antibiotics.48,49 However, the clinical development of curcumin is severely limited because of its poor aqueous solubility and low bioavailability.50 Fortunately, polymeric micelles could act as a decent nanocarrier to overcome the problems. Thus, combination of silver nanoparticles and curcumin within a polymeric micelle is highly desirable to fulfill the abovementioned purpose. Herein, we report a facile and cost-effective combination therapy to combat bacterial infections, utilizing a biodegradable polymeric micelle as the nanocarrier to simultaneously decorate silver nanoparticles in the shell and encapsulate curcumin in the core (Scheme 1). In this approach, the polymeric micelle was self-assembled from amphiphilic diblock copolymers, poly(ε-

2. MATERIALS AND METHODS 2.1. Materials. ε-Caprolactone (ε-CL, 99%) from Alfa Asear was dried with calcium hydride (CaH2) and then purified by distillation under reduced pressure before use. β-Benzyl L-aspartate-N-carboxyanhydride (BLA-NCA) was synthesized with the Fuchs−Farthing method using bis(trichloromethyl) carbonate (triphosgene) according to the literature procedure.54 Stannous octoate (Sn(Oct)2, 96%), tBoc-aminoethyl alcohol (99%), trifluoroacetic acid (TFA, 98%), trifluoromethanesulfonic acid (CF3SO3H, 98%), trimethylamine (TEA, 99%), sodium borohydride (NaBH4, 98%), and silver nitrate (AgNO3, 99.9%) were purchased from Alfa Asear and used as received. Curcumin (98%) and lipase from Pseudomonas cepacia were obtained from J&K Chemical Company (Beijing, China) and Sigma-Aldrich Chemical Co, respectively. All the organic solvents were redistilled before use. All aqueous solution were prepared with ultrapure Milli-Q water (resistance >18 MΩ cm−1). 2.2. Synthesis of the Block Copolymers. PAsp-b-PCL was synthesized as previously reported55 and the synthesis route was shown in Figure 1. First, PCL-NHBoc was synthesized by ring16881

DOI: 10.1021/acsami.7b03347 ACS Appl. Mater. Interfaces 2017, 9, 16880−16889

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Figure 1. Synthesis routes of PCL-b-PAsp diblock copolymers. opening polymerization (ROP) of ε-CL monomer with t-Bocaminoethyl alcohol as an initiator and Sn(Oct)2 as a catalyst in refluxed toluene. In brief, t-Boc-aminoethyl alcohol (0.16 g, 1 mmol) and ε-caprolactone (10.0 g, 87.8 mmol) were added into a roundbottom flask and dissolved in 20 mL of anhydrous toluene, followed by addition of a small amount of Sn(Oct)2 (0.1 wt %/wt). After three cycles of freeze−pump−thaw to remove moisture and oxygen, the reaction mixture was stirred at 110 °C for 12 h. The reaction mixture was then diluted with an appropriate amount of dichloromethane and precipitated into excess diethyl ether. The precipitate was filtered and dried under vacuum to obtain the PCL-NHBoc. Subsequently, the PCL-NH2 macroinitiator was achieved by the deprotection of the Boc group of PCL-NHBoc in the mixture of trifluoroacetic acid/dichloromethane (1/1, v/v). After stirring 12 h at room temperature, the solution was precipitated into excess diethyl ether and isolated by filtration. Then the precipitate was dissolved in the mixing solvent of dichloromethane/trimethylamine (1/1, v/v) and stirred at room temperature for 12 h, followed by precipitation into excess diethyl ether. The product PCL-NH2 was obtained as a white powder after drying under vacuum. Finally, PCL-b-PAsp was synthesized through deprotection of benzyl groups of poly(ε-caprolactone)-block-poly(β-benzyl-L-aspartate) (PCL-b-PBLA), which was prepared by ROP of β-benzyl Laspartate-N-carboxyanhydride (BLA-NCA) in the presence of PCLNH2. Briefly, prepared BLA-NCA (2.5 g, 10 mmol) and PCL-NH2 (2.0 g, 0.2 mmol) were dissolved in 15 mL of CH2Cl2. The reaction mixture was stirred for 24 h at 30 °C under a dry argon atmosphere. Then the solution was precipitated into excessive diethyl ether, filtered and dried under vacuum to obtain PCL-b-PBLA. Subsequently, 1.0 g of PCL-b-PBLA was treated with a mixture of trifluoroacetic acid/ trifluoromethanesulfonic acid/anisole to remove the benzyl group. The mixed solution was gently stirred for 2 h at 0 °C and precipitated into excessive diethyl ether to obtain PCL-b-PAsp. 2.3. Preparation of Different Polymeric Micelles. To obtain the polymeric micelles (PM), 5.0 mg PCL-b-PAsp was dissolved in 1 mL anhydrous DMF and the polymer solution was added dropwise into 9 mL phosphate buffer (1 mM, pH 7.4) under vigorous stirring. The PM was formed immediately, and the resulting solutions were stirred for 4 h at room temperature to make the micelles stable. The

solutions were then dialyzed against deionized water for 3 days to completely remove the DMF and the PM was thus obtained. Curcumin-loaded polymeric micelles (PM-Cur) were prepared by a similar method. In brief, 1.0 mg of curcumin and the polymer solution in DMF were mixed first and the resulting polymer/curcumin solution was added dropwise into 9 mL phosphate buffer (1 mM, pH 7.4), followed by the same procedure as described above. To prepare the silver-decorated polymeric micelles (PM-Ag), we added 100 μL of 5 mg/mL AgNO3 solution into the aqueous PCL-bPAsp micellar solution under magnetic stirring in an ice−water bath. After gently stirring for 30 min in the dark, 100 μL of 15 mg/mL freshly NaBH4 was quickly added and the color of solution turned from yellow to brown immediately. The resulting solution continued to stir for 4 h and was then dialyzed against deionized water for 2 days to obtain the PM-Ag. As for curcumin-oaded Ag micelles (PM-Ag-Cur), 1.0 mg of curcumin was dissolved in 0.5 mL of anhydrous DMF and added dropwise into PM-Ag solution under magnetic stirring. After being stirred for 2 h at room temperature, the solution was dialyzed against deionized water for 2 days to remove DMF and finally formed PM-AgCur. 2.4. Characterization. 1H NMR spectra of the block copolymers were recorded on a Varian UNITY-plus 400 M NMR spectrometer at room temperature using CDCl3 and DMSO-d6 as solvents. UV−vis absorption spectra were measured on a UV-2550 UV−visible spectrophotometer spectrophotometer (Shimadzu, Japan). Fluorescence spectra were recorded using a Hitachi F-4600 fluorescence spectrophotometer (Japan). Dynamic light scattering (DLS) experiments at a 90° scatter angle were performed on a laser light scattering spectrometer (BI-200SM) equipped with a digital correlator (BI9000AT) at 636 nm. All samples (about 1 mL) were obtained by filtering through 0.45 μm Millipore filter into a clean scintillation vial. Transmission electron microscopy (TEM) measurements were performed with a commercial Philips T20ST electron microscope at an acceleration voltage of 100 kV. To prepare the TEM samples, 10 μL sample solution was dropped onto a carbon-coated copper grid and dried in the air. The zeta potential values were measured on a Brookhaven ZetaPALS (Brookhaven Instrument, USA), using phosphate buffer solution (1 mM, pH 7.4) as the background buffer. 16882

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Figure 2. (A) Photographs of four kinds of polymeric micelles (PM, PM-Cur PM-Ag, and PM-Ag-Cur). (B) ζ-potentials of four kinds of polymeric micelles with different composition. (blue) PM, (orange) PM-Cur, (violet) PM-Ag, and (green) PM-Ag-Cur. (C) UV−vis absorption spectra and (D) fluorescence spectra of PM (blue line), PM-Cur (orange line), PM-Ag (violet line), and PM-Ag-Cur (green line) at a concentration of 0.5 mg/ mL. 2.5. Curcumin Release Study. The curcumin release of PM-Cur and PM-Ag-Cur were performed in phosphate buffer (10 mM, pH 7.4) with or without lipase. Briefly, 2 mL of the curcumin-load micelle solution with or without Pseudomonas lipase (at a final concentration of 1.0 mg/mL) was introduced into a dialysis bag (molecular weight cut off: 3500 Da) and then immersed in 20 mL of buffer solution containing Tween-80 (0.5% w/w) at 37 °C. At certain time intervals, 1 mL of the dialysis solution was taken out to measure the curcumin concentration and 1 mL fresh buffer solution was added. The cumulative release of curcumin was determined from the absorbance at 425 nm using UV-2550 UV−visible spectrophotometer (Shimadzu, Japan) and the release experiments were conducted in triplicate. 2.6. Antibacterial Activity. P. aeruginosa (Gram-negative bacteria) and S. aureus (Gram-positive bacteria) were used for antibacterial tests. First, a single colony of P. aeruginosa and S. aureus on the solid Luria−Bertani (LB) agar plate was transferred into 5 mL of liquid LB culture medium and grown overnight at 37 °C. The bacterial suspension was then diluted with LB culture medium to 1 × 105 CFU/mL. The different kinds of micelles at predetermined concentration were mixed with an equal volume of the diluted bacterial suspension and incubated at 37 °C for 12 h. Finally, the OD600 value of the solution was recorded by a Varioskan Flash (Thermo Scientific Company, USA). Samples treated with deionized water were used as controls and each assay was repeated three times. 2.7. Propidium Iodide Staining Assay Study. The 100 μL of bacterial suspension (5 × 108 CFU/mL) was mixed with 100 μL of different micelles (0.5 mg/mL) in sterile 1.5 mL Eppendorf tubes. After being cultured at 37 °C and 200 rpm for 4 h, 5 μL of PI solution (2 μg/mL) was added to the suspension and incubated for further 30 min in the dark. After that, 10 μL of the samples was dropped onto a glass slide grid with a coverslip and observed with an inverted fluorescence microscope (DMI6000B, Leica, Wetzlar, Germany).

2.8. Hemolysis Assay. Hemolysis assay was performed following the literature method with minor modification.8 First, fresh rat blood cells were harvested by centrifuging (5000 rpm for 5 min) and washing by PBS buffer for three times. Then the supernatant was discarded and the remaining red blood cells (RBCs) were resuspended in PBS buffer to obtain an approximate 5% (by volume) suspension. After that, 0.5 mL of the RBCs suspension was mixed with an equal volume of different micelles at predetermined concentration and incubated at 37 °C for 1 h. The mixtures were then centrifuged at 5000 rpm for 5 min. Finally, 100 μL of the supernatant was transferred to a 96-well plate and its absorbance at 570 nm was measured using a Varioskan Flash (Thermo Scientific Company, USA). RBCs suspensions incubated with PBS and 0.1% (by volume) Triton X-100 in PBS buffer were used as negative and positive control, respectively. All micelle samples as well as controls were placed into 3 wells to replicate in the plate. The percentage of hemolysis was calculated using the following formula hemolysis(%) = (absorbance of sample − absorbance of negative control) /(absorbance of positive control − absorbance of negative control) × 100 2.9. Cell Toxicity Assay. NIH 3T3 cells were cultured in DMEM (Gibco BRL) medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin in a humidified 5% CO2 environment at 37 °C. For the MTT assay, cells were plated at a density of 5000 cells per well on 96-well plates in fresh medium. After incubation of 24 h, different micelles (PM, PM-Cur, PM-Ag and PMAg-Cur) with predetermined concentration were added into each well. A total of 24 h later, 25 μL of MTT solution (5 mg/mL final concentration) were used to replace the mixture in each well and the cells were further incubated for another 4 h. Then, the solution was 16883

DOI: 10.1021/acsami.7b03347 ACS Appl. Mater. Interfaces 2017, 9, 16880−16889

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Figure 3. Size distributions and TEM images of four kinds of polymeric micelles with different composition. (A) PM, (B) PM-Cur, (C) PM-Ag, and (D) PM-Ag-Cur. replaced with 150 μL of DMSO and the plates were slightly shaken for 10 min. Absorbance values of formazan was measured at 570 nm using a Varioskan Flash (Thermo Scientific Company, USA). Cells without micelles were used as the control.

benzyl groups were completely removed and the target block copolymer PCL-b-PAsp was triumphantly obtained. 3.2. Preparation and Characterization of Concurrently Silver-Decorated and Curcumin-Loaded Polymeric Micelles. In the present study, the silver-decorated polymeric micelles which were simultaneously combined with curcumin (denoted as PM-Ag-Cur) were prepared in three steps. First, the amphiphilic block copolymer PCL-b-PAsp was selfassembled into uniform micelles (named as PM) in the aqueous solution, which consisted of hydrophobic PCL cores and hydrophilic PAsp shells. Attributed to the carboxyl groups (−COOH) of the PAsp chains, PM could display negative surface charges and long-term stability for a few months. In the second step, positively Ag+ ions were absorbed into the negatively surface of PM by mixing AgNO3 solution with PM and subsequently reduced into Ag nanoparticles by addition of NaBH4, resulting in the formation of silver-decorated polymeric micelles (named as PM-Ag). Finally, PM-Ag-Cur was obtained by encapsulating hydrophobic curcumin into the PCL core of PM-Ag. Meanwhile, curcumin-loaded polymeric micelles (named as PM-Cur) were prepared by self-assembly of PCLb-PAsp and curcumin to act as another control except for PM and PM-Ag. The appearance of four different micelles and curcumin aqueous solution were shown in Figure 2A and Figure S2, respectively. Curcumin could not be dissolved in pure water and formed a turbid yellow suspension, which finally precipitated at the bottom of the bottle. However, a clear and transparent solution of PM (colorless) and PM-Cur (yellow) could be observed, indicating that encapsulation of curcumin

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of the PCL-b-PAsp Diblock Copolymer. According to our previous work,55 the PCL-b-PAsp was synthesized through ROP procedure of ε-CL and BLA-NCA and subsequent deprotection of benzyl group. The chemical structures of the block copolymers were confirmed by 1H NMR. As shown in Figure S1A, the peaks at 4.05, 2.30, 1.63, and 1.38 ppm were assigned to the methylene protons a (−CH2O−), e (−CH2CO−), b+d and c in the PCL unit, respectively, and peak at 3.64 ppm was assigned to the methylene proton f (−CH2NH2), which was closed to terminal amino group. The degree of polymerization (DP) of the PCL in PCL-NH2 was calculated to be 80 by comparing the peak integration ratio of peak f and peak a. After the ROP of BLA-NCA using PCL-NH2 macroinitiator, the 1H NMR spectrum showed new peaks corresponding to the PBLA appeared at 2.6−3.1, 4.27, 5.0−5.1, and 7.2 ppm (Figure S1B), indicating the successfully synthesis of the block copolymer PCL-b-PBLA. Likewise, by comparing the peak intensities between the −CH2O− protons of PCL at 4.05 ppm and −NHCHCO− protons of the PBLA at 4.27 ppm, the DP of PBLA was calculated to be 45. After deprotection of PBLA block, peaks at 5.0−5.1 and 7.2 ppm disappeared in the 1H NMR spectrum of Figure S1C, which demonstrated that the 16884

DOI: 10.1021/acsami.7b03347 ACS Appl. Mater. Interfaces 2017, 9, 16880−16889

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was commonly known that PCL could be easily degraded in the presence of lipase.60 Therefore, it was hypothesized that the hydrophobic PCL core of our micelles would be hydrolyzed once interacting with the bacteria and thus fast released the encapsulated curcumin. To confirm this fact, the release behavior of curcumin from PM-Cur and PM-Ag-Cur were investigated in phosphate buffer at pH 7.4 both in the absence and presence of Pseudomonas lipase. As shown in Figure 4, the

into the PM rendered curcumin completely dispersible in the aqueous solution. In contrast, PM-Ag and PM-Ag-Cur displayed a brown color, which demonstrated the existence of Ag nanoparticles. The zeta potentials of these micelles were measured and the results were shown in Figure 2B. It could be found that the zeta potential of PM was −34.36 ± 0.97 mV because of the carboxyl groups in the micellar shell. Compared to the PM, the zeta potential of PM-Ag remarkably declined to −20.84 ± 0.55 mV because of the decoration of silver nanoparticles. Interestingly, there was also pronounced decrease of the zeta potential for PM-Cur (i.e., from −34.36 ± 0.97 mV to −20.53 ± 0.89 mV). According to the to the report by Pochan and co-workers,56 the phenolic OH groups of curcumin have pKa values of 8.38 (pKa1), 9.88 (pKa2) and 10.51 (pKa3) in aqueous solutions. At pH 7.4, curcumin would be in the neutral form and thus it could not bind to the PAsp shells through charge interactions. Moreover, the solubility of curcumin in aqueous solution is extremely low (2.99 × 10−8 M).57 Hence, the hydrophobic small molecule curcumin was mainly loaded into the PCL core through hydrophobic interaction. Therefore, the decline of zeta potential in PMCur was probably resulted from the weak hydrogen bonding interaction between the carboxyl groups in the PAsp shells and phenolic OH groups of encapsulated curcumin which exposed on the surface of PCL core. More importantly, PM-Ag-Cur displayed the lowest zeta potential with −9.38 ± 0.63 mV, which indicated that the micelles were decorated with Ag nanoparticles and encapsulated curcumin simultaneously. UV− vis spectroscopy was exploited to further confirm the fabrication of PM-Ag-Cur. As demonstrated in Figure 2C, compared with that no obvious absorbance for PM, PM-Cur and PM-Ag exhibited maximum absorption peak at around 425 and 400 nm, respectively, which were derived from the π−π* transition of curcumin and the surface plasmon resonance of Ag nanoparticles.9,58 As for PM-Ag-Cur, the absorption maximum was also located around 400 nm while the peak value showed obvious increase compared with that of the PM-Ag at same concentration, suggesting there was a strong interaction between PM-Ag and curcumin. The fluorescence spectra of the micelles were shown in Figure 2D. As can be seen, PM-Cur exhibited a broad band with a maximum emission at 520 nm with excitation of 420 nm, whereas the fluorescence intensity of PM-Ag-Cur dramatically decreased compared with that of PMCur, which was induced by the presence of Ag nanoparticles. This result was similar to to previous studies.59 Nevertheless, upon being treated with Triton X-100, the fluorescence of PMAg-Cur was significantly increased compared with that before adding Triton X-100 (Figure S3), implying that the curcumin capsulated in the PM-Ag-Cur was largely released because of the disruption of micellar core by the Triton X-100. The size distribution and morphology of the micelles were determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. As presented in Figure 3, the average hydrodynamic diameter (Dh) of these four different micelles were all approximately 90−95 nm with narrow particle size distributions. The TEM images revealed that the obtained micelles all possessed spherical structures with diameters which were close to the DLS results. 3.3. Lipase-Triggered Curcumin Release Study. According to the literature,51 there were a large number of different bacterial species, including P. aeruginosa, S. aureus, P. f luorescens, etc., could produce lipases which acted as an important virulence factor in bacterial infections. Meanwhile, it

Figure 4. Curcumin release profiles of PM-Cur and PM-Ag-Cur at pH 7.4 in 10 mM phosphate buffer in the absence or presence of lipase.

release rate of curcumin was very slow in the absence of lipase and only about 36% was released from PM-Ag-Cur in 48 h. However, when incubated with Pseudomonas lipase, the micelles demonstrated an obvious rapid release compared with that without lipase and the cumulative release of curcumin significantly increased to about 95% over 48 h of incubation. Similar results have been obtained for PM-Cur. These results revealed that the bacterial lipase indeed could facilitate the degradation of the PCL core of our designed micelles and thereby trigger the release of encapsulated curcumin. 3.4. Antibacterial Activity. The antibacterial activities of these micelles were evaluated by the optical density (OD600) measurement. P. aeruginosa and S. aureus were chosen as the Gram-negative and Gram-positive bacteria model respectively in this test. Bacteria were incubated with these four micelles with various concentrations at 37 °C for 12 h, and the absorbance at 600 nm after incubation were measured which had an inversely proportional relationship with antibacterial efficiency. The obtained OD600 values were subtracted by that of micelles self-to exclude any possible interference from them. As shown in Figure 5, compared with the nonantibacterial PM, the PM-Cur displayed weak inhibitory effect of bacterial growth when their concentration was over 250 μg/mL, which might be attributed to the drug loading content (DLC) of PM-Cur not being high enough (5.7 ± 0.6%, see Table S1) to completely kill the bacteria. However, the viabilities of P. aeruginosa and S. aureus significantly decreased with the increasing concentration of PM-Ag and PM-Ag-Cur, indicating that these two kinds of silver-decorated micelles had excellent antibacterial activity against both Gram-negative P. aeruginosa and Gram-positive S. aureus in a concentration-dependent manner. More importantly, PM-Ag-Cur exhibited greater inhibitory capability against bacterial proliferation than PM-Ag at the same dosage concentration, implying that the antibacterial activity was obviously enhanced when the silver-decorated polymeric micelles combined with curcumin. Furthermore, it was 16885

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Figure 5. Bacterial viability of (A) P.aeruginosa and (B) S.aureus incubated with four kinds of polymeric micelles (PM, PM-Cur, PM-Ag, and PM-AgCur) at different concentrations. Plots were obtained by optical density measurement and samples incubated with deionized water were used as the controls (100%). Data were presented as mean ± SD, n = 3.

Figure 6. Propidium iodide staining assays of P.aeruginosa and S.aureus after 4 h incubation with different micelles at 0.5 mg/mL via fluorescence microscopy observation. The scale bar equals 10 μm.

faint red fluorescence were observed which demonstrated that curcumin-loaded polymeric micelles showed weak toxicity on bacteria. However, the bacteria displayed strong red fluorescence after treatment with PM-Ag and PM-Ag-Cur for 4 h, suggesting that an abundant number of bacteria were dead and these two silver-decorated micelles could efficiently kill the bacteria. It should be point out that PI was a cell membrane impermeable fluorescent dye which could only enter into broken cell membranes and showed enhanced fluorescence after binding to nucleic acid.61,62 Therefore, these observations revealed that PM-Ag and PM-Ag-Cur possessed efficient ability to disrupt the membrane structure of bacteria. Taken together with the above results, it could be concluded that the enhanced

noteworthy that the amount of silver (see Table S1) in PM-AgCur was lower compared to that in PM-Ag at the same micelle concentrations, therefore the best antibacterial performance exerted by PM-Ag-Cur in these micelles suggested that there were synergic effect of PM-Ag and curcumin in inhibiting bacteria growth. To further investigate the antibacterial effect of these four micelles, we implemented propidium iodide (PI) staining assay to label the dead bacteria after incubated with these micelles. As seen in Figure 6, there was no any fluorescence when the P.aeruginosa and S.aureus treated with PM, signifying that all the bacteria were alive and no antibacterial activity of pure polymeric micelles. As for the PM-Cur, only sporadic and 16886

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Figure 7. Hemolysis of four kinds of polymeric micelles (PM, PM-Cur, PM-Ag, and PM-Ag-Cur) at different concentrations. RBCs were incubated with different micelles at predetermined concentration at 37 °C for 1 h and the mixtures were centrifuged to detect the cell-free hemoglobin in the supernatant. RBCs incubated with PBS and 0.1% (by volume) Triton X-100 in PBS buffer were used as negative (−) and positive (+) control, respectively. All micelle samples as well as controls were placed into 3 wells to replicate in the plate.

antibacterial efficiency of PM-Ag-Cur was ascribed to the membrane damage induced by PM-Ag and synchronously curcumin release caused by bacterial lipases. 3.5. Hemolysis and Cytotoxicity of the Micelles. Since the biocompatibility is of great importance for antibacterial material in clinical application, it was essential to assess the toxicity effect of the manufactured micelles on mammalian cells, especially red blood cells (RBCs). The hemolytic behavior of these micelles was therefore measured by exposing RBCs to a range of concentrations of micelles from 31 μg/mL to 500 μg/ mL. As shown in Figure 7 and Figure S4, there were no obvious hemolytic activity for these micelles even at the highest concentration tested (500 μg/mL), demonstrating the great biocompatibility of these micelles with RBCs. The cytotoxicity of these micelles against NIH3T3 cells was also investigated. Figure 8 showed the cell viability of NIH3T3 cells after 24 h culture with these micelles at different concentration. The results revealed that the cell viability of these micelles were all over 80% even at the highest concentration applied (500 μg/ mL), indicating that these micelles exhibited very low toxicity to mammalian cells. Thus, the PM-Ag-Cur had potential for in vivo use because of their good cytocompatibility and enhanced antibacterial activity.

Figure 8. Cytotoxicity of four different micelles (PM, PM-Cur, PMAg, and PM-Ag-Cur) against NIH3T3 cells after 24 h incubation. Cell viability was measured by MTT assay and the data were shown as the mean ± SD of 6 replicate groups.

membrane by decorated silver nanoparticles and synchronously antibacterial property of loaded curcumin. Morever, the polymeric micelles possessed great cytocompatibility toward mammalian cells and in particular red blood cells. Therefore, this combination system might be a promising and powerful weapon for combating the multiple bacteria-induced infections.

4. CONCLUSION In summary, we have successfully developed a new effective antibacterial agent based on the combination of sliverdecorated polymeric micelle and natural curcumin. Through hierarchical assembly strategy, silver nanoparticles are facilely decorated inside the micellar shell and curcumin is easily encapsulated into the micellar core at the same time. Compared with single sliver-decorated micelle and curcumin-loaded micelle, the fabricated combination system exhibited enhanced antibacterial activities against both Gram-negative P. aeruginosa and Gram-positive S. aureus based on the disruption of bacterial



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b03347. Characterizations of block copolymers and four different kinds of micelles (PM, PM-Cur, PM-Ag, and PM-AgCur), photographs of curcumin in water, fluorescence 16887

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ACS Applied Materials & Interfaces



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spectra of PM-Ag-Cur after the addition of Triton X-100, and hemolytic activity of these four micelles (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.L.). *E-mail: [email protected] (L.S.). *E-mail: [email protected] (J.L.). ORCID

Jinjian Liu: 0000-0001-5176-7613 Linqi Shi: 0000-0002-9534-795X Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51603231, 81471727), CAMS Initiative for Innovative Medicine (2016-I2M-3-022), the Natural Science Foundation of Tianjin, China (16ZXXYSY00040, 16JCQNJC02500, 16JCZDJC33000), PUMC Youth Fund and the Fundamental Research Funds for the Central Universities (3332016098), and the Fundamental Research Funds for CAMS & PUMC (2016ZX310079).



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Research Article

ACS Applied Materials & Interfaces

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