Surface-Adaptive Gold Nanoparticles with Effective Adherence and

Aug 14, 2017 - The surrounding healthy tissues showed no damage because the dispersed AuNPs had no photothermal effect under NIR light. In view of the...
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Surface-Adaptive Gold Nanoparticles with Effective Adherence and Enhanced Photothermal Ablation of Methicillin-Resistant Staphylococcus aureus Biofilm Dengfeng Hu,† Huan Li,† Bailiang Wang,‡ Zi Ye,‡ Wenxi Lei,† Fan Jia,† Qiao Jin,† Ke-Feng Ren,† and Jian Ji*,† †

MOE Key Laboratory of Macromolecule Synthesis and Functionalization of Ministry of Education, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China ‡ School of Ophthalmology & Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou 325027, China S Supporting Information *

ABSTRACT: Biofilms that contribute to the persistent bacterial infections pose serious threats to global public health, mainly due to their resistance to antibiotics penetration and escaping innate immune attacks by phagocytes. Here, we report a kind of surface-adaptive gold nanoparticles (AuNPs) exhibiting (1) a self-adaptive target to the acidic microenvironment of biofilm, (2) an enhanced photothermal ablation of methicillin-resistant Staphylococcus aureus (MRSA) biofilm under near-infrared (NIR) light irradiation, and (3) no damage to the healthy tissues around the biofilm. Originally, AuNPs were readily prepared by surface modification with pH-responsive mixed charged zwitterionic self-assembled monolayers consisting of weak electrolytic 11-mercaptoundecanoic acid (HS-C10COOH) and strong electrolytic (10-mercaptodecyl)trimethylammonium bromide (HS-C10-N4). The mixed charged zwitterion-modified AuNPs showed fast pH-responsive transition from negative charge to positive charge, which enabled the AuNPs to disperse well in healthy tissues (pH ∼7.4), while quickly presenting strong adherence to negatively charged bacteria surfaces in MRSA biofilm (pH ∼5.5). Simultaneous AuNP aggregation within the MRSA biofilm enhanced the photothermal ablation of MRSA biofilm under NIR light irradiation. The surrounding healthy tissues showed no damage because the dispersed AuNPs had no photothermal effect under NIR light. In view of the above advantages as well as the straightforward preparation, AuNPs developed in this work may find potential applications as a useful antibacterial agent in the areas of healthcare. KEYWORDS: biofilm, gold nanoparticles, mixed charged zwitterion, pH-responsive, photothermal therapy

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ellular bacterial DNA, and enzymes. The EPS serves as a guardian barrier against antibiotic infiltration and cellular attack by host innate immune cells.1,5−9 Due to the encapsulation of EPS, the microenvironment of biofilm lacks oxygen, resulting in anaerobic glycolysis, hypoxia, and ion channel turbulence, and is more acidic than that of healthy tissues.10 Furthermore, the biofilm is also the archcriminal of plenty of chronic and obstinate diseases.1,11−13 It is vital and urgent to develop novel strategies to fight against biofilms, especially biofilms of multidrug-resistant bacteria.

athogenic bacterial infections bring about the second largest cause of death with around 17 million victims all over the world every year, and plenty of pathogens have developed unparalleled resistance to drugs, due to the abuse of antibiotics in developed countries.1 In the last few decades, multi-drug-resistant bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), and so on, have become more general causes of intractable infections.2 The resistance to drugs of multi-drug-resistant bacteria not only arises from the structure transformation or gene mutation of planktonic bacteria3,4 but also results from the formation of biofilms.5 The biofilm is a hybrid of bacterial cells that is irreversibly adhered to the surface of material or tissue and packed in selfproduced extracellular polymeric substances (EPS) that are primarily composed of exopolysaccharides, proteins, extrac© 2017 American Chemical Society

Received: July 6, 2017 Accepted: August 14, 2017 Published: August 14, 2017 9330

DOI: 10.1021/acsnano.7b04731 ACS Nano 2017, 11, 9330−9339

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Scheme 1. Effect of an Acidic Trigger Approach onto the Effective Adherence and Enhanced Photothermal Ablation of MRSA Biofilma

a (A) Schematic illustration of the pH responsiveness of AuNPs. (B) AuNP-N-S, with negatively charged surfaces, dispersed stably in MRSA biofilm and healthy tissues but with no bactericidal effect under NIR light irradiation. (C) AuNP-N-C, with positively charged surfaces in acidic MRSA biofilm (pH ∼5.5), effectively adhered to bacteria and rapidly aggregated in MRSA biofilm, exhibiting great bactericidal effect under NIR light irradiation without damage to surrounding healthy tissues (pH ∼7.4).

poly(vinylpyrrolidone) sulfobetaine and polyaniline, providing the rapid and effective killing of 99.9% of the Gram-positive and Gram-negative bacteria.46 With many excellent properties such as bare biological toxicity, the gold nanoparticles,47,48 which can exhibit relatively high temperature increase via the surface plasma resonance under the advisable wavelength of light, also had been reported to ablate the bacterial infections.49,50 In photothermal therapy, however, it is still a tremendous problem that the nonlocalized heat generally causes great damage to healthy tissues when killing biofilms. In this article, we design a kind of surface-adaptive mixed charged zwitterionic 14 nm gold nanoparticle (abbreviated as AuNP-N-C), which was fabricated with mixed self-assembled monolayers (SAMs) consisting of strong electrolytic (10mercaptodecyl)trimethylammonium bromide (HS-C10-N4) and weak electrolytic 11-mercaptoundecanoic acid (HS-C10COOH). The mixed charged zwitterionic AuNPs with expected pH response ranges can be obtained by regulating the feed ratio of these two ligands. Here, we hypothesize that (1) in healthy tissues, with generally physiological conditions (pH ∼7.4), the AuNP-N-C with an abundant negatively charged surface

In order to solve severe biofilm infections, numerous strategies, not only involving chemotherapy by using antibiotics,14,15 antibacterial peptides,16,17 quaternary ammonium compounds,18 peptidopolysaccharides,19 cationic materials,20 antibacterial or antibiofilm coatings,21−24 and antibacterial polymers,25−36 but also containing nonchemotherapy, such as photodynamic therapy37,38 and photothermal therapy,39−41 have been put forward and researched. Compared with the chemotherapy, photothermal therapy which can destroy the structure of biofilm and kill bacteria by physical heat has tremendous advantages to treat the infections caused by biofilms. NIR light in the range of 700−1100 nm, which exhibits excellent capability of tissue penetration and minimal damage to healthy tissues, is the most favorable wavelength region suitable for photothermal therapy.42−45 More recently, photothermal therapy of bacterial infections based on NIR light irradiation have been widely applied. Ling et al. demonstrated that NIR light could promote graphene generating hyperpyrexia to kill Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli.42 Park et al. reported that NIR light triggers a sharp rise in photothermal heat of catechol-conjugated 9331

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Figure 1. (a) Digital images of AuNP-N-S and AuNP-N-C with the feed ratio of positively charged ligand and negatively charged ligand 1:1.5 when added to phosphate-buffered (PB, 10 mM) solution at different pH values. (b) Absorbance peak values of AuNP-N-S and AuNP-N-C detected by UV−vis spectrum once added to PB solution at different pH values. (c) ζ-Potential of AuNP-N-S, AuNP-N-C, and MRSA in PB solution at different pH values. (d) Size of AuNP-N-S and AuNP-N-C in different pH PB solution. (e) UV−vis spectrum of AuNP-N-C at pH 7.4 and 5.5 and detected after adjusting the AuNP-N-C from pH 5.5 to 7.4 again in PB, and the embedding graphs are the digital images of AuNP-N-C in different pH values. (f) Gel images of plasma protein adsorption assay. Line 1 and line 3: AuNP-N-S and AuNP-N-C mixed with PB solution (pH 7.4). Line 2 and line 4: AuNP-N-S and AuNP-N-C mixed with bovine serum albumin (BSA, 5 mg/mL, pH 7.4). (g) Thermographic images of AuNP-N-S and AuNP-N-C in PB solution at pH 5.5.

disperses stably. (2) While in MRSA biofilm, a dramatically acidic condition (pH ∼5.5), the negatively charged surfaces on AuNP-N-C are rapidly replaced by the positively charged surfaces, which show effective electrostatic adherence to

bacteria with negatively charged cell surfaces within biofilm. (3) Under NIR light irradiation, the aggregated gold nanoparticles within biofilm will exhibit outstanding photothermal ablation of MRSA biofilm, whereas the dispersed gold 9332

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Figure 2. (a) Digital images of AuNP-N-S (left, 0.5587 mM, 100 μL) and AuNP-N-C (right, 0.5587 mM, 100uL) when added to the MRSA biofilm at once. Aggregation quantities of AuNPs (b) with different concentrations for 60 min and (c) with the concentration of 0.5587 mM for different time intervals in MRSA biofilm evaluated by ICP-MS. (d) SEM images of MRSA biofilm treated with nothing, AuNP-N-S, and AuNP-N-C solutions (0.5587 mM, 100 μL) for 60 min (scale bar in images is 200 nm).

(Figure 1a). The pH-induced aggregation of AuNP-N-C was the result of ionization or protonation of carboxyl groups in weak electrolytic ligand at different pH values, which influenced the balance of forces on the surface of gold nanoparticles among van der Waals attraction, hydrogen-bonding attraction, hydration repulsion, electrostatic repulsion, and so on.51,52 AuNP-N-C containing weak carboxylic ligand electrolyte and strong quaternary ammonium ligand electrolyte exhibit peculiar zwitterionic characteristics. At high pH, the AuNP-N-C were dispersed stably in solution due to the ionization of carboxyl groups to a large extent, which provided a strong negative surface on the gold nanoparticles, resulting in the electrostatic repulsion, and hydration surpassed the attractions of the van der Waals force and hydrogen bonding. With the pH decreasing to a certain value, the carboxylic ligand partially protonated, and the quantities of negative charges declined, which resulted in the electrostatic repulsion and hydration becoming weak and the AuNP-N-C beginning to aggregate once the electrostatic repulsion and hydration weaken compared to van der Waals force and hydrogen bonding. As the pH continued to decrease,

nanoparticles show bare damage to surrounding healthy tissues. AuNP-N-S (14 nm gold modified with the SAMs of strong electrolytic (10-mercaptodecyl)trimethylammonium bromide (HS-C10-N4) and strong electrolytic 10-mercaptodecanesulfonic acid (HS-C10-SO3H)) exhibit no surface adaptability to acidic pH and were prepared as a control to investigate the effect of an acidic trigger approach onto the effective adherence and enhanced photothermal ablation of MRSA biofilm (Scheme 1).

RESULTS AND DISCUSSION Preparation and Characterization of pH-Responsive Zwitterionic AuNPs. In order to prepare AuNP-N-C, the 14 nm citrate-capped AuNPs were modified with mixed SAMs composed of strong electrolytic (10-mercaptodecyl)trimethylammonium bromide (HS-C10-N4) and weak electrolytic 11-mercaptoundecanoic acid (HS-C10-COOH) with various feed ratios. AuNP-N-C exhibited distinct aggregation transition pH value ranging from 5.5 to 6.5 when the feed ratio of quaternary ammonium ligand and carboxylic ligand was 1:1.5 9333

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Figure 3. (a) Temperature evolution curves of AuNP-N-S and AuNP-N-C solutions in MRSA biofilm under NIR light irradiation. (b) Number of bacterial colonies, (c) images of live/dead staining assay, and (d) SEM images after the MRSA biofilm treated with nothing, AuNP-N-S under NIR light irradiation, and AuNP-N-C under NIR light irradiation. (e) Cell viability of 3T3 fibroblasts when treated with AuNP-N-S, AuNP-N-C, and AuNP-N-S under NIR light irradiation and AuNP-N-C under NIR light irradiation. The concentration and volume of AuNP solutions were 0.5587 mM and 100 μL, respectively, and the intensity and irradiation time intervals of NIR light were 0.91 W/cm2 and 10 min, respectively.

surfaces in some pH ranges, obviously at pH 5.5 (Figure 1c), and the diameter of AuNP-N-C increased to several hundred nanometers (Figures 1d and S1). The apparent changes of size and surface potentials of gold nanoparticles demonstrated AuNP-N-C had excellent pH responsiveness at different pH

the carboxylic ligand protonated further and the amount of positive charges surpassed that of negative charges, leading to the gold nanoparticles dispersing again. Notably, before the complete redispersion, AuNP-N-C existed in solution in the form of aggregates (Figure 1a,b) with positively charged 9334

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Figure 4. (a) Thermographic images of rabbits treated by nothing, AuNP-N-S under NIR light, and AuNP-N-C under NIR light. (b) Digital photographs of MRSA infection sites on scarfskin and dermis with different treatments. (c) Photomicrographs of bacterial colony-forming units, obtained from infected tissue of rabbits treated under various experimental conditions. (d) Related quantitative results of standard plate counting assay (n = 6). (e) Histological photomicrographs of MRSA biofilm (red arrow) and its surrounding tissues (black arrow) that were treated with AuNP-N-C under NIR light on day 7 after treatment. All sections were stained with H&E. (f) Histological photomicrographs of skin tissue sections of infected rabbits with different treatments (on day 7).

values. In addition, the pH-responsive aggregation was reversible (Figure 1e), which could eliminate the perniciousness of aggregated gold nanoparticles to healthy tissues after ablating the biofilm. As a control, AuNP-N-S stabilized by strong

electrolytic positive and strong electrolytic negative ligands exhibited no evident pH responsiveness. Before the in vitro and in vivo antibacteria studies, the protein resistance in normal physiological microenvironment and the 9335

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bacteria would become denatured and metabolism would be disordered, eventually resulting in bacterial death above 50 °C.42 The standard plate counting assay showed that after treating with AuNP-N-C exposed to NIR light, the number of living bacteria decreased greatly (Figure 3b), indicating the outstanding photothermal bactericidal effect of AuNP-N-C under NIR light irradiation. To further evaluate the bactericidal effect, the live/dead stain assay was conducted. In this method, live bacteria with intact cell membranes would be stained green and dead bacteria with damaged membranes would be stained red when observed under a fluorescence microscope. The prevalence of red fluorescence of the MRSA biofilm in the presence of AuNP-N-C under NIR light indicated that the number of dead or compromised bacteria increased in quantity (Figure 3c), which was consistent with the results of a standard plate counting assay. After treatment with AuNP-N-C under NIR light irradiation, the MRSA biofilm presented obvious structural destruction (Figure 3d), and most of the bacteria were killed. As controls, the MRSA biofilm treated with nothing or AuNP-N-S in the presence NIR light had no apparent bactericidal effects. Moreover, the cell viability of 3T3 fibroblasts in the presence of AuNP-N-C whether exposed to NIR light or not was high and exhibited no obvious difference in pH 7.4 (Figure 3e), indicating that AuNP-N-C under NIR light irradiation showed no toxicity to healthy tissues. When it comes to the reason for the different photothermal effects of gold nanoparticles at different pH values, it can be attributed to the states of dispersion or aggregation. Compared with the dispersive 14 nm gold nanoparticles, the aggregated gold nanoparticles exhibit better photothermal effect in the presence of NIR light irradiation, not only because of a dramatic reduction (at least 2 orders of magnitude) in the bubble formation threshold, which is closely relevant to the local temperature of gold nanoparticles, but also because of the evident red-shifted absorption of light in aggregated nanoparticles.54,55 In Vivo Antibacterial Activity of AuNP-N-C Irradiated by NIR Light. In order to test the in vivo bactericidal effect, the AuNP solutions were injected into the subcutaneous abscess created in a rabbit model via the local infection of MRSA and then irradiated by NIR light. The AuNP-N-C that were injected in MRSA biofilm showed a rapid temperature increase and basically remained at about 55 °C irradiated by NIR light (Figure 4a). There was no doubt that this temperature was adequately high to kill bacteria, which was demonstrated in the previous study.42 Furthermore, the tissue surrounding MRSA biofilm showed no apparent temperature increase, implying no heat damage in healthy tissues. After treatment with AuNP-N-C under NIR light for 7 days, no evident inflammation was observed on scarfskin or dermis, suggesting great bactericidal effects, whereas there was severe inflammation and abscess on the rabbits treated by nothing and AuNP-N-S under NIR light, implying terrible results of killing bacteria (Figure 4b). Moreover, the effect of killing bacteria in each test group of rabbits was then conducted by using standard plate counting methods. The colony-forming unit counts exhibited an admirable bactericidal effect after receiving the treatment with AuNP-N-C under NIR light irradiation (Figure 4c,d). The histological analyses of skin sections using H&E stain were used to further evaluate the effect of photothermal therapy of the MRSA biofilm treated with AuNP-N-C in the presence of NIR light. Compared with the healthy tissues without any treatment, the histological analyses revealed signs of serious

enhanced photothermal effect of gold nanoparticles induced by pH responsiveness in acidic biofilm microenvironment were investigated carefully. With powerful negatively charged surfaces at pH 7.4 (Figure 1c), AuNP-N-C exhibited excellent protein resistance not only in single protein solution (BSA, 5 mg/mL) but also in biological complex media such as cell culture medium at pH 7.4 (Figures 1f and S2). After aggregation, AuNP-N-C aggregates exhibited intensive temperature increase under NIR light irradiation (Figures 1g and S3). It is trustworthy that the rapid and reversible pH-responsive transition of the AuNP-N-C will endow them an admirable pHsensitive performance in bactericidal applications. Effectively Adherence to Bacteria and Rapid Aggregation of AuNP-N-C in Biofilm. To investigate pH responsiveness of gold nanoparticles in acidic MRSA biofilm (Figure S4) that was prepared according to the previous method,53 the adherence and aggregation properties of AuNPN-C were evaluated meticulously. The biofilm was uniformly stained to bluish violet by crystal violet, which could combine nucleic acid specifically, indicating the MRSA biofilm prepared in this study was compact and homogeneous (Figure S4a). Moreover, the morphology observed by SEM was integrated (Figure S4b,c). After AuNP-N-C were added into the acidic MRSA biofilm (pH ∼5.5), a fast and remarkable color change from wine red to charcoal gray was observed, which demonstrated that the pH-induced aggregation of AuNP-N-C happened rapidly (Figure 2a). In addition, it was found that the retention quantities of AuNP-N-C were closely dependent on its concentration and contacting time intervals with MRSA biofilm (Figure 2b,c). With the concentration increasing, the retention amount of AuNP-N-C increased visibly. About onethird of the amount of AuNP-N-C remained in MRSA biofilm even though undergoing a three rinses with phosphate buffered saline (PBS, 10 mM) after adding them to biofilm for only 5 min, and the quantities of AuNP-N-C came up to about 60% of total adding amount after an hour. Nevertheless, as a control, there was no obvious adherence and aggregation of AuNP-N-S in MRSA biofilm regardless of its concentration or contacting time intervals with biofilm (Figure 2b,c). Next, the nature of the enhanced retention of AuNP-N-C in biofilm was demonstrated by relating the ζ-potentials of AuNPs and MRSA involved in this study (Figure 2c). As can be seen, the MRSA remained negatively charged from pH 5.0 to 7.4. Differently, the ζ-potentials of AuNP-N-C became positive when the pH decreased to 5.5. The electrostatic attraction promoted AuNP-N-C to adhere to MRSA effectively and resulted in intensive retention of gold nanoparticles in MRSA biofilm (Figures 2d and S5, red arrows show AuNP-N-C aggregates). This demonstrated that the AuNP-N-C with a suitable pH-responsive range admirably promoted the nanoparticles adhering to bacteria with negatively charged cell surfaces and largely aggregating in the biofilm, which provided great convenience for ablating the biofilm via photothermal effect. In Vitro Antibiofilm Activity of AuNP-N-C Irradiated by NIR Light. To evaluate the bactericidal effect, AuNP-N-C solution was added into the MRSA biofilm and then irradiated by the NIR light. Once irradiated by NIR light, the temperature of the MRSA biofilm containing AuNP-N-C increased rapidly in the first 2 min and showed a maximum temperature of approximately 60 °C (Figures 3a and S6), which demonstrated the excellent photothermal effect of AuNP-N-C even in MRSA biofilm. It was reported that the enzymes, proteins, and lipids in 9336

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Shimadzu UV-2505 spectrometer using 1 cm path length quartz cuvettes), and TEM (JEM-1230EX transmission electron microscope operating at 80 kV in bright-field mode) measurements. ζ-Potentials of AuNPs and MRSA and Hydrodynamic Diameters of AuNPs. The ζ-potentials of the AuNPs and MRSA were measured at 25 °C using a Delsa Nano C particle analyzer (Beckman Coulter Ireland Inc.) in PB (10 mM) from pH 5.0 to 7.4. The MRSA were suspended at a concentration 1 × 108 bacteria/mL. In addition, hydrodynamic radii of the AuNPs were measured using a Zetasizer. AuNP Interaction with BSA. The interaction of different AuNPs with BSA was measured by gel electrophoresis. The experiments were performed by mixing 5 μL of AuNPs with 5 μL of 10 mg/mL BSA. After incubation at room temperature for 10 min, the mixture was loaded on a 1% agarose gel buffered with 0.5× TBE (Tris-borateEDTA, pH 7.4). Gel electrophoresis was also performed in 0.5× TBE buffer at 120 V constant voltage for 15 min. Gel shift bands were directly recorded by a digital camera. Culturing and Harvesting MRSA Biofilm. MRSA (ATCC 43300, bought from Guangdong culture collection center) were employed in this study. For culturing MRSA biofilm, a 100 μL droplet of a MRSA suspension (108 bacteria/mL) and 100 μL of tryptone soy broth medium (TSB) were placed on 96-well plates to culture at 37 °C. After 24 h, TSB medium was replaced with fresh TSB, and the biofilm was grown for another 24 h. Finally, the TSB medium was removed and the MRSA biofilm attached on 96-well plates was harvested. Staining the MRSA Biofilm by Crystal Violet. The 5% crystal violet was added to the MRSA biofilm for 10 min, and then the dye was removed and rinsed by PBS five times. After being rinsed, 95% ethyl alcohol was added to the biofilm to dissolve and wash away the redundant crystal violet. Finally, the biofilm was observed and photographed under a microscope. Aggregation Quantity of AuNPs in Biofilm Detected by ICPMS. Aggregation of AuNPs in biofilm was measured by ICP-MS quantitatively. In order to determine the AuNP aggregation amount quantitatively, varying concentration of AuNP-N-S solutions (0.2974, 0.5587, 1.1173 mM) and AuNP-N-C solutions (0.2974, 0.5587, 1.1173 mM) were added into the biofilm cultured on 96-well plates for 5 min or other time intervals. At a determined time, the biofilms were washed three times with PBS and then treated with aqua regia (HNO3/HCL = 1:3, volume ratio) for 2 h. The treated solution was diluted 15 times to determine Au concentration by ICP-MS (Thermo Elemental Corporation of USA, X Series II). Aggregation and Adhesion Behavior of AuNPs in Biofilm Detected by SEM Analysis. For SEM biofilm section analysis, the MRSA were cultured to biofilm on a glass sheet according to the aforementioned method. After the biofilm was obtained, the two types of AuNPs with a Au atomic concentration of about 0.5587 mM were added to the biofilm for 60 min. At a determined time, the biofilms were washed three times with PBS and then fixed with 2.5% glutaraldehyde for 2 h at 4 °C. After fixation, the samples were dehydrated in an alcohol series. Finally, the samples were disposed by metal spraying and observed by SEM. NIR Photothermal Bactericidal Assay in Vitro. After being treated with AuNPs (0.5587 mM, 100 μL) for 60 min, the MRSA biofilms cultured in 96-well plates were irradiated using an 808 nm NIR light (0.91 W/cm2) for 10 min. The diode continuous wave light with a wavelength of 808 nm was from Hi-Tech Optoelectronics Co., Ltd. Standard Plate Counting Assays.57 After NIR irradiation, 100 μL of sterile PBS was added into each well of a 96-well plate. The plate was sonicated and then blown and inhaled by pipet carefully to disperse the biofilms. The suspensions were serially diluted with sterile PBS, and 100 μL diluted samples were spread on the tryptone soy agar plates. After incubation at 37 °C for 12 h, the colonies formed were counted. Live/Dead Staining Assays. After NIR irradiation, the wells in the 96-well plates were washed with sterile PBS slightly, and then biofilms in 96-well plates were incubated using BacLight live/dead dye

infection in the control groups, whereas a significant reduction of infiltration of inflammatory cells was observed following the treatment with AuNP-N-C irradiated by NIR light (Figure 4e), indicating excellent bactericidal effect. In addition, the in vivo biotoxicity of AuNP-N-C to healthy tissues was also demonstrated by histological analyses. There were only a small quantity of inflammatory cells (red arrow, Figure 4f) in the MRSA biofilm but no obvious foreign-body reaction (black arrow, Figure 4f) in the healthy tissues surrounded by the MRSA biofilm on the seventh day after treatment with AuNPN-C under NIR light, suggesting the AuNP-N-C with NIR irradiation effectively killed bacteria but caused no damage to surrounding healthy tissues.

CONCLUSIONS In summary, the surface-adaptive mixed charged zwitterionic AuNP-N-C, which can effectively adhere to bacteria and aggregate rapidly in acidic biofilm but disperse stably in healthy tissues, were successfully prepared. The aggregated AuNP-N-C with enhanced NIR absorbance can effectively convert NIR light energy into localized heat, resulting in thermal ablation of MRSA biofilm; at the same time, the dispersive AuNP-N-C exhibited no damage to healthy tissues. The pH-responsive gold nanoparticles described here, containing no conventional antibiotics, have great potential in the treatment of bacterial infection, including drug-resistant bacteria and their biofilms. Given the above advantages, AuNP-N-C developed in this study may provide great benefits in the areas of healthcare. MATERIALS AND METHODS Preparation of Thiol-Protected AuNPs. Citrate-modified AuNPs with a diameter of 14 nm and mixed charged zwitterion protected AuNPs were prepared according to previous reports.56 By adding the same total concentration of thiol ligands with the same feed ratios of strong electrolytic (10-mercaptodecyl)trimethylammonium bromide (HS-C10-N4) to weak electrolytic 11-mercaptoundecanoic acid (HS-C10-COOH) and HS-C10-N4 to strong electrolytic 10mercaptodecanesulfonic acid (HS-C10-SO3H) to the same amount of citrate-capped 14 nm AuNPs, two kinds of mixed charged SAMprotected AuNPs with different surface composition were prepared. Here, we refer to the AuNPs modified with HS-C10-N4 and HS-C10COOH and HS-C10-N4 and HS-C10-SO3H as AuNP-N-C and AuNPN-S, respectively. Taking AuNP-N-C as an example to illustrate the preparation routes, shortly, a mixed thiol ligand aqueous solution (20 mM, 1 mL) containing a 1:1.5 feed ratio of HS-C10-N4 and HS-C10COOH was poured into the initial citrate-capped 14 nm AuNP solution (10 mM, 20 mL). Then, 2 M NaOH was used to adjust the pH of the solution to pH ∼9 as HS-C10-COOH has a better solubility in alkaline conditions, besides, it is also beneficial for holding the AuNPs stable through the ligand exchange reaction. After being stirred for 24 h at room temperature, the mixed charged zwitterion-modified AuNPs were purified by centrifuging twice at 15000 rpm for 15 min. Afterward, the AuNPs were dispersed again in drops of water, and then a few drops of phosphate buffer solution (10 mM PB, pH 7.4) were added to regulate the pH of AuNP solution to ∼7.4. Analogously, the AuNPs protected by HS-C10-N4 and HS-C10-SO3H were prepared in the same way. Characterization of pH Responsiveness of AuNP-N-S and AuNP-N-C. To determine the pH responsiveness of AuNPs, 10 μL of AuNPs modified with ligands was added to 190 μL of phosphate buffer (PB, 10 mM) solutions with different pH values. For the pH responsiveness of AuNPs in biological media, AuNPs were added to cell culture media with 10% fetal bovine serum at different pH values. The pH responsiveness of gold nanoparticles was characterized by color change observation, DLS (90 plus particle size analyzer, Brookhaven Instruments Co.), UV−vis spectroscopy (UV−vis 9337

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ACS Nano in the dark for 15 min. After incubation, the redundant dye was washed by sterile PBS slightly for three times. At last, the biofilms in 96-well plates were observed under a fluorescence microscope. Cell Viability Assays. The cell viability of AuNPs was evaluated by MTT assays. 3T3 fibroblast cells were used as model cells and seeded into 96-well plates (6000 cells per well) with 180 μL of DMEM culturing medium in each well for 24 h. AuNP solution with concentrations from 0.2974, 0.5587, and 1.1173 mM were added to the cells and irradiated by NIR light. After being incubated for 48 h, 20 μL of MTT solution (0.1 mg/mL) was added to each well and cultured for another 4 h. Then the medium was removed, and 150 mL of DMSO was added to each well to dissolve the obtained crystals. The absorbance was recorded at 570 nm by a microplate reader (model 550, BioRad). Animal Studies. Animal experiments were performed according to the Guidelines for Animal Care and Use Committee, Wenzhou Medical University. Healthy New Zealand White Rabbits were used in the animal study. To evaluate the effect of the AuNP-N-C as a photothermal agent, a subcutaneous abscess was experimentally fabricated in each test rabbit. Briefly, the rabbits were first anesthetized using 2% sodium pentobarbital. After shaving and disinfecting with iodophor, a subcutaneous injection of MRSA (108 bacteria/mL, 100 μL) was conducted on the shaved back of the test rabbits. Ten rabbits were used in this study. After bacteria injection of 24 h, an obvious infected abscess had arisen subcutaneously in each test rabbit. The AuNP solution (0.5587 mM, 0.5 mL) was then directly injected into the infected abscess. Then an 808 nm NIR light (0.91 W/cm2) was used to irradiate the abscess for 10 min. During the NIR light irradiation, the rabbits were under anesthesia. The thermographic images of each test rabbit were taken by an IR thermal camera (ICI7320, infrared camera, Beaumont, TX, USA). After 7 days, the rabbits were euthanized by CO2 inhalation, and their skins were exposed to take photographs. To investigate their bactericidal efficiency, each test rabbit received three MRSA infections, and these were then treated under one of the following three experimental conditions: no treatment, injected with AuNP-N-S and exposed to the light, and treated with AuNP-N-C and exposed to the light (n = 6). Following treatments, the rabbits were sacrificed, and these abscesses were harvested for histological H&E staining analysis and analysis by standard plate count methods. Statistical Analysis. All data are expressed as means ± SD. Differences between groups were examined for statistical significance with two-tailed Student’s t test, accepting significance at p < 0.05.

gratefully acknowledged. We would like to thank Hua Wang for TEM, Suhua Cao for SEM, and Zigang Xu for ICP-MS.

REFERENCES (1) Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Bacterial Biofilms: A Common Cause of Persistent Infections. Science 1999, 284, 1318−1322. (2) Ladd, A. P.; Levy, M. S.; Quilty, J. Minimally Invasive Technique in Treatment of Complex, Subcutaneous Abscesses in Children. J. Pediatr. Surg. 2010, 45, 1562−1566. (3) Andersson, D. I.; Hughes, D. Antibiotic Resistance and Its Cost: Is It Possible to Reverse Resistance? Nat. Rev. Microbiol. 2010, 8, 260− 271. (4) Aminov, R. I. The Role of Antibiotics and Antibiotic Resistance in Nature. Environ. Microbiol. 2009, 11, 2970−2988. (5) Abee, T.; Kovacs, A. T.; Kuipers, O. P.; van der Veen, S. Biofilm Formation and Dispersal in Gram-Positive Bacteria. Curr. Opin. Biotechnol. 2011, 22, 172−179. (6) Chua, S. L.; Liu, Y.; Yam, J. K.; Chen, Y.; Vejborg, R. M.; Tan, B. G.; Kjelleberg, S.; Tolker-Nielsen, T.; Givskov, M.; Yang, L. Dispersed Cells Represent a Distinct Stage in the Transition from Bacterial Biofilm to Planktonic Lifestyles. Nat. Commun. 2014, 5, 4462. (7) Davies, D. Understanding Biofilm Resistance to Antibacterial Agents. Nat. Rev. Drug Discovery 2003, 2, 114−122. (8) Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Bacterial Biofilms: From the Natural Environment to Infectious Diseases. Nat. Rev. Microbiol. 2004, 2, 95−108. (9) Yang, L.; Liu, Y.; Wu, H.; Song, Z.; Hoiby, N.; Molin, S.; Givskov, M. Combating Biofilms. FEMS Immunol. Med. Microbiol. 2012, 65, 146−157. (10) Davies, D. G.; Marques, C. N. A Fatty Acid Messenger Is Responsible for Inducing Dispersion in Microbial Biofilms. J. Bacteriol. 2009, 191, 1393−1403. (11) Randal Bollinger, R.; Barbas, A. S.; Bush, E. L.; Lin, S. S.; Parker, W. Biofilms in the Large Bowel Suggest an Apparent Function of the Human Vermiform Appendix. J. Theor. Biol. 2007, 249, 826−831. (12) Spoering, A. L.; Lewis, K. Biofilms and Planktonic Cells of Pseudomonas Aeruginosa Have Similar Resistance to Killing by Antimicrobials. J. Bacteriol. 2001, 183, 6746−6751. (13) Stoodley, P.; Debeer, D.; Lewandowski, Z. Liquid Flow in Biofilm Systems. Appl. Environ. Microbiol. 1994, 60, 2711−2716. (14) Gu, H. W.; Ho, P. L.; Tong, E.; Wang, L.; Xu, B. Presenting Vancomycin on Nanoparticles to Enhance Antimicrobial Activities. Nano Lett. 2003, 3, 1261−1263. (15) Zhao, Y. Y.; Tian, Y.; Cui, Y.; Liu, W. W.; Ma, W. S.; Jiang, X. Y. Small Molecule-Capped Gold Nanoparticles as Potent Antibacterial Agents That Target Gram-Negative Bacteria. J. Am. Chem. Soc. 2010, 132, 12349−12356. (16) Qi, X.; Zhou, C.; Li, P.; Xu, W.; Cao, Y.; Ling, H.; Ning Chen, W.; Ming, L. C.; Xu, R.; Lamrani, M.; Mu, Y.; Leong, S. S.; Wook Chang, M.; Chan-Park, M. B. Novel Short Antibacterial and Antifungal Peptides with Low Cytotoxicity: Efficacy and Action Mechanisms. Biochem. Biophys. Res. Commun. 2010, 398, 594−600. (17) Saravanan, R.; Li, X.; Lim, K.; Mohanram, H.; Peng, L.; Mishra, B.; Basu, A.; Lee, J. M.; Bhattacharjya, S.; Leong, S. S. Design of Short Membrane Selective Antimicrobial Peptides Containing Tryptophan and Arginine Residues for Improved Activity, Salt-Resistance, and Biocompatibility. Biotechnol. Bioeng. 2014, 111, 37−49. (18) Pu, Y.; Hou, Z.; Khin, M. M.; Zamudio-Vazquez, R.; Poon, K. L.; Duan, H.; Chan-Park, M. B. Synthesis and Antibacterial Study of Sulfobetaine/Quaternary Ammonium-Modified Star-Shaped Poly[2(dimethylamino)ethyl methacrylate]-Based Copolymers with an Inorganic Core. Biomacromolecules 2017, 18, 44−55. (19) Su, Y.; Tian, L.; Yu, M.; Gao, Q.; Wang, D.; Xi, Y.; Yang, P.; Lei, B.; Ma, P. X.; Li, P. Cationic Peptidopolysaccharides Synthesized by ‘Click’ Chemistry with Enhanced Broad-Spectrum Antimicrobial Activities. Polym. Chem. 2017, 8, 3788−3800. (20) Li, P.; Poon, Y. F.; Li, W.; Zhu, H. Y.; Yeap, S. H.; Cao, Y.; Qi, X.; Zhou, C.; Lamrani, M.; Beuerman, R. W.; Kang, E. T.; Mu, Y.; Li,

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b04731. Figures S1−S6 (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Qiao Jin: 0000-0002-6584-4111 Ke-Feng Ren: 0000-0001-5456-984X Jian Ji: 0000-0001-9870-4038 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS Financial support from National Natural Science Foundation of China (21574114), Science and Technology Planning Project of Zhejiang Province (2016C04002), Science and Technology Planning Project of Zhejiang Province (2016C54003), and National Natural Science Foundation of China (51573160) is 9338

DOI: 10.1021/acsnano.7b04731 ACS Nano 2017, 11, 9330−9339

Article

ACS Nano C. M.; Chang, M. W.; Leong, S. S.; Chan-Park, M. B. A Polycationic Antimicrobial and Biocompatible Hydrogel with Microbe Membrane Suctioning Ability. Nat. Mater. 2011, 10, 149−156. (21) Gao, Q.; Yu, M.; Su, Y.; Xie, M.; Zhao, X.; Li, P.; Ma, P. X. Rationally Designed Dual Functional Block Copolymers for Bottlebrush-like Coatings: in vitro and in Vivo Antimicrobial, Antibiofilm, and Antifouling Properties. Acta Biomater. 2017, 51, 112−124. (22) Gu, J.; Su, Y.; Liu, P.; Li, P.; Yang, P. An Environmentally Benign Antimicrobial Coating Based on a Protein Supramolecular Assembly. ACS Appl. Mater. Interfaces 2017, 9, 198−210. (23) Su, Y.; Zhi, Z.; Gao, Q.; Xie, M.; Yu, M.; Lei, B.; Li, P.; Ma, P. X. Autoclaving-Derived Surface Coating with in Vitro and in Vivo Antimicrobial and Antibiofilm Efficacies. Adv. Healthcare Mater. 2017, 6, 1601173. (24) Zhi, Z.; Su, Y.; Xi, Y.; Tian, L.; Xu, M.; Wang, Q.; Padidan, S.; Li, P.; Huang, W. Dual-Functional Polyethylene Glycol-b-Polyhexanide Surface Coating with in Vitro and in Vivo Antimicrobial and Antifouling Activities. ACS Appl. Mater. Interfaces 2017, 9, 10383− 10397. (25) Bruenke, J.; Roschke, I.; Agarwal, S.; Riemann, T.; Greiner, A. Quantitative Comparison of the Antimicrobial Efficiency of Leaching versus Nonleaching Polymer Materials. Macromol. Biosci. 2016, 16, 647−654. (26) Mattheis, C.; Wang, H.; Meister, C.; Agarwal, S. Effect of Guanidinylation on the Properties of Poly(2-aminoethylmethacrylate)based Antibacterial Materials. Macromol. Biosci. 2013, 13, 242−255. (27) Mattheis, C.; Zhang, Y.; Agarwal, S. Thermo-Switchable Antibacterial Activity. Macromol. Biosci. 2012, 12, 1401−1412. (28) Tan, L.; Maji, S.; Mattheis, C.; Zheng, M.; Chen, Y.; CaballeroDiaz, E.; Gil, P. R.; Parak, W. J.; Greiner, A.; Agarwal, S. Antimicrobial Hydantoin-Containing Polyesters. Macromol. Biosci. 2012, 12, 1068− 1076. (29) Liu, R.; Chen, X.; Chakraborty, S.; Lemke, J. J.; Hayouka, Z.; Chow, C.; Welch, R. A.; Weisblum, B.; Masters, K. S.; Gellman, S. H. Tuning the Biological Activity Profile of Antibacterial Polymers via Subunit Substitution Pattern. J. Am. Chem. Soc. 2014, 136, 4410−4418. (30) Liu, R.; Chen, X.; Falk, S. P.; Masters, K. S.; Weisblum, B.; Gellman, S. H. Nylon-3 Polymers Active Against Drug-Resistant Candida Albicans Biofilms. J. Am. Chem. Soc. 2015, 137, 2183−2186. (31) Liu, C.; Liu, B.; Chan-Park, M. B. Synthesis of PolycaprolactonePolyimide-Polycaprolactone Triblock Copolymers via a 2-Step Sequential Copolymerization and Their Application as Carbon Nanotube Dispersants. Polym. Chem. 2017, 8, 674−681. (32) Liu, R.; Chen, X.; Falk, S. P.; Mowery, B. P.; Karlsson, A. J.; Weisblum, B.; Palecek, S. P.; Masters, K. S.; Gellman, S. H. StructureActivity Relationships among Antifungal Nylon-3 Polymers: Identification of Materials Active Against Drug-Resistant Strains of Candida Albicans. J. Am. Chem. Soc. 2014, 136, 4333−4342. (33) Bakhshi, H.; Agarwal, S. Dendrons as Active Clicking Tool for Generating Non-Leaching Antibacterial Materials. Polym. Chem. 2016, 7, 5322−5330. (34) Hauenstein, O.; Agarwal, S.; Greiner, A. Bio-Based Polycarbonate as Synthetic Toolbox. Nat. Commun. 2016, 7, 11862. (35) Landis, R. F.; Gupta, A.; Lee, Y. W.; Wang, L. S.; Golba, B.; Couillaud, B.; Ridolfo, R.; Das, R.; Rotello, V. M. Cross-Linked Polymer-Stabilized Nanocomposites for the Treatment of Bacterial Biofilms. ACS Nano 2017, 11, 946−952. (36) Wang, J.; Nguyen, T. D.; Cao, Q.; Wang, Y.; Tan, M. Y.; ChanPark, M. B. Selective Surface Charge Sign Reversal on Metallic Carbon Nanotubes for Facile Ultrahigh Purity Nanotube Sorting. ACS Nano 2016, 10, 3222−3232. (37) Xing, C.; Yang, G.; Liu, L.; Yang, Q.; Lv, F.; Wang, S. Conjugated Polymers for Light-Activated Antifungal Activity. Small 2012, 8, 524−529. (38) Yuan, H.; Chong, H.; Wang, B.; Zhu, C.; Liu, L.; Yang, Q.; Lv, F.; Wang, S. Chemical Molecule-Induced Light-Activated System for Anticancer and Antifungal Activities. J. Am. Chem. Soc. 2012, 134, 13184−13187.

(39) Hsiao, C. W.; Chen, H. L.; Liao, Z. X.; Sureshbabu, R.; Hsiao, H. C.; Lin, S. J.; Chang, Y.; Sung, H. W. Effective Photothermal Killing of Pathogenic Bacteria by Using Spatially Tunable Colloidal Gels with Nano-Localized Heating Sources. Adv. Funct. Mater. 2015, 25, 721− 728. (40) Zharov, V. P.; Mercer, K. E.; Galitovskaya, E. N.; Smeltzer, M. S. Photothermal Nanotherapeutics and Nanodiagnostics for Selective Killing of Bacteria Targeted with Gold Nanoparticles. Biophys. J. 2006, 90, 619−627. (41) Park, S. J.; Kang, E. B.; Sharker, S. M.; Lee, G.; In, I.; Park, S. Y. NIR-Mediated Antibacterial Clay Nanocomposites: Exfoliation of Montmorillonite Nanolayers by IR825 Intercalation. Macromol. Mater. Eng. 2016, 301, 141−148. (42) Wu, M. C.; Deokar, A. R.; Liao, J. H.; Shih, P. Y.; Ling, Y. C. Graphene-Based Photothermal Agent for Rapid and Effective Killing of Bacteria. ACS Nano 2013, 7, 1281−1290. (43) Huang, W. C.; Tsai, P. J.; Chen, Y. C. Multifunctional Fe3O4@ Au Nanoeggs as Photothermal Agents for Selective Killing of Nosocomial and Antibiotic-Resistant Bacteria. Small 2009, 5, 51−56. (44) Dong, K.; Ju, E.; Gao, N.; Wang, Z.; Ren, J.; Qu, X. Synergistic Eradication of Antibiotic-Resistant Bacteria Based Biofilms in Vivo Using a NIR-Sensitive Nanoplatform. Chem. Commun. 2016, 52, 5312−5315. (45) Zou, X.; Zhang, L.; Wang, Z.; Luo, Y. Mechanisms of the Antimicrobial Activities of Graphene Materials. J. Am. Chem. Soc. 2016, 138, 2064−2077. (46) Kim, S. H.; Kang, E. B.; Jeong, C. J.; Sharker, S. M.; In, I.; Park, S. Y. Light Controllable Surface Coating for Effective Photothermal Killing of Bacteria. ACS Appl. Mater. Interfaces 2015, 7, 15600−15606. (47) Bokern, S.; Gries, K.; Görtz, H.-H.; Warzelhan, V.; Agarwal, S.; Greiner, A. Precisely Designed Gold Nanoparticles by Surface Polymerization - Artificial Molecules as Building Blocks for Novel Materials. Adv. Funct. Mater. 2011, 21, 3753−3759. (48) Mitschang, F.; Schmalz, H.; Agarwal, S.; Greiner, A. Tea-Baglike Polymer Nanoreactors Filled with Gold Nanoparticles. Angew. Chem., Int. Ed. 2014, 53, 4972−4975. (49) Ramasamy, M.; Lee, S. S.; Yi, D. K.; Kim, K. Magnetic, Optical Gold Nanorods for Recyclable Photothermal Ablation of Bacteria. J. Mater. Chem. B 2014, 2, 981−988. (50) Ray, P. C.; Khan, S. A.; Singh, A. K.; Senapati, D.; Fan, Z. Nanomaterials for Targeted Detection and Photothermal Killing of Bacteria. Chem. Soc. Rev. 2012, 41, 3193−3209. (51) Yang, H.; Heng, X.; Hu, J. Salt- and pH-Resistant Gold Nanoparticles Decorated with Mixed-Charge Zwitterionic Ligands, and Their pH-induced Concentration Behavior. RSC Adv. 2012, 2, 12648−12651. (52) Liu, X. S.; Chen, Y. J.; Li, H.; Huang, N.; Jin, Q.; Ren, K. F.; Ji, J. Enhanced Retention and Cellular Uptake of Nanoparticles in Tumors by Controlling Their Aggregation Behavior. ACS Nano 2013, 7, 6244− 6257. (53) Liu, Y.; Busscher, H. J.; Zhao, B.; Li, Y.; Zhang, Z.; van der Mei, H. C.; Ren, Y.; Shi, L. Surface-Adaptive, Antimicrobially Loaded, Micellar Nanocarriers with Enhanced Penetration and Killing Efficiency in Staphylococcal Biofilms. ACS Nano 2016, 10, 4779− 4789. (54) Zharov, V. P.; Galitovskaya, E. N.; Johnson, C.; Kelly, T. Synergistic Enhancement of Selective Nanophotothermolysis with Gold Nanoclusters: Potential for Cancer Therapy. Lasers Surg. Med. 2005, 37, 219−226. (55) Zharov, V. P.; Letfullin, R. R.; Galitovskaya, E. N. MicrobubblesOverlapping Mode for Laser Killing of Cancer Cells with Absorbing Nanoparticle Clusters. J. Phys. D: Appl. Phys. 2005, 38, 2571−2581. (56) Liu, X.; Huang, H.; Jin, Q.; Ji, J. Mixed Charged Zwitterionic Self-Assembled Monolayers as A Facile Way to Stabilize Large Gold Nanoparticles. Langmuir 2011, 27, 5242−5251. (57) Zhang, P.; Li, S.; Chen, H.; Wang, X.; Liu, L.; Lv, F.; Wang, S. Biofilm Inhibition and Elimination Regulated by Cationic Conjugated Polymers. ACS Appl. Mater. Interfaces 2017, 9, 16933−16938.

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