Research Article Cite This: ACS Appl. Mater. Interfaces 2017, 9, 36606-36614
www.acsami.org
Strong Near-Infrared Absorbing and Biocompatible CuS Nanoparticles for Rapid and Efficient Photothermal Ablation of Gram-Positive and -Negative Bacteria Jiale Huang,† Jinfei Zhou,†,‡ Junyang Zhuang,‡ Hongzhi Gao,§ Donghong Huang,§ Lixing Wang,§ Wenlin Wu,‡ Qingbiao Li,†,‡ Da-Peng Yang,*,‡ and Ming-Yong Han*,‡,⊥ †
Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, P. R. China ‡ Fujian Province Key Laboratory for Preparation and Function Development of Active Substances from Marine Algae, College of Chemical Engineering and Materials Science, Quanzhou Normal University, Quanzhou 362000, Fujian Province, PR China § The Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, Fujian Province, Pr China ⊥ Institute of Materials Research and Engineering, Singapore 138634 S Supporting Information *
ABSTRACT: Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are the most common infectious bacteria in our daily life, and seriously affect human’s health. Because of the frequent and extensive use of antibiotics, the microbial strains forming drug resistance have become more and more difficult to deal with. Herein, we utilized bovine serum albumin (BSA) as the template to synthesize uniform copper sulfide (CuS) nanoparticles via a biomineralization method. The as-prepared BSA-CuS nanocomposites showed good biocompatibility and strong near-infrared absorbance performance and can be used as an efficient photothermal conversion agent for pathogenic bacteria ablation with a 980 nm laser at a low power density of 1.59 W/cm2. The cytotoxicity of BSA-CuS nanocomposite was investigated using skin fibroblast cells and displayed good biocompatibility. Furthermore, the antibacterial tests indicated that BSA-CuS nanocomposite showed no antibacterial activity without NIR irradiation. In contrast, they demonstrated satisfying killing bacterial ability in the presence of NIR irradiation. Interestingly, S. aureus and E. coli showed various antibacterial mechanisms, possibly because of the different architectures of bacterial walls. Considering the low cost, easy preparation, excellent biocompatibility and strong photothermal convention efficiency (24.68%), the BSA-CuS nanocomposites combined with NIR irradiation will shed bright light on the treatment of antibiotic-resistant pathogenic bacteria. KEYWORDS: copper sulfide, pathogenic bacteria, photothermal ablation, antibacterial, cytotoxicity
■
antibiotic resistance in bacteria.7 The drug-resistant strains are able to survive and even multiply in the presence of antibiotics.8 As such, antibiotics have been detected in groundwater. What is worse, the antibiotic drug has a limited service life, and it is very difficult to develop a new generation of drug.9 There may be another 1−2 decades left for people to use the existing antibiotics, which clearly indicates the developing new agents for killing bacteria is urgent.10,11 Nanomaterials have attracted great attention because of their intriguing physical and chemical properties.12 Especially, they have been explored as tools to kill pathogenic microbes.13−20 Such kind of nanomaterials mainly includes Ag nanoparticles
INTRODUCTION Bacterial infections are one of the most serious risks in the world that humans cannot avoid, whether in medicine, food, or other fields. It is reported that the worldwide food production industry loses about several billion per year due to the presence of food-borne bacteria.1 On the other hand, many patients die of postoperative infections annually.2 Both S. aureus and E. coli are the most common infectious bacteria in our daily life, and seriously affect the health of people. Several serious diseases including bacteremia, pneumonia, endocarditis, septic arthritis, osteomyelitis, and deep abscess formation are closely associated with bacterial infections.3 Therefore, great efforts have been actively devoted to curing these infection diseases.4−6 Currently, antibiotic has acted as an effective drug to inhibit the growth of bacteria. However, the overuse, misuse, and improper disposal of antibiotics lead to the evolution of © 2017 American Chemical Society
Received: July 26, 2017 Accepted: October 4, 2017 Published: October 4, 2017 36606
DOI: 10.1021/acsami.7b11062 ACS Appl. Mater. Interfaces 2017, 9, 36606−36614
Research Article
ACS Applied Materials & Interfaces (Ag NPs),21−24 C-based nanoparticles,26−28 Au nanoparticles29−33 and Zn,34,35 Cu-based36,25,37 nanoparticles. Of all reported antibacterial nanomaterials, Ag NPs are widely investigated as antibacterial agents in various fields. Ag pots and cups were also used in ancient time to protect food from decay.38 However, when Ag NPs are exposed to air or water, they will release Ag ions into the environment slowly, and undergo chemical and biochemical conversion, which is harmful to the biological systems.39 Additionally, Ag NPs are extremely unstable and cannot keep long-term activity.24 Graphene oxide (GO) is a hot 2D carbon nanomaterial for eradicating bacteria in vitro and in vivo.40−42 Unfortunately, the long-term toxicity of this material is still unknown.43 Although Au NPs are also efficient nanomaterials for antibacterial application,44 the high price limits its development in large scale. Compared with the above-mentioned nanomaterials, Cu-based NPs with low cost, easy preparation, high stability, and good biocompatibility45 have emerged as new class of nanomaterials in electronic, optical, catalysis and biomedical fields. Since the d-d energy band transition of Cu2+ ions, CuS NPs can transform light into heat. Therefore, CuS NPs with NIR absorption property are being actively explored as novel photothermal agents for tumor ablation. For example, Hu et al. synthesized hydrophilic flowerlike CuS superstructures as an efficient 980 nm laser-driven photothermal agent for ablation of HeLa cells.46 Huang et al. prepared ultrasmall and uniform CuS nanoparticles using ferritin (Fn) as the template. The resulting CuS-Fn nanocomposites can fully destroy the U87MG tumor cells with a low laser irradiation dose (808 nm, 0.8 W cm−2). Although these work has demonstrated on the tumor ablation application of CuS NPs, less work focus on the killing of pathogenic bacteria using the material under NIR irradiation.7,47 In our previous work, we reported the highly branched gold nanocrosses as novel antibacterial agents that exhibited efficient antibacterial activity.8 To reduce the high cost of raw material and enhance the biocompatibility, in this work, low-cost NIR absorbing and biocompatible CuS NPs were synthesized using BSA as the template via a facile biomineralization method,48 as shown in Scheme 1. The as-prepared materials showed good
low CuS NPs concentration (50 ppm). Thus, CuS NPs with excellent photothermal antibacterial ability and much less toxicity to human cells stand for a promising disinfection nanomaterial in medical treatment for bacterial infections.
■
EXPERIMENTAL SECTION
Materials and Reagents. Copper(II) nitrate trihydrate (Cu (NO3)2·3H2O) and nitric acid (HNO3) were obtained from Sinopharm Chemical Reagent Co. Ltd. (China). Bovine serum albumin (BSA) was purchased from Shanghai Shisheng Sibas Advanced Technology Co. Ltd. Thioacetamide (CH3CSNH2) and phosphate buffer saline were acquired from Sinopharm Chemical Reagent Co. Ltd. LIVE/DEAD Baclight Bacterial Viability and Counting Kit was purchased from Thermo Fisher Scientific (China) Co. Ltd. BacT/ALERT SN broth medium was purchased from Biomerieux. Skin fibroblast cells were obtained from the cell bank of Chinese Academy of Sciences. Bacteria were provided by the Second Affiliated Hospital of Fujian Medical University. Deionized (DI) water was used throughout this work. Synthesis of CuS Nanoparticles. In a typical synthesis of CuS NPs, 250 mg of BSA powder and 120 mg of Cu(NO3)2·3H2O were separately dissolved in 50 mL of deionized water. The two solutions were then mixed together under magnetic stirring at 1200 rpm/min for 30 min. A nitric acid solution was further added dropwise into the resulting light blue solution to adjust pH to 3. Thereafter, 5 mL of 0.2 M CH3CSNH2 solution was injected and the color of the solution turned to yellow gradually. After heating in a water bath at 90 °C for 1 h, the color of the solution turned to dark green, indicating the formation of BSA-encapsulated CuS (i.e., BSA-CuS) NPs. The precipitate was collected by centrifugation, washed with deionized water at least three times, and dried under vacuum for further characterization and application. Photothermal Performance Test of BSA-CuS NPs. One mL of BSA-CuS NPs solution with different Cu ions concentrations (0, 10, 30, 50, and 100 ppm) in PBS was respectively exposed to a 980 nm laser (MW-GX-980/3000Mw China) at a power density of 1.59 W/ cm2 for 10 min. One thermometer (Beckmann, China) was inserted into the solution to record the temperature change in order to test the photothermal performance of BSA-CuS NPs. Photothermal Ablation against S. aureus and E. coli. S. aureus (ATCC 25923) and E. coli (ATCC 25922) were incubated in agar plates at 37 °C for 12 h in a biochemical incubator. The photothermal ablation experiment was carried out against S. aureus and E. coli after culturing them in BacT/ALERT SN broth medium. Appropriate amounts of S. aureus and E. coli were individually inoculated on agar plates and then exposed to BSA-CuS NPs (50 ppm) for photothermal ablation testing. The detailed experimental procedure is as following: the agar plates were evenly covered with a mixture of 50 μL of broth medium and 50 μL of bacteria supernatant (106 CFU) in the presence of 50 μL (50 ppm) BSA-CuS NPs, incubated at 37 °C for 16 h accompanied by and without 980 nm laser irradiation (a power density of 1.59 W/cm2, 5 min). As a control, agar plates were evenly covered with a mixture of 50 μL of broth medium and 50 μL of bacteria supernatant in the absence of BSA-CuS NPs, incubated at 37 °C for 16 h accompanied by and without 980 nm laser irradiation (power density of 1.59 W/cm2, 5 min). Alternatively, 2 mL of S. aureus or E. coli with 1 × 106 colonyforming unit (CFU) was inoculated into tubes after mixing 2 mL of BacT/ALERT SN broth medium, and then exposed to 2 mL of BSACuS NPs at different concentrations (0, 10, 20, 30, 40, 50 ppm) for testing the photothermal ablation of bacterial in solution. And then, the tubes were placed in the biochemical incubator, incubated for 16 h. For S. aureus, six samples in the tubes labeled by a1−a6 (without 980 nm irradiation) and b1−b6 (under 980 nm irradiation for 45 min before incubation) corresponding to different concentrations of BSACuS NPs from 0, 10, 20, 30, 40, 50 ppm were completed. The growth rates of the strains were determined by measuring the optical density of 150 μL of solution (sampled from the tubes) at 450 nm wavelength using a microplate reader (Rayto RT-6100 China).
Scheme 1. Schematic Diagram Illustrating the Fabrication and Bactericidal Process of BSA-CuS NPs As a Kind of Photothermal Agent
biocompatibility to skin fibroblast cells. Likewise, antibacterial test showed that there is no antibacterial activity against both S. aureus and E. coli upon incubation of BSA-CuS NPs alone, even at a high concentration. Upon irradiation, BSA-CuS NPs exhibited excellent killing ability of pathogenic bacteria. Over 80% bacteria (either S. aureus or E. coli) were eradicated with 36607
DOI: 10.1021/acsami.7b11062 ACS Appl. Mater. Interfaces 2017, 9, 36606−36614
Research Article
ACS Applied Materials & Interfaces
Figure 1. (A) TEM image and (B) XRD pattern of BSA-CuS NPs. Inset is the histogram of BSA-CuS NPs. (C) UV−vis−NIR absorption spectra of BSA-CuS NPs with original solution (black line) and after NIR irradiation 1 h (red line). (D) Temperature change of the 1 mL of aqueous solution containing BSA-CuS NPs with different concentrations and irradiation time.
■
For the measurement of the survival rate of the strains, 2 mL of the strains (S. aureus and E. coli) inoculated with 1 × 106 CFU were added to BacT/ALERT SN broth media tubes (2 mL) and the tubes were incubated at 37 °C for 12 h in a biochemical incubator. For each strain, four tubes were labeled as following: (a) adding 2 mL, 50 ppm BSACuS NPs and exposing to NIR irradiation for 45 min; (b) adding 2 mL, 50 ppm BSA-CuS NPs without NIR irradiation; (c) adding 2 mL PBS and exposing to NIR irradiation for 45 min; (d) adding 2 mL PBS without NIR irradiation. The tubes continued to be incubated for 12 h. Then, 1 mL of solution was sampled from each tube and centrifuged three times to remove the culture. And then, the precipitates were redispersed in 1 mL normal saline (NS). Subsequently, a 10 μL sample was taken from the solution and added to the flow cytometry tube. Then, 987 μL of normal saline and 3 μL of staining solution (by LIVE/DEAD Baclight Bacterial Viability and Counting Kit) were added into the flow cytometry tube. Finally, the flow cytometry tube was tested by the flow cytometer (Beckman USA). The dye solution was dropped onto a glass slide and observed by fluorescence microscope (LEICA DM 2500, Germany). Furthermore, the Cu2+ ions concentration in the bacteria was determined by inductively coupled plasma mass spectrometry (ICP-MS Thermo Fisher Scientific USA). Characterizations of BSA-CuS NPs and Bacteria. Transmission electron microscopy (TEM) characterization of BSA-CuS NPs was performed on an electron microscope (Tecnai F30, FEI; Netherlands) with an accelerating voltage of 300 kV. The sizes of CuS NPs were measured on the basis of TEM images. After centrifugation, the precipitates were dried at −20 °C in vacuum and taken for X-ray diffraction (XRD) analysis, which was performed on a Rigaku Ultima IV X-ray Diffractometer (Rigaku, Japan). The morphology of E.coil and S. aureus after contact with the samples were observed by the scanning electron microscope (ZEISS, Germany) with an accelerating voltage of 1.2 kV. The bacterial suspensions were first rinsed by using normal saline (NS). Then, they were fixed with 35% glutaraldehyde for 0.5 h and dehydrated in graded ethanol series (30−95%). The suspension was dropped onto a clean silicon wafer and dried naturallu, not sputter-coated with platinum for SEM observation.
RESULTS AND DISCUSSION Characterization of BSA-CuS NPs. BSA had been widely selected as a soft template for the synthesis of nanomaterials
Figure 2. Bacterial growth after incubation in broth medium accompanied by NIR irradiation for 5 min: (A, E) in the presence of BSA-CuS NPs, (B, F) in the presence of PBS. Bacterial growth after incubation in broth medium without irradiation: (C, G) in the presence of BSA-CuS NPs, (D, H) in the presence of PBS.
because of its low cost, excellent biocompatibility and abundant functional groups including thiol, amine, and carboxyl groups. It has been reported that albumin can enhance the stability of the nanostructures.49 Furthermore, it can be employed as a viable drug carrier for anticancer and antibiotic drugs.50−52 In this work, BSA-CuS NPs were synthesized in aqueous solution by reacting Cu (NO3)2 and CH3CSNH2 in the presence of BSA at 90 °C for 60 min. It should be noted that Qu et al. synthesized hydrophilic Cu2−xS NPs through two-step ligands transformation in different solvents and a high thermal decomposition condition (180 °C), which is more complex than our method.12 Figure 1A shows the representative TEM images of the as-prepared products. The nanoparticles are almost platelike with a narrow size distribution. From the histogram of size distribution (inset in Figure 1A), it can be seen that the 36608
DOI: 10.1021/acsami.7b11062 ACS Appl. Mater. Interfaces 2017, 9, 36606−36614
Research Article
ACS Applied Materials & Interfaces
Figure 3. (A) Turbidity changes with different dosages of CuS NPs (0, 10, 20, 30, 40, and 50 ppm) incubated with S. aureus and E. coli for 12 h with/ without NIR irradiation at a power density of 1.59 W/cm2 for 45 min. Growth curves of S. aureus (B) and E. coli (C) with different concentrations of BSA-CuS NPs encountering NIR irradiation (blue line) and lacking NIR irradiation (green line).
heat quickly under NIR irradiation for effective photothermal ablation of bacteria. Antibacterial Activity of the BSA-CuS NPs. To evaluate photothermal ablation of BSA-CuS NPs against S. aureus and E. coli, both bacteria in the agar plates are incubated at 37 °C for 12 h with different conditions. As shown in Figure 2, the small white spots stand for the living bacteria on the culture plates. No bacterial colonies with BSA-CuS NPs under NIR irradiation for 5 min (prior to the incubation) were observed even after 3 days (Figure 2A, E). However, many small white spots were seen with only NIR irradiation (Figure 2B, F), indicating that bacteria can survive after NIR irradiation. Compared with the results of Figure 2A, E, the addition of BSA-CuS NPs in the presence of NIR irradiation was essential for the survival of bacteria. Next, to determine whether the only addition of BSACuS NPs in the absence of NIR irradiation can also have the same effect in viability of bacteria, we first tested the cytotoxicity of BSA-CuS NPs. MTT assays showed that over 90% skin fibroblast cells viability was observed as far as the concentration of 50 ppm BSA-CuS NPs (Figure S2). In the subsequent antibacterial tests, we found that there are a lot of white small spots on the plates (Figure 2C and 2G). Bacteria can grow and proliferate well along the plates. Furthermore, we measured the influence of different concentrations of BSA-CuS NPs regarding the bacteria growth (Figure S3). Surprisingly, E. coli exhibited superior tolerance to BSA-CuS NPs even at an ultrahigh concentration (450 ppm). These results showed that BSA-CuS NPs are not cytotoxic and have no antibacterial activity at certain concentration and in the absence of external NIR irradiation. To rule out the factors of bacterial death or apoptosis, the control group in the presence of PBS without NIR irradiation and BSA-CuS NPs addition was done. As shown in Figure 2D, H, there are a large number of white spots
diameters of particles ranged from 12 to 28 nm. Furthermore, the statistic particle diameter is 19.2 ± 2.8 nm. The XRD patterns of BSA-CuS NPs indicate that all the diffraction peaks could be assigned to covellite-phase CuS, corresponding to the standard data (PDF card No.: 00-001-1281). The diffraction peaks located at 2θ of 29.04, 31.96, 33.22, 47.86, 59.34 are attributed to (102), (103), (006), (110), and (116) planes, respectively (Figure 1B). The albumin plays a key role in directing the growth of CuS NPs. Such a small size distribution determines that BSA-CuS NPs have a good dispersibility and stability, and even remain unchanged after storing them in water for three months. In order to demonstrate the photothermal stability of BSA-CuS NPs, the UV−vis absorbance spectra before and after NIR irradiation were measured. As shown in Figure 1C, the absorbance spectra of the BSA-CuS NPs slightly decreased after using 980 nm laser irradiation (power density of 1.59 W cm−2 ; 1 h), implying that the materials have good photothermal performance and can be used repeatedly. Furthermore, the heating rate experiments were recorded with different concentrations of BSA-CuS NPs. As illustrated in Figure 1D, the temperature of BSA-CuS samples increased rapidly with the increase of sample concentrations and irradiation time. For example, the temperature change of 1 mL solution (BSA-CuS, 50 ppm) could easily reach 24 °C within 10 min irradiation. In contrast, 1 mL of pure water exhibited insignificant temperature change under the same condition. The photothermal conversion efficiency of the as-prepared BSA-CuS NPs was measured by a modified method similar to the report by Roper et al.53 Thus, the 980 nm laser heat conversion efficiency (η) of as-prepared BSA-CuS NPs was calculated to be 25.68% (Figure S1), which is higher than other nanomaterials. These results show that BSA-CuS NPs could efficiently convert light energy into heat. The unique optical property allows the BSA-CuS NPs to generate localized 36609
DOI: 10.1021/acsami.7b11062 ACS Appl. Mater. Interfaces 2017, 9, 36606−36614
Research Article
ACS Applied Materials & Interfaces
value decreased with the increase of BSA-CuS NPs concentrations and kept a steady status when the concentrations beyond a certain value (S.aureus, 30 ppm; E. coli, 20 ppm). To further explore the antibacterial mechanism, we employed SEM to directly observe the morphological changes of the bacteria. Figure 4A−D showed the outer morphologies of S. aureus treated with different conditions. In the case of BSA-CuS NPs addition and NIR irradiation (Figure 4A), it can be seen that many small nanoparticles adhered to the surfaces of bacteria (white dots).This can be attributed to the strong interactions between BSA-CuS NPs and bacteria cell wall. Zeta potential tests showed that BSA-CuS NPs exhibit negative charge, while S. aureus showed an opposite charge (Figure S4). S. aureus secrete protein belonging to the MSCRAMM (microbial surface components recognizing adhesive matrix molecules) family,54 which can enhance the surface viscosity of the cell wall, and enhance the adsorption of copper ions. Furthermore, the content of copper ions in S. aureus irradiated for 45 min was 8263.00 ng/g measured by ICP-MS, which is given in Table S1. Next, we observed the morphology of S. aureus incubated with BSA-CuS NPs without NIR irradiation (Figure 4B), finding that it did not differ much from the morphology presented in Figure 4A. However, the content of copper ions (9785.65 ng/g) in S. aureus is a little higher than the one with NIR irradiation. The difference in the content of copper ions might be attributed to the loss of some copperbinding molecules caused by local high-temperature ablation. In contrast, the surface of bacteria incubated with PBS under NIR irradiation was smooth (Figure 4C), and the whole cell structure of the S. aureus remains intact. The control group treated with PBS in the absence of NIR irradiation (Figure 4D) was also carried out. The result is the same as the one observed in Figure 4C. Interestingly, significant morphological changes occurred in E. coli in the same conditions (Figure 4E−H). Herein, it is worth noting that the cell membrane of E. coli is much thinner than the one of S. aureus. Figure 4E gives a clear view of E. coli treated with BSA-CuS NPs under NIR irradiation. Unlike in Figure 4A, one can see few particles adhered on the surfaces of E. coli. However, some holes were found on the surfaces of bacteria, indicating that the membrane integrity was compromised in such a case. It might be attributed to the local high temperature generated by BSA-CuS NPs. Next, we measured the content of copper ions in E. coli. The value is 3843.00 ng/g, which is much lower than S. aureus (8263.0 ng/g). Considering the charge difference between E. coli (−28.5 mv) and S. aureus (−32 mv) as well as surface viscosity, the binding ability of BSA-CuS NPs to them is different. S. aureus has stronger binding capacity than E. coli in attracting BSA-CuS NPs. This is further identified in Figure 4F. No particles are observed on the surfaces of E. coli and the content of copper ions is 685.80 ng/g (Table S1). The value is much lower than E. coli with NIR irradiation, revealing that NIR irradiation is efficient in helping BSA-CuS NPs enter the interior of the bacteria through the ablation-formed pores. In fact, direct NIR irradiation without BSA-CuS NPs can also give rise to some pores on the surfaces of E. coli (Figure 4G), but the number of the injured bacteria is less than the one in Figure 4E. It was also observed that there was fracturing and breaking. The control group (in Figure 4H) showed the original structure of E. coli. The morphology is rodshaped and integral. Considering the complex influencing factors caused by nanoparticle explosion, shock waves, bubble
Figure 4. SEM images of (A−D) S. aureus and (E−H) E. coli treated with different conditions. (A, E) BSA-CuS NPs with NIR irradiation; (B, F) BSA-CuS NPs without NIR irradiation; (C, G) PBS with NIR irradiation; (D, H) PBS without NIR irradiation.
in the vision. Taken together, both NIR irradiation and BSACuS NPs addition are indispensable for bacteria survival. To further evaluate the effects of BSA-CuS NPs and NIR irradiation on the bacteria activity, a series of experiments are carried out. Figure 3A shows the turbidity changes with different dosages of BSA-CuS NPs (0, 10, 20, 30, 40, and 50 ppm) incubated with S. aureus and E. coli for 12 h with or without NIR irradiation. For S. aureus, the bacterial suspension incubated with BSA-CuS NPs without NIR irradiation was turbid, indicating that the bacteria can survive well. In comparison, the solution without NIR irradiation began to become clear when BSA-CuS NPs exceeded 30 ppm, indicating most bacteria have loosed their biological activity. Therefore, we estimated that the minimal inhibition concentration of BSACuS NPs is 30 ppm under NIR irradiation. Similar phenomena were also observed in the case of E. coli except that the minimal inhibition concentration is 20 ppm. Compared with S. aureus, E. coli is much more sensitive in the same treating condition. Figure 3B, C shows the relationship between the OD450 value (stand for the density of bacteria in a medium) and BSA-CuS NPs concentrations. Obviously, NIR irradiation was essential for the survival of S. aureus and E. coli in the presence of BSA-CuS NPs. Additionally, the OD450 36610
DOI: 10.1021/acsami.7b11062 ACS Appl. Mater. Interfaces 2017, 9, 36606−36614
Research Article
ACS Applied Materials & Interfaces
Figure 5. Flow cytometer dot plot of (A−D) S. aureus and (E−H) E. coli.
stood for the particular fluorescence signal of S. aureus (Figure 5A−D) and E. coli (Figure 5E−H) doubly stained with PI and SYTO9. The red and blue regions represent the proportion of dead/live bacteria, respectively.58 For S. aureus, 13.7% of bacteria with NIR and BSA-CuS NPs fell in the blue region
formation and thermal disintegration etc.,55−57 it is not clear for the molecular mechanism at present. It is worth exploring for us next. Next, we used flow cytometry analysis to accurately verify bacteria mortality. As shown in Figure 5A−H, these pictures 36611
DOI: 10.1021/acsami.7b11062 ACS Appl. Mater. Interfaces 2017, 9, 36606−36614
Research Article
ACS Applied Materials & Interfaces
CuS NPs and NRI irradiation can induce the death of bacteria. While, a great number of green dots were observed in Figure 6 B, indicating that BSA-CuS NPs without NIR irradiation had little impacts on bacterial biological activity. In contrast, the green dots observed (in Figure 6C) are weaker than those incubated with only BSA-CuS NPs. That is to say, some bacteria are killed under simple NIR irradiation. The control group (Figure 6D) shows only green dots, indicating that no bacteria apoptosis or death occurred in this case. As to E. coli, we found the similar results (Figure 6E−H).
■
CONCLUSION In summary, the BSA-CuS NPs have been successfully synthesized via a facile hydrothermal method. The assynthesized products have excellent photostability and biocompatibility. The solution of the BSA-CuS NPs exhibits an intense absorbance in the NIR region, and the temperature of the 50 ppm solution can easily reach up 24 °C within 10 min under irradiation. More importantly, incubated with BSA-CuS NPs at a low concentration, bacterial biological activity can be efficiently destroyed under NIR irradiation, and bacterial lethality can reach 80%. Because of their high photothermal conversion efficiency, low toxicity, as well as good biocompatibility, the novel material combined with NIR irradiation will give us a valuable insight into the effective ablation of pathogenic bacteria in medicine.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b11062. Photothermal property of BSA-CuS NPs, cytotoxicity assays of BSA-CuS NPs to skin fibroblast cells, cytotoxicity assays of BSA-CuS NPs to bacteria, zeta potential measurements (PDF)
Figure 6. Fluorescence microscopy images of bacteria under different treatments by SYTO9/PI staining. (A−D) Proliferation of S. aureus with BSA-CuS NPs under NIR irradiation, with BSA-CuS NPs and no NIR irradiation, PBS with NIR irradiation, and PBS without NIR irradiation, respectively. (E−H) stand for the proliferation of E. coli with BSA-CuS NPs under NIR irradiation, with BSA-CuS NPs and no NIR irradiation, PBS with NIR irradiation, and PBS without NIR irradiation. Scale bar = 20 μm.
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Da-Peng Yang: 0000-0003-3509-2825
(Figure 5A), whereas the other values were 93.3%, 48.1% and 99.8% for bacteria treated with BSA-CuS NPs without NIR irradiation, PBS with NIR irradiation and PBS without NIR irradiation, respectively (Figure 5B−D). In the case of E. coli, the blue region presented 16.2% live bacteria (Figure 5E), and BSA-CuS NPs without NIR irradiation, PBS with NIR irradiation and PBS without NIR irradiation exhibited 87.3, 41.7, and 93.4% of survival (Figures 5F−H). In the case of BSA-CuS NPs and NIR irradiation, the live bacterial amounts determined by flow cytometry analysis (against S. aureus and E. coli) were much lower than other groups. It showed that BSACuS NPs and NIR irradiation had synergistic antibacterial effects. Next, confocal fluorescence microscopy was used to identify the living status of bacterial cells. S. aureus and E. coli suspensions stained by SYTO9 plus PI were dropped to glass slide and observed by confocal fluorescence microscopy (Figure 6A−H). Obvious bacteria death (overwhelming red dots, in Figure 6A) was observed when S. aureus was treated with BSACuS NPs under NIR irradiation. The result implies that BSA-
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (81472001, 31400851, and 21536010), the Minjiang Scholars Program of Fujian Province, the Tongjiang Scholars Program of Quanzhou City, the Fourth Health Education Joint Development Project of Fujian Province (WKJ-2016-2-36).
■
REFERENCES
(1) 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 (8), 3193−3209. (2) Weitz, J. S.; Poisot, T.; Meyer, J. R.; Flores, C. O.; Valverde, S.; Sullivan, M. B.; Hochberg, M. E. Phage-Bacteria Infection Networks. Trends Microbiol. 2013, 21 (2), 82−91. (3) Enright, M. C.; Day, N. P.; Davies, C. E.; Peacock, S. J.; Spratt, B. G. Multilocus Sequence Typing for Characterization of Methicillin-
36612
DOI: 10.1021/acsami.7b11062 ACS Appl. Mater. Interfaces 2017, 9, 36606−36614
Research Article
ACS Applied Materials & Interfaces Resistant and Methicillin-Susceptible Clones of Staphylococcus aureus. J. Clin. Microbiol. 2000, 38 (3), 1008−1015. (4) Pihl, M.; Bruzell, E.; Andersson, M. Bacterial Biofilm Elimination Using Gold Nanorod Localised Surface Plasmon Resonance Generated Heat. Mater. Sci. Eng., C 2017, 80, 54−58. (5) 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 (1), 51−56. (6) Yang, D. P.; Oo, M.; Deen, G. R.; Li, Z.; Loh, X. J. Nano-StarShaped Polymers for Drug Delivery Applications. Macromol. Rapid Commun. 2017, 1700410. (7) Li, L.-L.; Xu, J.-H.; Qi, G.-B.; Zhao, X.; Yu, F.; Wang, H. Core− Shell Supramolecular Gelatin Nanoparticles for Adaptive and “OnDemand” Antibiotic Delivery. ACS Nano 2014, 8 (5), 4975−4983. (8) Teng, C. P.; Zhou, T.; Ye, E.; Liu, S.; Koh, L. D.; Low, M.; Loh, X. J.; Win, K. Y.; Zhang, L.; Han, M. Y. Effective Targeted Photothermal Ablation of Multidrug Resistant Bacteria and Their Biofilms with NIR-Absorbing Gold Nanocrosses. Adv. Healthcare Mater. 2016, 5 (16), 2122−2130. (9) Zhang, W.; Li, Y.; Niu, J.; Chen, Y. Photogeneration of Reactive Oxygen Species on Uncoated Silver, Gold, Nickel, and Silicon Nanoparticles and Their Antibacterial Effects. Langmuir 2013, 29 (15), 4647−4651. (10) 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 (2), 1281−1290. (11) Dai, X.; Fan, Z.; Lu, Y.; Ray, P. C. Multifunctional Nanoplatforms for Targeted Multidrug-Resistant-Bacteria Theranostic Applications. ACS Appl. Mater. Interfaces 2013, 5 (21), 11348−11354. (12) Liu, Z.; Liu, X.; Du, Y.; Ren, J.; Qu, X. Using Plasmonic Copper Sulfide Nanocrystals as Smart Light-Driven Sterilants. ACS Nano 2015, 9 (10), 10335−10346. (13) Javani, S.; Lorca, R.; Latorre, A.; Flors, C.; Cortajarena, A. L.; Somoza, A. Antibacterial Activity of DNA-Stabilized Silver Nanoclusters Tuned by Oligonucleotide Sequence. ACS Appl. Mater. Interfaces 2016, 8 (16), 10147−10154. (14) Jiang, S.; Ma, B. C.; Reinholz, J.; Li, Q.; Wang, J.; Zhang, K. A.; Landfester, K.; Crespy, D. Efficient Nanofibrous Membranes for Antibacterial Wound Dressing and UV Protection. ACS Appl. Mater. Interfaces 2016, 8 (44), 29915−29922. (15) Ma, S.; Zhan, S.; Jia, Y.; Zhou, Q. Superior Antibacterial Activity of Fe3O4-TiO2 Nanosheets under Solar Light. ACS Appl. Mater. Interfaces 2015, 7 (39), 21875−21883. (16) Shao, W.; Liu, X.; Min, H.; Dong, G.; Feng, Q.; Zuo, S. Preparation, Characterization, and Antibacterial Activity of Silver Nanoparticle-Decorated Graphene Oxide Nanocomposite. ACS Appl. Mater. Interfaces 2015, 7 (12), 6966−6973. (17) Song, J.; Kang, H.; Lee, C.; Hwang, S. H.; Jang, J. Aqueous Synthesis of Silver Nanoparticle Embedded Cationic Polymer Nanofibers and Their Antibacterial Activity. ACS Appl. Mater. Interfaces 2012, 4 (1), 460−465. (18) Tang, J.; Chen, Q.; Xu, L.; Zhang, S.; Feng, L.; Cheng, L.; Xu, H.; Liu, Z.; Peng, R. Graphene Oxide-Silver Nanocomposite as A Highly Effective Antibacterial Agent with Species-Specific Mechanisms. ACS Appl. Mater. Interfaces 2013, 5 (9), 3867−3874. (19) Tang, Q.; Liu, J.; Shrestha, L. K.; Ariga, K.; Ji, Q. Antibacterial Effect of Silver-Incorporated Flake-Shell Nanoparticles under DualModality. ACS Appl. Mater. Interfaces 2016, 8 (29), 18922−18929. (20) Wang, X.; Zhu, S.; Liu, L.; Li, L. Flexible Antibacterial Film Based on Conjugated Polyelectrolyte/Silver Nanocomposites. ACS Appl. Mater. Interfaces 2017, 9 (10), 9051−9058. (21) Black, K. C.; Sileika, T. S.; Yi, J.; Zhang, R.; Rivera, J. G.; Messersmith, P. B. Bacterial Killing by Light-Triggered Release of Silver from Biomimetic Metal Nanorods. Small 2014, 10 (1), 169− 178. (22) Huo, D.; Gao, J.; Guo, B.; Ding, Y.; He, J.; Yu, H.; Zhou, Z.; Hu, Y. Silver Nanoshells as Tri-mode Bactericidal Agents Integrating Long Term Antibacterial, Photohyperthermia and Triggered Ag+ Release Capabilities. RSC Adv. 2013, 3 (27), 10632−10638.
(23) Yuan, X.; Setyawati, M. I.; Leong, D. T.; Xie, J. Ultrasmall Ag +-Rich Nanoclusters as Highly Efficient Nanoreservoirs for Bacterial Killing. Nano Res. 2014, 7 (3), 301−307. (24) Chaudhuri, S. K.; Chandela, S.; Malodia, L. Plant Mediated Green Synthesis of Silver Nanoparticles Using Tecomella undulata Leaf Extract and Their Characterization. Nano Biomed. Eng. 2016, 8 (1), 1−8. (25) Li, C.; Wang, X.; Chen, F.; Zhang, C.; Zhi, X.; Wang, K.; Cui, D. The Antifungal Activity of Graphene Oxide-Silver Nanocomposites. Biomaterials 2013, 34 (15), 3882−3890. (26) Lin, D.; Qin, T.; Wang, Y.; Sun, X.; Chen, L. Graphene Oxide Wrapped SERS tags: Multifunctional Platforms Toward Optical Labeling, Photothermal Ablation of Bacteria, and The Monitoring of Killing Effect. ACS Appl. Mater. Interfaces 2014, 6 (2), 1320−1329. (27) Yang, X.; Li, Z.; Ju, E.; Ren, J.; Qu, X. Reduced Graphene Oxide Functionalized with A Luminescent Rare-Earth Complex for The Tracking and Photothermal Killing of Drug-Resistant Bacteria. Chem. Eur. J. 2014, 20, 394−398. (28) 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 (2), 619−627. (29) Khan, S. A.; Singh, A. K.; Senapati, D.; Fan, Z.; Ray, P. C. Targeted Highly Sensitive Detection of Multi-Drug Resistant Salmonella DT104 Using Gold Nanoparticles. Chem. Commun. 2011, 47, 9444−9446. (30) Khan, S. A.; Singh, A. K.; Senapati, D.; Fan, Z.; Ray, P. C. BioConjugated Popcorn Shaped Gold Nanoparticles for Targeted Photothermal Killing of Multiple Drug Resistant Salmonella DT104. J. Mater. Chem. 2011, 21, 17705−17709. (31) Ray, P. C.; Khan, S. A.; Fan, Z.; Senapati, D. Gold Nanotechnology for Targeted Detection and Killing of Multiple Drug Resistant Bacteria from Food Samples. ACS Symp. Ser. 2013, 1143, 1−19. (32) Balamurugan, M.; Kaushik, S.; Saravanan, S. Green Synthesis of Gold Nanoparticles by Using Peltophorum Pterocarpum Flower Extracts. Nano Biomed. Eng. 2016, 8 (4), 213−218. (33) Liu, Y.; He, L.; Mustapha, A.; Li, H.; Hu, Z. Q.; Lin, M. Antibacterial Activities of Zinc Oxide Nanoparticles Against Escherichia coli O157:H7. J. Appl. Microbiol. 2009, 107 (4), 1193− 1201. (34) Raghupathi, K. R.; Koodali, R. T.; Manna, A. C. Size-Dependent Bacterial Growth Inhibition and Mechanism of Antibacterial Activity of Zinc Oxide Nanoparticles. Langmuir 2011, 27 (7), 4020−4028. (35) Raffi, M.; Mehrwan, S.; Bhatti, T. M.; Akhter, J. I.; Hameed, A.; Yawar, W.; ul Hasan, M. M. Investigations into The Antibacterial Behavior of Copper Nanoparticles Against Escherichia coli. Ann. Microbiol. 2010, 60 (1), 75−80. (36) Tian, X. B.; Wang, Z. M.; Yang, S. Q.; Luo, Z. J.; Fu, R. K. Y.; Chu, P. K. Antibacterial Copper-Containing Titanium Nitride Films Produced by Dual Magnetron Sputtering. Surf. Coat. Technol. 2007, 201 (19−20), 8606−8609. (37) Yadav, L.; Tripathi, R. M.; Prasad, R.; Pudake, R. N.; Mittal, J. Antibacterial Activity of Cu Nanoparticles against E. coli, Staphylococcus aureus and Pseudomonas aeruginosa. Nano Biomed. Eng. 2017, 9 (1), 9−14. (38) Ivask, A.; ElBadawy, A.; Kaweeteerawat, C.; Boren, D.; Fischer, H.; Ji, Z.; Chang, C. H.; Liu, R.; Tolaymat, T.; Telesca, D.; et al. Toxicity Mechanisms in Escherichia coli Vary for Silver Nanoparticles and Differ from Ionic Silver. ACS Nano 2014, 8 (1), 374−386. (39) Alarcon, E. I.; Udekwu, K.; Skog, M.; Pacioni, N. L.; Stamplecoskie, K. G.; Gonzalez-Bejar, M.; Polisetti, N.; Wickham, A.; Richter-Dahlfors, A.; Griffith, M.; Scaiano, J. C. The Biocompatibility and Antibacterial Properties of Collagen-Stabilized, Photochemically Prepared Silver Nanoparticles. Biomaterials 2012, 33 (19), 4947−4956. (40) He, J.; Zhu, X.; Qi, Z.; Wang, C.; Mao, X.; Zhu, C.; He, Z.; Li, M.; Tang, Z. Killing Dental Pathogens Using Antibacterial Graphene Oxide. ACS Appl. Mater. Interfaces 2015, 7 (9), 5605−5611. 36613
DOI: 10.1021/acsami.7b11062 ACS Appl. Mater. Interfaces 2017, 9, 36606−36614
Research Article
ACS Applied Materials & Interfaces (41) Tegou, E.; Magana, M.; Katsogridaki, A. E.; Ioannidis, A.; Raptis, V.; Jordan, S.; Chatzipanagiotou, S.; Chatzandroulis, S.; Ornelas, C.; Tegos, G. P. Terms of Endearment: Bacteria Meet Graphene Nanosurfaces. Biomaterials 2016, 89, 38−55. (42) Ye, S.; Shao, K.; Li, Z.; Guo, N.; Zuo, Y.; Li, Q.; Lu, Z.; Chen, L.; He, Q.; Han, H. Antiviral Activity of Graphene Oxide: How Sharp Edged Structure and Charge Matter. ACS Appl. Mater. Interfaces 2015, 7 (38), 21571−21579. (43) Seabra, A. B.; Paula, A. J.; de Lima, R.; Alves, O. L.; Duran, N. Nanotoxicity of Graphene and Graphene Oxide. Chem. Res. Toxicol. 2014, 27 (2), 159−168. (44) Huang, W.-C.; Tsai, P.-J.; Chen, Y.-C. Functional Gold Nanoparticles as Photothermal Agents for Selective-Killing of Pathogenic Bacteria. Nanomedicine 2007, 2 (6), 777−787. (45) Prodi, A.; Filon, F. L. Nano-Scaled Particles and Fibres Occupational Exposure Assessment: An Integrated Approach from Air Sampling to Skin and Surface Contamination. Nano Biomed. Eng. 2016, 8 (2), 91−104. (46) Tian, Q.; Tang, M.; Sun, Y.; Zou, R.; Chen, Z.; Zhu, M.; Yang, S.; Wang, J.; Wang, J.; Hu, J. Hydrophilic Flower-like CuS Superstructures as An Efficient 980 nm Laser-Driven Photothermal Agent for Ablation of Cancer Cells. Adv. Mater. 2011, 23 (31), 3542− 3547. (47) Guo, L.; Yan, D. D.; Yang, D.; Li, Y.; Wang, X.; Zalewski, O.; Yan, B.; Lu, W. Combinatorial Photothermal and Immuno Cancer Therapy Using Chitosan-Coated Hollow Copper Sulfide Nanoparticles. ACS Nano 2014, 8 (6), 5670−5681. (48) Pan, U. N.; Khandelia, R.; Sanpui, P.; Das, S.; Paul, A.; Chattopadhyay, A. Protein-Based Multifunctional Nanocarriers for Imaging, Photothermal Therapy, and Anticancer Drug Delivery. ACS Appl. Mater. Interfaces 2017, 9 (23), 19495−19501. (49) Huang, J.; Lin, L.; Sun, D.; Chen, H.; Yang, D.; Li, Q. BioInspired Synthesis of Metal Nanomaterials and Applications. Chem. Soc. Rev. 2015, 44 (17), 6330−6374. (50) Lu, W.; Zhang, Y.; Tan, Y. Z.; Hu, K. L.; Jiang, X. G.; Fu, S. K. Cationic Albumin-Conjugated Pegylated Nanoparticles as Novel Drug Carrier for Brain Delivery. J. Controlled Release 2005, 107 (3), 428− 448. (51) Sugibayashi, K.; Akimoto, M.; Morimoto, Y.; Nadai, T.; Kato, Y. Drug-Carrier Property of Albumin Microspheres in Chemotherapy. III. Effect of Microsphere-Entrapped 5-Fluorouracil on Ehrlich Ascites Carcinoma in Mice. J. Pharmacobio-Dyn. 1979, 2 (6), 350−355. (52) Gao, C.; Lu, Z.; Liu, Y.; Zhang, Q.; Chi, M.; Cheng, Q.; Yin, Y. Highly Stable Silver Nanoplates for Surface Plasmon Resonance Biosensing. Angew. Chem., Int. Ed. 2012, 51 (23), 5629−5633. (53) Tian, Q.; Jiang, F.; Zou, R.; Liu, Q.; Chen, Z.; Zhu, M.; Yang, S.; Wang, J.; Wang, J.; Hu, J. Hydrophilic Cu9S5 Nanocrystals: A Photothermal Agent with A 25.7% Heat Conversion Efficiency for Photothermal Ablation of Cancer Cells in Vivo. ACS Nano 2011, 5 (12), 9761−9771. (54) Foster, T. J.; Hö ö k, M. Surface Protein Adhesins of Staphylococcus aureus. Trends Microbiol. 1998, 6 (12), 484−488. (55) Norman, R. S.; Stone, J. W.; Gole, A.; Murphy, C. J.; SaboAttwood, T. L. Targeted Photothermal Lysis of The Pathogenic Bacteria, Pseudomonas Aeruginosa, with Gold Nanorods. Nano Lett. 2008, 8 (1), 302−306. (56) Muzzio, N. E.; Pasquale, M. A.; Diamanti, E.; Gregurec, D.; Moro, M. M.; Azzaroni, O.; Moya, S. E. Enhanced Antiadhesive Properties of Chitosan/Hyaluronic Acid Polyelectrolyte Multilayers Driven By Thermal Annealing: Low Adherence for Mammalian Cells and Selective Decrease In Adhesion for Gram-Positive Bacteria. Mater. Sci. Eng., C 2017, 80, 677−687. (57) Rau, J. V.; Wu, V. M.; Graziani, V.; Fadeeva, I. V.; Fomin, A. S.; Fosca, M.; Uskoković, V. The Bone Building Blues: Self-Hardening Copper-Doped Calcium Phosphate Cement and Its In Vitro Assessment Against Mammalian Cells and Bacteria. Mater. Sci. Eng., C 2017, 79, 270−279. (58) Berney, M.; Hammes, F.; Bosshard, F.; Weilenmann, H. U.; Egli, T. Assessment and Interpretation of Bacterial Viability by Using the
LIVE/DEAD BacLight Kit in Combination with Flow Cytometry. Appl. Environ. Microbiol. 2007, 73 (10), 3283−3290.
36614
DOI: 10.1021/acsami.7b11062 ACS Appl. Mater. Interfaces 2017, 9, 36606−36614
