Peptide-Conjugated CuS Nanocomposites for NIR-Triggered Ablation

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Peptide Conjugated CuS Nanocomposites for NIR Triggered Ablation of Pseudomonas aeruginosa Biofilm Xiaomei Dai, Yunjian Yu, Xiaosong Wei, Xijuan Dai, Xiaozhuang Duan, Cong Yu, Xinge Zhang, and Chaoxing Li ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00033 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 2, 2019

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ACS Applied Bio Materials

Peptide Conjugated CuS Nanocomposites for NIR Triggered Ablation of Pseudomonas aeruginosa Biofilm

Xiaomei Dai, Yunjian Yu, Xiaosong Wei, Xijuan Dai, Xiaozhuang Duan, Cong Yu, Xinge Zhang, Chaoxing Li



Corresponding author:

The Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Weijin Road 94, Tianjin 300071, China Tel: +86-22-23501645; Fax: +86-22-23505598 E-mail: [email protected]

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ABSTRACT The Gram-negative bacteria Pseudomonas aeruginosa is one famous bacterial strain owing to its ability to effectively form biofilms, which is a front-line mechanism of bacterial tolerance. Herein, the near-infrared-induced nanocomposites were one-step prepared by modifying copper sulfide nanoparticle with peptide to effectively eradicate Pseudomonas aeruginosa biofilm through electrostatic interaction, photodynamic effect and photothermal effect. These nanocomposites could rapidly adhere to the surface of bacteria, and irreversible damage the bacterial membrane under near-infrared laser irradiation. Furthermore, the nanocomposites could selectively eliminate bacteria over mammalian cell without distinct toxicity to NIH 3T3 cells. The nanocomposites will exert a far-reaching impact on the future design of biocompatible near-infrared-induced antibacterial agents, exhibiting its potential applications in Gram-negative bacteria and biofilm infections. Keywords: copper sulfide nanoparticles, near-infrared laser, photothermal and photodynamic effect, Gram-negative bacterial biofilm


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INTRODUCTION Pseudomonas aeruginosa (P. aeruginosa) is infamous for its intrinsic resistance mechanism and highly sophisticated intrinsic.1,2 P. aeruginosa shows poor membrane permeability, limiting the penetration of most antibacterial agents into the cells, leading to secondary adaptive resistance.3,4 Some antibiotic, for example, vancomycin is a glycopeptides antibiotic widely utilized for therapy of infection caused by Gram-positive bacteria, but ineffectiveness for Gram-negative strains owing to its large molecular size and limited penetration into the bacterial membrane.5 Furthermore, due to the formation of biofilm, the antibacterial tolerance of P. aeruginosa is enhanced.6,7 Therefore, there is sorely in need of new design agents to fight P. aeruginosa, especially P. aeruginosa biofilm. Transition metal chalcogenides (TMCs, such as CuS, MoS2, WSe2, and MoSe2) have been studied in diversified fields ranging from environment remediation,8 sensors,9 catalysis,10 energy storage11 and nanomedicine.12 Particularly, TMC nanoparticles have a large amount of advantages in biomedical applications: large surface area, high biocompatibility, excellent biological stability, and strong near-infrared (NIR, 780 – 1100 nm) absorption. Hence, they are ideal nanocarriers for photo-induced therapy and photo-controlled drug delivery.13,14 To data, most efforts have been made toward photodynamic and photothermal dual-modal therapy. Copper sulfide nanoparticles (CuSNPs)

exhibited

both

NIR-induced

photodynamic

therapy

(PDT)

and

photothermal therapy have aroused concern.15,16 However, van der Waals force between CuSNPs results in their strong aggregation,17 which restricts its application in



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vivo. To overcome this limitation, our previously study found that cationic polymers could be utilized to modify with CuSNPs to enhance its stability and antibacterial activity.12 Nevertheless, cationic polymers depend on complex chemical synthesis, and most of them are hard to be degraded into nontoxic species for clearance from the body. Peptides are promising building agents due to their diverse capabilities such as binding to specific receptors,18 blocking or stimulating signaling cascades, or forming structures that interact with membranes.19 ε-Polylysine (EPL), a class of biological peptides that consist of a repeat unit of L-lysine, which are nontoxic and can be biologically degraded into essential amino acid nutrients for the body.20 Furthermore, EPL possess a wide-spectrum of antibacterial activity.21 Hence, EPL could be used to decorate with CuSNPs to form nanocomposites. To obtain the efficient nanocomposites, EPL@CuSNPs with different molar ratios of EPL and CuSNPs were prepared, and their selective antibacterial activity, antibacterial mechanism, antibiofilm activity and biocompatibility were systematic studied. This work offers careful understanding of the antimicrobial behaviors of the NIR-induced nanocomposites, further highlighting its potential as effectively antimicrobial agents with high selectivity and low toxicity to mammalian cells. EXPERIMENTAL METHODS Materials ε-Polylysine (EPL, 3500 Da), ο-nitrophenyl-β-D-galactopyranoside (ONPG) and 2,7-dichlorofluorescein diacetate (DCFH-DA) were gained from Tianjin Heowns Biochem

Technologies

LLC

(Tianjin,

China).


Ethidium

bromide

(EB),

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4’,6-diamidino-2-phenylindole (DAPI) and acridine orange (AO) were obtained from Alfa Aesar (Beijing, China). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) was purchased from J&K China Chemical Ltd. (Beijing, China). Synthesis of EPL@CuSNPs The nanocomposites were synthesized as the previously reported method with minor modification.22 In brief, CuCl2·2H2O (0.1 mmol, 17.0 mg) and different amount of EPL were added into deionized water (50 mL). Then, Na2S·9H2O (0.1 mmol, 24.0 mg) was added and stirred at 60 °C for further 2 h. The mixture was dialyzed for 3 d to obtain the EPL@CuSNPs nanocomposites. The formation of EPL@CuSNPs was monitored using UV-vis-NIR spectroscopy (Shimadzu UV-3600). Transmission electron microscopy (TEM; Tecnai G2 F20) was utilized to evaluate the morphology and size of the nanocomposites. The Zeta potential and hydrodynamic diameters of EPL@CuSNPs were evaluated using dynamic light scattering (DLS, Malvern Nano-ZS90). PTT Effect of EPL@CuSNPs Solutions containing different concentrations of EPL@CuSNPs were irradiated under 980 nm light for different periods. The infrared thermal imaging system (E50) was utilized to determine the temperature changes induced by EPL@CuSNPs. The temperature was evaluated over 5 min. The phosphate buffer solution (PBS) was utilized as a control. ROS Production of EPL@CuSNPs Solutions containing different concentrations of EPL@CuSNPs were mixed with



DCFH-DA. All samples were irradiated under 980 nm for a different period. Fluorescence images of the above samples were acquired under Caliper IVIS Lumina II (excitation wavelengths: 465 nm, emission wavelengths: 520 nm) Antimicrobial Activity Here, four isolates of P. aeruginosa were utilized to determine the antimicrobial activity of EPL@CuSNPs. The mixture containing different concentrations of EPL@CuSNPs (0 - 100 μg/mL) was added into the equal volume of bacteria (2.0 × 107 CFU/mL). After the mixture was cultured at 37 °C for 1 h, all bacteria were exposed to 980 nm (1.5 W/cm2 and 3 min). After being cultured at 37 °C for 8 h, the antimicrobial properties of EPL@CuSNPs was investigated by determining the value of optical density at 600 nm (OD600): OD600(bacteria) = OD600(bacteria + nanocomposites) – OD600(nanocomposites) Live/Dead Assay EPL@CuSNPs solution (12.5 μg/mL) was mixed with four isolates of P. aeruginosa suspensions, respectively. After being cultured at 37 °C for 1 h, bacteria were exposed to 980 nm for 3 min, and stained with EB and AO for 15 min. Then, all bacteria were observed by the fluorescence microscope (Axio Imager Z1). In reference, the bacteria were treated with PBS at 37 °C for 1 h, and then exposed to 980 nm for 3 min. The Morphological Characterization of Bacteria EPL@CuSNPs solution (12.5 μg/mL) was mixed with four isolates of P. aeruginosa suspensions, respectively. After being cultured at 37 °C for 1 h, bacteria were exposed to 980 nm for 3 min. All cells were gently placed on the glass sides, and fixed with


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2.5% glutaraldehyde for 12 h. After being washed with PBS, all cells were observed using scanning electron microscopy (SEM; JSM-7500F). In reference, the bacteria were treated with PBS at 37 °C for 1 h, and then exposed to the NIR laser for 3 min. Inhibitory Activity of EPL@CuSNPs against β-Galactosidase EPL@CuSNPs (0.5 mL) was mixed with the solution (0.5 mL) containing cytoplasm. ONPG (10 mg/mL, 1.0 mL) was added after the solution was cultured at 37 °C for 30 min. The produced ortho-nitrophenol (ONP) was determined by investigating the OD420. The inhibition ratio of β-galactosidase was evaluated as follows: Inhibition% =

OD420(control) - OD420(sample) OD420(control)

× 100%

Static Biofilm Assays The biofilm was synthesized as previous method with slight modifications.23 Bacterial suspensions (100 μL, 1 × 106 CFU/mL) were seeded into 96-well plates and cultured at 37 °C for 24 h. After being washed with PBS, fresh growth media (120 μL) containing antibacterial agents (EPL@CuSNPs, EPL, CuSNPs and Tob) was added. After being cultured at 37 °C for 24 h, biofilms were exposed to 980 nm for 3 min. The biomass of biofilms was investigated according to crystal violet assay.24 In reference, the bacterial biofilm was treated with PBS and subjected to NIR laser. All experiments were carried out five times. The Morphological Characterization of Bacterial Biofilm The bacterial biofilm was formed on glass coverslips according to previously study.25 All biofilms were treated with agents (EPL@CuSNPs, EPL, CuSNPs and Tob, 12.5 μg/mL) exposed to 980 nm for 3 min. Biofilms were dyed by EB and ConA-FITC,



and observed under confocal laser scanning microscopy (CLSM, Leica TCS SP8). In reference, the bacterial biofilm was treated with PBS and subjected to NIR laser. Cytotoxicity Assay The biocompatibility of EPL@CuSNPs was investigated via the MTT assay. NIH 3T3 cells (104) were uniform added into a 96-well plate. After being incubated at 37 °C for 24 h, the medium was replaced with nanocomposites. After being incubated at 37 °C for 24 h, MTT solution (10 μL) was added into 96-well plate. After being cultured for 4 h, the original medium was replaced with DMSO (100 μL). OD490 was determined to evaluate cell viability. Selective Interaction between Bacteria and Mammalian Cells Here, NIH 3T3 cells were utilized as research mode to investigate the selective interaction of EPL@CuSNPs between bacteria and mammalian cells. Cells (2 × 104) were uniform added into a 24-well plate. After being incubated for 24 h, the original medium was replaced with the fresh medium without antibiotics. Then, FITC-labeled bacterial cells (5 × 107 CFU/mL, 100 μL) were seeded into the plate and cultured for further 30 min. Then, EPL@CuSNPs (12.5 μg/mL) was added and cultured for 1 h. After being exposed to 980 nm (3 min), all cells were dyed by DAPI, finally imaged under CLSM. In reference, the samples were treated with PBS and subjected to 980 nm. RESULT AND DISCUSSION Synthesis and Characterization of EPL@CuSNPs CuSNPs as a novel class of p-type semiconductors have become attractive


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photothermal and photodynamic agents for ablation properties against tumor or microbes.12,26-28 However, the poor stability of CuSNPs in aqueous solution was restricted their application in vivo. EPL, a homopolymer of the naturally occurring peptide lysine,29 could be utilized as a building agent to combine with CuSNPs. For a one-step synthesis of EPL@CuSNPs, a green solution was formed after mixing EPL, CuCl2 and Na2S together at 60 °C for 2 h (Figure 1). After treatment with EPL@CuSNPs for 1 h, the nanocomposites could adhere on the surface of bacteria through electrostatic interaction, which could increase the local concentration of EPL@CuSNPs. Then, exposure of EPL@CuSNPs and bacterial aggregation to NIR laser for 3 min, the generated hyperthermia and ROS disrupted the bacterial membrane, resulting in bacterial death.

Figure 1. The one-step preparation of EPL@CuSNPs, and bacterial elimination of EPL@CuSNPs under NIR laser irradiation. To investigate the effect of the surface chemistry of EPL@CuSNPs on antimicrobial activity, nanocomposites with different molar ratios of CuSNPs to EPL (1:0.05, 1:0.1, 1:0.2 and 1:0.4) were synthesized. P. aeruginosa is particularly infamous for its



capacity to rapidly form a biofilm and its inherent ability to develop resistance to antibiotics.1,2 Here, four isolates of P. aeruginosa were utilized to study the antimicrobial activity of EPL@CuSNPs nanocomposites. The minimum inhibitory concentration (MIC) of nanocomposites was measured. As displayed in Figure 2, the MIC values of EPL@CuSNPs with different molar ratios of CuSNPs to EPL (1:0.05, 1:0.1, 1:0.2 and 1:0.4) were 50, 1.6, 6.3 and 25 μg/mL, respectively. The nanocomposites with 1:0.1 molar ratios of CuSNPs to EPL showed the excellent antibacterial activity against four isolates of P. aeruginosa. Previously study indicated that the hydrophobic/hydrophilic balance of antibacterial agents plays the vital part in their biological property.30,31 EPL@CuSNPs (1:0.1) may be obtained the appropriate hydrophilic/hydrophobic balance to adhere on the bacterial membrane, and increase the local concentration, then generate sufficient heat and ROS to kill the bacteria. Therefore, EPL@CuSNPs (1:0.1) was used in the following research.


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Figure 2. Antimicrobial activity of EPL@CuSNPs with different molar ratios of CuSNPs to EPL (A: 1:0.05, B: 1:0.1, C: 1:0.2 and D: 1:0.4) against four isolates of P. aeruginosa. The nanocomposites could be well dispersed in aqueous solution for up to three months without aggregations, ascribing to the successful surface modification of EPL for high stability and splendid dispersity (Figure 3A). The size and morphology of EPL@CuSNPs nanocomposites were investigated using TEM. As demonstrated in Figure 3B, all nanocomposites were monodisperse and sphere, with diameter of about 18 nm. Furthermore, the optical property of aqueous dispersions of EPL@CuSNPs was determined by UV-vis-NIR spectroscopy. As displayed in Figure 3B (inset), the nanocomposites exhibited a wide absorption in the NIR region, which favors their light-induced properties.32 The Zeta potential and average hydrodynamic diameter of EPL@CuSNPs was measured using DLS (Figure 3C). The average hydrodynamic



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diameter of CuSNPs was about 30 nm (PDI 0.12), and the Zeta potential was about -10 mV. After being coated with EPL, the average hydrodynamic diameters of nanocomposites were approximately 100 nm (PDI 0.1), and the Zeta potential turned into positive charge (15 - 35 mV). The increased diameter of EPL@CuSNPs in aqueous solution was possibly owing to the EPL layers, which are invisible under TEM. The surface electronic state of EPL@CuSNPs was analyzed using X-ray photoelectron spectroscopy (XPS, Axis Ultra DLD). As shown in Figure 3D, the peaks of S 2p, O 1s, Cu 2p, N 1s, and C1s were observed in the XPS spectrum. The peaks of C, N and O were ascribed to EPL. The Cu and S peaks were attributed to CuSNPs. The binding energies of Cu 2P3/2 and Cu 2p1/2 peaks were 931.5 and 951.5 eV, respectively, which is attributed to the Cu2+ in CuSNPs.33,34 Also, for PTT therapy, the temperature changes of EPL@CuSNPs solution after NIR laser irradiation were monitored using an infrared thermal imaging system. As shown in Figure 3E, the temperature

rise

of

the

aqueous

solution

containing

EPL@CuSNPs

is

concentration-dependent and increased from 18 to 62 °C. The PBS solution showed virtually no temperature increase (from 18 to 19 °C) within the same irradiation time. The results indicate that EPL@CuSNPs can efficiently convert NIR laser into heat, which demonstrates their great potential in PTT against bacteria. To investigate the PDT property of EPL@CuSNPs, DCFH-DA was utilized to determine the production of ROS, since non-fluorescent DCFH-DA can be oxidized by ROS to produce fluorescent 2,7-dichlorofluorescein (DCF).34,35 As displayed in Figure 3F, after treatment of EPL@CuSNPs under NIR laser irradiation for different periods, all


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samples showed fluorescence, suggesting the generation of ROS. The density of fluorescence was enhanced with increasing the concentrations of nanocomposites and irradiation time (Figure S1). Furthermore, CuSNPs could generate larger amounts of ROS than EPL@CuSNPs nanocomposites at the same condition. The results confirmed that EPL@CuSNPs possess excellent photothermal and photodynamic effect, and can be used as promising PDT and PTT agents to ablate the Gram-negative bacteria as well as biofilms.

Figure 3. Photographs of EPL@CuSNPs with different molar ratios in aqueous



solution (A); TEM images of EPL@CuSNPs, inset: UV-vis-NIR spectrum of EPL@CuSNPs (B); diameter distribution and Zeta potential of EPL@CuSNPs (C); XPS pattern of EPL@CuSNPs (D); temperature changes of EPL@CuSNPs at different concentrations (E); and solution containing EPL@CuSNPs treated with DCFH-DA (F). Antibacterial Mechanism of EPL@CuSNPs Binding of antibacterial agents to the bacterial membrane leads to permeabilization and finally ablation of the bacteria.36 To investigate whether EPL@CuSNPs interacts with the bacterial membrane, AO and EB were utilized to stain the bacterial cells. The bacteria with damage/intact membrane exhibited red/green fluorescence. As shown in Figure S2, after treatment with EPL@CuSNPs, most of bacteria were displayed red fluorescence, indicating that most of cells are damaged. The bacteria treated only with NIR laser displayed green fluorescence, suggesting that all bacteria are live (Figure 4A). All bacteria incubated with EPL@CuSNPs showed red fluorescence after NIR laser irradiation, verifying that all bacteria are killed. Furthermore, the bacterial aggregation could obviously observe under the fluorescence microscope. It is attributed to the electrostatic interaction between the positive-charged EPL@CuSNPs and negative-charged bacteria, which benefits the eradication of the bacteria via PTT and PDT. The result above was also verified by the morphological study of bacterial cells using SEM. As displayed in Figure 4B and Figure S2, after treatment with EPL@CuSNPs under NIR laser irradiation, destroyed bacterial morphologies and debris were obviously observed. In contrast, the bacterial cells uniformly displayed clear edges and smooth cell walls after NIR laser irradiation only. Furthermore, the nanocomposites were obviously observed on the bacterial membrane after being


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treated with nanocomposites. These results confirmed that EPL@CuSNPs could adhere to the bacterial membrane, and irreversible disrupt bacterial membrane by electrostatic interaction, photothermal and photodynamic effect, leading to the bacterial death.

Figure 4. Fluorescence micrographs (A) and SEM images (B) of bacteria before and after treatment with EPL@CuSNPs exposed to NIR laser. EPL@CuSNPs could adhere to the bacterial membrane and damage them; then the intracellular enzyme would leak out. Ling et al. confirmed that the enzymes were damaged and would inhibit necessary intracellular reactions when the solution temperature was up to 50 °C.37 β-Galactosidase was utilized as the research model to investigate whether EPL@CuSNPs could inhibit the activity of intracellular substances. As depicted in Figure 5, after the mixture was incubated with EPL@CuSNPs, the activity of β-galactosidase was significant inhibited. In detail,



over 55% of β-galactosidase was inhibited when the concentration of EPL@CuSNPs was 100 μg/mL. This is possible because that EPL@CuSNPs could produce adequate heat and ROS under NIR laser irradiation, which may destroy the activity of an intracellular enzyme. The results confirmed that EPL@CuSNPs could inhibit the activity of enzymes, resulting in the death of bacteria.

Figure 5. Inhibition ratio of β-galactosidase activity after being treated with different concentrations of EPL@CuSNPs under NIR laser irradiation. Cytotoxicity Assay Biocompatibility is a major criterion for antibacterial materials which are developed for the biological field. To investigate the biocompatibility of EPL@CuSNPs, nanocomposites were cultured with NIH 3T3 cells. NIH 3T3 cells were selected because they frequently utilized in studies of functions such as cell adhesion, movement, proliferation and shape change.38 The viability of NIH 3T3 cells is concentration-dependent as demonstrated in Figure 6. In detail, EPL@CuSNPs showed negligible cytotoxicity when the concentration was 100 μg/mL. The selective cytotoxicity might be explained by the difference between the membrane of bacteria and mammalian cells. Positively charged nanocomposites possess strong interactions


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with bacterial membrane due to bacteria contain more negative charges than mammalian cells.39,40 The results suggested that the EPL@CuSNPs with selective toxicity might be promising antibacterial agents in vivo.

Figure 6. Cell viabilities of NIH 3T3 after treatment with EPL@CuSNPs.

Antibiofilm Activity of EPL@CuSNPs Bacterial biofilm display obviously resistance against conventional antibiotics, which largely implicated in chronic and intractable infections.7,41 For example, the Gram-negative bacteria P. aeruginosa is famous for its ability to rapidly and effectively form biofilms, which is a front-line mechanism of bacterial tolerance.42,43 To evaluate the capability of EPL@CuSNPs to eliminate bacterial biofilm, four isolates of P. aeruginosa were used in this work. After the formation of bacterial biofilms, EPL@CuSNPs solutions with different concentrations were added. Tobramycin (Tob) is often utilized in treatment of P. aeruginosa infection in vivo.44 Herein, Tob was used as model control. As displayed in Figure 7, after treatment with Tob, CuSNPs, EPL and EPL@CuSNPs (100 μg/mL), approximately 40%, 40%, 60% and 75% bacterial biofilms were eliminated. Among them, EPL@CuSNPs showed



excellent antibiofilm activity. When the concentration of EPL@CuSNPs was up to 25 μg/mL, over 70% of the established biofilms was eliminated, which demonstrates the excellent electrostatic interaction, photothermal and photodynamic effect of EPL@CuSNPs even in the bacterial biofilm.

Figure 7. Eradication percentages of biofilms after being incubated with antibacterial agents at different concentrations: P. aeruginosa 14 (A), P. aeruginosa 68 (B), P. aeruginosa O1 (C) and P. aeruginosa K (D). To further evaluate the antibiofilm activity of nanocomposites, the mature bacterial biofilm were characterized by CLSM. As shown in Figure 8, the biofilms treated with Tob, CuSNPs, EPL and EPL@CuSNPs under NIR laser irradiation displayed a sharp reduction. Also, the biofilms treated with EPL@CuSNPs showed more significant destruction than that of EPL, Tob or CuSNPs treatment. It is possible because that the positive-charged EPL@CuSNPs can adhere on the negative-charged bacteria,


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improving the local concentration, and then producing enough heat and ROS to eradicate bacterial biofilm as well as kill the bacteria in the biofilm. The results confirmed that EPL@CuSNPs possess excellent electrostatic interaction, PDT and PTT effects against biofilms.

Figure 8. CLSM images of biofilms stained with EB (red) and ConA-FITC (green) after treatment with different materials. Cellular Morphology and Viability after Bacterial Infection and EPL@CuSNPs Treatment EPL@CuSNPs were utilized in the bacteria-mammalian cells infection model to investigate the selectively antibacterial activity of EPL@CuSNPs over mammalian cells. As demonstrated in Figure 9, the mammalian cells were damaged and a large



number of bacteria can be observed in the control groups. However, after treatment with EPL@CuSNPs, all mammalian cells keep their structure and morphology, and few or no bacteria can be observed, indicating that EPL@CuSNPs can selectively eliminate bacteria over mammalian cells. It is responsible for the difference between the surface of mammalian cells and bacteria, and bacteria have more negative charges than cells,40 which obtains strong interactions with the positively charged nanocomposites. The results certified that EPL@CuSNPs can selectively ablate bacteria over mammalian cells.

Figure 9. CLSM images of NIH 3T3 infected by P. aeruginosa 14 (A) and P. aeruginosa 68 (B) treated with or without EPL@CuSNPs. (The scare bar 20 μm)


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CONCLUSION In summary, multifunctional EPL@CuSNPs were synthesized for selectively ablate Gram-negative bacteria over mammalian cells. Upon irradiation under NIR laser, EPL@CuSNPs can generate heat and ROS to kill the bacteria. Analysis of antimicrobial mechanism confirmed that EPL@CuSNPs can irreversibly damage bacterial membrane, and distinctly inhibit the activity of intracellular enzymes, ultimately resulting in the bacterial death. Furthermore, EPL@CuSNPs possess excellent activity against Gram-negative bacteria biofilm by the electrostatic interaction, photothermal and photodynamic effect in vitro. EPL@CuSNPs will exert a far-reaching impact on the future design of antibacterial agents, exhibiting its potential applications in Gram-negative bacteria and biofilm infections. ASSOCIATED CONTENT Supporting Information Figure S1: EPi-fluorescence efficiency of CuSNPs and EPL@CuSNPs at different concentrations; Figure S2: Fluorescence micrographs (A) and SEM images (B) of bacteria after treatment with EPL@CuSNPs without NIR laser irradiation. ORCID Xinge Zhang: 0000-0003-3399-1659 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The work was supported by the National Natural Science Foundation of China (Grant



Nos. 21774062, 21474055 and 51673102) and the Natural Science Foundation of Tianjin, China (Grant No. 18JCYBJC29300). REFERENCES [1] Oliver, A.; Mulet, X.; Lopez-Causape, C.; Juan, C. The Increasing Threat of Pseudomonas aeruginosa High-Risk Clones. Drug Resist. Updates 2015, 21-22, 41-59. [2] Wagner, S.; Sommer, R.; Hinsberger, S.; Lu, C.; Hartmann, R. W.; Empting, M.; Titz, A. Novel Strategies for the Treatment of Pseudomonas aeruginosa Infections. J. Med. Chem. 2016, 59, 5929-5969. [3] Breidenstein, E. B.; de la Fuente-Nunez, C.; Hancock, R. E. Pseudomonas aeruginosa: All Roads Lead to Resistance. Trends. Microbiol. 2011, 19, 419-426. [4] Nikaido, H. Prevention of Drug Access to Bacterial Targets Permeability Barriers and Active Efflux. Science 1994, 264, 382-388. [5] Fernandes, M. M.; Ivanova, K.; Hoyo, J.; Pérez-Rafael, S.; Francesko, A.; Tzanov, T. Nanotransformation of Vancomycin Overcomes the Intrinsic Resistance of Gram-Negative Bacteria. ACS Appl. Mater. Interfaces 2017, 9, 15022-15030. [6] Flemming, H.-C.; Wingender, J. The Biofilm Matrix. Nat. Rev. Microbiol. 2010, 8, 623-633 [7] Davies, D. Understanding Biofilm Resistance to Antibacterial Agents. Nat. Rev. Drug Discov. 2003, 2, 114-122. [8] Basu, M.; Sinha, A. K.; Pradhan, M.; Sarkar, S.; Negishi, Y.; Pal, G. T. Evolution of Hierarchical Hexagonal Stacked Plates of CuS from Liquid-Liquid Interface and its


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Peptides

SAAP-145

and

SAAP-276

Prevents

Experimental

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TOC


Peptide-Conjugated CuS Nanocomposites for NIR-Triggered Ablation

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