Carbon Dot-Based Platform for Simultaneous Bacterial

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Carbon Dot-Based Platform for Simultaneous Bacterial Distinguishment and Antibacterial Applications Jingjing Yang, Xiaodong Zhang, Yong-Hao Ma, Ge Gao, Xiaokai Chen, Hao-Ran Jia, Yan-Hong Li, Zhan Chen, and Fu-Gen Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10398 • Publication Date (Web): 27 Oct 2016 Downloaded from http://pubs.acs.org on October 28, 2016

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ACS Applied Materials & Interfaces

Carbon Dot-Based Platform for Simultaneous Bacterial Distinguishment and Antibacterial Applications Jingjing Yang,†,‡,# Xiaodong Zhang,†,# Yong-Hao Ma,† Ge Gao,† Xiaokai Chen,† Hao-Ran Jia,† Yan-Hong Li,† Zhan Chen,*,§ and Fu-Gen Wu*,†

†

State Key Laboratory of Bioelectronics, School of Biological Science and Medical

Engineering, Southeast University, Nanjing 210096, P. R. China ‡

Beijing Research Center for Agricultural Standards and Testing, Beijing Academy of

Agriculture and Forestry Sciences, Beijing 100097, P. R. China §

Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor,

Michigan 48109, United States

1

ACS Paragon Plus Environment


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ABSTRACT In this work, we prepared quaternized carbon dots (CDs) with simultaneous antibacterial and bacterial differentiation capabilities using a simple carboxyl–amine reaction between lauryl betaine and amine-functionalized CDs. The obtained quaternized CDs have several fascinating properties/abilities: (1) A long fluorescence emission wavelength ensures the exceptional bacterial imaging capability, including the super-resolution imaging ability; (2) The polarity-sensitive fluorescence emission property leads to significantly enhanced fluorescence when the quaternized CDs interact with bacteria; (3) The presence of both hydrophobic hydrocarbon chains and positively charged quaternary ammonium groups makes the CDs selectively attach to Gram-positive bacteria, realizing the bacterial differentiation; (4) Excellent antimicrobial activity against Gram-positive bacteria with a minimum inhibitory concentration of 8 µg/mL for S. aureus. Besides, the quaternized CDs are highly stable in various aqueous solutions, and exhibit negligible cytotoxicity, suggesting that they hold great promise for clinical applications. Compared to the traditional Gram staining method, the selective Gram-positive bacterial imaging achieved by the quaternized CDs provides a much simpler and faster method for bacterial differentiation. In summary, by combining selective Gram-positive bacterial recognition, super-resolution imaging, and exceptional antibacterial activity into a single system, the quaternized CDs represent a novel kind of metal-free nanoparticle-based antibiotics for bacterial drug resistance reversion and a new type of reagents for efficient bacterial differentiation. KEYWORDS: quaternized carbon dots, metal-free NP-based antibiotics, bacterial differentiation, polarity-sensitive fluorescence, super-resolution imaging

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1. INTRODUCTION Bacterial infection is one of the biggest global challenges to human health.1,2 and fast diagnosis is vital for treatment. Thus, extensive efforts have been devoted to developing methods for bacterial infection diagnosis and bacterial detection. The standard means for characterizing unknown bacteria is Gram staining, which differentiates bacterial species into two categories (Gram-positive and Gram-negative).3 However, this method has several disadvantages such as laborious procedures and proneness to generate false positive results.4 Therefore, other ways such as caustic alkali5 and sulfuric acid6 methods have also been developed. Nevertheless, all these protocols need a certain amount of bacterial cells and do not work when both of the two kinds of bacteria exist in one sample. Other methods such as standard plate count (SPC),7 biochemical staining,8 and polymerase chain reaction (PCR)9 have been proposed to detect and combat with bacterial contamination and infection. Despite their success in many aspects, these technologies still have limitations such as low reproducibility, laborious procedures, and long operation time.10 Quantitative real-time PCR (qPCR) has been considered to be a highly sensitive tool for bacterial species identification,11 but this technique is too expensive for many places in developing countries, which significantly limits its practical application.12 Therefore, fast bacterial infection diagnosis and efficient bacterial identification methods without complicated preparations are in urgent need. Recently, fluorescence analysis has been demonstrated to be an effective method to identify bacteria in different categories.13–16 Sizemore et al. used fluorescence-labeled wheat germ agglitinin (WGA) to specifically image Gram-positive bacteria.17 However, some studies reported that Gram-negative bacteria could also be stained by other WGA conjugates.18 Adhya et al. developed a rapid and simple method for high-sensitivity bacterial detection using biotin-tagged phage and streptavidin-coated quantum dots.19 Antibiotics (such as vancomycin and daptomycin) modified fluorescent nanoparticles (NPs) were also prepared for Gram-positive bacteria targeting.20–22 However, such strategy may lead to unexpected drug resistance. In addition to bacterial identification, developing new strategy to fight against bacterial infection and drug resistance is also important.23–27 Unlike conventional antibiotics which target microbial metabolism, quaternary ammonium compounds (QACs) exhibit their 3



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antimicrobial activity by directly disrupting the microbial cell membranes,28,29 which make bacteria difficult to develop resistance.30 Meanwhile, since antibacterial materials can be coated on nanoparticles (NPs), NPs can be used as excellent drug carriers for antibacterial-related applications.31–35 Up to now, NPs, by loading drugs into these NPs through noncovalent interaction36 or covalent conjugation37, exhibit enhanced antibacterial activity with the minimum inhibitory concentration (MIC) values significantly lower than those of the free drug counterparts.37,38 Most of these NPs are not fluorescent, it is therefore difficult to use such NPs for simultaneous bacterial detection (by fluorescence) and inhibition. On the other hand, carbon dots (CDs), as carbon-based fluorescent nanomaterials, have received much attention recently due to their outstanding photoluminescence properties, low/nontoxic features, and tunability of the surface functionality.39–43 This new class of nanomaterials is considered to be a potential alternative to semiconductor quantum dots (which may have severe long-term toxicity) and other currently available fluorescent nanomaterials in many fields such as biological imaging,44–47 gene/drug delivery,48–51 light-emitting devices,52,53 catalysts,54,55 and antibacterial agents.56,57 CDs usually have the unique excitation-dependent fluorescence emission property, centering mainly in the blue or green region. Extensive attempts have been carried out to synthesize functional CDs with such a multicolor fluorescence emission property.58,59 However, up to now, CDs with multifunctional properties such as bacterial imaging, bacterial classification, and bacterial killing are still lacking. In this work, we developed novel quaternized CDs by conjugating CDs with the quaternary ammonium compound lauryl betaine (abbreviated as BS-12). BS-12 contains a quaternary ammonium group, a long hydrocarbon chain, and a carboxyl group. The quaternary ammonium group has been well known as effective antibacterial moiety. The long hydrocarbon chain enables the successful membrane insertion of the reagent and can significantly enhance the antibacterial performance. The carboxyl group makes the molecule be able to conjugate with other amine-containing molecules or nanoparticles such as the CDs. The thus obtained CDs-C12 have the multicolor fluorescence emission capability for simultaneous detection and inhibition of Gram-positive bacteria. The schematics showing the synthesis of CDs-C12 and the proposed principle of CDs-C12 for killing, selective recognition, and imaging of 4


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Gram-positive bacterial strains are illustrated in Scheme 1. CDs covered with amino groups were prepared by a solvothermal method, in which glycerol acted as both the carbon source and

the

reaction

medium,

while

3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane (AEEA) was used as the surface passivation reagent. Then the BS-12 molecules were covalently conjugated onto the surface of the amine-functionalized CDs via amino–carboxyl reaction, producing CDs-C12 with the multicolor fluorescence emission property and enhanced antibacterial activity. Because of the structural difference in the cell walls of Gram-positive and Gram-negative bacteria, CDs-C12 showed selective fluorescence imaging and excellent antibacterial capability for Gram-positive bacteria. Since CDs-C12 had multicolor fluorescence emission, stimulated emission depletion (STED) microscopy was also employed to investigate the localization of CDs-C12 in S. aureus cells. The most significant finding of this work is that we developed quaternized CDs-C12 in a very simple and highly efficient method to selectively identify, and image (with multicolors and super-resolution) Gram-positive bacteria and simultaneously kill such bacteria. To the best of our knowledge, this is the first time to report the development of CDs with such important multiple functions.

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Scheme 1. Schematic illustrating the preparation of multicolor CDs-C12 and their application in selective Gram-positive bacterial imaging and killing.

2. EXPERIMENTAL SECTION Synthesis of CDs. The carbon dot (CD) precursor solution was prepared by adding 0.75 mL AEEA to 5 mL glycerol, and then degassed with nitrogen gas for 5 min. The resultant solution was transferred into an oven and heated at a temperature of 260 oC for 12 h. After naturally cooling to room temperature, the CDs were obtained. Before further modification, the raw CD solution was centrifuged to remove precipitates and dialyzed against pure water using a dialysis membrane (MWCO 1000) for 12 h. Synthesis

of

CDs-C12.

BS-12

(100

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

mg)

was

hydrochloride

activated

with

(EDC·HCl)

and

N-hydroxysulfosuccinimide (Sulfo-NHS) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution (0.1 M, pH = 6.0). The molar ratio of BS-12 : EDC·HCl : Sulfo-NHS was 1:4:4. After activation for 0.5 h, the mixture was added to 4 mL CD solution (50 mg/mL) in

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phosphate-buffered saline (PBS, 0.1 M, pH = 7.4) and reacted for another 12 h. Then pure CDs-C12 were obtained after dialysis (MWCO 1000) and freeze-drying. The determination of the quantum yields (QYs) of CDs or CDs-C12 was similar to the previous work.60,61 Evaluation of the Interaction Between CDs-C12 and Bacterial Model Membranes. Bacterial model membranes were prepared according to the previously reported method.31 Briefly, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) were dissolved in chloroform at a molar ratio of 3:1. After drying under nitrogen stream and vacuum drying, the mixture was dispersed in 0.1 M PBS buffer. The bacterial liposomes were then obtained by extrusion through a polycarbonate membrane (100 nm) for 21 times. To evaluate the interaction between CDs-C12 and bacterial model membranes, CD-C12 solutions with a fixed concentration of 50 µg/mL were mixed with different concentrations (0–2.0 mg/mL) of liposome solutions in PBS solution. After incubation for 2 h, the corresponding fluorescence spectra were collected. Cell Imaging. A549 cells were cultured in confocal dishes at 2×104/dish at 37 oC under 5% CO2 for 24 h. Then, the cells were treated with CDs-C12 (20 µg/mL) at 37 oC for 2 h. Afterward, they were washed with PBS solution for three times to remove free CDs-C12. Cell imaging was performed using a confocal laser scanning microscopy (CLSM, TCS-SP8, Leica, Germany) and excited at 405, 488, and 552 nm, respectively. The emission windows were set as the range of 409–480, 493–547, and 557–640 nm for the multicolor imaging of CDs-C12, respectively. Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Proteusbacillus vulgaris (P. vulgaris) as Gram-negative bacteria, and Staphylococcus aureus (S. aureus), Micrococcus luteus (M. luteus), and Bacillus subtilis (B. subtilis) as Gram-positive bacteria, were selected to evaluate the bacterial imaging ability of CDs-C12. All the six species of bacteria were cultured in lysogeny broth (LB) medium (containing 10 mg/mL tryptone, 5 mg/mL yeast extract, ,and 0.5 mg/mL NaCl) and incubated in a shaking incubator for 12 h at 37 oC. The bacteria were collected and diluted (about 1 × 106 to 1 × 107 CFU/mL), then mixed 7



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with the CDs-C12 (30 µg/mL). After incubation at 37 oC in a shaking incubator (200 rpm) for 1 h, the bacteria were collected by centrifugation at 5000 rpm for 5 min, washed with PBS buffer for three times, and then imaged on CLSM. A flow cytometer (NovoCyteTM 2060, ACEA, USA) was used to quantify the fluorescence intensities of the bacterial cells.. CLSM and STED Imaging. The STED imaging experiments were performed on a TCS SP8 STED microscope (Leica, Germany). A continuous doughnut-shaped stimulated emission depletion (STED) laser emitting at λSTED = 660 nm was coupled to λex = 552 nm for the excitation of CDs-C12. In order to compare CLSM and STED imaging, the acquisitions were carried out in sequential mode: only the 552 nm laser was applied for CLSM imaging in the first channel; while both the 552 nm laser and the STED laser (λSTEDex = 660 nm) were applied in the second channel. For both CLSM and STED imaging, the emission window was 560–650 nm. Other Experimental Details and Procedures. Detailed experimental procedures for the evaluation of in vivo and in vitro antibacterial activity, determination of minimal inhibitory concentration (MIC), cell viability assay of mammalian cells, and histological staining were similar to the previous work,31 which can be found in the Supporting Information.

3. RESULTS AND DISCUSSION Preparation and Characterization of CDs-C12. A series of measurements were carried out to characterize the synthesized CDs-C12. Transmission electron microscopy (TEM) image revealed that CDs-C12 had an average size of ~4 nm with good dispersity and uniformity (Figure 1A), which was slightly larger than that of the original CDs (~2.7 nm) (Figure S1). The hydrodynamic diameter (measured by DLS) of CDs-C12 in water and PBS was ~8 nm, which was a little larger than that of the bare CDs (~6 nm) (Figure S2). For the zeta potential experiments, we have tried many times to measure the zeta potentials of CDs and CDs-C12 in water. However, the zeta potential values could not be detected, indicating that the zeta potentials of small nanoparticles such as carbon dots may not be suitable for measurement using the Zetasizer instrument, possibly due to the unstability of the electrical double layer of

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these small-sized CDs in pure water. When dispersed in PBS, the zeta potentials of CDs and CDs-C12 could be measured (see the results in Figure S3) and these values might be used for reference. It was found that CDs had an average zeta potential of –16.0 mV in PBS, which may be attributed to the presence of carboxyl groups on the surface. After conjugation with BS-12, the CDs became less negatively charged with a zeta potential of –11.6 mV, indicating the successful conjugation of BS-12. Fourier transform infrared (FTIR) spectra were also collected to confirm that BS-12 was successfully linked to CDs to prepare CDs-C12. As shown in Figure 1B, similar spectra feature in the range of ~1010–1160 cm−1, attributed to Si−O and C−O stretching vibration, was observed from the spectra of both CDs and CDs-C12. Similar strong signals could also be observed from both CDs-C12 and BS-12 at 1467 cm−1 (CH2 bending vibration), 2922 cm−1 (CH2 asymmetric stretching vibration), and 2852 cm−1 (CH2 symmetric stretching vibration), coming from the long methylene chains of BS-12 moiety. The relatively weak signals in the range of 2800–3000 cm–1 might come from the CH stretching vibration of the AEEA and glycerol moieties in the CDs. The broad band between 3300 and 3500 cm−1 in the spectra of CDs and CDs-C12 may be attributed to the OH stretching vibration band partially overlapped with the NH stretching vibration band. The large number of OH groups in BS-12 is due to the very hygroscopic property of the sample, while the massive OH groups in CDs and CDs-C12 may be derived from the reactant glycerol molecules. These results showed clearly that BS-12 was successfully linked to the CDs and the formed CDs-C12 have plentiful functional groups. XPS spectra were collected to further determine the chemical bonds and functional groups of CDs-C12. The detailed N 1s spectrum of CDs displayed two peaks at 398.7 eV (C−N−C) and 400.3 eV (N−H) (Figure S4). For the N 1s spectrum of CDs-C12, two peaks located at 399.3 and 402.1 eV were observed, which are assigned to C−N−C and quaternary ammonium group, respectively (Figure 1C). The XPS data, together with the FTIR results, provided strong evidence for successful synthesis of CDs-C12. Furthermore, the UV−vis absorption of CDs, BS-12, and CDs-C12 were also measured. As shown in Figure 1D, free BS-12 had no absorption peak, while the spectrum of CDs exhibited two broad peaks centered at 280 and 340 nm, which could be ascribed to the π−π* transition of an aromatic π system and the n−π* transition of carbonyl groups, respectively, as observed 9



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previously in various types of CDs prepared using different methods.44,48 Differently, CDs-C12 had a new absorption band at around 510 nm, which extended to the longer wavelength region (to ~800 nm). This may result from the change of surface functional groups that induces other types of electron transition.59 The change in the UV-vis absorption of CDs and CDs-C12 was also in accordance with the change of the solution color from yellow (CDs) to brown (CDs-C12) (inset, Figure 1D). Besides, the fluorescence properties of CDs and CDs-C12 were also investigated. The CDs exhibited excitation wavelength-dependent emission when excited from 320 to 540 nm (Figure S5), and the results showed that the fluorescence intensity of CDs was very low under the excitation wavelengths from 540 to 600 nm. In contrast, CDs-C12 showed continuous excitation-dependent emission, covering a larger wavelength region (Figure 1E). The emission peaks of CDs-C12 gradually red-shifted from 450 to 630 nm when excited from 320 to 600 nm. It has been reported that the relative amount and type of C atoms binding with N and O atoms may alter the electronic structure and energy gap of carbon dots and then tune their fluorescence emission frequency.59 Therefore we speculated that the differences in fluorescence properties between CDs and CDs-C12 may be ascribed to the distinct functional groups on their surfaces after CDs were conjugated with BS-12 molecules. CD-C12 and CD solutions emitted green and blue fluorescence, respectively, under 302 nm UV irradiation (inset, Figure 1D). The photoluminescence quantum yields (PLQYs) of CDs and CDs-C12 were calculated to be 14.5% and 3.5%, respectively, using quinine sulfate (PLQY = 54% in 0.1 M H2SO4) as a reference. The larger excitation/emission wavelength range of CDs-C12 could endow the CDs-C12 to be used for multicolor imaging, which will be discussed later in this work. Meanwhile, to investigate the stability of synthesized CDs-C12, they were dispersed in different aqueous solutions including deionized water, normal saline, PBS solution, and LB medium. Clear solutions without any precipitates were observed within 6 months (Figure S6), indicating that CDs-C12 are very stable and suitable for biomedical application. The excellent water-dispersibility of CDs-C12 may be mainly attributed to the abundant hydroxyl groups on the surface derived from the reactant glycerol molecules. Besides, the AEEA molecule is also important for the improved stability of CDs after quaternization. We have proved that by replacing AEEA with (3-aminopropyl)trimethoxysilane (APTMS) in the synthesis of 10


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amine-functionalized CDs, the resultant CDs were not stable after dialysis. This indicates that the AEEA molecule, with the unique structure containing three amine groups, plays an important role in the stability of the CD sample. Besides, the fluorescence emission spectra of CDs and CDs-C12 dissolved in various solvents (including water, ethanol, dichloromethane, and acetone) were collected. CDs presented the strongest fluorescence intensity in ethanol and the weakest intensity in acetone (Figure S7). However, CDs-C12 in acetone presented the strongest fluorescence intensity, possibly due to the best solubility of CDs-C12 in acetone (Figure 1F). The results indicated that the BS-12 moiety had a large influence on the polarity of CDs-C12. More importantly, CDs-C12 are sensitive to the polarity of the solvent, and their fluorescence intensity may significantly increase as CDs-C12 are moved from water to a less polar environment, e.g., when it is accumulated on the anionic cell membrane. To further reveal the mechanism of the fluorescence increase of CDs-C12 in bacterial cells, the membrane of Gram-positive bacteria was modeled using liposomes prepared according to a previous method.31,62 The obtained liposomes had good dispersity in PBS solution with an average hydrodynamic diameter of 119.2 ± 24.7 nm. After mixing with increasing concentrations of liposomes (0.1 to 2.0 mg/mL), the fluorescence intensity of CDs-C12 gradually increased (Figure S8), suggesting that CDs-C12 could have significantly enhanced fluorescence emission upon interacting with the bacterial membranes.

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Figure 1. (A) TEM image and size distribution (inset) of CDs-C12. (B) FTIR spectra of CDs-C12, CDs, and BS-12. (C) N1s XPS spectrum showing nitrogen-related bonds in CDs-C12. (D) UV−vis absorption spectra of BS-12, CDs, and CDs-C12. The insets are photographs of CDs and CDs-C12 dispersed in water. (E) Fluorescence emission spectra of CDs-C12 under varied excitation wavelengths. The insets are photographs of CD-C12 and CD aqueous solutions under a UV lamp (302 nm). (F) Fluorescence spectra of CDs-C12 dispersed in various solvents.

Selective CLSM and STED Imaging of Gram-Positive Bacteria. Considering the 12


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excellent fluorescence properties of CDs-C12, the fluorescence imaging ability of CDs-C12 towards bacteria was explored. Typically, as shown in Figure 2A, after incubating S. aureus (a typical example of Gram-positive bacteria) with CDs-C12 for 1 h, different emission colors of the CDs within the bacteria were clearly visualized under different excitation wavelengths. In contrast, E. coli cells displayed no noticeable fluorescence signal under the same condition. Flow cytometry experiments were performed to quantitatively analyze the fluorescence signals of CDs-C12-treated S. aureus and E. coli cells, respectively. As shown in Figure 2B, for the FL1 channel (FITC channel), the fluorescence intensity of S. aureus was significantly enhanced after the treatment with CDs-C12 for 1 h, which was in good agreement with that observed using confocal laser scanning microscopy (CLSM). For E. coli, the mean fluorescence intensity of CDs-C12-treated group showed no obvious difference compared to the control group. To further confirm the selectivity of CDs-C12 toward S. aureus, the zeta potentials of S. aureus and E. coli with and without treatment with CDs-C12 (30 µg/mL) were measured (Figure S9). After incubation with CDs-C12, S. aureus became more negatively charged (from –12.7 to –15.3 mV) in PBS, indicating the interaction between CDs-C12 and the bacterial surface. In contrast, no apparent change was observed between the zeta potentials of E. coli before and after treatment with CDs-C12. Thus, the interaction between S. aureus cells and CDs-C12 may be dictated by the localized interaction between the positively charged quaternary ammonium group (bearing a long hydrocarbon chain) of the BS-12 moiety and the negatively charged bacterial surface, while the zeta potential of the whole carbon dots may have little contribution to the interaction. These data suggested the specific binding interaction between CDs-C12 and Gram-positive bacteria. To directly visualize the selective binding, a mixture containing both S. aureus and E. coli bacteria was incubated with 30 µg/mL CDs-C12, and examined by CLSM. As shown in Figure 2C, after the treatment with CDs-C12 for 1 h, S. aureus cells were stained and emitted red fluorescence signal under 552 nm excitation. In contrast, negligible fluorescence signal was detected for E. coli cells under the same experimental condition, proving that CDs-C12 could selectively image and identify Gram-positive S. aureus, but not E. coli. Two other species of Gram-positive bacteria (M. luteus and B. Subtilis) and two other species of Gram-negative bacteria (P. vulgaris and P. aeruginosa) were also selected to confirm selective imaging property of CDs-C12 toward 13



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Gram-positive bacteria. As shown in Figure 3, the CLSM and flow cytometry results both confirmed that the Gram-positive bacteria could be selectively stained by CDs-C12. Therefore, we provide a new method for bacterial distinguishment using fluorescent CDs-C12. The selective bacterial imaging effect may be attributed to the different cell wall structures and/or composition diversity. It has been reported that Gram-positive bacteria are much more sensitive to lipophilic molecules than Gram-negative ones.63 Although both types of bacteria possess similar cytoplasmic membrane comprising phospholipids and membrane proteins, Gram-negative bacteria have a more complicated cell surface structure than Gram-positive ones. In general, Gram-positive bacteria have mesh-like thick but simple-structured cell walls, consisting of an interconnected peptidoglycan layer that lies outside of the plasma membrane. The negatively charged teichoic acids on the peptidoglycan layer provide many anionic sites, facilitating the electrostatic binding of cationic molecules or nanoparticles to the bacterial cell surface. Because of the more negatively charged cell surface, a Gram-positive bacterium could more easily interact with the BS-12 moiety of CDs-C12 with both quaternary ammonium group and long hydrocarbon chains compared to a Gram-negative bacterium, leading to the disruption of the cell wall. This could enhance the penetration of CDs-C12 and result in the accumulation of CDs-C12 onto the less polar environment of the cell plasma membrane. Such CDs-C12 accumulation on the Gram-positive bacterial cell membrane could enhance the fluorescence imaging performance of CDs-C12. For Gram-negative bacteria, the cell wall is composed of a thin peptidoglycan layer between the protective outer cell membrane and the cytoplasmic membrane. The outer membrane is rich in lipopolysaccharides which are cross-bridged by divalent cations. Such a unique structure and composition of the outer membrane makes the membrane more impermeable to lipophilic molecules or NPs, resulting in weak fluorescence staining of CDs-C12 for Gram-negative bacteria. The interaction of cationic NPs with bacterial wall has also been studied by Bunz et al. using hydrophobic/cationic Au NPs as a model.64 It was found that the cationic NPs display varied aggregation patterns on Gram-positive and Gram-negative bacteria: The cationic NPs aggregated more on the surface of Gram-positive bacteria B. subtilis than that of Gram-negative bacteria E. coli. When excited at 552 nm, CDs-C12 showed a broad emission band at around 580 nm and it 14


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extended to above 660 nm (Figure S10). Therefore CDs-C12 interacting with bacteria can be studied using super-resolution STED imaging at 660 nm. For STED experiments, S. aureus cells were incubated with CDs-C12 (30 µg/mL) for 1 h, and then washed with normal saline for three times to remove residual CDs-C12. Both STED images and corresponding CLSM images (for comparison) were obtained (Figure 2D). Clearly the STED image confirmed that most of CDs-C12 gathered at the surface of S. aureus, whereas from the CLSM result the CDs-C12 seemed to be dispersed around the whole thalli. More evidence could be seen in the closer view (Figure 2D (c,d)). Unlike the CLSM result that showed a blurred and conglobate image of a S. aureus cell, the STED image revealed the CDs-C12 distributed around the membrane surface clearly with small aggregates. The ability of STED to increase the imaging resolution of S. aureus was also demonstrated by comparing the fluorescence line profiles. Figure 2D (e) showed a representative example of the line profiles of CLSM and STED spots (marked by green lines) in Figure 2D (c,d). Different from the CLSM image that showed only one unresolved spot, the STED picture exhibited three separated spots. As presented by Gaussian fitted profiles of the representative intensity plot of fluorescent spots in CLSM and STED images, the full width at half maximum (FWHM) value in CLSM mode was 327 nm, while the three peaks in STED mode were measured to be 136, 144, and 129 nm (peak left to right, respectively). The minimal lateral resolution in STED images was much higher than that (200–300 nm) in CLSM images. As far as we know, this is the first report of applying STED microscopy for imaging the interaction between CDs and bacteria.

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Figure 2. (A) CLSM images of E. coli and S. aureus bacteria after incubation with CDs-C12 (30 µg/mL) for 1 h under excitation of 405 nm (a,e), 488 nm (b,f), 552 nm (c,g), and the overlay channel (d,h), respectively. (B) Flow cytometry assay to quantify the fluorescence intensity of S. aureus and E. coli before and after treatment with CDs-C12. (C) CLSM images of the mixture of E. coli and S. aureus exposed to CDs-C12 for 1 h under 552 nm laser irradiation and the overlay channel merged from the CLSM channel (552 nm) and the bright field channel, respectively. (D) CLSM (a) versus STED (b) imaging of S. aureus after treatment with CDs-C12 for 1 h. The closer views of the regions in the white squares in (a) and (b) are presented in (c) and (d), respectively. (e) Representative intensity plots of fluorescent spots extracted from CDs-C12 on the surface of S. aureus after Gaussian fitting in CLSM and STED modes. 16


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Figure 3. (A) CLSM images of M. luteus, B. subtilis, P. aeruginosa, and P. vulgaris after incubation with CDs-C12 (30 µg/mL) for 1 h under 405, 488, and 552 nm laser excitations, and the overlay channel, respectively. (B) The corresponding flow cytometric results.

SEM Characterization of Bacteria. To further investigate the influence of CDs-C12 on bacterial cells, the morphological changes of bacteria with and without the treatment of CDs-C12 were observed by scanning electron microscopy (SEM). Gram-positive S. aureus and Gram-negative E. coli were chosen as model bacteria for SEM evaluation. As shown in Figure 4, untreated S. aureus cells showed clear edges and smooth cell walls. After incubation with CDs-C12 for 2.5 h, their cell surfaces became damaged and wrinkled, and the leakages of intracellular contents could be observed clearly for most of the cells, suggesting that CDs-C12 could be used in antibacterial application and the antimicrobial mechanism involved the disruption of bacterial cell walls and/or membranes, similar to the previous reports.63 However, after the treatment with BS-12 at the same concentration as CDs-C12 on S. aureus cells, almost no morphological changes could be observed for S. aureus cells as compared to the control groups (Figure S11), indicating the markedly enhanced antibacterial activity of CDs-C12 compared to that of free BS-12. As for E. coli, after treatment with CDs-C12 (Figure 4) or BS-12 (Figure S12), the morphology of most cells still remained unchanged with unbroken surfaces, confirming that CDs-C12 or BS-12 did not have noticeable interactions with E. coli cells.

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Figure 4. Typical SEM images of S. aureus (A) and E. coli (B) cells without (left) and with CDs-C12 (right) treatment at 60 µg/mL for 2.5 h.

Evaluation of In Vitro and In Vivo Antibacterial Capability. Based on the above CLSM, STED, and SEM results, we believe that CDs-C12 could effectively kill Gram-positive bacteria. In the following, we will report the antibacterial properties of CDs-C12 and compared such properties to those of pure CDs and free BS-12 tested by optical density (OD) measurement, colony counting method (CFU), and cell counting kit-8 (CCK-8) assay. Figure 5A showed the time-dependent antibacterial testing results of CDs-C12 at different doses by measuring the OD values at 600 nm (OD600). For S. aureus, it was observed that when the concentration of CDs-C12 reached 20 µg/mL, the bacterial growth was significantly inhibited in the first 12 h. At 30 µg/mL, the growth of the bacteria was completely inhibited by CDs-C12. CDs and free BS-12 at the concentration of 30 µg/mL showed no antibacterial effects for S. aureus (Figure S13). We also explored the antibacterial effect of free BS-12 in solution at different concentrations (Figure S14). Our experimental results indicated that BS-12 could not completely inhibit the proliferation of S. aureus within the first 24 h until the concentration was up to 500 µg/mL (or higher), which is nearly 20-fold higher than that of CDs-C12 (Figure 5). The CCK-8 assay results also confirmed that free BS-12 and CDs showed no obvious effect on the S. aureus viability at 30 µg/mL (Figure 5B). The viability of S. aureus after the treatment of CDs, free BS-12, or CDs-C12 at the same concentration (10 µg/mL) was also 18


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studied by the CFU counting method to further demonstrate the elevated antibacterial properties of CDs-C12. As showed in Figure 5C, the amount of viable bacterial cells after the treatment with CDs or free BS-12 is almost the same as the control group, in good agreement with the above CCK-8 assay and OD600 measurement results. In contrast, after the treatment with 10 µg/mL CDs-C12, few bacterial colonies were observed with only 1.5% of viable S. aureus (Figure 5D). These results demonstrated the significantly enhanced antibacterial activity of CDs-C12 is not simply the additive effects of CDs and BS-12 but may be due to the synergistic effect of the two components, especially the unique multivalency effect of the surface ligands on CDs-C12. Likely the concentrated BS-12 molecules on CDs-C12 surface can more effectively inhibit bacterial growth than the same amount of free BS-12 molecules. For Gram-negative bacteria E. coli, the time-dependent antibacterial testing results showed that the growth of E. coli could only be slowed down slightly within several hours even when the concentration of CDs-C12 reached 200 µg/mL (Figure S15A). For BS-12, even when its concentration reached 1 mg/mL, the growth of E. coli was only slightly inhibited (Figure S15B). The results from CCK-8 assay also confirmed that only 20% of the cells were inhibited by 200 µg/mL CDs-C12, while neither CDs nor BS-12 had antibacterial activity against E. coli (Figure S11). The results confirmed that the antibacterial activity of CDs-C12 toward E. coli was much weaker than that toward S. aureus, similar to other surface-quaternized NPs.36 To generalize our conclusion, the MIC values of two other types of Gram-positive bacteria (M. luteus and B. subtilis) and two other types of Gram-negative bacteria (P. vulgaris and P. aeruginosa) were also measured. The MIC is defined as the lowest concentration at which an antimicrobial agent can completely inhibit the proliferation of bacteria at the concentration of 1×105~1×106 CFU/mL, by measuring the OD600 value after incubation at 37 oC over 14 h. As shown in Table 1, the MIC values of CDs-C12 against Gram-positive bacteria were much lower than those of BS-12, indicating the enhanced antibacterial activity of CDs-C12. Whereas for Gram-negative bacteria, even when the concentration of CDs-C12 reached 200 µg/mL, it still could not completely inhibit the growth of bacteria. The variations in the MICs of CDs-C12 for different bacterial types indicated their distinct interaction mechanisms with CDs-C12, and the results further confirmed that only Gram-positive bacteria could be completely inhibited by CDs-C12. Note that the concentration 19



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of CDs-C12 includes the whole nanoparticle (including the CD core and the BS-12 ligand) and the antibacterial BS-12 ligand only accounts for a small part of the nanoparticle. Therefore, the still much smaller MICs of CDs-C12 than those of free BS-12 against Gram-positive bacteria suggests the importance of multivalency effect of CDs-C12 after surface conjugation with BS-12.

Table 1. MIC values of CDs-C12 and BS-12 for six species of bacteria. Species

Gram type

CDs-C12 (µg/mL)

BS-12 (µg/mL)

S. aureus

G+

8

80

M. luteus

G+

6

70

B. subtilis

G+

12

80

E. coli

G–

> 200

> 200

P. vulgaris

G–

> 200

> 200

P. aeruginosa

G–

> 200

> 200

To further assess the antibacterial efficacy of CDs-C12 in vivo, the S. aureus-infected mice were established as a model (Figure 5E–G). We divided the mice into two groups: PBS solution-treated group and CDs-C12-treated group. The therapeutic efficacy was evaluated by enumerating and comparing the bacterial counts in the infectious site. After the treatment, quantification of the bacteria in the infectious tissue showed 2.2 × 108 CFU/g for the PBS buffer-treated mice group. Differently, mice treated with CDs-C12 resulted in a bacterial burden of 2.8 × 106 CFU/g, a significant reduction in the bacterial number (with a bacterial killing efficiency of 98.7%) as compared to the control group.

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Figure 5. In vitro and in vivo antibacterial testing results of CDs-C12 in comparison with CDs and free BS-12. (A) Inhibition effect of CDs-C12 on the growth of S. aureus as a function of CD-C12 concentration. (B) Cell viability evaluation of S. aureus after treatment with different concentrations of CDs, free BS-12, or CDs-C12 at 2.5 h. (C&D) Typical photographs of S. aureus colonies and the corresponding statistical histograms after the treatment with CDs, BS-12, or CDs-C12 at the final concentration of 10 µg/mL for 2.5 h. (E) Typical photographs of S. aureus-infected mice treated with PBS buffer (control) or CD-C12 solution. (F) Photographs and (G) statistical histograms (with error bars) of bacterial colonies counted from the dilutions of homogenized infectious tissue.

Biocompatibility Assay. To verify that CDs-C12 are suitable for biomedical applications, 21



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we carried out the safety evaluation of CDs-C12 in vitro and in vivo. As shown in Figure 6A, after the treatment with 20 µg/mL of CDs-C12 for 24 h, more than 90% of A549 and AT II cells remained alive (while over 90% of the S. aureus cells were killed within 2.5 h at the same dose as reported above). Even the concentration was up to 60 µg/mL, over 65% cells still remained viable. CLSM was used as a complementary technique to further demonstrate the feasibility of using CDs-C12 in real biomedical applications. As shown in Figure 6B, due to the broad tunability of the fluorescence emission as well as the remarkably high photostability of the CDs-C12, after incubation with CDs-C12 for 2 h at 20 µg/mL, A549 cells emitted intense blue, green, and red signals under 405, 488, and 552 nm laser excitations, respectively, proving that the CDs-C12 are also suitable for cell imaging. Last, we evaluated the in vivo toxicity of CDs-C12. The pathomorphology of the main organs (heart, liver, lung, kidney, and spleen) was evaluated using hematoxylin and eosin (H&E) stain. The tissues from CDs-C12-treated mice maintained an undisturbed structure as compared to those from control group (Figure 6C). This demonstrates that CDs-C12 should be a promising antibacterial agent with negligible toxicity in vivo because it can effectively kill Gram-positive bacteria and do not affect any main organs.

Figure 6. In vitro and in vivo safety testing results of CDs-C12. (A) Relative viabilities of A549 and AT II cells exposed to different concentrations of CDs-C12 for 24 h. (B) CLSM images of A549 cells after the treatment with CDs-C12 at the concentration of 20 µg/mL for 2 22


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h. Scale bar is 50 µm. (C) Typical H&E stained images of main organs from mice treated with PBS solution or CDs-C12 (scale bar is 200 µm).

4. CONCLUSIONS In summary, we first synthesized a new type of amine-functionalized CDs that can ensure excellent water dispersibility after conjugation with quaternary ammonium compound (BS-12) bearing a long hydrophobic hydrocarbon chain. The thus obtained quaternized CDs (CDs-C12) exhibit many excellent properties such as high brightness, superior photostability, multicolor fluorescence emission, selective Gram-positive bacterial identification and inactivation ability, good water solubility, and excellent biocompatibility. The main contributions of this research can be summarized as follows: (1) We developed a facile preparation method of a novel type of metal-free NP-based antibiotics, CDs-C12, by conjugating BS-12 to the one-step synthesized amine-functionalized CDs via a simple carboxyl–amine reaction. Such a simple, low-cost preparation method can readily realize large scale production. (2) CDs-C12, coated with BS-12 moieties, possess the continuous excitation-dependent emission property. The variation of the surface chemistry compared to the precursor CDs extends the emission light to a longer wavelength region. Therefore CDs-C12 can be used for multicolor fluorescence imaging using CLSM as well as super-resolution imaging using STED microscopy. The present work is the first example in which STED microscopy is used to reveal the exact interaction site of the CDs within bacteria. (3) CDs-C12 can effectively distinguish Gram-positive and Gram-negative bacteria due to the different cell walls/membranes of the two types of bacteria. Compared to free BS-12, CDs-C12 have a significantly increased local positive charge density and number of hydrophobic chains, leading to greatly enhanced therapeutic index on bacteria. We believe that the new CDs-C12 material developed in this research which can selectively image and kill Gram-positive bacteria will advance the applications for carbon dots. Such a material holds great potential for use to combat bacterial infections and overcome bacterial drug resistance.

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Details of the reagents, instruments, and experimental procedures; TEM image, size distribution, XPS spectrum, and fluorescence emission spectra of precursor CDs; Hydrodynamic diameters and zeta potentials of CDs and CDs-C12; Typical photographs of CDs-C12 in different aqueous solutions; Fluorescence emission spectra of CDs under different excitation wavelengths and in different solvents; Fluorescence responses of CDs-C12 after mixing with liposomes mimicking bacterial membranes; Zeta potentials of S. aureus and E. coli before and after treatment with CDs-C12; SEM images of E. coli and S. aureus cells with the treatment of BS-12; other antibacterial results of CDs, BS-12, and CDs-C12; Cell viability of E. coli after the treatment with CDs, BS-12, or CDs-C12.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected].

Author Contributions #

J.J.Y. and X.D.Z. contributed equally.

Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (21303017 and 21673037), the National High Technology Research & Development Program of China (2015AA020502), Six Talents Peak Project in Jiangsu Province (2015-SWYY-003), Fundamental Research Funds for the Central Universities (2242015R30016), and Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. ZC thanks the University of Michigan for the support.

REFERENCES (1) Levy, S. B.; Marshall, B. Antibacterial Resistance Worldwide: Causes, Challenges and Responses. Nat. Med. 2004, 10, S122–S129. (2) Gao, W. W.; Thamphiwatana, S.; Angsantikul, P.; Zhang, L. F. Nanoparticle Approaches against Bacterial Infections. Wiley Interdiscip Rev. Nanomed. Nanobiotechnol. 2014, 6, 532–547. (3) Nugent, R. P.; Krohn, M. A.; Hillier, S. L. Reliability of Diagnosing Bacterial Vaginosis is Improved by a Standardized Method of Gram Stain Interpretation. J. Clin. Microbiol. 1991, 29, 297–301. (4) Oethinger, M.; Warner, D. K.; Schindler, S. A.; Kobayashi, H.; Bauer, T. W. Diagnosing Periprosthetic Infection: False-Positive Intraoperative Gram Stains. Clin. Orthop. Relat. Res. 2011, 469, 954–960. (5) Ryu, E. On the Gram-Differentiation of Bacteria by the Simplest Method. J. Jpn. Soc. Vet. Sci. 1938, 17, 31. (6) Ryu, E. On the Gram-Differentiation of Bacteria by the Simplest Method. III. The Sulfuric Acid Methods. J. Jpn. Soc. Vet. Sci. 1940, 2, 491–496. (7) Reasoner, D. J.; Geldreich, E. E. A New Medium for the Enumeration and Subculture of Bacteria from Potable Water. Appl. Environ. Microb. 1985, 49, 1–7. (8) Gao, J. H.; Li, L. H.; Ho, P. L.; Mak, G. C.; Gu, H. W.; Xu, B. Combining Fluorescent Probes and Biofunctional Magnetic Nanoparticles for Rapid Detection of Bacteria in Human Blood. Adv. Mater. 2006, 18, 3145–3148. (9) Mothershed, E. A.; Whitney, A. M. Nucleic Acid-Based Methods for the Detection of Bacterial Pathogens: Present and Future Considerations for the Clinical Laboratory. Clin. Chim. Acta 2006, 363, 206–220. (10) Petti, C. A. Detection and Identification of Microorganisms by Gene Amplification and Sequencing. Clin. Infect. Dis. 2007, 44, 1108–1114. (11) Loman, N. J.; Constantinidou, C.; Chan, J. Z.; Halachev, M.; Sergeant, M.; Penn, C. W.; Robinson, E. R.; Pallen, M. J. High-Throughput Bacterial Genome Sequencing: an Embarrassment of Choice, a World of Opportunity. Nat. Rev. Microbiol. 2012, 10, 599–606. 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(12) Loman, N. J.; Misra, R. V.; Dallman, T. J.; Constantinidou, C.; Gharbia, S. E.; Wain, J.; Pallen, M. J. Performance Comparison of Benchtop High-Throughput Sequencing Platforms. Nat. Biotechnol. 2012, 30, 434–439. (13) Tao, Y.; Ran, X.; Ren, J. S.; Qu, X. G. Array-Based Sensing of Proteins and Bacteria by Using Multiple Luminescent Nanodots as Fluorescent Probes. Small 2014, 10, 3667–3671. (14) Chen, W. W.; Li, Q. Z.; Zheng, W. S.; Hu, F.; Zhang, G. X.; Wang, Z.; Zhang, D. Q.; Jiang, X. Y. Identification of Bacteria in Water by a Fluorescent Array. Angew. Chem. Int. Ed. 2014, 53, 13734–13739. (15) Li, Y. Q.; Zhu, B. W.; Li, Y. G.; Leow, W. R.; Goh, R.; Ma, B.; Fong, E.; Tang, M.; Chen, X. D. A Synergistic Capture Strategy for Enhanced Detection and Elimination of Bacteria. Angew. Chem. Int. Ed. 2014, 53, 5837–5841. (16) Lan, M. H.; Wu, J. S.; Liu, W. M.; Zhang, W. J.; Ge, J. C.; Zhang, H. Y.; Sun, J. Y.; Zhao, W. W.; Wang, P. F. Copolythiophene-Derived Colorimetric and Fluorometric Sensor for Visually Supersensitive Determination of Lipopolysaccharide. J. Am. Chem. Soc. 2012, 134, 6685–6694. (17) Sizemore, R. K.; Caldwell, J. J.; Kendrick, A. S. Alternate Gram Staining Technique Using a Fluorescent Lectin. Appl. Environ. Microb. 1990, 56, 2245–2247. (18) Rojas, E.; Theriot, J. A.; Huang, K. C. Response of Escherichia coli Growth Rate to Osmotic Shock. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 7807–7812. (19) Edgar, R.; McKinstry, M.; Hwang, J.; Oppenheim, A. B.; Fekete, R. A.; Giulian, G.; Merril, C.; Nagashima, K.; Adhya, S. High-Sensitivity Bacterial Detection Using Biotin-Tagged Phage and Quantum-Dot Nanocomplexes. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4841–4845. (20) Li, L. L.; Xu, J. H.; Qi, G. B.; Zhao, X. Z.; Yu, F. Q.; Wang, H. Core–Shell Supramolecular Gelatin Nanoparticles for Adaptive and “On-Demand” Antibiotic Delivery. ACS Nano 2014, 8, 4975–4983. (21) Qi, G. B.; Li, L. L.; Yu, F. Q.; Wang, H. Vancomycin-Modified Mesoporous Silica Nanoparticles for Selective Recognition and Killing of Pathogenic Gram-Positive Bacteria over Macrophage-Like Cells. ACS Appl. Mater. Interfaces 2013, 5, 10874–10881. (22) Zhong, D.; Zhuo, Y.; Feng, Y. J.; Yang, X. M. Employing Carbon Dots Modified with Vancomycin for Assaying Gram-Positive Bacteria like Staphylococcus aureus. Biosens. Bioelectron. 2015, 74, 546–553. (23) Fischbach, M. A.; Walsh, C. T. Antibiotics for Emerging Pathogens. Science 2009, 325, 1089–1093. (24) Fernandes, P. Antibacterial Discovery and Development—the Failure of Success? Nat. Biotechnol. 2006, 24, 1497–1503. (25) Wang, L. S.; Gupta, A.; Rotello, V. M. Nanomaterials for the Treatment of Bacterial Biofilms. ACS Infect. Dis. 2016, 2, 3–4. (26) Bao, Q.; Zhang, D.; Qi, P. Synthesis and Characterization of Silver Nanoparticle and Graphene Oxide Nanosheet Composites as a Bactericidal Agent for Water Disinfection. J. Colloid Interface Sci. 2011, 360, 463–470. (27) Wang, Y.; Ding, X. L.; Chen, Y.; Guo, M. Q.; Zhang, Y.; Guo, X. K.; Gu, H. C. 26


Page 26 of 30

Page 27 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Antibiotic-Loaded, Silver Core-Embedded Mesoporous Silica Nanovehicles as a Synergistic Antibacterial Agent for the Treatment of Drug-Resistant Infections. Biomaterials 2016, 101, 207–216. (28) Zhu, C. L.; Yang, Q.; Liu, L. B.; Lv, F. T.; Li, S. Y.; Yang, G. Q.; Wang, S. Multifunctional Cationic Poly(p-phenylene vinylene) Polyelectrolytes for Selective Recognition, Imaging, and Killing of Bacteria over Mammalian Cells. Adv. Mater. 2011, 23, 4805–4810. (29) Li, X. N.; Robinson, S. M.; Gupta, A.; Saha, K.; Jiang, Z. W.; Moyano, D. F.; Sahar, A.; Riley, M. A.; Rotello, V. M. Functional Gold Nanoparticles as Potent Antimicrobial Agents Against Multi-Drug-Resistant Bacteria. ACS Nano 2014, 8, 10682–10686. (30) Wang, H. Y.; Hua, X. W.; Wu, F. G.; Li, B. L.; Liu, P. D.; Gu, N.; Wang, Z. F.; Chen, Z. Synthesis of Ultrastable Copper Sulfide Nanoclusters via Trapping the Reaction Intermediate: Potential Anticancer and Antibacterial Applications. ACS Appl. Mater. Interfaces 2015, 7, 7082–7092. (31) Zhang, X. D.; Chen, X. K.; Yang, J. J.; Jia, H. R.; Li, Y. H.; Chen, Z.; Wu, F. G. Quaternized Silicon Nanoparticles with Polarity-Sensitive Fluorescence for Selectively Imaging and Killing Gram-Positive Bacteria. Adv. Funct. Mater. 2016, 26, 5958–5970. (32) Wang, F.; Fang, R. H.; Luk, B. T.; Hu, C. M. J.; Thamphiwatana, S.; Dehaini, D.; Angsantikul, P.; Kroll, A. V.; Pang, Z. Q.; Gao, W. W.; Lu, W. Y.; Zhang, L. F. Nanoparticle-Based Antivirulence Vaccine for the Management of Methicillin-Resistant Staphylococcus aureus Skin Infection. Adv. Funct. Mater. 2016, 26, 1628–1635. (33) Thamphiwatana, S.; Gao, W. W.; Obonyo, M.; Zhang, L. F. In Vivo Treatment of Helicobacter pylori Infection with Liposomal Linolenic Acid Reduces Colonization and Ameliorates Inflammation. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 17600–17605. (34) Tian, T. F.; Shi, X. Z.; Cheng, L.; Luo, Y. C.; Dong, Z. L.; Gong, H.; Xu, L. G.; Zhong, Z. T.; Peng, R.; Liu, Z. Graphene-Based Nanocomposite as an Effective, Multifunctional, and Recyclable Antibacterial Agent. ACS Appl. Mater. Interfaces 2014, 6, 8542–8548. (35) Tang, J.; Chen, Q.; Xu, L. G.; Zhang, S.; Feng, L. Z.; 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, 3867–3874. (36) Ahangari, A.; Salouti, M.; Heidari, Z.; Kazemizadeh, A. R.; Safari, A. A. Development of Gentamicin-Gold Nanospheres for Antimicrobial Drug Delivery to Staphylococcal infected foci. Drug Deliv. 2013, 20, 34–39. (37) 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. (38) Li, P.; Poon, Y. F.; Li, W. F.; Zhu, H. Y.; Yeap, S. H.; Cao, Y.; Qi, X. B.; Zhou, C. C.; Lamrani, M.; Beuerman, R. W.; Kang, E. T.; Mu, Y. G.; Li, 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. (39) Ding, H.; Yu, S. B.; Wei, J. S.; Xiong, H. M. Full-Color Light-Emitting Carbon Dots with a Surface-State-Controlled Luminescence Mechanism. ACS Nano 2016, 10, 484–491. (40) Zhu, S. J.; Meng, Q. N.; Wang, L.; Zhang, J. H.; Song, Y. B.; Jin, H.; Zhang, K.; Sun, H. C.; Wang, H. Y.; Yang, B. Highly Photoluminescent Carbon Dots for Multicolor Patterning, Sensors, and Bioimaging. Angew. Chem., Int. Ed. 2013, 52, 3953–3957. 27



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(41) Long, Y. M.; Zhou, C. H.; Zhang, Z. L.; Tian, Z. Q.; Bao, L.; Lin, Y.; Pang, D. W. Shifting and Non-Shifting Fluorescence Emitted by Carbon Nanodots. J. Mater. Chem. 2012, 22, 5917–5920. (42) Sun, Y. P.; Zhou, B.; Lin, Y.; Wang, W.; Fernando, K. A. S.; Pathak, P.; Meziani, M. J.; Harruff, B. A.; Wang, X.; Wang, H. F. Quantum-Sized Carbon Dots for Bright and Colorful Photoluminescence. J. Am. Chem. Soc. 2006, 128, 7756–7757. (43) Huang, Y. F.; Zhou, X.; Zhou, R.; Zhang, H.; Kang, K. B.; Zhao, M.; Peng, Y.; Wang, Q.; Zhang, H. L.; Qiu, W. Y. One-Pot Synthesis of Highly Luminescent Carbon Quantum Dots and Their Nontoxic Ingestion by Zebrafish for In Vivo Imaging. Chem. Eur. J. 2014, 20, 5640–5648. (44) Zheng, M.; Ruan, S. B.; Liu, S.; Sun, T. T.; Qu, D.; Zhao, H. F.; Xie, Z. G.; Gao, H. L.; Jing, X. B.; Sun, Z. C. Self-Targeting Fluorescent Carbon Dots for Diagnosis of Brain Cancer Cells. ACS Nano 2015, 9, 11455–11461. (45) Kong, W. Q.; Liu, R. H.; Li, H.; Liu, J.; Huang, H.; Liu, Y.; Kang, Z. H. High-Bright Fluorescent Carbon Dots and Their Application in Selective Nucleoli Staining. J. Mater. Chem. B 2014, 2, 5077–5082. (46) Liu, J. H.; Cao, L.; LeCroy, G. E.; Wang, P.; Meziani, M. J.; Dong, Y.; Liu, Y.; Luo, P. G.; Sun, Y. P. Carbon "Quantum" Dots for Fluorescence Labeling of Cells. ACS Appl. Mater. Interfaces 2015, 7, 19439–19445. (47) Song, Y. B.; Zhu, S. J.; Yang, B. Bioimaging Based on Fluorescent Carbon Dots. RSC Adv. 2014, 4, 27184–27200. (48) Gong, X. J.; Zhang, Q. Y.; Gao, Y. F.; Shuang, S. M.; Choi, M. M.; Dong, C. Phosphorus and Nitrogen Dual-Doped Hollow Carbon Dot as a Nanocarrier for Doxorubicin Delivery and Biological Imaging. ACS Appl. Mater. Interfaces 2016, 8, 11288–11297. (49) Liu, C. J.; Zhang, P.; Zhai, X. Y.; Tian, F.; Li, W. C.; Yang, J. H.; Liu, Y.; Wang, H. B.; Wang, W.; Liu, W. G. Nano-Carrier for Gene Delivery and Bioimaging Based on Carbon Dots with PEI-Passivation Enhanced Fluorescence. Biomaterials 2012, 33, 3604–3613. (50) Pierrat, P.; Wang, R.; Kereselidze, D.; Lux, M.; Didier, P.; Kichler, A.; Pons, F.; Lebeau, L. Efficient In Vitro and In Vivo Pulmonary Delivery of Nucleic Acid by Carbon Dot-Based Nanocarriers. Biomaterials 2015, 51, 290–302. (51) Wang, J.; Zhang, Z. H.; Zha, S.; Zhu, Y. Y.; Wu, P. Y.; Ehrenberg, B.; Chen, J. Y. Carbon Nanodots Featuring Efficient FRET for Two-Photon Photodynamic Cancer Therapy with a Low fs Laser Power Density. Biomaterials 2014, 35, 9372–9381. (52) Zhang, X. Y.; Zhang, Y.; Wang, Y.; Kalytchuk, S.; Kershaw, S. V.; Wang, Y. H.; Wang, P.; Zhang, T. Q.; Zhao, Y.; Zhang, H. Z. Color-Switchable Electroluminescence of Carbon Dot Light-Emitting Diodes. ACS Nano 2013, 7, 11234–11241. (53) Li, Y.; Hu, Y.; Zhao, Y.; Shi, G. Q.; Deng, L. E.; Hou, Y. B.; Qu, L. T. An Electrochemical Avenue to Green-Luminescent Graphene Quantum Dots as Potential Electron-Acceptors for Photovoltaics. Adv. Mater. 2011, 23, 776–780. (54) Liu, J.; Liu, Y.; Liu, N. Y.; Han, Y. Z.; Zhang, X.; Huang, H.; Lifshitz, Y.; Lee, S. T.; Zhong, J.; Kang, Z. H. Metal-Free Efficient Photocatalyst for Stable Visible Water Splitting via a Two-Electron Pathway. Science 2015, 347, 970–974. (55) Li, H.; Guo, S.; Li, C.; Huang, H.; Liu, Y.; Kang, Z. Tuning Laccase Catalytic Activity with Phosphate Functionalized Carbon Dots by Visible Light. ACS Appl Mater Interfaces 28


Page 28 of 30

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2015, 7, 10004–10012. (56) Meziani, M. J.; Dong, X. L.; Zhu, L.; Jones, L. P.; LeCroy, G. E.; Yang, F.; Wang, S. Y.; Wang, P.; Zhao, Y. P.; Yang, L. J.; Tripp, R. A.; Sun, Y. P. Visible-Light-Activated Bactericidal Functions of Carbon "Quantum" Dots. ACS Appl. Mater. Interfaces 2016, 8, 10761–10766. (57) Ge, J. C,; Lan, M. H.; Liu, W. M.; Jia, Q. Y.; Guo, L.; Zhou, B. J.; Meng, X. M.; Niu, G. L.; Wang, P. F. Graphene Quantum Dots as Efficient, Metal-free, Visible-Light-Active Photocatalysts. Sci. China Mater. 2016, 59, 12–19 (58) Bao, L.; Liu, C.; Zhang, Z. L.; Pang, D. W. Photoluminescence-Tunable Carbon Nanodots: Surface-State Energy-Gap Tuning. Adv. Mater. 2015, 27, 1663–1667. (59) Nie, H.; Li, M. J.; Li, Q. S.; Liang, S. J.; Tan, Y. Y.; Sheng, L.; Shi, W.; Zhang, S. X. A. Carbon Dots with Continuously Tunable Full-Color Emission and Their Application in Ratiometric pH Sensing. Chem. Mater. 2014, 26, 3104–3112. (60) Zhang, X. D.; Chen, X. K.; Kai, S. Q.; Wang, H. Y.; Yang, J. J.; Wu, F. G.; Chen, Z. Highly Sensitive and Selective Detection of Dopamine Using One-Pot Synthesized Highly Photoluminescent Silicon Nanoparticles. Anal. Chem. 2015, 87, 3360–3365. (61) Wu, F. G.; Zhang, X. D.; Kai, S. Q.; Zhang, M. Y.; Wang, H. Y.; Myers, J. N.; Weng, Y. X.; Liu, P. D.; Gu, N.; Chen, Z. One-Step Synthesis of Superbright Water-Soluble Silicon Nanoparticles with Photoluminescence Quantum Yield Exceeding 80%. Adv. Mater. Interfaces 2015, 2, 1500360. (62) Lee, J.; Jung, S. W.; Cho, A. E. Molecular Insights into the Adsorption Mechanism of Human beta-Defensin-3 on Bacterial Membranes. Langmuir 2016, 32, 1782–1790. (63) Lien, E. J.; Hansch, C.; Anderson, S. M. Structure-Activity Correlations for Antibacterial Agents on Gram-Positive and Gram-Negative Cells. J. Med. Chem. 1968, 11, 430–441. (64) Hayden, S. C.; Zhao, G. X.; Saha, K.; Phillips, R. L.; Li, X. N.; Miranda, O. R.; Rotello, V. M.; El-Sayed, M. A.; Schmidt-Krey, I.; Bunz, U. H. Aggregation and Interaction of Cationic Nanoparticles on Bacterial Surfaces. J. Am. Chem. Soc. 2012, 134, 6920–6923.

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