Selective Labeling and Growth Inhibition of Pseudomonasaeruginosa by Aminoguanidine Carbon DotsGil Otis,† Sagarika Bhattacharya,† Orit Malka,† Sofiya Kolusheva,‡ Priyanka Bolel,§ Angel Porgador,§

and Raz Jelinek*,†,‡

†Department of Chemistry, ‡Ilse Katz Institute for Nanotechnology, and §The Shraga Segal Department of Microbiology,Immunology and Genetics, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel

*S Supporting Information

ABSTRACT: Pseudomonas aeruginosa is a highly virulentbacterium, particularly associated with the spread of multidrugresistance. Here we show that carbon dots (C-dots),synthesized from aminoguanidine and citric acid precursors,can selectively stain and inhibit the growth of P. aeruginosastrains. The aminoguanidine-C-dots were shown both totarget P. aeruginosa bacterial cells and also to inhibit biofilmformation by the bacteria. Mechanistic analysis points tointeractions between aminoguanidine residues on the C-dots’surface and P. aeruginosa lipopolysaccharide moieties as thelikely determinants for both antibacterial and labelingactivities. Indeed, the application of biomimetic membraneassays reveals that LPS-promoted insertion and bilayer permeation constitute the primary factors in the anti-P. aeruginosa effectof the aminoguanidine-C-dots. The aminoguanidine C-dots are easy to prepare in large quantities and are inexpensive andbiocompatible and thus may be employed as a useful vehicle for selective staining and antibacterial activity against P. aeruginosa.KEYWORDS: carbon dots, Pseudomonas aeruginosa, antibacterial materials, bacterial labeling, aminoguanidine

Pseudomonas aeruginosa is a prominent pathogen responsiblefor varied nosocomial infections. This bacterial species isassociated with pneumonia, urinary tract infections, andsurgical wound infections.1,2 P. aeruginosa infections areparticularly hard to treat because the bacteria are capable ofdeveloping sophisticated resistance mechanisms againstcommon antibiotics,1−3 and in many instances, resistance iseven acquired during treatment.1−4 P. aeruginosa, for example,exhibits significant resistance rates for fluoroquinolones,ciprofloxacin, and levofloxacin (first-line antibiotics), rangingfrom 20 to 35%.1 Such multidrug resistance (MDR)characteristics underscore an urgent need for the developmentof alternative antibacterial substances against P. aeruginosa.Various antibacterial materials including novel antibiotics,5,6

antimicrobial peptides (AMPs),7,8 metal-based nanoparticles(NPs),9−11 and others have been employed as inhibitors andantibacterial substances against P. aeruginosa. Metallic NPs,specifically Ag NPs, have been pursued in antibacterialstrategies.9,12,13 However, Ag NPs, as well as other inorganicNPs, are toxic and in many cases induce “collateral damage” tohost cells and useful human microbiota.13,14

Carbon dots (C-dots) are a new class of carbon nanoma-terials generally exhibiting sizes of <10 nm.15−22 C-dots exhibitremarkable physicochemical properties, including tunablefluorescence, low photobleaching, and biocompatibil-ity.15,20,23,24 C-dots have been employed in diverse applica-tions, including chemical and biological sensing, optics,

catalysis, and others.25−30 Importantly, because C-dots canbe essentially synthesized from any carbonaceous precursorsthey are generally nontoxic and environmentally friendly, theyhave attracted significant interest as vehicles for biological andbiomedical applications, including bioimaging,15−17,19,30,31

drug delivery and targeting,32−34 and therapeutics.32,35,36

Recently, promising microbiological applications of C-dotshave been reported. C-dots, for example, have been utilized asfluorescent probes for bacterial detection and labeling,31,37

bacterial biofilm staining,38 and bacterial viability testing.39

The synthesis pathways mostly pursued in such systems havefocused on conjugating as-prepared C-dots with bacterialtargeting agents.40,41 These strategies have shown certainlimitations, however, such as specifically complex synthesispathways and often insufficient selectivity. C-dots directedagainst Gram-positive bacteria have been reported in whichbacterial targeting was accomplished through electrostaticinteraction between the anionic bacterial membrane andcationic residues on the C-dots’ surface.40,41 Another studyhas shown similar electrostatically based antibacterial activityagainst Porphyromonas gingivalis, an anerobic Gram-negativepathogen related to periodontitis.20

Here, we demonstrate construction, for the first time, of C-dots that are selective toward P. aeruginosa. The biocompatible

Received: October 14, 2018Published: December 27, 2018

Article

pubs.acs.org/journal/aidcbcCite This: ACS Infect. Dis. XXXX, XXX, XXX−XXX

© XXXX American Chemical Society A DOI: 10.1021/acsinfecdis.8b00270ACS Infect. Dis. XXXX, XXX, XXX−XXX

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C-dots were prepared through a simple one-pot synthesisscheme from a mixture of aminoguanidine (AG) and citric acidas the carbon source.25 We show that the AG units constitutethe primary targeting vehicle of the C-dots toward the P.aeruginosa bacterial cells. Notably, an important property of C-dots, which is exploited here, is the retaining of structural andfunctional characteristics of the C-dot precursors (such as AGemployed here) on the C-dots’ surfaces.16,18,39,42 Indeed, theexperiments presented here demonstrate that AG moietiesdisplayed on the C-dots’ surface play a major role in targetingthe C-dots to P. aeruginosa cells, affecting cell staining, bacterialgrowth inhibition, and bacterial biofilm formation disruption.Mechanistic analysis reveals that the AG/CA-C-dots’ selectiv-ity toward P. aeruginosa is likely due to specific interactionsbetween the AG residues and LPS moieties displayed on the P.aeruginosa cell surface.

■ EXPERIMENTAL SECTION

Materials. Amino guanidine hydrochloride, citric acid, PBStablets (pH 7.4), 10,12-tricosadiynoic acid, lipopolysaccharide(LPS) from P. aeruginosa, lipopolysaccharide (LPS) from E.coli, osmium tetroxide, glutaraldehyde, and ethanol absolutewere purchased from Sigma-Aldrich (St. Louis, MO, USA).Hexamethyldisilazane (HMDS) and poly-L-lysine-coated glasscoverslips were purchased from Electron Microscopy Sciences(Hatfield, PA, USA). LB broth Lennox (pH range 6.8−7.2)and BHI medium (pH 7.4) were purchased from BD Difco;LB agar was purchased from Pronadisa (Spain), quininehemisulfate monohydrate 99% was purchased from Alfa Aesar;methanol, ethanol, sulfuric acid 98%, and chloroform werepurchased from Bio-Lab Ltd. (Jerusalem, Israel); and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoylsn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG)were purchased from Avanti Polar Lipids.Carbon Dot Synthesis and Characterization. Carbon

dots were prepared using different weight ratios of theaminoguanidine (AG) and citric acid (CA) precursors.Specifically, AG/CA (2:1) C-dots were prepared by using 50mg of AG/25 mg of CA in the hydrothermal synthesis, AG/CA (1:1) C-dots were prepared by using 50 mg of AG and 50mg of CA, and AG/CA (1:2) C-dots were prepared by using25 mg of AG/50 mg of CA. In all synthesis schemes,aminoguanidine hydrochloride 98% was mixed with citric acidand dissolved in 200 μL of distilled water. The solution wastightly sealed using a Teflon film and then heated in an oven to150 °C for 2 h. After the reaction was completed, the resultantmixture was allowed to cool to room temperature, yielding abrown precipitate indicating the formation of carbon dots.Then the precipitate was redispersed in 2 mL of methanolthrough sonication for 2 min and centrifuged at 10 000 rpm for10 min to remove high-weight precipitate and agglomeratedparticles. After this, methanol was evaporated under reducedpressure to obtain an orange solid. This was dissolved indistilled water and dialyzed (MWCO 2000) against distilledwater for 24 h, and after complete purification, the clear,orange carbon dot solution was sampled for further character-ization and use.The quantum yield (QY) of the AG/CA-C-dots in deionized

water was determined by placing the C-dots inside a quartzcuvette and measuring the integrated photoluminescenceintensity (in the range of 380−650 nm) upon excitation at350 nm. The absorbance values of the C-dots at 350 nm were

also measured with respect to a standard solution of quininesulfate dissolved in 0.2 N H2SO4 (Φ = 58).Finally, the quantum yield of each C-dot solution was

calculated using eq 1

ikjjjjj

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ikjjjjj

y{zzzzz

i

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{zzzzzz

PlAbs

AbsPlsm st

sm

sm

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Φ is the quantum yield, Pl is the integrated photo-luminescence, Abs is the absorbance at λext, η is the refractiveindex of the solvent, st is the quinine sulfate standard, and smis the C-dot sample.The following techniques were applied to C-dot character-

ization:

Fluorescence spectroscopy. AG/CA-C-dot solutions indeionized water were placed inside quartz cuvettes, andthe fluorescence emission spectra were recorded on anFL920 spectrofluorimeter (Edinburgh Instruments,U.K.).High-resolution transmission electron microscopy (HR-TEM). The AG/CA-C-dot solution was placed on agraphene-coated copper grid, and HR-TEM images wererecorded on a 200 kV JEOL JEM-2100F microscope(Tokyo, Japan). The sample was dried overnight beforethe measurement.X-ray photoelectron spectroscopy (XPS). The AG/CA-C-dot solution was placed on a silicon wafer and driedovernight. Once dried, the samples were measured usingan ESCALAB 250 ultrahigh vacuum (1 × 10−9 bar)-typeX-ray photoelectron spectrometer fitted with an Al KαX-ray source and a monochromator. The beam diameterwas 500 μm with a pass energy (PE) of 150 eV forrecording survey spectra, while for high-energy-reso-lution spectra the recorded pass energy (PE) was 20 eV.The AVANTAGE program was used to process the XPSresults.

Bacterial Growth. Two Gram-positive bacterial strains{Staphylococcus aureus (S. aureus) provided by Dr. MarisaManzano (The National Institute of Health, Italy) and Bacilluscereus (B. cereus)} and four additional Gram-negative bacterialstrains {Escherichia coli K12 (E. coli K12), Escherichia coliDIHO B (E. coli DIHO B), Salmonella enteritidis (S.enteritidis),and Salmonella typhimurium strain ATCC14028 (S.typhimurium)} were used in this work, provided by thelaboratory of A. Kushmaro (BGU). Two P. aeruginosa PAO-1wild type species were kindly provided by the laboratory of M.Meijler (BGU), denoted as P. aeruginosa PAO 1 A, and P.aeruginosa PAO 1 B was provided by A. Kushmaro.B. cereus was grown in a brain−heart infusion (BHI)

medium containing 7.7 g of calf brains (infusion from 200 g),9.8 g of beef heart (infusion from 250 g), 10 g of proteasepeptone, 2 g of dextrose, 5 g of sodium chloride, and 2.5 g ofdisodium phosphate per 1 L of medium. Some of the bacteriawere grown in 30 °C for 12 h, and some of the bacterial specieswere grown in Lennox medium containing 10 g of tryptone, 5g of yeast extract, and 5 g of sodium chloride per 1 L ofmedium. The bacteria were grown for 12 h at 37 °C.

Antibacterial Activity of the C-dots. The antibacterialactivity of AG/CA-C-dots was evaluated using a broth dilutionassay in which the bacteria were initially grown in mediumovernight until full growth was achieved (OD600 = 1), followedby dilution of the bacteria to OD600 = 0.05 (CFU = 4 × 107)

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incubation with AG/CA-C-dots at different concentrations (1,0.5, 0.25, 0.125, and 0.0625 mg/mL). Growth curvesdescribing the change in OD 600 with time were collectedduring 24 h of incubation at 37 °C. The growth curves weremeasured using 96-well plates on a Biotek Synergy H1 platereader (Biotek, Winooski, VT, USA), and bacterial viabilitywas determined after 18 h of growth.Minimal Inhibitory Concentration (MIC) of Bacterial

Growth. The MIC values were determined using a brothdilution method. Briefly, all bacterial cells were grown underoptimum growth conditions with increasing concentrations ofC-dots in the medium. OD600 values were recorded after 24 hof incubation, and the C-dot concentrations in which there isno bacterial growth observed by the unaided eye weredetermined.Scanning Electron Microscopy (SEM). Bacterial sol-

utions at OD600 = 0.05 were incubated together with AG/CA-C-dots for 12 h at 37 °C, following which the bacterial pelletwas collected and washed several times with PBS buffer (0.01M phosphate buffer, 0.0027 M potassium chloride, 0.137 Msodium chloride, pH 7.4). Subsequently, the bacteria wereresuspended in PBS buffer and fixed for the SEM experiments.Bacterial strains were fixed on a poly-L-lysine cover glass,initially using glutaraldehyde 2.5% solution in buffer for 2 h,and then incubated with osmium tetroxide 1% solution,followed by dehydration by rinsing with ethanol/HMDSmixtures. The fixed bacteria were spray coated with a thin goldlayer and placed in the microscope for measurements. SEMimages were recorded using a JSM-7400 SEM (JEOL LTD,Tokyo, Japan).Biofilm Analysis. P. aeruginosa PAO 1 A biofilm was grown

in a clear-glass-bottomed 96-well plate, typically throughplacing 300 μL of the medium in each well, followed by theaddition of 5 μL of bacterial solutions at OD600 = 1 (CFU = 8× 108). The resulting solution was grown for 24 h at 37 °C toyield P. aeruginosa biofilms. The biofilm was washed severaltimes with PBS buffer and imaged by confocal microscopy. In asimilar manner, a P. aeruginosa biofilm was grown in a mediumcontaining 1 mg/mL AG/CA-C-dots. The P. aeruginosabiofilm was imaged using a Plan-Apochromat 20x/0.8 M27confocal laser scanning microscope (CLSM; Zeiss LSM880,Germany). Image processing to obtain 3-D images and toevaluate the biofilm total volume was obtained using IMARISsoftware (Bitplane, Zurich, Switzerland).Bacterial Cell Labeling. Bacterial cell labeling was

conducted by initially growing all bacteria until full growth(OD600 = 1; CFU = 8 × 108), followed by centrifugation of thebacterial solution and separation of the supernatant from thebacterial pellet. The bacterial pellet was washed several timeswith PBS buffer and then resuspended in a solution of 1 mg/mL amino guanidine C-dots dissolved in PBS buffer. Theresulting solution was incubated for 3 h at 37 °C, followed bywashing of the bacterial pellet in PBS buffer in order to discardC-dots that were not attached to the bacteria. After the finalwashing, the C-dot-labeled bacterial pellets were resuspendedin PBS buffer and imaged with a Plan-Apochromat 20x/0.8M27 confocal laser scanning microscope.Cell Culture and Cytotoxicity of AG/CA-C-dots. HeLa,

the human cervical adenocarcinoma cell line, and HEK293T,the human embryonic kidney cell line, were grown in DMEMsupplanted with 10% FBS and maintained at 37 °C, 95%relative humidity, and 5% CO2 in a CO2 incubator understerile conditions. In a 96-well U-bottomed microtiter plate,

2.5 × 105 cells in 1 mL of media/well (n = 3 for each sample)were seeded and left in the CO2 incubator for 2 h until thecells became recumbent. Cells are incubated for 18 and 48 hseparately with increasing titrated concentrations of AG/CA-C-dots (0, 25, 50, 100, 150, and 200 μg/mL).

Measurement of Cell Death by Flow Cytometry. Celldeath was measured in a Gallios flow cytometer from BeckmanCoulter Inc., with side-scattered light (SSC) analysis combinedwith 7-AAD uptake recorded in a multiwell plate using anexcitation 488 nm/emission 695 nm channel on a logarithmicscale. The flow cytometry experiments utilized 2.5 × 105 cellsboth for the HeLa and HEK293T cell lines that were incubatedwith varying titrated concentrations of AG/CA-C-dots (0, 25,50, 100, 150, and 200 μg/mL). After 18 and 48 h of incubationseparately, all cells were collected for each sample to check forcell death using flow cytometry. Cells were washed withPBS1X, treated with 7-AAD, and incubated on ice for 30 minin the dark for the assessment of apoptotic cells. A flowcytometry measurement was recorded in a density plot of theintensities of side-scattered light (SSC) that is proportional tocell granularity or internal complexity vs 7-AAD, which has ahigh DNA binding constant and is useful for dead celldiscrimination during flow cytometry while being efficientlyexcluded by intact cells.43 In an SSC vs 7-AAD dot plot, 1 ×104 events were recorded,44 the data were analyzed withKaluza Analysis version 2.1 software from Beckman CoulterInc., and the percentage of dead cells was plotted.

Zeta Potential. C-dot solutions in PBS buffer were placedinside a Malvern DTS 1070 disposable capillary cuvette, andthe zeta potential was measured with a Zetasizer Nano ZS(Malvern; Worcestershire).

Elemental Analysis. Powders of AG/CA-C-dots wereanalyzed for carbon, hydrogen, nitrogen, and sulfur (CHNS)contents. Data were collected using an OEA 2000 ThermoScientific Flash Smart elemental analyzer.

Lipid/Polydiacetylene Vesicle Assay. Two types ofvesicles were prepared: DMPG/DMPC/PDA in a 1:1:3 moleratio and LPS/DMPC/PDA in a 0.2:2:3 mole ratio. Thevesicles were prepared according to established protocols.46

Briefly, lipid stocks were dissolved in chloroform/ethanol 1:1,mixed together in the appropriate ratios, and dried in vacuo ata constant pressure of 40 mbar for 6 h until a dry powder wasobtained. (P. aeruginosa LPS was prepared in doubly distilledwater and added to the lipid mixture after drying.) Next, 2 mLof deionized water was added to the lipids to a final lipidconcentration of 1 mM. The solution was then probe-sonicated at 70 °C for 3 min to produce vesicular particles,and the vesicles were cooled at room temperature for fewhours and placed in a 4 °C environment overnight. Thevesicles were then polymerized using UV irradiation at 254 nm(energy flow = 0.3 J/s) for 10−20 s, with the resultingsolutions exhibiting an intense blue appearance. For thepreparation of the LPS/DMPC/PDA vesicles in which the LPSsource was E. coli, the same experimental steps were carriedout, with the difference that the E. coli LPS stock was dissolvedin chloroform/ethanol 1:1 and mixed with the phospholipidsand diacetylene monomers prior to drying.The fluorescence response assay was used to evaluate the

interaction of C-dots with the PDA-containing vesicles, usingexcitation at 485 nm, yielding emission at 555 nm.Accordingly, the percentage fluorescent chromatic response(FCR%) in 555 nm was calculated using eq 2:45−49

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FCR%Em Em

Em Em100%sample blue

red blue=

−−

×(2)

Data were collected on a Fluoroskan Ascent Thermo Scientificfluorescence microplate reader.

■ RESULTS AND DISCUSSIONCarbon Dot Synthesis and Characterization. Figure 1

presents the simple synthesis scheme and characterization of

the aminoguanidine C-dots (AG/CA-C-dots). As shown inFigure 1A, the AG-dots were prepared via hydrothermaltreatment of aminoguanidine and citric acid at a 2:1 mol ratio(150 °C, 2 h). Importantly, the chemical features of themolecular building blocks (in the case here, AG and citric acid)are retained on the C-dot surface (Figure 1A). The AG/CA-C-dots exhibited the typical excitation-dependent emissionspectra (Figure 1B) with the maximal emission (uponexcitation at 390 nm) at 480 nm (i.e., green-yellowappearance). A representative high-resolution transmittingelectron microscopy (HR-TEM) image of the AG/CA-C-dots in Figure 1C underscores the crystalline nature of the C-dots’ carbon core, showing a lattice spacing of 0.32 nmcorresponding to graphitic carbon.53−55 The size distributionof the AG/CA-C-dots, determined by the HR-TEM analysis,

was relatively narrow (4.3 ± 0.5 nm, Figure S1). The quantumyield of the AG/CA-C-dots was approximately 3%.X-ray photoelectron spectroscopy (XPS) data in Figure 1D

illuminate the functional units on the AG/CA-C-dots’ surface,confirming the retention of the aminoguanidine and carboxylicacid residues. Specifically, the C 1s spectrum in Figure 1Dfeatures deconvoluted peaks at 284, 285.2, 286.5, and 288.5 eVthat correspond to CC, C−N, C−O/CN, and CObonds, respectively. The N 1s XPS peak in Figure 1D similarlydisplays deconvoluted signals at 399.7 and 400.7 eV ascribedto the C−N and CN bonds, respectively. The XPS resultsare further corroborated by Fourier transform infrared (FTIR)spectroscopy, showing peaks in areas attributed to C−N, CN, CO, and O−H bonds (Figure S1B). The absorbancespectrum of the AG/CA-C-dots was also measured, displayingpeaks corresponding to the carbonyl residues on the C-dots(Figure S1C). The crystallinity of the carbon dots was alsoestimated by X-ray diffraction (XRD), and the d spacing of thecarbon core was found to be 0.366 nm, corresponding to the(002) plane of graphite (Figure S1D).

Antibacterial Effects of AG/CA-C-dots. Figure 2illustrates the selective antibacterial properties of the AG/CA-C-dots. In the experiments summarized in Figure 2, AG/CA-C-dots were added to the bacterial growth medium, andbacterial proliferation was monitored. The concentration-dependent bactericidal effects of the AG/CA-C-dots againstP. aeruginosa PAO 1 A are depicted in Figure 2A,demonstrating that the proliferation of P. aeruginosa PAO 1A was inhibited with increasing C-dot concentration. Indeed,no bacterial growth was apparent at a C-dot concentration of0.5 mg/mL (Figure 2A). A concentration-dependent anti-bacterial effect against P. aeruginosa PAO 1 A was also apparentin the agar dilution method, in which the AG/CA-C-dots wereincorporated within the agar matrix (Figure S3). Growthcurves of all other bacterial strains are presented in Figures S4and S5.The bar diagram in Figure 2B demonstrates that the

antibacterial effect of AG/CA-C-dots is selective toward P.aeruginosa. Indeed, Figure 2B demonstrates that the AG/CA-C-dots had a significant inhibitory effect on P. aeruginosa PAO1 A and P. aeruginosa PAO1 B, while the C-dots appeared toexhibit no bactericidal effects in the cases of Salmonellaenteritidis, Staphylococcus aureus, E. coli K12, and Bacillus cereus(Figure 2B). A small antibacterial effect at a high C-dotconcentration was apparent in the case of the E. coli DHIO Bstrain. Table 1 summarizes the minimum concentrationsinhibiting cell growth (MIC), further demonstrating thebactericidal selectivity of the AG/CA-C-dots toward P.aeruginosa bacterial strains.Figure 3 presents scanning electron microscopy (SEM)

images showing the effect of the AG/CA-C-dots upon differentbacterial cells. Notably, Figure 3 reveals that significantmorphology alteration occurred when P. aeruginosa PAO 1 Acells were incubated with the AG/CA-C-dots, specifically, the“flattening” and elimination of cell surface smoothness (Figure3A). This observation likely indicates the leakage of intra-cellular fluid due to bacterial cell wall permeability induced bythe AG/CA-C-dots, similar to membrane perforation inducedby other bactericidal compounds.7,49,50 In contrast to thesevere morphology transformation of the P. aeruginosa PAO 1A cells following incubation with the AG/CA-C-dots, the SEMimages in Figure 3B show no discernible effect of the AG/CA-C-dots upon S. aureus cells, consistent with the data in Figure

Figure 1. Aminoguanidine carbon dot (AG/CA-C-dots) synthesisand characterization. (A) Synthesis scheme showing the one-pothydrothermal route generating AG/CA-C-dots from a mixture of AGand citric acid. (B) Excitation-dependent fluorescence emissionspectra of the AG/CA-C-dots; different excitation wavelengths areindicated. (C) High-resolution transmission electron microscopy(HR-TEM) image of AG/CA-C-dots. (D) Deconvoluted C 1s and N1s X-ray photoelectron spectra (XPS) of the AG/CA-C-dots; thefunctional units are indicated.

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2B and Table 1 pointing to no antibacterial activity of the AG/CA-C-dots against Gram-positive bacteria.

We further investigated the impact of the AG/CA-C-dotsupon biofilm formation (Figure 4). Bacterial biofilms arelargely impermeable saccharide/proteinaceous matrixes enclos-ing bacterial cells, constituting a prominent pathogenicfactor.51−53 The microscopy images in Figure 4 show asignificant decrease in biofilm abundance when P. aeruginosaPAO 1 A bacteria were incubated with the AG/CA-C-dots for24 h. Biofilm volume determination, carried out by the analysisof the three-dimension images, revealed an almost 50%decrease when AG/CA-C-dots were added to the bacterialgrowth medium (Figure 4B).

Fluorescence Staining of P. aeruginosa with Amino-guanidine Carbon Dots.While the experiments presented inFigures 2−4 highlight the selective bactericidal effects of theAG/CA-C-dots against P. aeruginosa, we further examined thefeasibility of bacterial cell labeling by the fluorescent C-dots(Figure 5). Indeed, the fluorescence confocal microscopyimages in Figure 5 demonstrate that the AG/CA-C-dots can beemployed for the selective staining of P. aeruginosa. Toaccomplish bacterial cell staining, the bacterial suspensionswere grown to saturation (OD600 = 1), pelleted, and incubatedwith the AG/CA-C-dots (1 mg/mL) for 3 h. The fluorescencemicroscopy images (excitation = 405 nm) dramaticallydemonstrate that P. aeruginosa cells were labeled by the C-dots, while no fluorescence labeling was apparent in the case ofS. aureus. The fluorescence microscopy results in Figure 5corroborate the selectivity profile of the AG/CA-C-dots,confirming the specific targeting of P. aeruginosa. Furthermore,Figure 5 demonstrates that bacterial staining (rather thanbacterial inhibition) can be attained through tailoring theexperimental parameters (C-dot concentration, point of C-dotaddition to the bacterial suspension, and incubation time).

Toxicity of AG/CA-C Dots toward Mammalian Cells.To evaluate the cytotoxicity of the AG/CA-C-dots, we carriedout flow cytometry analysis utilizing HeLa and HEK293T cells(Figure 6; raw data provided in Figure S6). In the experiments,the cells were incubated for 18 and 48 h with increasingtitrated concentrations of AG/CA-C-dots, and cell death wasmeasured by flow cytometry. The percentages of dead cells,presented in Figure 6, confirm that for both cell types there isno apparent effect of the AG/CA-C-dots on cell viability, up toa high AG/CA-C-dot concentration of 200 μg/mL.

Figure 2. Antibacterial activities of the aminoguanidine-C-dots. (A) Growth curves of Pseudomonas aeruginosa PAO 1 A recorded through the brothdilution method in different concentrations of AG/CA-C-dots. (B) Bacterial viabilities of different bacterial strains as a function of AG/CA-C-dotconcentrations. Viabilities were evaluated following incubation of the bacteria with the AG/CA-C-dots for 18 h.

Table 1. MIC Values for the AG/CA-C Dots Applied to theGrowth of Different Bacteria

species gram type MIC (mg/mL)

P. aeruginosa PAO 1 A _ 0.5P. aeruginosa PAO 1 B _ 1S. enteritidis _ >1S. typhimurium _ >1E. coli K12 _ >1E. coli DIHO B _ >1B. cereus + >1S. aureus + >1

Figure 3. Effects of aminoguanidine-C-dots upon bacterial cellmorphology. Scanning electron microscopy (SEM) images ofPseudomonas aeruginosa PAO 1 A and Staphylococcus aureus incubatedwith AG/CA-C-dots (0.5 mg/mL) for 12 h. Scale bars correspond to1 μm.

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Mechanistic Analysis. While the experiments outlined inFigures 2−5 show the selective antibacterial and stainingcapabilities of the AG/CA-C-dots against P. aeruginosa, wefurther aimed to elucidate the mechanism for P. aeruginosatargeting, staining, and bactericidal activity by the AG/CA-C-dots (Figures 7 and 8). To probe the relationship between theC-dot surface properties and bactericidal activity, wesynthesized C-dots with different mass ratios between theaminoguanidine and citric acid precursors and assessed theirantibacterial activities (Figure 7). Specifically, in addition to

the AG/CA-C-dots exhibiting a 2:1 mass ratio between the AGand citric acid (CA) precursors, we also examined C-dots with1:1 and 1:2 AG/CA mass ratios. {A detailed characterization ofthe AG/CA-C-dots (1:1) and AG/CA-C-dots (1:2) isprovided in Figures S7 and S8.} Importantly, many studieshave demonstrated that functional units of the precursormolecules are retained on the surfaces of the C-dots generatedfrom those precursors.54−56 As such, the experimentspresented in Figure 7 examine the relationship between therelative abundance of aminoguanidine residues on the C-dots’surface and the bactericidal activity of those C-dots.Figure 7A presents zeta potential measurements recorded for

the C-dots prepared from the AG and CA precursors at thethree different mass ratios. The C-dots were negatively chargedbecause they display abundant carboxylic residues formedthrough the hydrothermal synthesis process.40 However,Figure 7A demonstrates that the negative charge on the C-dots’ surface, reflected by the zeta potential values, wasreciprocal to the relative concentration of AG (which exhibitspositive amine residues) in the reaction mixture. Fouriertransform infrared (FTIR) spectral analysis of the three C-dotspecies (Figure S9A) corroborates the zeta potential data,indicating that the AG abundance on the C-dots depended onthe AG/CA precursor ratio.To further assess the relationship between the AG display on

the C-dots and their antibacterial effects against P. aeruginosa,we quantitatively correlated the percentage of nitrogen (whichsolely originates from the AG precursor) and the bacterial

Figure 4. Effect of the aminoguanidine carbon dots upon P. aeruginosa biofilms. (A) Bright-field confocal microscopy images of the P. aeruginosaPAO 1 A biofilm (24 h growth) with and without incubation with AG/CA-C-dots. (B) Biofilm total volume (μm3).

Figure 5. Selective labeling of Pseudomonas aeruginosa by AG/CA-C-dots. Confocal fluorescence microscopy images of Pseudomonasaeruginosa PAO 1 A and Staphylococcus aureus by 1 mg/mL AG/CA-C-dots. Fluorescent images are presented with 405 nm excitation.

Figure 6. Cytotoxicity of AG/CA-C dots. Percentage of dead cell determined by flow cytometry using a 7-AAD viability assay for HeLa (2.5 × 105cells/mL) and HEK293T (2.5 × 105 cells/mL) cells with increasing titrated concentrations of AG/CA-C-dots. (A) 18 h of incubation and (B) 48h of incubation of the cells with the AG/CA-C-dots.

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viability (Figure 7B). The results indicate a dramatic reciprocalrelationship between the percentage of nitrogen in the C-dotsand the P. aeruginosa viability: lower nitrogen concentration(i.e., lower AG display on the C-dots’ surface) yielded greaterbacterial viability and vice versa. The full data set regarding theC, N, and H atomic percentages in all of the C-dots tested ispresented in Figure S9B.The correlation between AG concentration and antibacterial

properties of the C-dots is similarly clearly manifested in the P.aeruginosa growth curves, recorded in the presence of identicalconcentrations (1 mg/mL) of the three types of C-dots, each

prepared from the two precursors at different mass ratios(Figure 7C). For example, the total inhibition of P. aeruginosaPAO 1 A growth was achieved upon addition of the C-dotsexhibiting a 2:1 mass ratio between AG and CA (Figure 7C,red curve). In comparison, C-dots prepared from a lower AGconcentration (1:1 mass ratio between AG and CA) had asmaller antibacterial effect (blue curve in Figure 7C), while C-dots synthesized from a 1:2 AG/CA mixture did not inhibit thegrowth of P. aeruginosa PAO 1 A (Figure 7C, green curve).Crucially, the P. aeruginosa PAO 1 A growth analysis in

Figure 7C reveals that the addition of aminoguanidine alone

Figure 7. Surface properties and antibacterial activities of C-dots prepared from the aminoguanidine (AG) and citric acid (CA) precursors atdifferent mass ratios. (A) Zeta potential of the AG/CA-C-dots measured in PBS buffer at pH 7.4. (B) Weight percentage of nitrogen in the AG/CA-C-dots determined by elemental analysis in relation to the bacterial cell viability of P. aeruginosa PAO 1 A after 18 h of growth at 37 °C togetherwith the AG/CA-C-dots. (C) Growth curves of Pseudomonas aeruginosa PAO 1 A in the presence of the AG/CA-C-dots prepared at different massratios. The C-dot concentration was 1 mg/mL.

Figure 8. Interactions of AG/CA-C-dots with different bilayer compositions. Fluorescence emissions induced by AG/CA-C-dots in lipid/PDAvesicles exhibiting different compositions. Black: DMPG/DMPC/PDA vesicles. Red: LPS/DMPC/PDA vesicles. (A) Kinetic analysis. (The C-dotconcentration was 0.071 mg/mL.) (B) Fluorescence emission recorded after 30 min via different concentrations of the AG/CA-C-dots. (C)Comparison of the fluorescent chromatic responses of lipid/PDA vesicles containing LPS extracted from P. aeruginosa vs lipid/PDA vesiclescontaining LPS extracted from E. coli.

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did not have a discernible antibacterial effect (Figure 7C, light-purple curve). This important observation indicates that thedisplay of the AG units on the C-dots’ surface makes a criticalcontribution to the antibacterial properties of the particles. Theenhanced bactericidal functionality of the C-dot-displayed AGmay be ascribed to both the presentation of a large number ofthe AG units on the C-dots as well as the surfaceimmobilization of biologically active AG conformations.Previous studies have similarly demonstrated enhancedactivities of therapeutic molecules immobilized on the surfacesof nanoparticles.40,41,57

While the data in Figure 7 confirm the correlation betweenthe bactericidal properties and the display of aminoguanidineresidues on the C-dots’ surfaces, we further elucidated thelikely molecular targets of the AG/CA-C-dots on the bacterialcell surfaces (Figure 8). Figure 8 presents fluorescenceemissions of lipid/polydiacetylene (PDA) vesicles followingthe addition of AG/CA-C-dots. Lipid/PDA vesicles constitutea useful biomimetic membrane model which has been widelyemployed for studying bilayer interactions of membrane-activemolecules.45,46,48,58−60 In general, lipid/PDA vesicles mimicbilayer membranes, and the conjugated PDA units constitutesensitive fluorescent reporters for the binding of soluble speciesonto the bilayer surface.45,46,48

In the experiments depicted in Figure 8A,B, we recorded theAG/CA-C-dot-induced fluorescence emitted by lipid/PDAvesicles comprising two distinct compositions: DMPG/DMPC/PDA vesicles (1:1:3 mol ratio) designed to mimicthe outer membranes of Gram-positive bacteria that arerelatively rich in phosphatidylglycerol (PG)61−63 and P.aeruginosa-extracted lipopolysaccharide (LPS)/DMPC/PDAvesicles (0.2:2:3 mol ratio) mimicking the outer membranesof P. aeruginosa.64−67 Importantly, LPS molecules are notpresent in the membranes of Gram-positive bacteria, which areenclosed by a thick peptidoglycan layer.62,63

The data in Figure 8A,B shows significant differencesbetween the fluorescence response of the LPS/DMPC/PDAvesicles induced upon addition of AG/CA-C-dots compared toDMPG/DMPC/PDA vesicles. Specifically, the kinetic analysisin Figure 8A demonstrates that the fluorescence emitted byDMPG/DMPC/PDA vesicles on AG/CA-C-dots additionincreased much more rapidly and to a higher fluorescencevalue than for the LPS/DMPC/PDA vesicles. Previous studieshave linked enhanced fluorescence emission in lipid/PDAvesicles to bilayer surface interactions of membrane-activemolecules.69−71 Accordingly, the results in Figure 8A areindicative of more pronounced surface binding of the AG/CA-C-dots in the DMPG/DMPC/PDA vesicles, while deeperbilayer penetration of the AG/CA-C-dots likely occurred in thecase of the LPS/DMPC/PDA vesicles.45,70

We also measured the fluorescence response following theaddition of the AG/CA-C-dots to lipid/PDA vesiclescomprising LPS extracted from E. coli compared to lipid/PDA vesicles containing LPS derived from P. aeruginosa(Figure 8C). The chromatic response curves of the twovesicles upon addition of the AG/CA-C-dots (at the sameconcentration) were significantly different; a much steepercurve (more pronounced color/fluorescence transformation)was observed in the case of the vesicles displaying E. coli LPS,reflecting more pronounced surface localization of the AG/CA-C-dots. Accordingly, the PDA vesicle assay results inFigure 8C further illuminate the probable mechanismaccounting for the AG-C-dot selectivity: the LPS moieties

displayed on the P. aeruginosa cell surface promote theinsertion of the AG-C-dots into the bacterial membranebilayer, which permeate the cell membrane, thereby exhibitingbactericidal action (akin to the effect of pore-formingantimicrobial peptides). This model may also explain therelatively high MIC values (on the milligram range) presentedin Table 1 because sufficiently high concentrations ofmembrane-internalized C-dots are likely required to achievea perforation threshold. In the case of E. coli (or other Gram-negative bacteria), the PDA vesicle chromatic data in Figure8C points to the preferred accumulation of the AG/CA-C-dotsat the membrane surface; this translates to lesser bilayerinsertion, which explains the significantly lower bactericidaleffect.The role of LPS in promoting the insertion of the AG/CA-

C-dots into the biomimetic membrane bilayer is furtherhighlighted in the bar diagram in Figure 8B, depicting thefluorescence responses of the two types of lipid/PDA vesicles,recorded 30 min after the addition of different concentrationsof the AG/CA-C-dots. Figure 8B demonstrates that at all AG/CA-C-dots concentrations, the fluorescence signals emitted bythe LPS/DMPC/PDA vesicles were significantly lower thanthe corresponding values recorded for DMPG/DMPC/PDAvesicles. These results confirm that P. aeruginosa LPS induceddeeper penetration of the AG/CA-C-dots into the lipid bilayer.Indeed, membrane insertion and permeation are keyantibacterial determinants in varied compounds and nano-particles.10,12,49,72

Figure 9 outlines a mechanism for the cell staining andbactericidal activity of the AG/CA-C-dots toward P.

aeruginosa, based upon the data presented in Figures 2−8.The aminoguanidine units on the C-dots’ surface hone ontothe LPS residues displayed on the outer membrane of P.aeruginosa bacterial cells, likely through electrostatic attraction.Indeed, guanidinium thiocyanate has been reported to targetLPS.57,73 The LPS-bound AG/CA-C-dots penetrate the

Figure 9. Schematic model for P. aeruginosa targeting by theaminoguanidine-C dots. AG/CA-C-dots attach to the LPS unitswithin the bacterial membrane, consequently penetrating anddisrupting the bilayer.

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bacterial cell membrane, resulting in bilayer permeability andcell death. This interpretation is consistent with the SEM datain Figure 3, showing pronounced leaching of the intracellularcontents upon incubation of P. aeruginosa cells with the AG/CA-C-dots. Importantly, while LPS is a universal membranemarker of Gram-negative bacteria, P. aeruginosa (as well asother Gram-negative bacterial species) exhibits distinctorganization and structural features of LPS,65,67,74 likelyaccounting for the significant selectivity of the AG/CA-C-dots observed here. The small difference in inhibitory activityof the AG/CA-C-dots toward the two P. aeruginosa strains (i.e.,Figure 3B) likely reflects the known genetic and phenotypicdivergence of P. aeruginosa PAO-1.68 Selective binding to theLPS moieties of P. aeruginosa may also account for thefluorescence labeling properties of the AG/CA-C-dots (Figure5) because LPS-mediated attachment to the P. aeruginosa cellsurface facilitates bacterial staining.

■ CONCLUSIONSNew carbon dots were synthesized from aminoguanidine andcitric acid molecular precursors, exhibiting remarkable labelingand antibacterial activities toward P. aeruginosa. Dependingupon the experimental conditions, we demonstrated bothspecific staining of P. aeruginosa bacterial cells and theinhibition of P. aeruginosa growth and biofilm formation.Mechanistic analysis reveals that P. aeruginosa targeting by theAG-displaying C-dots was likely due to interactions betweenthe AG units and LPS residues on the P. aeruginosa cell surface.The aminoguanidine-C-dots are biocompatible and easilyprepared from inexpensive reagents and could be employedas a useful diagnostic platform and effective weapon against P.aeruginosa.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsinfec-dis.8b00270.

Size distribution; FTIR, UV−vis, and XRD spectra; andcytotoxicity of AG/CA-C-dots (2:1). UV−vis andfluorescence spectra and FTIR and AFM images ofAG/CA-C-dots (1:1) and AG/CA-C-dots (1:2). Agardilution of AG/CA-C-dots (2:1) and growth curves ofStaphylococcus aureus, Bacillus cereus, Escherichia coli K12,Escherichia coli DIHO B, Salmonella enteritidis, Salmonellatyphimurium strain ATCC14028, and Pseudomonasaeruginosa PAO 1 B in the presence of AG/CA-C-dots(2:1). (PDF)

■ AUTHOR INFORMATIONCorresponding Author*E-mail: razj@bgu.ac.il.ORCIDSagarika Bhattacharya: 0000-0003-2641-0246Raz Jelinek: 0000-0002-0336-1384NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors are grateful to Nicholas Zerby and Dr. Rina Jegerfor help with SEM imaging.

■ ABBREVIATIONS

AG/CA-C dots, aminoguanidine carbon dots; CFU, colony-forming unit; LPS, lipopolysaccharide; MIC, minimuminhibitory concentration; MDR, multidrug resistance.

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