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Selective labeling and growth inhibition of Pseudomonas aeruginosa by aminoguanidine carbon dots gil otis, Sagarika Bhattacharya, orit malka, Sofiya Kolusheva, priyanka bolel, Angel Porgador, and Raz Jelinek ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00270 • Publication Date (Web): 27 Dec 2018 Downloaded from http://pubs.acs.org on January 2, 2019

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ACS Infectious Diseases

Selective labeling and growth inhibition of Pseudomonas aeruginosa by aminoguanidine carbon dots Gil Otis a, Sagarika Bhattacharya a, Orit Malka a, Sofiya Kolusheva b, Priyanka Bolel c, Angel Porgador c and Raz Jelinek a,b* a Department of Chemistry, Ben Gurion University of the Negev, Beer Sheva 84105, Israel b Ilse Katz Institute for Nanotechnology, Ben Gurion University of the Negev, Beer Sheva 84105, Israel c The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, BenGurion University of the Negev, Beer Sheva 84105, Israel * [email protected]

Pseudomonas aeruginosa is highly virulent bacteria, particularly associated with the spread of multi-drug resistance.

Here we show that carbon dots (C-dots), synthesized from

aminoguanidine and citric acid precursors, can selectively stain and inhibit the growth of Pseudomonas aeruginosa strains.

The aminoguanidine-C-dots were shown to both target

Pseudomonas aeruginosa bacterial cells, and also inhibit biofilm formation by the bacteria. Mechanistic analysis points to interactions between aminoguanidine residues upon the C-dots' surface and Pseudomonas aeruginosa lipopolysaccharide moieties as the likely determinants for both antibacterial and labeling activities. Indeed, application of biomimetic membrane assays reveals that LPS-promoted insertion and bilayer permeation constitute the primary factors for the anti-Pseudomonas aeruginosa effect of the aminoguanidine-C-dots. The aminoguanidine C-dots

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are easy to prepare in large quantities, are inexpensive, biocompatible, thus may be employed as a useful vehicle for selective staining and antibacterial activity against Pseudomonas aeruginosa.

Keywords: carbon dots; Pseudomonas aeruginosa; antibacterial materials; bacterial labeling; aminoguanidine

Pseudomonas aeruginosa is a prominent pathogen responsible for varied nosocomial infections. This bacterial species is associated with pneumonia, urinary tract infections, and surgical wound infections.1,2 P. aeruginosa infections are particularly hard to treat because the bacteria are capable of developing sophisticated resistance mechanisms against common antibiotics,1-3 and in many instances resistance is even acquired during treatment.1-4 P. aeruginosa, for example, exhibits significant resistance rates for fluoroquinolones, ciprofloxacin, and levofloxacin (first line antibiotics), ranging from 20 to 35%.1 Such multi-drug resistance (MDR) characteristics underscore an urgent need for development of alternative antibacterial substances against P. aeruginosa. Various antibacterial materials ranging from novel antibiotics,5,6 antimicrobial peptides (AMP’s),7,8 metallic based nanoparticles (NPs),9-11 and others have been employed as inhibitors and antibacterial substances against pseudomonas aeruginosa. Metallic NPs, specifically Ag NPs, have been pursued in antibacterial strategies.9,12,13 However, Ag NPs, as well as other inorganic NPs, are toxic and in many cases induce "collateral damage" to host cells and useful human microbiota.13,14 Carbon dots (C-dots) are a new class of carbon nanomaterials, generally exhibiting sizes under 10 nm.15-22

C-dots exhibit remarkable physico-chemical properties, including tunable 2 ACS Paragon Plus Environment

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fluorescence, low photo-bleaching, and biocompatibility.15,20,23,24 C-dots have been employed in diverse applications, including chemical and biological sensing, optics, catalysis, and others.25-30 Importantly, because C-dots can be essentially synthesized from any carbonaceous precursors they are generally non-toxic and environmentally-friendly; as such C-dots have attracted significant interest as vehicles for biological and biomedical 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-dots have been reported. C-dots, for example, have been utilized as fluorescent probes for bacterial detection and labelling,31,37 bacterial biofilm staining,38 and bacterial viability testing.39 The synthetic pathways mostly pursued in such systems have focused of conjugating as-prepared C-dots with bacterial targeting agents.40,41 These strategies have shown certain limitations, however, specifically complex synthetic pathways and often insufficient selectivity. C-dots directed against Gram-positive bacterial have been reported, in which bacterial targeting was accomplished through electrostatic interaction between the anionic bacterial membrane and cationic residues upon the C-dots' surface.40,41 Another study has shown similar electrostatically-based antibacterial activity against Porphyromonas gingivalis, an anerobic Gram-negative pathogen related to periodontitis.20 Here, we demonstrate construction, for the first time, of C-dots that are selective towards P. aeruginosa.

The biocompatible C-dots were prepared through a simple one-pot synthesis

scheme from a mixture of aminoguanidine (AG) and citric acid as the carbon source.25 We show that the AG units constitute the primary targeting vehicle of the C-dots towards the P. aeruginosa bacterial cells. Notably, an important property of C-dots, exploited here, is the retaining of structural and functional characteristics of the C-dot precursors (such as AG employed here) upon the C-dots' surfaces.16,18,39,42

Indeed, the experiments presented here



demonstrate that AG moieties displayed upon the C-dots' surface play a major role in targeting the C-dots to P. aeruginosa cells, affecting cell staining, inhibition of bacterial growth, disruption of bacterial biofilm formation. Mechanistic analysis reveals that the AG/CA-C-dots selectivity towards P. aeruginosa is likely due to specific interactions between the AG residues and LPS moieties displayed upon P. aeruginosa cell surface.

Experimental Section Materials.

Amino guanidine hydrochloride, citric acid, PBS tablets (pH =7.4), 10,12-

tricosadiynoic acid, lipopolysaccharide (LPS) from P. aeruginosa, lipopolysaccharide (LPS) from E. coli, osmium tetroxide, glutaraldehyde and ethanol absolute were purchased from Sigma Aldrich, St. Louis, MO, USA. Hexamethyldisilazane (HMDS) and Poly-L-lysine-coated glass cover slips were purchased from Electron Microscopy Sciences, Haterfield, USA. L.B broth Lennox (pH range: 6.8-7.2), BHI medium (pH = 7.4) were purchased from BD DifcoTM, LB agar was purchased from Pronadisa (Spain), Quinine hemi sulfate monohydrate 99% was purchased from alfa aesar, methanol, ethanol, sulfuric acid 98% and chloroform were purchased from BioLab Ltd (Jerusalem, Israel), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2dimyristoylsn-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 the aminoguanidine (AG) and citric acid (CA) precursors. Specifically, AG:CA (2:1) C-dots were prepared by using 50 mg AG / 25 mg CA in the hydrothermal synthesis, AG-CA 4 ACS Paragon Plus Environment

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(1:1) C-dots were prepared by using 50 mg AG and 50 mg CA, and AG:CA (1:2) C-dots by using 25 mg AG / 50 mg CA. In all synthesis schemes aminoguanidine hydrochloride 98% was mixed with citric acid and dissolved in 200 µL of distilled water. The solution was tightly sealed using a Teflon film and then heated in an oven to 150°C for 2 hours. After the reaction was completed, the resultant mixture was allowed to cool to room temperature yielding a brown precipitate indicating the formation of carbon dots, the precipitate was re-dispersed in 2 mL methanol through sonication for 2 minutes and centrifuged at 10,000 rpm for 10 min to remove high-weight precipitate and agglomerated particles. After this, methanol was evaporated under reduced pressure to obtain an orange color solid. This was dissolved in distilled water and dialyzed (MWCO 2000) against distilled water for 24 hours, after complete purification, the clear orange carbon dots solution was taken for further characterization and use. The quantum yield (QY) of the AG/CA-C-dots in deionized water was determined by placing the C-dots inside a quartz cuvette and the integrated photoluminescence intensity (in the range of 380−650 nm) was measured upon excitation at 350 nm. The absorbance values of the C-dots at 350 nm were measured as well with respect to a standard solution of quinine sulfate dissolved in 0.2N H2SO4 (

).

Finally, quantum yield of each C-dots solution was calculated using the equation:

Equation 1: Quantum yield calculation. where

is quantum yield,

is integrated photoluminescence,

is absorbance at λext,

refractive index of the solvent, st is quinine sulfate standard and sm is C-dots sample.


is


The following techniques were applied for C-dot characterization: -

Fluorescence spectroscopy. AG/CA-C-dots solutions in deionized water were placed inside quartz cuvettes and the fluorescence emission spectra were recorded on an FL920 spectrofluorimeter (Edinburgh Instruments, UK).

-

High Resolution Transmission Electron Microscopy (HR-TEM). The AG/CA-C-dots solution was placed upon a graphene-coated copper grid and HR-TEM images were recorded on a 200 kV JEOL JEM-2100F microscope (Tokyo, Japan). The sample was dried overnight before the measurement.

-

X-ray Photoelectron Spectroscopy (XPS). The AG/CA-C-dots solution was placed upon a silicon wafer and dried overnight. Once dried, the samples were measured using an Xray photoelectron spectrometer type ESCALAB 250 ultrahigh vacuum (1 × 10−9 bar) apparatus fitted with an Al Kα X-ray source and a monochromator. The beam diameter was 500 μm with pass energy (PE) of 150 eV for recording survey spectra, while for high energy resolution spectra the recorded pass energy (PE) was 20 eV. The AVANTAGE program was used to process the XPS results.

Bacterial growth. Two Gram-positive bacterial strains - Staphylococcus aureus (S. aureus) provided by Dr. Marisa Manzano (The National Institute of Health, Italy), Bacillus cereus (B. cereus), and four additional Gram negative bacterial strains Escherichia coli K12 (E. coli K12), Escherichia coli DIHO B (E. coli DIHO B), Salmonella enteritidis (S. enteritidis) and Salmonella Typhimurium strain ATCC14028 (S. Typhimurium) were used in this work, provided by the laboratory of A. Kushmaro (BGU). Two Pseudomonas aeruginosa PAO-1 wild type species were kindly provided by the laboratory of M. Meijler (BGU), denoted Pseudomonas


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aeruginosa PAO 1 A, and the second, denoted Pseudomonas aeruginosa PAO 1 B, was provided by A. Kushmaro. B. cereus was grown in a brain heart infusion (BHI) medium containing 7.7 gr calf brains (infusion from 200 gr), 9.8 beef heart (infusion from 250 gr), 10 gr protease peptone, 2 gr dextrose, 5 gr sodium chloride and 2.5 gr disodium phosphate per 1 liter medium, the bacteria was grown in 300C for 12 hours, all the rest of the bacterial species were grown in Lennox medium containing 10 gr Tryptone, 5 gr yeast extract and 5 gr sodium chloride per 1 liter medium. The bacteria were grown for 12 hours at 370C.

Antibacterial activity of the C-dots. The antibacterial activity of AG/CA-C-dots was evaluated using a broth dilution assay in which the bacteria were initially grown in medium overnight until full growth was achieved (O. D600 =1) followed by dilution of the bacteria to O.D600 = 0.05 (CFU =

and were incubated with AG/CA-C-dots in different concentrations (1, 0.5, 0.25,

0.125, 0.0625 mg/mL), growth curves describing the change in O.D 600 with time were collected for 24 hours of incubation at 370C. the growth curves were measured in 96-well plates on a Biotek Synergy H1 plate reader (Biotek, Winooski, VT, USA).

Bacterial viability was

determined after 18-hour growth.

Minimal Inhibitory Concentration (MIC) of bacterial growth. The MIC values were determined using a broth dilution method. Briefly, all bacterial cells were grown in optimum growth conditions with increasing concentrations of C-dots in the medium. O.D600 values were recorded after 24 hours of incubation, and the C-dot concentrations in which there is no bacterial growth observed by the unaided eye were determined. 7 ACS Paragon Plus Environment


Scanning electron microscopy (SEM). Bacterial solutions at O.D600 = 0.05 were incubated together with AG/CA-C-dots for 12 hours at 370C, following which the bacterial pellet was collected and washed several times with PBS buffer (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride, pH = 7.4). Subsequently, the bacteria were resuspended in PBS buffer and fixated for the SEM experiments. Bacterial strains were fixed upon a Poly-L-lysine cover glass, initially using glutaraldehyde 2.5% solution in buffer for 2 hours, then incubated with osmium tetroxide 1% solution, followed by dehydration by rinsing with ethanol/HMDS mixtures. The fixed bacteria were spray-coated with a thin gold layer and placed in the microscope for measurements. SEM images 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 bottom 96 well plate, typically through placing 300 µL of the medium in each well followed by addition of 5 µL bacterial solutions at O.D600 = 1 (CFU =

. The resulting solution was grown for 24

hours in 370C to yield P. aeruginosa biofilms. The biofilm was washed several times by PBS buffer and imaged by confocal microscopy. In a similar manner, P. aeruginosa biofilm was grown in a medium containing 1 mg/mL of AG/CA-C-dots. The P. aeruginosa biofilm was imaged using confocal laser scanning microscopy (CLSM) Plan-Apochromat 20x/0.8 M27, Zeiss LSM880, Germany. Image processing to obtain 3-D images and to evaluate biofilm total volume was obtained using the IMARIS software (Bitplane, Zurich, Switzerland).


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Bacterial Cell Labelling. Bacterial cell labeling was conducted by initially growing all bacteria until full growth (O.D600 = 1; CFU =

, followed by centrifugation of the bacterial

solution and separation of the supernatant from the bacterial pellet. The bacterial pellet was washed several times by PBS buffer and then re-suspended in a solution of 1 mg/mL amino guanidine C-dots dissolved in PBS buffer. The resulting solution was incubated for 3 hours in 370C followed by washing of the bacterial pellet in PBS buffer in order to discard C-dots that were not attached to the bacteria. After final washing the C-dot-labelled bacterial pellets were re-suspended in PBS buffer and imaged by confocal laser scanning microscopy (CLSM) PlanApochromat 20x/0.8 M27, Zeiss LSM880, Germany.

Cell Culture and cytotoxicity of AG/CA-C-dots. HeLa, human cervical adenocarcinoma cell line and HEK293T, human embryonic kidney cell lines were grown in DMEM supplanted with 10% FBS and maintained at 370C, 95% relative humidity, and 5% CO2 in a CO2 incubator at sterile conditions. In a 96-well U-bottom microtiter plate, 2.5 × 105 cells in 1ml of media/well (n = 3 for each sample) were seeded and left in the CO2 incubator for 2 h until the cells became recumbent. Cells are incubated for 18h and 48h separately 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. Cell death was measured in a Gallios flow cytometer from Beckman Coulter Inc., side scattered light (SSC) analysis combined with 7-AAD uptake recorded in a multiwall plate using an excitation 488 nm/emission 695 nm channel, on a logarithmic scale. The flow cytometry experiments utilized 2.5 × 105 cells both for HeLa and HEK293T cell lines that were incubated with varying titrated concentrations of AG/CA-C-dots (0, 25, 50, 100, 150 and 200 µg/ml). After 18 and 48 h incubation separately, all cells were 9 ACS Paragon Plus Environment


collected for each sample to check for cell death using flow cytometry. Cells were washed with PBS1X, treated with 7-AAD, and incubated on ice for 30 min in the dark for assessment of apoptotic cells. Flow cytometry measurement was recorded in a density plot of the intensities of side scattered light (SSC) that is proportional to cell granularity or internal complexity, vs 7AAD, which has a high DNA binding constant and is useful for dead cell discrimination during flow cytometry, while efficiently excluded by intact cells.43 In a SSC vs 7-AAD dot plot, 1 × 104 events were recorded,44 the data were analyzed in Kaluza Analysis version 2.1 software from Beckman Coulter Inc and the percentage of dead cells were plotted.

Zeta potential. C-dot solutions in PBS buffer were placed inside of a Malvern DTS 1070 disposable capillary cuvette and the zeta potential was measured by Zetasizer Nano ZS, Malvern, Worcestershire.

Elemental analysis. Powders of AG/CA-C-dots were analyzed for carbon, hydrogen, nitrogen, and sulfur (CHNS) contents. Data were collected using Thermo Scientific Flash Smart elemental analyzer OEA 2000.

Lipid/polydiacetylene vesicle assay. Two types of vesicles were prepared: DMPG/DMPC/PDA at a 1:1:3 mole ratio and LPS/DMPC/PDA at a 0.2:2:3 ratio. The vesicles 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 in a constant pressure of 40 millibars for 6 hours until a dry powder was obtained (P. aeruginosa LPS was prepared in double distilled water and added to the lipid mixture after drying). Next, 2 mL of


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deionized water was added to the lipids to a final lipid concentration of 1 mM. The solution was then probe-sonicated at 700C for 3 minutes to produce vesicular particles; the vesicles were then cooled at room temperature for few hours and placed in 40C overnight. The vesicles were then polymerized using UV irradiation at 254 nm (energy flow = 0.3 j/sec) for 10-20 sec, with the resulting solutions exhibiting an intense blue appearance.

For the preparation of the

LPS/DMPC/PDA vesicles in which the LPS source was E. coli the same experimental steps were carried out, with the difference that the E. coli LPS stock was dissolved in chloroform/ethanol 1:1 and mixed with the phospholipids and diacetylene monomers prior to drying. The fluorescence response assay was used to evaluate the interaction of C-dots with the PDAcontaining vesicles, using excitation at 485 nm, yielding emission at 555 nm. Accordingly, the percentage fluorescent chromatic response (FCR%) in 555 nm was calculated using the equation:45-49

Equation 2: Fluorescent chromatic response calculation. Data were collected on a fluorescence microplate reader - Fluoroskan Ascent™ Thermo Scientific.

Results and Discussion 1. Carbon dot synthesis and characterization Figure 1 presents the simple synthesis scheme and characterization of the aminoguanidine C-dots (AG/CA-C-dots).

As shown in Figure 1A, the AG-dots were prepared via hydrothermal 11 ACS Paragon Plus Environment


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treatment of aminoguanidine and citric acid at a 2:1 mole ratio (150 0C, 2 hours). Importantly, the chemical features of the molecular building blocks (in the case here AG and citric acid) are retained upon the C-dot surface (Figure 1A).

The AG/CA-C-dots exhibited the typical

excitation-dependent emission spectra (Figure 1B) with the maximal emission (upon excitation at 390 nm) at 480 nm (i.e. green-yellow appearance).

A representative high-resolution

transmitting electron microscopy (HR-TEM) image of the AG/CA-C-dots in Figure 1C underscores the crystalline nature of the C-dots' carbon core, showing the lattice spacing of 0.32 nm corresponding to graphitic carbon.53-55 Size distribution of the AG/CA-C-dots, determined by the HR-TEM analysis, was relatively narrow (4.3 +/- 0.5 nm, Figure S1). The quantum yield of the AG/CA-C-dots was approximately 3%.


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Figure 2: Aminoguanidine carbon dot (AG/CA-C-dots) synthesis and characterization. A. Synthetic scheme, showing the one-pot hydrothermal route generating AG/CA-C-dots from a mixture of AG and citric acid. B. Excitation-dependent fluorescence emission spectra of the AG/CA-C-dots; different excitation wavelengths are indicated. C. High resolution transmission electron microscopy (HR-TEM) image of AG/CA-C-dots. D. Deconvoluted C 1s and N 1s x-ray photoelectron spectra (XPS) of the AG/CA-C-dots; the functional units are indicated.

X ray photoelectron spectroscopy (XPS) data in Figure 1D illuminate the functional units upon the AG/CA-C-dots' surface, confirming retention of the aminoguanidine and carboxylic acid residues. Specifically, the C 1s spectrum in Figure 1D features deconvoluted peaks at 284 eV, 285.2 eV, 286.5 eV, and 288.5 eV which correspond to C=C, C-N, C-O/C=N, and C=O bonds, respectively. The N 1s XPS peak in Figure 1D similarly displays deconvoluted signals at 399.7 eV and 400.7 eV ascribed to the C-N and C=N bonds, respectably. The XPS results are further corroborated by Fourier transform infrared (FTIR) spectroscopy, showing peaks in areas attributed to C-N, C=N, C=O and O-H bonds (Figure S1,B). The absorbance spectrum of the AG/CA-C-dots was also measured, displaying peaks corresponding to the carbonyl residues upon the C-dots (Figure S1,C), the crystallinity of the carbon dots was also estimated by X-ray diffraction (XRD) and the d spacing of the carbon core was found to be 0.366 nm, corresponding to the (002) plane of graphite (Figure S1,D).

2. Antibacterial effects of AG/CA-C-dots. Figure 2 illustrates the selective antibacterial properties of the AG/CA-C-dots. In the experiments summarized in Figure 2, AG/CA-C-dots 13 ACS Paragon Plus Environment


were added to the bacterial growth medium and bacterial proliferation was monitored. The concentration-dependent bactericidal effects of the AG/CA-C-dots against P. aeruginosa PAO 1 A are depicted in Figure 2A, demonstrating that proliferation of P. aeruginosa PAO 1 A was inhibited upon increasing C-dot concentration. Indeed, no bacterial growth was apparent at a Cdot concentration of 0.5 mg/mL (Figure 2A). Concentration-dependent anti-bacterial effect against P. aeruginosa PAO 1 A was also apparent in the agar dilution method, in which the AG/CA-C-dots were incorporated within the agar matrix (Figure S3). Growth curves of all other bacterial strains are presented in Figure S4 and Figure S5.

Figure 2.

Antibacterial activities of the aminoguanidine-C-dots. A. Growth curves of

Pseudomonas aeruginosa PAO 1 A recorded through the broth dilution method in different concentrations of AG/CA-C-dots; B. bacterial viabilities of different bacterial strains as a function of AG/CA-C-dot concentrations. Viabilities were evaluated following incubation of the bacteria with the AG/CA-C-dots for 18 hours.


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The bar diagram in Figure 2B demonstrates that the antibacterial effect of AG/CA-C-dots is selective towards P. aeruginosa. Indeed, Figure 2B demonstrates that the AG/CA-C-dots had significant inhibitory effect upon P. aeruginosa PAO 1 A and P. aeruginosa PAO1 B, while the C-dots appeared to exhibit no bactericidal effects in case of Salmonella enteritidis, Staphylococcus aureus, E. coli K12 or Bacillus cereus (Figure 2B). A small antibacterial effect at a high C-dot concentration was apparent in case of the E. coli DHIO B strain. Table 1 summarizes the minimum concentrations inhibiting cell growth (MIC), further demonstrating the bactericidal selectivity of the AG/CA-C-dots towards P. aeruginosa bacterial strains.

Species

Gram type

MIC (mg/mL)

P. aeruginosa PAO 1 A

_

0.5

P. aeruginosa PAO 1 B

_

1

S. enteritidis

_

>1

S. typhimurium

_

>1

E. coli K12

_

>1

E. coli DIHO B

_

>1

B. cereus

+

>1

S. aureus

+

>1

Table 1: MIC values for the AG/CA-C-dots applied to growth of different bacteria.



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Figure 3 presents scanning electron microscopy (SEM) images showing the effect of the AG/CA-C-dots upon different bacterial cells. Notably, Figure 3 reveals that significant morphology alteration occurred when P. aeruginosa PAO 1 A cells were incubated with the AG/CA-C-dots, specifically "flattening" and elimination of cell surface smoothness (Figure 3A). This observation likely indicates leakage of intracellular fluid due to bacterial cell wall permeability induced by the AG/CA-C-dots, similar to membrane perforation induced by other bactericidal compounds.7,49,50 In contrast to the severe morphology transformation of the P. aeruginosa PAO 1 A cells following incubation with the AG/CA-C-dots, the SEM images in Figure 3B show no discernable effect of the AG/CA-C-dots upon S. aureus cells, consistent with the data in Figure 2B and Table 1 pointing to no antibacterial activity of the AG/CA-C-dots against Gram-positive bacteria.

Figure 3: Effects of aminoguanidine-C-dots upon bacterial cell morphology.

Scanning

electron microscopy (SEM) images of Pseudomonas aeruginosa PAO 1 A and Staphylococcus aureus incubated with AG/CA-C-dots (0.5 mg/mL) for 12 hours. Scale bars correspond to 1 µm.


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We further investigated the impact of the AG/CA-C-dots upon biofilm formation (Figure 4). Bacterial biofilms are largely-impermeable saccharide/proteinaceous matrixes enclosing bacterial cells, constituting a prominent pathogenic factor.51-53 The microscopy images in Figure 4 show a significant decrease in biofilm abundance when P. aeruginosa PAO 1 A bacteria were incubated with the AG/CA-C-dots for 24 hours. Biofilm volume determination, carried out by analysis of the three-dimension images, revealed an almost 50% decrease when AG/CA-C-dots were added to the bacterial growth medium (Figure 4B).

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

3. Fluorescence staining of P. aeruginosa with aminoguanidine carbon dots. While the experiments presented in Figures 2-4 highlight the selective bactericidal effects of the AG/CA-C-dots against P. aeruginosa, we further examined the feasibility of bacterial cell labeling by the fluorescent C-dots (Figure 5). Indeed, the fluorescence confocal microscopy 17 ACS Paragon Plus Environment


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images in Figure 5 demonstrate that the AG/CA-C-dots can be employed for selective staining of P. aeruginosa. To accomplish bacterial cell staining, the bacterial suspensions were grown to saturation (OD600 =1), pelleted, and incubated with the AG/CA-C-dots (1 mg/mL) for 3 hours. The fluorescence microscopy images (excitation = 405 nm) dramatically demonstrate that P. aeruginosa cells were labeled by the C-dots, while no fluorescence labeling was apparent in case of S. aureus. The fluorescence microscopy results in Figure 5 corroborate the selectivity profile of the AG/CA-C-dots, confirming specific targeting of P. aeruginosa. Furthermore, Figure 5 demonstrates that bacterial staining (rather than bacterial inhibition) can be attained through tailoring the experimental parameters (C-dot concentration, point of C-dot addition to the bacterial suspension, and incubation time).

Figure 5: Selective labelling of Pseudomonas aeruginosa by AG/CA-C-dots.

Confocal

fluorescence microscopy images of Pseudomonas aeruginosa PAO 1 A and Staphylococcus aureus by AG/CA-C-dots 1 mg/mL, fluorescent images are presented with 405 nm excitation.


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4. Toxicity of AG/CA-C-dots towards mammalian cells. To evaluate the cytotoxicity of the AG/CA-C-dots we carried out flow cytometry analysis utilizing HeLa and HEK293T cells (Figure 6; raw data provided in Figure S6).

In the

experiments, the cells were incubated for 18h and 48h, respectively, with increasing titrated concentrations of AG/CA-C-dots, and cell death was measured by flow cytometry. The percentages of dead cells, presented in figure 6, confirm that for both cell types there is no apparent effect of the AG/CA-C-dots on cell viability, up to a high AG/CA-C-dot concentration of 200 µg/mL.

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 × 105 cells/ml) and HEK293T (2.5 × 105 cells/ml) cells with increasing titrated concentrations of AG/CA-C-dots. A. 18 hours incubation and B. 48 hours incubation of the cells with the AG/CA-C-dots.



5. Mechanistic analysis While the experiments outlined in Figures 2-5 show the selective antibacterial and staining capabilities of the AG/CA-C-dots against P. aeruginosa, we further aimed to elucidate the mechanism for P. aeruginosa targeting, staining, and bactericidal activity by the AG/CA-C-dots (Figures 7-8). To probe the relationship between C-dot surface properties and bactericidal activity we synthesized C-dots with different mass ratios between the aminoguanidine and citric acid precursors and assessed their antibacterial activities (Figure 7). Specifically, in addition to the AG/CA-C-dots exhibiting 2:1 mass ratio between the AG and citric acid (CA) precursors, we also examined C-dots with 1:1 and 1:2 AG:CA mass ratios [detailed characterization of the AG/CA-C-dots (1:1) and AG/CA-C-dots (1:2) is provided in Figures S7 and S8]. Importantly, many studies have demonstrated that functional units of the precursor molecules are retained upon the surfaces of the C-dots generated from those precursors.54-56 As such, the experiments presented in Figure 7 examine the relationship between the relative abundance of aminoguanidine residues upon 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 the three different mass ratios. The C-dots were negatively charged since they display abundant carboxylic residues formed through the hydrothermal synthesis process.40 However, Figure 7A demonstrates that the negative charge upon the C-dots' surface, reflected by the zeta-potential values, was reciprocal to the relative concentration of AG (which exhibits positive amine residues) in the reaction mixture. Fourier transform infrared (FTIR) spectral analysis of the three C-dot species (Figure S9, A) corroborates the zeta potential data, indicating that the AG abundance upon the C-dots depended upon the AG:CA precursor ratio.


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Figure 7: Surface properties and antibacterial activities of C-dots prepared from the aminoguanidine (AG) and citric acid (CA) precursors at different mass ratios. A. Zeta potential of the AG/CA-C-dots measured in PBS buffer pH = 7.4. B. Weight percentage of nitrogen in the AG/CA-C-dots determined by elemental analysis in relation to bacterial cell viability of P. aeruginosa PAO 1 A after 18 hours growth at 370C together with the AG/CA-Cdots. C. Growth curves of Pseudomonas aeruginosa PAO 1 A in the presence of the AG/CA-Cdots prepared at different mass ratios. C-dot concentrations were 1 mg/mL.

To further assess the relationship between AG display upon the C-dots and their antibacterial effects against P. aeruginosa, we quantitatively correlated the percentage of nitrogen (which solely originates from the AG precursor) and bacterial viability (Figure 7B). The results indicate a dramatic reciprocal relationship between the percentage of nitrogen in the C-dots and P. aeruginosa viability: lower nitrogen concentration (i.e. lower AG display upon the C-dots' surface) yielded greater bacterial viability and vice versa. The full data regarding the C,N,H atomic percentage in all the C-dots tested is presented in figure S9,B.



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 identical concentrations (1 mg/mL) of the three types of C-dots, each prepared from the two precursors at different mass ratios (Figure 7C). For example, total inhibition of P. aeruginosa PAO 1 A growth was achieved upon addition of the C-dots exhibiting 2:1 mass ratio between AG and CA (Figure 7C, red curve). In comparison, C-dots prepared from a lower AG concentration (1:1 mass ratio between AG and CA) had a lesser antibacterial effect (blue curve in Figure 7C), while C-dots synthesized from a 1:2 AG:CA mixture did not inhibit the growth of P. aeruginosa PAO 1 A (Figure 7C, green curve). Crucially, the P. aeruginosa PAO 1 A growth analysis in Figure 7C reveals that addition of aminoguanidine alone did not have a discernable antibacterial effect (Figure 7C, light purple curve). This important observation indicates that display of the AG units upon the C-dots' surface has a critical contribution to the antibacterial properties of the particles. The enhanced bactericidal functionality of the C-dot-displayed AG may be ascribed to both presentation of large number of the AG units upon the C-dots, as well as surface immobilization of biologicallyactive AG conformations. Previous studies have similarly demonstrated enhanced activities of therapeutic molecules immobilized upon surfaces of nanoparticles.40,41,57 While the data in Figure 7 confirm the correlation between the bactericidal properties and the display of aminoguanidine residues upon the C-dots' surfaces, we further elucidated the likely molecular targets of the AG/CA-C-dots upon the bacterial cell surfaces (Figure 8). Figure 8 presents fluorescence emissions of lipid/polydiacetylene (PDA) vesicles following addition of AG/CA-C-dots. Lipid/PDA vesicles constitute a useful biomimetic membrane model which has been widely employed for studying bilayer interactions of membrane-active molecules.45,46,48,58-60 22 ACS Paragon Plus Environment

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In general, lipid/PDA vesicles mimic bilayer membranes, and the conjugated PDA units constitute sensitive fluorescent reporters for binding of soluble species onto the bilayer surface.45,46,48 In the experiments depicted in Figure 8A-B we recorded the AG/CA-C-dots-induced fluorescence

emitted

by lipid/PDA

vesicles

comprising

two

distinct

compositions:

DMPG/DMPC/PDA vesicles (1:1:3 mole ratio) designed to mimic the outer membranes of Gram-positive bacteria that are relatively rich in phosphatidylglycerol (PG),61-63 and P. aeruginosa-extracted lipopolysaccharide (LPS)/DMPC/PDA vesicles (0.2:2:3 mole ratio), mimicking the outer membranes of P. aeruginosa.64-67 Importantly, LPS molecules are not present in the membranes of Gram-positive bacteria, which are enclosed by a thick peptidoglycan layer.62,63



Figure 8: Interactions of AG/CA-C-dots with different bilayer compositions. Fluorescence emissions induced by AG/CA-C-dots in lipid/PDA vesicles exhibiting different compositions. Black: DMPG/DMPC/PDA vesicles, red: LPS/DMPC/PDA vesicles. A. Kinetic analysis (Cdot concentration was 0.071 mg/mL); B. fluorescence emission recorded after 30 minutes by 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 vesicles containing LPS extracted E. coli.

The data in Figure 8A-B show significant differences between the fluorescence response of the LPS/DMPC/PDA vesicles induced upon addition of AG/CA-C-dots, compared to DMPG/DMPC/PDA vesicles. Specifically, the kinetic analysis in Figure 8A demonstrates that the fluorescence emitted by DMPG/DMPC/PDA vesicles upon AG/CA-C-dots addition increased much more rapidly, and to a higher fluorescence value, compared to the LPS/DMPC/PDA vesicles. Previous studies have linked enhanced fluorescence emission in lipid/PDA vesicles to bilayer surface interactions of membrane-active molecules.69-71 Accordingly, the results in Figure 8A are indicative of more pronounced surface binding of the AG/CA-C-dots in the DMPG/DMPC/PDA vesicles, while deeper bilayer penetration of the AG/CA-C-dots likely occurred in case of the LPS/DMPC/PDA vesicles.45,70 We also measured the fluorescence response following addition of the AG/CA-C-dots to lipid/PDA vesicles comprising LPS extracted from E. coli compared to lipid/PDA vesicles containing LPS derived from P. aeruginosa (Figure 8C). The chromatic response curves of the two vesicles upon addition of the AG/CA-C-dots (at the same concentration) were significantly different – a much steeper curve (more pronounced color/fluorescence transformation) was 24 ACS Paragon Plus Environment

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observed in 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 in Figure 8C further illuminate the probable mechanism accounting for the AG-C-dot selectivity: the LPS moieties displayed upon the P. aeruginosa cell surface promote insertion of the AG-C-dots into the bacterial membrane bilayer, which permeate the cell membrane thereby exhibiting bactericidal action (akin to the effect of pore forming antimicrobial peptides). This model may also explain the relatively high MIC values (in the milligram range) presented in Table 1, since sufficiently high concentrations of membrane-internalized C-dots are likely required to achieve a "perforation threshold". In the case of E. coli (or other Gram-negative bacteria), the PDA vesicle chromatic data in Figure 8C point to preferred accumulation of the AG/CA-C-dots at the membrane surface; this translates into lesser bilayer insertion which explains the significantly lower bactericidal effect. The role of LPS in promoting insertion of the AG/CA-C-dots into the biomimetic membrane bilayer is further highlighted in the bar diagram in Figure 8B, depicting the fluorescence responses of the two types of lipid/PDA vesicles, recorded 30 minutes after addition of different concentrations of the AG/CA-C-dots. Figure 8B demonstrates that in all AG/CA-Cdots concentrations, the fluorescence signals emitted by the LPS/DMPC/PDA vesicles were significantly lower than the corresponding values recorded for DMPG/DMPC/PDA vesicles. These results confirm that P. aeruginosa LPS induced deeper penetration of the AG/CA-C-dots into the lipid bilayer.

Indeed, membrane insertion and permeation are key antibacterial

determinants in varied compounds and nanoparticles.10,12,49,72 Figure 9 outlines a mechanism for the cell staining and bactericidal activity of the AG/CA-C-dots towards P. aeruginosa, based upon the data presented in Figures 2-8. The 25 ACS Paragon Plus Environment


aminoguanidine units upon the C-dots' surface hone onto the LPS residues displayed upon the outer membrane of P. aeruginosa bacterial cells, likely through electrostatic attraction. Indeed, guanidinium thiocyanate has been reported to target LPS.57,73 The LPS-bound AG/CA-C-dots penetrate into the bacterial cell membrane, resulting in bilayer permeability and cell death. This interpretation is consistent with the SEM data in Figure 3, showing pronounced leaching of the intracellular contents upon incubation of P. aeruginosa cells with the AG/CA-C-dots. Importantly, while LPS is a universal membrane marker of Gram-negative bacteria, P. aeruginosa (as well as other Gram-negative bacterial species) exhibits distinct organization and structural features of LPS,65,67,74 likely accounting to the significant selectivity of the AG/CA-Cdots observed here. The small difference in inhibitory activity of the AG/CA-C-dots towards the two P. aeruginosa strains (i.e. Figure 3B) likely reflects the known genetic and phenotypic divergence of P. aeruginosa PAO-1.68 Selective binding to the LPS moieties of P. aeruginosa may also account for the fluorescence labeling properties of the AG/CA-C-dots (Figure 5), as LPS-mediated attachment onto the P. aeruginosa cell surface facilitates bacterial staining.


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Figure 9. Schematic model for P. aeruginosa targeting by the aminoguanidine-C-dots. AG/CA-C-dots attach onto the LPS units within the bacterial membrane, consequently penetrating and disrupting the bilayer.

Conclusions New carbon dots were synthesized from aminoguanidine and citric acid molecular precursors, exhibiting remarkable labeling and antibacterial activities towards P. aeruginosa. Depending upon the experimental conditions, we demonstrated both specific staining of P. aeruginosa bacterial cells, and inhibition of P. aeruginosa growth and biofilm formation. Mechanistic analysis reveals that P. aeruginosa targeting by the AG-displaying C-dots was likely due to interactions between the AG units and LPS residues upon the P. aeruginosa cell surface. The aminoguanidine-C-dots are biocompatible, easily prepared from inexpensive reagents, and could be employed as a useful diagnostic platform and effective weapon against P. aeruginosa.

Abbreviations AG/CA-C-dots, aminoguanidine carbon dots; CFU, colony forming unit; LPS, lipopolysaccharide; MIC, minimum inhibitory concentration; MDR, Multi-drug resistance.

Acknowledgments The authors are grateful to Nicholas Zerby and Dr. Rina Jeger for help with SEM imaging.

Supporting information Size distribution, FTIR, UV-VIS, XRD spectra and cytotoxicity of AG/CA-C-dots (2:1), UV-VIS, Fluorescence spectra, FTIR and AFM images of AG/CA-C-dots (1:1) and AG/CA-C-dots (1:2), Agar dilution of AG/CA-C-dots (2:1) and growth curves of Staphylococcus aureus, Bacillus cereus, Escherichia coli K12, Escherichia coli DIHO B, 27 ACS Paragon Plus Environment


Salmonella enteritidis, Salmonella Typhimurium strain ATCC14028 and Pseudomonas aeruginosa PAO 1 B in the presence of AG/CA-C-dots (2:1).

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