SYNTHESIS OF ANTIMICROBIAL CARBON DOTS AND USES THEREOF

Abstract

The present invention relates to antibacterial carbon dots, having aminoguanidine functionality on their outermost surface. The invention further relates to the synthesis of said antibacterial carbon dots, and to the uses of these carbon dots for inhibiting a biofilm formation in the presence of said carbon dots and as fluorescent labeling markers for specific bacteria.

Claims

1. Antimicrobial carbon-dots (C-dots) wherein at least one aminoguanidine functional group on their outer surface, wherein said C-dots are characterized in having a maximal emission at a wavelength of about 480 nm upon excitation at a wavelength of 390 nm, and wherein said antimicrobial C-dots inhibit the growth of P. aeruginosa.

2. The antimicrobial C-dots according to claim 1, wherein the size of said C-dots is between 3.5 and 5 nm in diameter.

3. The antimicrobial C-dots according to 1, wherein said C-dots are characterized by having an XPS spectrum as demonstrated in FIG. 1D.

4. The antimicrobial C-dots according to 1, wherein said C-dots are characterized by having quantum yield of about 3% in deionized water.

5. The antimicrobial C-dots according to 1, wherein said C-dots are characterized by having a zeta potential value higher than about −16 mV in PBS buffer at pH=7.4.

6. The antimicrobial C-dots according to claim 5, wherein said C-dots have a zeta potential between −1 to −15 mV in PBS buffer at pH=7.4.

7. The antimicrobial C-dots according to claim 6, wherein said C-dots have a zeta potential of about −2 mV in PBS buffer at pH=7.4.

8. A method for inhibiting the formation of a biofilm and/or disrupting the propagation of a biofilm comprising contacting antimicrobial C-dots having at least one aminoguanidine functional group on their outer surface with biofilm forming bacterial cells.

9. A method for selective bacterial cell labeling, comprising the steps of A) contacting bacterial cells sought to be labeled with fluorescent C-dots having at least one aminoguanidine functional group on their outer surface at a concentration range of between about 0.1 mg/ml to about 1 mg/ml; and B) removing excess C-dots; thereby providing precise cell labeling.

10. The method according to claim 8, wherein the bacterial cells are P. aeruginosa cells.

11. The method according to claim 8, wherein the C-dots are characterized by having a zeta potential value higher than about −16 mV in PBS buffer at pH=7.4.

12. The method according to claim 11, wherein said C-dots have a zeta potential between −1 to −15 mV in PBS buffer at pH=7.4.

13. The antimicrobial C-dots according to claim 6, wherein said C-dots have a zeta potential of about −2 mV in PBS buffer at pH=7.4.

14. The method of claim 9, wherein the concentration of the bacteria in step A is ranging between about O.D.sub.600 0.1 to about 1.

15. A method for preparing antimicrobial C-dots wherein at least one aminoguanidine functional group on their outer surface, wherein said method comprising the step of reacting a carbonaceous molecule with aminoguanidine precursor in aqueous media under heating, further wherein the mass ratio between aminoguanidine precursor and said carbonaceous molecule is between about 2.5:1 to about 1.5:1.

16. The method according to claim 15, wherein said carbonaceous molecule is selected from the group consisting of carboxylic acids, sugars, amino acids, ascorbic acid and peptides.

17. The method according to claim 15, wherein said carbonaceous molecule is a carboxylic acid.

18. The method according to claim 17, wherein the carbonaceous molecule is citric acid.

19. The method of claim 14, wherein the aminoguanidine precursor is aminoguanidine hydrochloride.

20. The method according to claim 18, wherein the mass ratio between aminoguanidine precursor and citric acid is about 2:1.

21. The method according to claim 15, wherein the heating occurs at a temperature of between about 120 to about 180 degrees Celsius.

22. An antimicrobial composition comprising C-dots wherein at least one aminoguanidine functional group on their outer surface, wherein said C-dots are characterized in having a maximal emission at a wavelength of about 480 nm upon excitation at a wavelength of 390 nm, and wherein said antimicrobial C-dots inhibit the growth of P. aeruginosa, or as prepared by a method according to claim 15.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1: A. Synthetic scheme, showing the one-pot hydrothermal route generating AG-C-dots from a mixture of AG and citric acid. B. Excitation-dependent fluorescence emission spectra of the AG-C-dots; different excitation wavelengths are indicated 350 nm Black, 370 nm Red, 390 nm Blue, 410 nm Green, 430 nm Pink, 450 nm Brown. C. High resolution transmission electron microscopy (HR-TEM) image of AG-C-dots. D. Deconvoluted C 1s and N 1s x-ray photoelectron spectra (XPS) of the AG-C-dots; the functional units are indicated.

[0023] FIG. 2. Antibacterial activities of the aminoguanidine-C-dots. A. Growth curves of Pseudomonas aeruginosa PAO 1 luxCDABE recorded through the broth dilution method in different concentration AG-C-dots of the invention (AG: CA is 2:1). B. bacterial viabilities of different strains as a function of AG-C-dots concentrations upon incubation for 18 hours. Color code for both A and B: (black)—control, (red)—0.0625 mg/mL, (blue)—0.125 mg/mL, (green)—0.25 mg/mL, (pink)—0.5 mg/mL C. agar dilution method representing the growth of P. aeruginosa PAO 1 luxCDABE in different AG-C-dots (2:1) concentrations. I—control, II—0.0625 mg/mL, III—0.125 mg/mL, IV—0.25 mg/mL, V—0.5 mg/mL.

[0024] FIG. 3: Effects of aminoguanidine-C-dots upon bacterial cell morphology. Scanning electron microscopy (SEM) images of Pseudomonas aeruginosa PAO 1 luxCDABE and Staphylococcus aureus incubated with AG-C-dots (0.5 mg/mL) for 12 hours. Scale bars correspond to 1 μm.

[0025] FIG. 4: Effect of the aminoguanidine carbon dots on P. aeruginosa biofilms. A. Brightfield confocal microscopy images of P. aeruginosa PAO 1 luxCDABE biofilm (24 hours growth) with and without incubation with AG-C-dots. B. Biofilm total volume (μm.sup.3).

[0026] FIG. 5: Selective labelling of Pseudomonas aeruginosa by AG-C-dots. Confocal fluorescence microscopy images of Pseudomonas aeruginosa PAO 1 luxCDABE and Staphylococcus aureus by AG-C-dots 1 mg/mL, fluorescent images are presented with 405 nm excitation.

[0027] FIG. 6: Surface properties and antibacterial activities of C-dots prepared from the aminoguanidine (AG) and citric acid (CA) precursors at different mass ratios. A. Weight percentage of nitrogen in the AG-C-dots determined by elemental analysis in relation to bacterial cell viability of P. aeruginosa PAO 1 luxCDABE after 18 hours growth at 37° C. together with the AG-C-dots. Red squares represent bacterial viability and black dots represent % nitrogen B. Growth curves of Pseudomonas aeruginosa PAO 1 luxCDABE in the presence of the AG-C-dots prepared at different mass ratios. Black: control, Red: AG:CA 2:1, Blue: AG:CA 1:1, Green: AG:CA 1:2, Pink: free aminoguanidine C. Growth curves of Pseudomonas aeruginosa PAO 1 luxCDABE in the presence of the AG-C-dots prepared at 4:1 mass ratios (Red) vs control (Black). C-dot concentrations were 1 mg/mL.

[0028] FIG. 7: Surface charges of the C-dots as measured in zeta potential measurements. Zeta potential of different C-dots having varied AG:CA ratios measured in PBS buffer pH=7.4.

EXAMPLES

Example 1—Synthesis of Antibacterial C-Dots

[0029] Antimicrobial carbon dots were prepared using aminoguanidine (AG) and citric acid (CA) precursors. Specifically, AG:CA (2:1) C-dots were prepared by hydrothermal synthesis: 50 mg aminoguanidine hydrochloride 98% was mixed with 25 mg 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. The solid obtained was 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.

[0030] Other C-dots were obtained utilizing an identical synthetic procedure, utilizing different weight ratios of the aminoguanidine (AG) and citric acid (CA) precursors in order to validate the effect of the unique ratio of precursors on the resultant antibacterial activity of the C-dots produced. (I) AG-CA (1:1) C-dots were prepared by using 50 mg AG and 50 mg CA; (II) AG:CA (1:2) C-dots by using 25 mg AG/50 mg CA; and (III) AG:CA (4:1) C-dots by using 60 mg AG/15 mg CA.

[0031] Physical characterization of antimicrobial C-dots as prepared in section A: the quantum yield (QY) of the AG-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 H.sub.2SO.sub.4 (Φ=58).

[0032] Finally, quantum yield of each C-dots solution was calculated using the equation:

[00001]${\Phi }_{sm}={\Phi }_{st}\times \left(\frac{P{l}_{sm}}{Ab{s}_{sm}}\right)\times \left(\frac{Ab{s}_{st}}{P{l}_{st}}\right)\times \left(\frac{{\eta }_{sm}^2}{{\eta }_{st}^2}\right)$

where Φ=quantum yield, Pl=integrated photoluminescence, Abs=absorbance at λ.sub.ext, η=refractive index of the solvent, st=quinine sulfate standard and sm=C-dots sample. The quantum yield of the AG-C-dots of the invention was approximately 3% in deionized water.

[0033] Fluorescence spectroscopy. AG-C-dot solutions in deionized water were placed inside quartz cuvettes and the fluorescence emission spectra were recorded on an FL920 spectrofluorimeter (Edinburgh Instruments, UK). The AG-C-dots exhibited the typical excitation-dependent emission spectra (FIG. 1B) with the maximal emission (upon excitation at 390 nm) at 480 nm (i.e. green-yellow appearance).

[0034] High Resolution Transmission Electron Microscopy (HR-TEM). The AG-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. Size distribution of the AG-C-dots, determined by the HR-TEM analysis, gave rise to 4.3+/−0.5 nm.

[0035] X-ray Photoelectron Spectroscopy (XPS). The AG-C-dot solution was placed upon a silicon wafer and dried overnight. Once dried, the samples were measured using an X-ray 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.

[0036] X ray photoelectron spectroscopy (XPS) data is depicted in FIG. 1D. According to the results one can see the functional units on the AG-C-dots' surface, confirming retention of the aminoguanidine and carboxylic acid residues. Specifically, the C 1s spectrum in FIG. 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 is XPS peak in FIG. 1D similarly displays deconvoluted signals at 399.7 eV and 400.7 eV ascribed to the C—N and C═N bonds, respectably.

[0037] Zeta potential. C-dot solutions in PBS buffer (pH=7.4) were placed inside of a Malvern DTS 1070 disposable capillary cuvette and the zeta potential was measured by Zetasizer Nano ZS, Malvern, Worcestershire.

[0038] The results are depicted in FIG. 7 for the comparison of different C-dots having distinct aminoguanidine: citric acid precursor ratio, as measured in PBS buffer. It can be seen that the 2:1 C-dots of the invention have relatively neutral surface charge compared to other precursor ratio dots. The measured values of 4:1 C-dots gave rise to a negatively charged particles having a surface charge of about −18 mV in PBS buffer pH=7.4. In comparison, the 2:1 C-dots gave rise to a light surface charge of −2 mV due to a lower excess of amino groups which was mostly neutralized by negatively charged carboxyl groups.

Example 2—Antibacterial Activity

[0039] Bacterial growth: Two Gram positive bacterial strains—Staphylococcus aureus (S. aureus) and Bacillus cereus (B. cereus) and five Gram negative bacterial strains Escherichia coli K12 (E. coli K12), Salmonella enteritidis (S. enteritidis), Salmonella typhimurium strain ATCC14028 (S. typhimurium), Pseudomonas aeruginosa PAO 1 luxCDABE and Pseudomonas aeruginosa PAO 1 were used.

[0040] B. cereus were 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 30 C 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 37° C.

[0041] The antibacterial activity of AG-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. D.sub.600=1), followed by dilution of the bacteria to O.D.sub.600=0.05 (CFU=1×10.sup.6) and were incubated with AG-C-dots in different concentrations (1, 0.5, 0.25, 0.125, 0.0625 mg/mL), growth curves describing the change in O.D.sub.600 with time were collected for 24 hours of incubation at 37 C. The growth curves were measured in 96-well plates on a Biotek Synergy H1 plate reader (Biotek, Winooski, Vt., USA).

[0042] B) The MIC50 values referring to the concentrations in which the bacterial cell viability was 50% after 24 hours incubation with C-dots, were determined using a broth dilution method. All bacterial cells were grown in optimum growth conditions with increasing concentrations of C-dots in the medium. O.D.sub.600 values were taken after 24 hours of incubation and the C-dot concentrations in which the bacterial viability was reduced to 50% were determined.

[0043] C) Inhibition on agar plates containing different concentrations of C-dots as prepared in section 1A were prepared by initially autoclaving L.B agar, after cooling the agar to approximately 60 C dry C-dots powder was added to the warm agar and diluted in order to create the following dilution series: 0.0625, 0.125, 0.25, 0.5 and 1 mg/mL, the agar plates were than left to cool down and harden for further use.

[0044] After hardening, 2 microliters of P. aeruginosa solution (O.D.sub.600=1) was placed on each agar plate and the agar plates were incubated for 8 hours in 37 C.

[0045] D) Scanning electron microscopy (SEM). Bacterial solutions at O.D.sub.600=0.05 were incubated together with AG-C-dots for 12 hours at 37 C, 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 re-suspended 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).

Results

[0046] Antibacterial effects of AG-C-dots. FIG. 2 illustrates the selective antibacterial properties of the AG-C-dots. In the experiments summarized in FIG. 2, AG-C-dots were added to the bacterial growth medium and bacterial proliferation was monitored. The concentration-dependent bactericidal effects of the AG-C-dots against P. aeruginosa PAO 1 luxCDABE are depicted in FIG. 2A, demonstrating that the proliferation of P. aeruginosa PAO 1 luxCDABE was inhibited upon increasing C-dot concentration. No bacterial growth was apparent at a C-dot concentration of 0.5 mg/mL (FIG. 2A). Concentration-dependent anti-bacterial effect against P. aeruginosa PAO 1 luxCDABE was also apparent in the agar dilution method, in which the AG-C-dots were incorporated within the agar matrix (FIG. 2 C).

[0047] The bar diagram in FIG. 2B demonstrates that the antibacterial effect of AG-C-dots is selective towards P. aeruginosa sp. FIG. 2B demonstrates that the AG-C-dots had significant inhibitory effect upon P. aeruginosa PAO 1 luxCDABE and P. aeruginosa PAO1, while the C-dots appeared to exhibit no antibactericidal effects in case of Salmonella enteritidis, Staphylococcus aureus, E. coli K12 or Bacillus cereus (FIG. 2B).

[0048] Table 1 summarizes the minimum concentrations required for inhibiting 50% cell growth (MIC50), further demonstrating the bactericidal selectivity of the AG-C-dots towards P. aeruginosa bacterial strains.

TABLE-US-00001 TABLE 1 Species Gram type MIC50 (mg/mL) P. aeruginosa PAO 1 luxCDABE − 0.156 P. aeruginosa PAO 1 − 0.335 S. enteritidis − >1 S. typhimurium − >1 E. coli K12 − >1 B. cereus + >1 S. aureus + >1

[0049] FIG. 3 presents scanning electron microscopy (SEM) images showing the effect of the AG-C-dots upon different bacterial cells. Notably, FIG. 3 reveals that significant morphology alteration occurred when P. aeruginosa PAO 1 luxCDABE cells were incubated with the AG-C-dots, specifically “flattening” and elimination of cell surface smoothness (FIG. 3A). This observation likely indicates leakage of intracellular fluid due to bacterial cell wall permeability induced by the AG-C-dots. In contrast to the severe morphology transformation of the P. aeruginosa PAO 1 luxCDABE cells following incubation with the AG-C-dots, the SEM images in FIG. 3B show no discernable effect of the AG-C-dots upon S. aureus cells, consistent with the data in FIG. 2B and Table 1, pointing to no antibacterial activity of the AG-C-dots against Gram-positive type bacteria.

[0050] The effect that the C-dot surface groups composition has on their bactericidal activity was shown to be pronounced. As reported in section 1B herein above, C-dots having different mass ratios between the aminoguanidine and citric acid precursors were synthesized. Their corresponding antibacterial activity was assessed in p. aeruginosa cells. As can be seen in FIG. 6, the C-dots having a mass ratio of 2:1 (AG:CA) demonstrated an improved antimicrobial activity. As can be detected in FIG. 6A, there is a direct correlation between the content of nitrogen and the antibacterial activity over a certain rang, however, according to FIG. 6C, C-dots having a high ratio of 4:1. (AG:CA) demonstrated only a low antibacterial efficiency in comparison to C-tots having 2:1 ratio (FIG. 6B).

Example 3—Disruption to Biofilm Propagation

[0051] P. aeruginosa PAO 1 luxCDABE 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.D.sub.600=1 (CFU=1×10.sup.8). The resulting solution was grown for 24 hours in 37° C. 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-dots. The P. aeruginosa biofilm was imaged using confocal laser scanning microscopy (CLSM) Plan-Apochromat 20×/0.8 M27, Zeiss LSM880, Germany. Image processing to obtain 3-D images and to evaluate biofilm total volume was obtained using the IMAMS software (Bitplane, Zurich, Switzerland).

Results

[0052] The microscopy images in FIG. 4 show a significant decrease in biofilm abundance when P. aeruginosa PAO 1 luxCDABE bacteria were incubated with the AG-C-dots for 24 hours. Biofilm volume determination, carried out by analysis of the three-dimension images, revealed close to 50% decrease upon AG-C-dots addition to the bacterial growth medium (FIG. 4B).

Example 4—Bacterial Cell Labeling

[0053] Bacterial cell labeling was conducted by initially growing all bacteria until full growth (O.D.sub.600=1; CFU=1*10.sup.8), 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 37° C. 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) Plan-Apochromat 20×/0.8 M27, Zeiss LSM880, Germany.

Results

[0054] To accomplish bacterial cell staining, the bacterial suspensions were grown to saturation (OD600=1), pelleted, and incubated with the AG-C-dots (1 mg/mL) for 3 hours. The fluorescence microscopy images (excitation=405 nm) demonstrated that P. aeruginosa cells were labeled by the C-dots of the invention while no fluorescence labeling was apparent in case of S. aureus. The fluorescence microscopy results in FIG. 5 demonstrated the selectivity profile of the AG-C-dots, confirming specific targeting of P. aeruginosa. Furthermore, FIG. 5 demonstrates that bacterial staining (rather than killing) can be attained through tailoring the experimental parameters (C-dot concentration, point of C-dot addition to the bacterial suspension, and incubation time).

[0055] While the present disclosure has been illustrated and described with respect to a particular embodiment thereof, it should be appreciated by those of ordinary skill in the art that various modifications to this disclosure may be made without departing from the spirit and scope of the present disclosure.