Engineered Bright Blue- and Red-Emitting Carbon Dots Facilitate

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Imaging and Diagnostics

Engineered Bright Blue and Red Emitting Carbon Dots Facilitate Synchronous Imaging and Inhibition of Bacterial and Cancer Cell Progression via 1O2 Mediated DNA Damage under Photo-irradiation Shanka Walia, Ashish K Shukla, Chandni Sharma, and Amitabha Acharya ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00149 • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 21, 2019

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ACS Biomaterials Science & Engineering

Engineered Bright Blue and Red Emitting Carbon Dots Facilitate Synchronous Imaging and Inhibition of Bacterial and Cancer Cell Progression via 1O2 Mediated DNA Damage under Photo-irradiation

Shanka Walia,1,2 Ashish K. Shukla,1,2, # Chandni Sharma,1,2,# and Amitabha Acharya1,2,* 1Biotechnology

Division, CSIR-Institute of Himalayan Bioresource Technology (CSIR-IHBT), Palampur (H.P.) 176061, India

2Academy

of Scientific and Innovative Research (AcSIR), CSIR-Institute of Himalayan

Bioresource Technology (CSIR-IHBT), Palampur (H.P.) 176061, India #Contributed *Author

equally

to whom the correspondence should be addressed, E-mail: [email protected];

[email protected]; Tel (off): +91-1894-233339; Extn. 397; Fax: +91-1894-230433

Title running head: Nitrogen doped blue and red emitting carbon dots for synchronous in vitro imaging and therapeutic applications.

Brief:

Bright blue and red emitting CDs were synthesized, and characterized by various

spectroscopic and microscopic techniques. The developed CDs showed comparatively strong anti-bacterial efficacy against E.coli, than S.aureus. MTT assay and confocal microscopy data of HeLa cells reflected that these CDs can also be used as cancer cell targeting agents.

Keywords: Carbon dots, quantum yield, anti-bacterial efficacy, anti-cancer activity, cellular imaging. 1

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Abstract The development of biocompatible, widely applicable fluorescent imaging probe, with emission beyond the cellular and tissue auto-fluorescence interference, is a challenging task. In this regard, a series of 28 different fluorescent carbon dots (CDs) were synthesized using carbohydrates as carbon, and cysteine (Cys) and o-phenylenediamine (OPD) as nitrogen source. The screened CDs showed photostability with bright blue (~505-520 nm) and red (~588-596 nm) emission and high ﬂuorescence quantum yield (QY, 72.5±4.5 %). FTIR and NMR studies suggested presence of carboxylate and ester group for Cys and OPD based CDs, respectively. HRTEM results showed particle size of ~3.3 - 5.8 nm for all the developed CDs. The antibacterial studies suggested that the developed CDs showed preferential anti-bacterial activity against E.coli, with IC50 value of ~200µg/ml. Cytotoxicity and confocal microscopy studies of HeLa cells reflected that these CDs showed both anti-cancer activity and imaging ability. Agarose gel electrophoresis, together with SOSG assay and thiol estimation studies suggested oxidative stress induced DNA degradation to be the primary cause for cell death.

These

hemocompatible CDs can thus be used as simultaneous imaging probe and photo dynamic therapeutic agent for both anti-bacterial and anti-cancer activity.

2


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Introduction Molecular imaging allows the direct visualization of biological processes at the cellular and subcellular level and has gained tremendous attention in recent past with the developing concept of personalized medicine. Fluorescent molecular imaging probe with added therapeutic potential can serve the dual purpose of both detecting the disease site accurately and killing the same. Developing these theragnosis probe is indeed a challenging task, keeping the cytotoxicity issues under control. In this context, fluorescent carbon dots (CDs) with tunable size parameters, water solubility, photostability and above all high degree of biocompatibility and low toxicity, attracted worldwide interests in the field of bioimaging, analyte sensing, drug delivery etc1-6. Such properties make these new class of nanoparticles (NP) superior to quantum dots (high toxicity concerns) and conventional dyes (photobleaching)7-10.

Since their accidental discovery in

200411, different strategies have been applied for the synthesis of CDs which include pyrolysis, electrochemical, laser ablation, oxidation, microwave and hydrothermal synthesis using different carbon sources12-19.

However, the major problem still lies the same i.e., how to get the

fluorescent CDs with longer λmax value and high quantum yield? It has now been proved that the fluorescence emission profile can be improved by doping CDs with heteroatoms such as N, S, P, B etc12, 20-26. Antibiotic resistance of the bacterial pathogens has become a growing concern, and hence the development of alternative anti-bacterial substitutes is of utmost importance27.

In past,

researchers were interested in developing metallic nanomaterials viz., silver, iron oxide, copper oxide, nickel, zinc oxide etc. with improved antibacterial activities but their toxicity concerns remain a serious threat to human health28-33. Such inherent problems can be overcome by using biocompatible fluorescent CDs with enhanced therganostic properties which includes simultaneous diagnosis and therapy of the disease sites. However, to the best of our knowledge 3



no reports are available where the same CDs were used for simultaneous anti-bacterial and anticancerous efficacy studies together with strong cellular imaging applications. In the present work, bright blue and red emitting CDs were synthesized, and characterized by various spectroscopic and microscopic techniques. The developed CDs when tested against two bacteria viz., Gram-negative bacterium Escherichia coli (E.coli, MTCC-43) and Gram-positive Staphylococcus aureus (MTCC-3196) showed preferential anti-bacterial efficacy against the former. MTT assay and confocal microscopy data of HeLa cells reflected that these CDs showed differential anti-cancerous activity and imaging ability; thus can be used as an advanced theragnosis probe.

Experimental Method General materials and characterization part has been included in the Supporting Information. CD synthesis under acidic condition. Acid treated CDs were prepared by following literature report, with added modifications34. Briefly, 400 mg of each of the precursor carbohydrate source viz., D-glucose (G), D-fructose (F), D-sucrose (S) and chitosan (CS) were diluted in 2 ml of double distilled water (DDW), followed by addition of 5 ml of orthophosphoric acid (PA) and the reaction mixture was refluxed at 80˚C for 6 h. After cooling, the pellet obtained was centrifuged at 10,000 rpm for 15 min. Next, the pellet was washed several times with DDW and dialyzed to remove excess acid or any other reactants. Finally, the brown colored CD solutions viz., G-PA, F-PA, S-PA and CS-PA were obtained, which were then dried in oven at 60˚C for 48 h to obtain solid mass. CD synthesis by Maillard reaction. Nitrogen (N) doped CDs were prepared via Maillard reaction, using cysteine (Cys), glutathione (GSH) and ortho-phenylenediamine (OPD) as nitrogen source, under alkaline condition using 0.6 and 1.0 M of NaOH35. Maillard reaction 4


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involves simultaneous condensation and polymerization, along with the carbonization and aromatization process, which finally lead to the formation of CDs. Briefly, 10 mM of each of the carbohydrate moieties were mixed with 120 mM of different nitrogen source in 20 ml of double distilled water. To these solutions, 0.6 and 1.0 M NaOH was added and the solutions were refluxed at 150˚C for 24 h. After cooling, the samples were dialyzed for 24 h and dried using rotary evaporator. The as synthesized CDs were abbreviated as G-Cys0.6, G-Cys1.0, F-Cys0.6, FCys1.0, S-Cys0.6, S-Cys1.0, CS-Cys0.6, CS-Cys1.0, G-GSH0.6, G-GSH1.0, F-GSH0.6, F-GSH1.0, SGSH1.0, S-GSH1.0, CS-GSH0.6, CS-GSH1.0, G-OPD0.6, G-OPD1.0, F-OPD0.6, F-OPD1.0, S-OPD0.6, S-OPD1.0, CS-OPD0.6, and CS-OPD1.0. Anti-bacterial studies. The anti-bacterial activity of the synthesized CDs was evaluated by disc diffusion method, growth inhibition curve kinetics, confocal microscopy, TEM, MTT and SOSG assay. Bacterial culture. To prepare bacteria inocula, single E.coli and S.aureus colonies from their respective media plates were incubated overnight in 10 ml luria broth (LB) and Mueller Hinton Broth (MHB) medium, at pre-specified growth conditions of temperature at 37˚C with continuous shaking at 200 rpm. The pre-culture was diluted to obtain the fixed OD600 value at 0.08-0.1 and this stock was used for further antibacterial studies. Disc diffusion method. Anti-bacterial activity of CDs was evaluated by disc diffusion method. In brief, different concentrations i.e. 25, 50, 100, 200 and 300 µg/ml of CDs (G-Cys1.0, F-Cys1.0, G-OPD1.0 and F-OPD1.0) were loaded on 6 mm (for 25 and 50 µg/ml) and 10 mm (for 100, 200 and 300 µg/ml) discs placed on LB and MHB agar plates which were seeded with 10 µl of cell suspension having cell density of 108 CFU/ml of both the bacterial strains viz., E.coli and S.aureus, respectively. After incubation at 37˚C for 12 h, width of the bacteria-free areola was measured. 5



Bacterial growth inhibition monitoring. The growth kinetic studies of E.coli and S.aureus in presence of the prepared CDs were assessed by using cell growth measurements based on the optical density at 600 nm. Briefly, E.coli and S.aureus cells were inoculated in LB and MHB media with varying concentrations (100, 200 and 300 µg/ml) of each of the carbon dots (GCys1.0, F-Cys1.0, G-OPD1.0 and F-OPD1.0) at 37˚C and the growth inhibition was monitored by measuring the absorbance at 600 nm at different time intervals of 0, 2, 4, 6, 8 and 10 h. Animal cell culture. In vitro studies were performed using human embryonic kidney 293 cells (HEK-293) and HeLa (cervical cancer cell) cells grown in DMEM supplemented with 10% FBS and 1% antimycotic-antibiotic solution in 5% CO2 at 37˚C. For all the studies PBS of 1X strength with pH 7.4 was used. MTT assay to monitor cyto-compatibility.

Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) assay was used to quantify the effect of CDs on HeLa cell viability. Briefly, both cell lines viz., HEK-293 and HeLa cells were incubated in 96 well plates at a concentration of 15000 cells/ml for 24 h. After 24 h of incubation, the cells were washed twice using PBS and exposed to different concentration of CDs viz., 100, 200, 300, 500 and 1000 µg/ml. For HEK-293 cells two additional concentrations of CDs viz., 10 and 25 µg/ml were also used. Separately, the cells treated with dulbecco's modified eagle medium (DMEM) were taken as control. The plates were incubated at 37°C for 24 and 48 h. At the end of the treatment, cells were washed twice with PBS; then 10 µl of MTT solution (5 mg/ml) along with 90 µl PBS was added to each well and the solutions were further incubated for 4 h at 37˚C. After incubation, the formazan crystals formed were dissolved in 100 µl DMSO and incubated in dark for 15 min. The intensity of the color was measured using micro plate reader at 570 nm. All experiments were performed in triplicate, and the cell viability (%) was calculated using eqn (1). MTT assay

6


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data was statistically analyzed by ordinary two-way ANOVA. Similar protocol of MTT assay was followed for bacterial cells (supporting information, Page S5).  Absorbance of test sample    100 Cell viability (%)    Absorbance of control sample 

(1)

Hemocompatibility studies. The isolation of the human blood was done according to the standard protocol approved by Institutional Ethics Committee (IHBT Project No.-IEC/IHBTP5/Dec-2015). The freshly collected blood was centrifuged at 1000 g for 5 min at 4˚C; the supernatant was discarded and the RBC pellet was washed thrice with PBS (10mM, pH 7.4). The stock solution was prepared by diluting RBC pellet in 20 ml PBS. The synthesized CDs solution (500 µl) of different concentrations viz., 0.3125, 0.625 and 1.25 mg/ml were dissolved in PBS and incubated for 1h at 37˚C. Additionally, 1% triton X-100 and PBS were also used as positive and negative control, respectively. All these samples were further incubated with the isolated RBCs (500 µl) at 37˚C for 3h at a continuous shaking speed of 60 rpm. After incubation RBCs suspensions were centrifuged at 1000 g for 5 min and the supernatants were collected carefully. Then, the absorbance values of the released hemoglobin (Hb) in the supernatants (100 μl) were measured at 540 nm using microplate reader. The hemolytic activity of CDs treated RBCs were calculated by comparing the absorbance values of the tested supernatants to that of the positive control (i.e., 100% hemolysis). The calculation of % hemolysis is given in eqn (2); Hemolysis (%) 

 Absorbance of test sample  Absorbance of

postive control   Absorbance of postive control  Absorbance of negative control 

(2)

Confocal microscopy studies with HeLa cells. HeLa cells were seeded in DMEM at density of ~30,000 cells/well on poly L-lysine coated cover slips in 6 wells cell culture plate and incubated for 24 h. After incubation the cells were washed twice using PBS and treated with 300 µg/ml of CDs and further incubated for 3 h. After incubation, 20 µl freshly prepared PI solution (1mg/ml 7



stock) was added and the total solution was kept in dark for 15 min.

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Finally, cellular

internalization of CDs was examined through confocal microscope. The band pass used for the studies were same as previously documented for bacterial studies. Different excitation and emission band pass viz., PI channel (PI, Ex. 477 nm, band pass: 560-615 nm), nanoparticle emission channel (for G-Cys1.0 and F-Cys1.0 Ex. 458 nm, band pass: 505-550 nm, blue emission; for G-OPD1.0 and F-OPD1.0 Ex. 477 nm, band pass: 530-600 nm, yellow emission) were used to capture the images. Similar protocol was followed for bacterial confocal studies (supporting information, Page S5-S6). Singlet oxygen detection assay. Singlet oxygen sensor green (SOSG) reagent was used as the singlet oxygen tracking agent whereas rose bengal (RB) was used as standard photosensitizer. Stock solution of 165 µM of SOSG was prepared by dissolving 100 µg SOSG in methanol (33 µl), followed by diluting the solution with 970 µl Milli-Q water. A calibration curve was generated using a stock solution of 1M RB dissolved in 1X PBS (1mg/ml). Further, the CDs diluted in 1X PBS (300 µg/ml) were treated with 10 µl of SOSG (1.65 µM) in dark. The negative control was prepared by dissolving 10 µl SOSG in 990 µl 1X PBS. The samples were then irradiated at 525 nm and absorbance was measured at every 20 min for 3h, using microplate reader. All the experiments were performed in triplicates. The same protocol was followed for SOSG assay with two bacterial strains (E.coli and S.aureus) having cell density of 108 CFU/ml treated with 25, 50, 100, 200 and 300 µg/ml of synthesized CDs. In case of HeLa cells (15000 cells/well), the concentrations of different CDs were varied at 100, 200 and 300 µg/ml. Total thiol estimation. Trypsinized HeLa cells (5x105 cells) were grown in 100 mm cell culture petri dish for 24 h. The HeLa cells were treated with 300 µg/ml of CDs for 24 h and harvested by centrifugation (300 x g for 5 min). The untreated cells were used for control studies. The isolated pellets were washed with 1X PBS twice and resuspended in 500 µL RIPA buffer. 8


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Suspended cells were placed on ice for 10 min followed by 2 min of sonication at 35% amplitude for lysis. The suspension was then centrifuged at 10,000 g for 1 min and the supernatant was collected for total thiol estimation. Total thiol estimation of the treated and untreated HeLa cells was done via Ellman’s reagent (DTNB). For this, dilution buffer (30 mM Tris HCl, 3 mM EDTA, pH 8.2) and DTNB (3 mM in methanol) solution were prepared. N-acetyl cysteine solution was prepared in serial dilution from 1000 to 31.25 µM, to obtain the standard curve. In each of the thiol estimating reaction tube, containing 75 µl of dilution buffer, 25 µl DTNB and 400 µL methanol, 20 µl of cell lysate supernatant was added. All the reactions were kept for ~510 min at room temperature, followed by centrifugation at 3000 x g for 5 min. Each reaction mixture (100 µl) was taken into 96 wells plate and the corresponding absorbance was measured at 412 nm. The observed OD value was fitted to the standard curve to quantify thiol present in each sample. The experiments were repeated in triplicates and data were statistically analyzed by two way ANOVA. Isolation of genomic DNA (gDNA) from bacterial cells. Both E.coli and S.aureus, having cell density of 1x108 CFU/ml, were treated with the synthesized CDs at a fixed concentration of 300 µg/ml and incubated for 12 h. The incubated solution was centrifuged at 6800g for 10 min. To the isolated pellet, 800 µl of Tris HCl-EDTA (TE, pH 8.0) buffer was added to resuspend the cells. The solution was mixed with sodium dodecyl sulfate (SDS) (100µl; 10%) and 5µl Proteinase K (10 mg/ml), followed by incubation for 1 h at 37ºC in a shaker incubator. Next, to this, 1 ml of phenol: chloroform: isoamyl alcohol mixture (25:24:1) was added and again incubated for 5 min at room temperature. The collected pellet was further centrifuged at 4˚C (10,000 rpm for 10 min). The viscous supernatant obtained after centrifugation, was collected in freshly autoclaved tube and to this, mixture containing phenol, chloroform and isoamyl alcohol treatment was repeated. After this, the solution was treated with chloroform to remove the 9



phenolic contamination. The nucleic acid was obtained in aqueous phase and collected in a 2 ml autoclaved tube. The obtained nucleic acids were mixed thoroughly with 100 µl of sodium acetate (3 M), followed by addition of 1 ml of isopropanol and mixed gently, by repeatedly inverting the tube until the white strands of nucleic acid precipitated out, which was again centrifuged at 5000 rpm for 10 min. Next, RNAase A (2µl; 50mg/ml) was added to each tube and the solutions were incubated at 37˚C for 30 min, followed by RNAse degradation at 80˚C for 10 min. To this, ethanol (1 ml; 70%) was added, followed by centrifugation (5000 rpm, 10 min). The collected pellet was again washed with 70% ethanol followed by air drying of the tubes at room temperature for 15 min. The isolated pellet was finally mixed with 200µl of TE buffer (pH 8.0).

The concentrations of the isolated DNA for each samples were determined using

spectrophotometer by monitoring absorbance at 260/280 nm. Isolation of nuclear DNA (nDNA) from HeLa cells. Trypsinized HeLa cells (2x106 cells) were grown in 100 mm cell culture petridish for 24 h. Grown monolayer HeLa cells treated with 300 µg/ml of CDs were incubated for 24 h and harvested by centrifugation (300 g for 5 min). Untreated cells were used as control. The isolation of nuclear DNA from both the treated and untreated cells were done according to previously published report36. DNA fragmentation analysis. Equal proportions of isolated gDNA (from bacterial cells) and nDNA (from HeLa cells) were run on 1.5% agarose gel to observe the fragmentation pattern in CDs treated and control samples.

Results and Discussions The chemical synthesis of CDs was achieved by using carbohydrates as main carbon source. The selection of carbohydrates was made based on the presence of number of sugar units in each motif viz., monosaccharides [D-glucose (G) and D-fructose (F)], disaccharide [D-sucrose (S)] 10


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and polysaccharide [chitosan (CS)] (Figure 1a). The reactions were carried out by using Lcysteine (Cys), L-glutathione (GSH) or ortho-phenylenediamine (OPD), under acidic or basic conditions. Based on these reactants, a library of total of 28 CDs were synthesized and these were summarized in Supplementary Information 01 (Table S1). Absorption and Fluorescence Studies. The absorption and fluorescence spectra of all the synthesized CDs were monitored to screen the best fluorescent probe amongst all the prepared nanomaterials. CDs with strong emission maxima (λmax) beyond 500 nm were preferred, since their emission can be easily distinguished from cell and tissue auto-fluorescence under in vivo condition. In case of Cys and GSH based CDs, the absorbance peaks were observed in the range of ~280-350 nm due to π → π* and n → π* transitions of conjugated carbons,2, 37 whereas, for OPD synthesized CDs, an additional peak at ~ 460 nm was also observed, reason might be due to the presence of different surface states

38.

However, in case of acid treated CDs, no specific

absorbance peaks were detected (Figure S1). The fluorescence emission profile of all the CDs were studied by exciting them at different wavelengths ranging from 280 to 500 nm, at an interval of 20 nm.

The emission spectra of all the synthesized CDs displayed excitation

wavelength-dependency and the emission wavelength was found to red-shift at higher excitation wavelength. In literature, the variable emission profile of the CDs has been reported to be the result of different size of the particles or due to the existence of different emissive sites present on the surfaces of the CDs 39. The selected excitation and emission wavelength of all the CDs were documented in Table S1 Supplementary Information. The emission spectra suggested that on excitation at 380 nm G-PA, F-PA and S-PA showed emission only upto 440 nm, whereas, CS-PA showed weak emission at ~516 nm. Similarly, in case of GSH based CDs, emission maxima were observed only upto ~494 nm. Hence, both the acid and GSH treated CDs were not selected for further studies due to the weak emission profile along with the presence of multiple 11



peaks in most of the samples. It should be mentioned here that apart from the acid treated samples, other CS based CDs were either insoluble or did not show any prominent emission spectra, and thus all the CS based CDs were also not considered for further studies. In case of Cys samples, S-Cys CDs showed emission in the range of 417-440 nm on excitation between 340 to 360 nm, and these were also not used for future studies. However, on excitation at 440 nm, both G-Cys and F-Cys showed emission maxima between 505-520 nm, with strong fluorescence intensity.

Similarly, OPD based samples viz., G-OPD and F-OPD, showed

emission maxima between 585-596 nm upon excitation at 480 nm (Figure S2). It was also observed that at higher NaOH concentration viz., 0.6 to 1.0 M, ~8-10 nm red shift in λmax with increased fluorescence intensity was obtained for both Cys and OPD based samples. Comparative fluorescence studies revealed that the emission profile (based on longer λmax and higher intensity) of the synthesized CDs followed the trend of G>F>S>CS for carbohydrates, OPD>Cys>PA>GSH amongst reactants and 1.0 M>0.6 M NaOH for basic condition. Thus, on the basis of emission profile, 4 synthesized CDs viz., G-Cys1.0, F-Cys1.0, G-OPD1.0 and F-OPD1.0 were finally selected for the future studies (Figure 1b,c). The quantum yields of G-Cys1.0, FCys1.0, G-OPD1.0 and F-OPD1.0 were found to be ~60.5±1.6, 72.5±4.5, 6.5±1.1 and 5.3±1.4 %, respectively (Table S2). Comparative literature survey on carbohydrate based CDs suggested that our synthesized CDs have longer λmax value with higher quantum yield percentage (Table S3). Further, when these CDs were irradiated at 366 nm, G-Cys1.0 and F-Cys1.0 showed blue emission, whereas light red emission was observed for G-OPD1.0 and F-OPD1.0 (Figure 1d,e). Effect of Long Time Storage, Temperature and pH Variation on the Fluorescence Emission of the CDs. Time dependent kinetic stability studies for the screened CDs were done for a period of 20 days. No appreciable changes, either in the fluorescence intensity, peak position or in shape of the spectrum was observed for Cys based CDs whereas, for OPD based CDs, 12


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minimal decrease in the fluorescence intensity were observed (Figure S3a). Similarly, the temperature dependent fluorescence studies of the selected CDs were done by recording the corresponding emission profile at different temperature ranging from 10 to 70˚C with an increment of 5˚C each time. Results suggested that with increase in temperature, Cys based CDs showed decrease in their emission intensity; possibly due to increase in the non-radiative decay rate, resulting in decreased quantum efficiency and corresponding fluorescence intensity

40-41.

However, the trend was found to be reverse for OPD based CDs, where increase in the temperature, also increased the fluorescence emission intensity of the respective CDs. The increase in fluorescence intensity with increasing temperature might have originated from a carrier transfer mechanism between the N-dopant induced state (energy level) and emitting state of CDs42. The hydrogen-bonding interactions and surface functional groups of CDs could also be responsible for such changes43. It should be noted that all the screened CDs showed excellent fluorescence intensity at physiological temperature (37°C) (Figure S3b). The biological milieu surrounding any particular disease site may have different pH environment compared to the rest of body. Hence, an ideal molecular imaging probe should be able to retain its fluorescence intensity at the target site pH. In this respect, G-Cys1.0 and F-Cys1.0, showed decrease in the fluorescence intensity at strong acidic pH (pH 3), whereas, under neutral and basic conditions (pH 7.5 - 10), these showed higher emission intensity. However, in case of OPD based CDs, higher fluorescence intensity was observed only at acidic pH (pH 3); though at higher pH, the intensity gradually decreased. However, no change in λmax was observed for any of the CDs either under acidic or basic conditions (Figure S3c). The protonation/deprotonation of the functional groups present on the surface of CDs can lead to shift in fluorescence intensity of the CDs44-46. Also, pH-sensitive strengthening or weakening of π−π aggregation of CDs may cause changes in the emission profile of respective CDs47. All these results suggested that under 13



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physiological condition, Cys based CDs may be used as molecular imaging probe to target neutral or basic pH sites, whereas, OPD based CDs can be used for acidic sites. Competitive Fluorescence Emission Studies in Presence of High Concentrations of NaCl and Chelating Metal Ions. The fluorescence emission profile of the selected CDs was also monitored at different concentrations of NaCl solution to observe their stability under high ionic strength environment (Figure S3d (a)). The obtained data suggested that even at 1.0 M NaCl concentration, the fluorescence emission intensity of all the CDs did not show any significant quenching (only ~15% quenching was observed for G-Cys1.0).

Again, to observe the

interference of metal ions on the fluorescence intensity, 100 µM of different monovalent, divalent and trivalent metal ions were titrated against the CDs (Figure S3d (b)). It was observed that both G-Cys1.0 and F-Cys1.0 showed fluorescence quenching of ~38 and 21, 27 and 15, 13 and 8 % for Mn2+, Cu2+ and Pb2+, respectively. Interestingly, for the same CDs, Zn2+ and Cd2+ showed ~39 – 45 and 25 – 36 % of fluorescence enhancement. The enhancement in fluorescence intensity of Cys based CDs in presence of Zn2+ and Cd2+ ions possibly have occurred due to the formation of ZnS and CdS sphere. These metal sulphide complexes generally served as surface passivation reagents and thus avoid non radiative recombination pathway48. No other metal ions showed any obvious interference in the fluorescence intensity of the Cys based CDs. It should be further mentioned that G-OPD1.0 and F-OPD1.0 did not show any change in fluorescence intensity in presence of these metal ions. In literature, the fluorescence intensity of CDs was found to quench in presence of either Cu2+ or Fe3+. Fluorescence quenching in presence of Cu2+ takes place through the charge transfer process when they are close to the surface of the CDs. In case of Fe3+, direct interaction between Fe3+ and –COOH/and -NH2 groups, present on the surface of the cysteine based CDs might have resulted the fluorescence quenching49-51. All these

14


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studies suggested that the developed CDs can retain their fluorescence intensity in biological milieu. FTIR and NMR Spectroscopic Studies to Identify the Chemical Composition of the Synthesized CDs. FTIR studies were done to identify the chemical composition and functional groups present in the synthesized CDs (Figure 2a, Figure S4). G and F showed –C-H and –O-H vibrations bands between 3500 - 2900 cm-1 whereas, characteristic -C-O and -C-C groups vibration modes were observed in the region between ~1500 - 600 cm-1.

Cys showed

characteristic -O-H peak at ~3165 cm-1, -N-H peak at ~2953 cm-1, -S-H peak at ~2542 cm-1, COO- peak between 1579 – 1531 cm-1 and –C-S peak at ~632 cm-1. In case of OPD, the triplet absorption bands appearing at ~3385, 3363 and 3186 cm−1 were assigned to the asymmetric stretching vibration for –N-H group whereas, peaks at ~1271 cm-1 were attributed to symmetric stretching vibration of -C–N. The appearance of sharp bands between ~1155 - 925 cm-1 and broad absorption band at ~742 cm-1 were attributed to out of plane deformation of –C-H for 1,2disubstituted benzene ring52. FTIR spectra of G-Cys1.0 and F- Cys1.0 showed broad band of -O-H stretching at ~3340 cm-1, single sharp peaks in the range ~1573 - 1568 cm-1 due to -COO- groups whereas, symmetric stretching vibration of -C–N was observed between ~1392 – 1381 cm-1. The peaks between ~1226 and 1138 cm-1 indicated the presence of –C-O group and a broad peak at ~670 - 640 cm-1 suggested –C-S bond was intact.

Interestingly, the absence of strong

characteristic peak at ~2550 cm-1 reflected that –S-H bond of Cys was no longer present in the synthesized CDs. The FTIR spectra of G-OPD1.0 and F-OPD1.0 showed broad peaks between ~3385 - 3028 cm-1 which were attributed to the stretching of –OH and –NH, respectively. The sharp peaks between ~1630 cm-1 suggested presence of –COO- group, peaks at ~ 1591 cm-1 were attributed to the aromatic C=C bending and the sharp peak at ~1360 cm-1 was recognized as stretching vibration of –C-N bond. It should be mentioned here that in literature, peak at ~ 1360 15



Page 16 of 50

cm-1 was also attributed to the defects related to the D mode in graphitic carbon53. The peaks at ~1055 and 1031 cm-1 were indicative of –C-O stretching vibration. Thus, the comparative FTIR spectra suggested the presence of carbonyl groups for both Cys and OPD based CDs whereas, strong aromatic carbons were found for only OPD based CDs.

Additionally, it was also

concluded that both type of CDs contains –C-N and C-O-C functional groups in their core structure. The FTIR spectra were further supported by 1H and 13C NMR studies. Typical, 1H and 13C NMR chemical shifts of carbohydrate ring protons and carbons were observed between ~3 – 4 and 60 – 110 ppm, respectively for both G and F (Figure S5). Similarly, Cys showed 13C peak for –C=O at ~172 ppm whereas the aromatic carbon peaks of OPD were observed between ~120 – 134 ppm. The 1H NMR spectra suggested appearance of new peaks for G-Cys1.0 and F- Cys1.0 in the range of ~1 – 2 ppm which may have originated due to the presence of electronegative atoms attached to H. Both G-Cys1.0 and F- Cys1.0 showed 13C peak for –C=O at ~ 178 ppm whereas, additional

13C

peaks were appeared between ~ 20 – 60 ppm. These peaks were indicative of

presence of new sp3 hybridized carbon atoms in both the samples. Further, relatively less

13C

NMR peaks were observed between 60 – 110 ppm which suggested that the carbohydrate backbone has lost its structural integrity due to the chemical reaction. For OPD based CDs, aromatic carbon peaks were observed between ~ 117 – 134 ppm. Interestingly, appearance of 13C

NMR peak at ~ 168 ppm suggested the formation of ester –C=O for both the CDs.

Therefore, NMR studies suggested presence of –COOH group for Cys based CDs and aromatic – C=C and ester –C=O group for OPD based CDs. Thus it can be speculated that the higher fluorescence intensity of Cys based CDs under neutral or basic condition and OPD based CDs under acidic condition, was due to the availability of free –COO- group in the solution.

16


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Studies on Size, Morphology and Crystalline Nature of the Synthesized CDs. DLS and zeta potential studies were performed to measure the hydrodynamic diameter and net zeta potential of the synthesized CDs. The DLS results reported the sizes of G-Cys1.0, F-Cys1.0, G-OPD1.0 and FOPD1.0 to be ~ 215 ± 24, 252 ± 47, 127 ± 2 and 101 ± 1.3 nm, with their corresponding zeta potential values of ~ -22.6 ± 0.84, -24.36 ± 1.14, -34.36 ± 0.7 and -48.4 ± 1.9 mV, respectively (Figure S6, Table S4). The fact that comparatively higher hydrodynamic sizes and negative zeta potential values were obtained for the synthesized CDs, suggested strong electrostatic interaction between the surface exposed negatively charged functional groups with surrounding water molecules. X-ray diffraction patterns (XRD) for the synthesized CDs were displayed in Figure 2b (Figure S7). Both, G-Cys1.0 and F-Cys1.0 showed a broad peak between 2θ = 16° – 34°, centered at ~21°, which was similar to the graphite lattice spacing, and was attributed to highly disordered carbon atoms54, 55. In case of G-Cys1.0, F-Cys1.0 broad diffraction band was observed at ~21˚ which indicated the presence of turbostratic carbon phase or might have resulted from the small/thin nanostructures and weak layer−layer interactions of CDs44, 56. Also, in literature the width of XRD spectrum has been reported to be inversely proportional to particle size; broader the peak, smaller the size of carbon dots, which was also observed in HRTEM studies44, 57-60. The presence of number of peaks in the XRD patterns of G-OPD1.0 and F-OPD1.0 might be due to the ordered stacking of prepared carbon dots61. The presence of number of peaks in the XRD patterns of G-OPD1.0 and F-OPD1.0 revealed a complex mixture of crystalline components for both the samples46. The TEM and HRTEM results (Figure 2c-f), showed that the particle size of all the CDs fall in the range of ~3.3 to 5.8 nm; additionally, few bigger size particles of ~10.2±1.5 nm were also observed for G-OPD1.0 and F-OPD1.0 (Figure 2e,f). The lattice spacing was found to be in the range of ~ 0.20-0.26 nm for all the CDs, which corresponds to that of graphite structure50,62,63. The SAED pattern of the CDs suggested poly crystalline nature (Figure 17



Page 18 of 50

S8a, b). The EDX studies confirmed the presence of S in case of G-Cys1.0 whereas the same was missing in G-OPD1.0 (Figure S8c, d). Assessment of Anti-bacterial Activity of the Synthesized CDs.

Since one of the most

common antibiotic kanamycin, contain sugar moieties in its backbone, it would be interesting to evaluate the anti-bacterial potential of these developed CDs. Further, in literature the antibacterial activity of the CDs was studied by conjugating the corresponding nanomaterials with known antibiotics64,65. Two different class of bacteria viz., E.coli and S.aureus were chosen for the studies. The results reflected that the zone of inhibition (ZOI) was directly proportional to the concentration of the CDs used (Figure 3a,b,d). The maximum ZOI was observed for E.coli and S. aureus at 300 µg/ml concentration for both Cys and OPD based CDs. It was interesting to note that all the CDs showed enhanced anti-bacterial activity compared to their precursor molecules. It should be noted that control OPD showed comparable ZOI data with that of GOPD1.0. This observation can be explained by the fact that the concentration of OPD was ~13 times lower in case of OPD based CDs as compared to the control OPD. A closer look into the plates, along with the associated bar diagrams suggested that the anti-bacterial efficacy of the CDs followed the trend F-OPD1.0>G-OPD1.0>G-Cys1.0>F-Cys1.0.

Comparative studies also

reflected that the developed CDs exhibited preferential killing of E.coli. The data of growth curve plotted at different time intervals reflected that the developed CDs can inhibit the growth of both the bacteria (Figure 3c, e; Figure. S9). It was further noticed that all the CDs at 300 µg/ml concentration showed best activity, whereas the control molecules at the same concentration did not have much effect on bacterial growth inhibition. The fact that G-OPD1.0300 and F-OPD1.0-300 documented best anti-bacterial activity, supported our previous ZOI results. The growth inhibition curve also suggested that OPD based CDs showed stronger antibacterial activity towards E.coli compared to S.aureus. 18


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Confocal Microscopy and TEM Studies to Confirm Bacterial Killing and CD Localization Inside Bacterial Cells. The localization of the CDs inside bacterial cells was monitored by confocal microscopy studies (Figure 4; Figure S10). The experiments were carried out using three different concentrations of CDs viz., 100, 200 and 300 µg/ml and the results were compared with appropriate controls. The dead bacterial cells (treated with PI) were marked in red whereas, Cys and OPD based CDs were marked in blue and yellow, respectively. Confocal micrographs suggested the internalization of both types of CDs inside bacterial cells, though the emission was found to be strong for Cys based CDs. The merged images further showed superimposed red and blue dots for G-Cys1.0 and F-Cys1.0 and red and yellow dots for G-OPD1.0 and F-OPD1.0, which proved that the bacterial killing has occurred due to the internalization of CDs. The fact that comparatively less number of dead bacterial cells were observed for S.aureus suggested that the developed CDs have preference towards E.coli; a fact which supports our previous results. Concentration dependent confocal microscopy studies reflected increase in bacterial killing at higher concentration of CDs (Figure S10). To monitor the bacterial killing mechanism, TEM studies were also performed (Figure 4). The characteristic rod and round shaped morphology was observed for E.coli and S.aureus, respectively (Figure S10). When E.coli was treated with CDs, the cytoplasmic components were found to ooze through bacterial cell membrane and simultaneously these lost the regular cell morphology. In case of S.aureus though, the killing mechanism was found to involve elongation of bacterial cell membrane, followed by disruption of the same. TEM studies also reflected that at the same time interval, CDs showed better anti-bacterial activity towards E.coli compared to S.aureus. Quantitative Measurement of Anti-bacterial Activity by MTT Assay. To quantitatively evaluate the anti-bacterial efficacy of the synthesized CDs, MTT assay was performed. It was observed that in both the cases, the cell viability % decreased with increase in CDs concentration 19



(Figure S11). Results suggested that at all the concentrations, F-Cys1.0 showed highest cell viability for both the bacteria whereas F-OPD1.0 showed maximum cell inhibition. Comparative results indicated that the CDs showed more preference towards E.coli at all the concentrations. The IC50 value for E.coli was found to be ~200 µg/ml whereas the same was calculated to be ~300µg/ml, for S.aureus in case of G-Cys1.0, G-OPD1.0, F-OPD1.0, whereas for F-Cys1.0 it was slightly higher.

Thus, all the bacterial studies reflected preferential inhibition of E.coli in

presence of both types of CDs. Cyto-compatibility Assay. To check the suitability of the developed CDs as molecular imaging probe towards animal system and also to monitor the effect of the same on the normal cell line and cancer cells, MTT assay was performed with HEK-293 and HeLa. The experiments were carried out at two different time intervals viz., 24 and 48 h, using different concentrations viz., 100, 200, 300, 500 and 1000 µg/ml of G-Cys1.0, F-Cys1.0, G-OPD1.0 and F-OPD1.0. For HEK-293 cells, two additional concentrations of CDs viz., 10 and 25 µg/ml were also used (Figure 5a,b and Figure S12). It was observed that the developed CDs did not show any appreciable toxicity after 24 h at 100 µg/ml concentration in HeLa cells. After 24 h of incubation with the CDs, both GCys1.0 and F-Cys1.0 documented cell viability of ~68 and 75 % at 1000 µg/ml of concentration, whereas the same was found to be only ~34 and 48 % for G-OPD1.0 and F-OPD1.0, respectively. The cell viability % dropped significantly after 48 h, documenting ~33 and 46 % for G-Cys1.0 and F-Cys1.0 and ~7 and 4 % for G-OPD1.0 and F-OPD1.0, respectively. Further, it was also observed that for both types of CDs, the cell viability % decreased at higher concentrations. The cytocompatibility studies with HEK-293 suggested both G-Cys1.0 and F-Cys1.0 did not show any toxicity up to 300 µg/ml even after 24 h whereas the same limit for G-OPD1.0 and F-OPD1.0 was found to be 25 µg/ml (Figure S12).

20


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Hemocompatibility Assay.

The hemocompatible nature of CDs was evaluated through

hemolysis assay at three different concentrations viz., 0.3125, 0.625 and 1.25 mg/ml (Figure 5ce, Table S5). The spectrophotometric results along with the photographic images suggested that the all the developed CDs were hemocompatible, even at a concentration of as high as 1.25 mg/ml. Monitoring Internalization of CDs by Confocal Microscopy Studies.

The cellular

internalization of CDs was monitored by confocal microscopy studies using HeLa cells (Figure 6). The cells were treated with fixed CD concentration of 300 µg/ml for 3 h and incubated further with PI, to monitor the dead cell localization. The internalization of Cys and OPD based CDs in HeLa cells were confirmed by blue and yellow emission, respectively, whereas, the merged images reflected presence of CDs inside cells. It was interesting to note that both GOPD1.0 and F-OPD1.0 showed strong intracellular fluorescence response as compared to their Cys counterparts. This might have happened due to the acidic environment surrounding the cancer cells which favors increased fluorescence emission of OPD based CDs. Interestingly, a closer look into the confocal images also suggested that the localization of Cys based CDs were mostly restricted in the cytoplasmic milieu whereas, for OPD based CDs, the fluorescence response was also observed from nucleus. In vitro Quantification of Singlet Oxygen Production by CDs. To monitor whether the developed CDs can produce oxidative stress under laser irradiation, SOSG assay was performed. When the developed CDs were irradiated at 525 nm, the concentration of generated singlet oxygen was found to be ~0.7, 0.9, 4.15 and 6.8 µM for G-Cys1.0, F-Cys1.0, G-OPD1.0 and FOPD1.0, respectively (Figure S13 a). These results indicated that singlet oxygen generation was minimal in case of Cys based CDs as compared to OPD based CDs. This observation can be correlated with the fact that OPD based CDs showed higher killing in both bacterial and HeLa 21



cells. Previously in literature it was reported that the CDs produce singlet oxygen species, due to the energy transfer on the CDs surfaces, by the oxygen molecules involved in oxidation66. To cross check whether the developed CDs can produce singlet oxygen species under in vitro conditions, the SOSG studies were extended to bacterial cells viz., E.coli (Figure S13 b) and S.aureus (Figure S13 c) at 25, 50, 100, 200 and 300 µg/ml and for HeLa cells at 100, 200 and 300 µg/ml of CDs (Figure 7a). For bacterial studies, it was observed that G-OPD1.0 and FOPD1.0 showed marked increase in the amount of singlet oxygen species generation, as compared to G-Cys1.0 and F-Cys1.0 (Figure S13b,c). It was further observed that both the OPD based CDs, produced ~46% more singlet oxygen species in case of E.coli compared to S.aureus under similar conditions. Results of HeLa cells suggested that both G-Cys1.0 and F-Cys1.0 showed marginal increase (~1.5 – 1.9 times) in the singlet oxygen species concentration compared to control, whereas for G-OPD1.0 and F-OPD1.0, the same was observed to be ~ 12 – 25 times. Again, G-OPD1.0 and F-OPD1.0 showed ~3.9 – 8 times increase in the singlet oxygen species concentration compared to G-Cys1.0 and F-Cys1.0. It was also observed that the singlet oxygen species generation was directly proportional to the concentrations of OPD based CDs. All these studies suggested that the higher cytotoxicity of the OPD based CDs might have originated due to their strong ability to produce oxidative stress towards bacterial and HeLa cells. In addition, ROS quantification studies were performed to monitor the oxidative stress created by the developed CDs in both the bacteria as well as cancer cells. Approximately 2-2.25 times increase in the oxidative stress was observed for OPD based CDs compared to their Cys counterparts (Figure S14). All these results suggested that the developed CDs can generate ROS in both the bacteria and cancer cells, which might result in their killing. Total Thiol Estimation of CDs Treated HeLa Cells. To confirm whether there is any chemical structural correlation associated with the intracellular localization of CDs, total thiol estimation 22


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of both the treated and untreated cells were done using Ellman’s reagent (Figure 7b). Results suggested that compared to control (untreated sample), ~1.5 – 2 times increase in the total thiol concentration was observed for F-Cys1.0 and G-Cys1.0 treated samples respectively, whereas the same was found to be only ~0.25 times for G-OPD1.0 and F-OPD1.0. Previously, FTIR studies suggested absence of –SH functional group for Cys based CDs, though –C-S- bond was found to be intact. Thus it can be speculated that due to aerial oxidation, -SH groups were mostly converted to –S-S- bond. It is well known that the intracellular glutathione reductase catalyzes the reduction of glutathione disulfide (GSSG) to the sulfhydryl form glutathione (GSH). The fact that higher amount free thiol content was observed for Cys based CDs, suggested that the oxidized –S-S- coating present on these CDs were unstable under intracellular glutathione presence, which was also proved by their occurrence only at cytoplasmic content. The OPD based CDs though, can easily bypass such degradation process due to the unavailability of suitable functional groups on their surfaces and can be tracked in both cytoplasm and nuclear µµregion. Plausible Mechanism for Anti-bacterial and Anti-cancer Activity. To find out the possible molecular mechanism responsible for the anti-bacterial and anti-cancer activity of the developed CDs, the genomic DNA (gDNA) of both the bacterial cell lysates and nuclear DNA (nDNA) of HeLa cells were isolated. Gel electrophoresis studies suggested that CDs treated E.coli and S.aureus did not produce any noticeable DNA fragments, though higher intensity smears of degraded DNA were observed for E.coli, treated with OPD based CDs (Figure 7c and 7d). These results confirmed our previous observations that the developed CDs were more selective towards E.coli. However, for HeLa cell lysates, both G-Cys1.0 and F-Cys1.0 showed nDNA fragments of ~ 4500 kb which were absent in control sample (Figure 7e). Further, to our surprise, no DNA bands could be located for both G-OPD1.0 and F-OPD1.0. 23


It should be


mentioned here that the amount of nDNA isolated from G-Cys1.0 and F-Cys1.0 treated samples were found to be approximately ten times higher the concentration of G-OPD1.0 and F-OPD1.0 treated samples. The lower amount of nDNA concentration of G-OPD1.0 and F-OPD1.0 treated samples could have resulted from the higher cytotoxic potential of these CDs, compared to their Cys counterparts. All these results lead to the speculation that the anti-bacterial activity of the developed CDs involves three synchronous steps viz., rupturing of cell membrane through oxidative stress induced lipid peroxidation, followed by internalization of CDs along with DNA degradation and oozing out of the cytoplasmic content. It should be mentioned that Gramnegative bacteria are more prone to mechanical breakage due to the presence of thin layer of peptidoglycans on bacterial cells67-68. However, in HeLa cells, nuclear DNA damage could be speculated as one of the possible reason behind the anti-cancer activity of the CDs (Figure 7f). Conclusion In conclusion, using carbohydrates as major carbon source, we have synthesized 28 different CDs. The fluorescence emission of all the CDs suggested that under similar reaction condition, monosaccharides resulted better emission profile for CDs compared to disaccharides or polysaccharides. This observation can be supported by the fact that glycosidic ether linkage between sugars reduces the reactivity of both disaccharides or polysaccharides and hence the carbonization process was comparatively incomplete for sucrose and chitosan. The fact that OPD based CDs showed longer λmax values (beyond 520 nm), reflected that incorporation of both high degrees of aromaticity and nitrogen content may increase the possibility of getting nearinfra red (NIR) emitting CDs.

Chemical characterization followed by microscopic studies

suggested presence of sp2 hybridized graphite like structures for the developed CDs. Antibacterial studies, reflected, OPD based CDs were more effective towards E.coli with IC50 value of ~200 µg/ml, compared to Cys based CDs, though the fluorescence emission was strong for the 24


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later CDs. The confocal microscopy studies together with TEM results showed internalization of CDs inside bacterial cells and subsequent release of cytoplasmic content could have been the possible reason for bacterial killing. MTT assay studies on HeLa and HEK-293 cells showed Cys based CDs were more cyto-compatible compared to OPD based CDs. The thiol estimation studies on HeLa cell lysates documented increased thiol content for Cys based CDs. This allows the respective cells to resist oxidative stress, created by the internalized CDs, by maintaining the reducing environment of the cells, which can be a possible reason behind higher cytocompatibility % of Cys based CDs. HeLa cells treated G-OPD1.0 and F-OPD1.0 showed strong fluorescence emission, suggesting cellular acidic environment favors their use as molecular imaging probe. These developed CDs thus can find their role in advanced healthcare applications. Associated Content Supporting Information The Supporting Information is available free of charge on the Publications website at DOI: Absorption and fluorescence, stability analysis, FTIR, NMR, DLS and zeta potential, TEM, XRD,

anti-bacterial

efficacy

studies,

confocal

imaging,

cytocompatibility

studies,

hemocompatibility studies. Conflict of interest The author declares no competing interest. Acknowledgements The authors are thankful to the Director, CSIR-IHBT for infrastructural facilities.

AA

acknowledges the financial support from CSIR (MLP0201) and DST (GAP0214, EMR/2016/003027). SW, AKS and CS acknowledge CSIR for their respective fellowships. The CSIR-IHBT communication number of this manuscript is 4309. 25



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Page 36 of 50

Figure 1 (a)

CH2OH

CH2OH CH2OH

O

CH2OH

O

OH

D-glucose (G)

OH

D-sucrose (S) OH

OH

OH O

CH2OH

OH

D-fructose (F)

OH HO HO

O

OH

OH

OH

OH

OH

OH

NH2

NH2

O

O HO

O

O HO

O

OH

OH

OH

CH2OH

O

OH

P

OH

O NH2

n

Chitosan (CS)

OH

o-phosphoric acid (PA) SH

O

O

O

NH2

O H N

HS

OH

N H

HO

OH

NH2

NH2

L-cysteine (Cys)

NH2

O

L-glutathione (GSH)

o-phenylenediamine (OPD)

700

1.2

(b)

Fluorescence intensity

Absorbance

1.0

G-Cys 1.0 F-Cys 1.0 G-OPD1.0 F-OPD1.0

0.8 0.6 0.4 0.2

0.0 200 250 300 350 400 450 500 550 600

600

White light

F-Cys 1.0 G-OPD1.0 F-OPD1.0

500 400 300 200 100

0 400 450 500 550 600 650 700 750 800

Wavelength (nm)

Wavelength (nm)

(d) G-Cys1.0

G-Cys 1.0

(c)

(e) F-Cys1.0

G-OPD1.0

F-OPD1.0

G-Cys1.0

366 nm

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

36


F-Cys1.0

G-OPD1.0

F-OPD1.0

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


Figure 1.

(a) Chemical structures of different reagents used for the synthesis of CDs.

Absorbance and fluorescence spectra of G-Cys1.0, F-Cys1.0, G-OPD1.0 and F-OPD1.0 were depicted in (b) and (c), respectively. Excitation wavelength of G-Cys1.0 and F-Cys1.0 was 440 nm and the same was 480 nm for G-OPD1.0 and F-OPD1.0. Photographic images of TLC spotting (d) and UV light irradiation of CD solutions at 366 nm (e) suggested fluorescence behavior for the selected CDs.

37



Figure 2 (b)

(a)

F-OPD1.0

F-OPD1.0

G-OPD1.0 F-Cys 1.0

G-OPD1.0

G-Cys 1.0

Intensity

% Transmittance

OPD

F-Cys 1.0

Cys

F

G-Cys 1.0

G 4000 3500 3000 2500 2000 1500 1000 500

Wavelength (nm) 60

10 20 30 40 50 60 70 80 2-theta (degree) 30

(d)

50

Percentage (%)

Percentage (%)

(c)

40 30 20 10 0

4

6

8 10 Size (nm)

20 15 10 5 2

4

6 8 Size (nm)

10

12

7

8

d=0.26 nm

5 nm

5 nm 60

50

(f)

50 40

Percentage (%)

(e)

25

0

12

d=0.23 nm

Percentage (%)

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

Page 38 of 50

30 20 10 0

40 30 20 10

4 5 6 7 8 9 10 11 12 Size (nm)

0

2

3

4 5 6 Size (nm)

d=0.22 nm

d=0.20 nm 10 nm

5 nm

38


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


Figure 2. FTIR (a) and powder XRD (b) spectra of the synthesized CDs. The corresponding TEM micrographs of G-Cys1.0 (c), F-Cys1.0 (d), G-OPD1.0 (e) and F-OPD1.0 (f) suggested smaller particle size for the developed CDs. The particle size analysis graph and high magnification HRTEM images were given in inset for the respective CDs. Circles reflected CD localization in the TEM grids. The scale bar for TEM images were 100 nm whereas the corresponding scales for HRTEM were 5 nm, except (f), which is 10 nm.

39



Figure 3 50 E. coli

Cys-25

Cys-100

G-Cys1.0-25

G-Cys1.0-50

F-Cys1.0-25

Cys-50

Cys-200

F-Cys1.0-50

OPD-25

OPD-100

G-OPD1.0-25

OPD-200

G-OPD1.0-50

(b)

40

Cys OPD G-Cys 1.0

30

G-OPD1.0 F-OPD1.0

OPD-50

Zone of inhibition (mm)

(a)

F-Cys 1.0

20 10 0

50

25

10

0

20

0

30

Concentration (g/ml ) G-Cys1.0-100

G-Cys1.0-200

F-Cys1.0-100

F-Cys1.0-200

G-OPD1.0-100 G-OPD1.0-200

1.4 1.2

(c)

F-OPD1.0-50

Cys-300

OPD-300

F-Cys1.0-300

E.coli Cys-300 G-Cys 1.0-300 OPD-300 G-OPD1.0-300

OD600

F-OPD1.0-25

0

F-Cys 1.0-300

1.0

F-OPD1.0-300

0.8 0.6

F-OPD1.0-100 F-OPD1.0-200

G-Cys1.0-300 G-OPD1.0-300

0.4

F-OPD1.0-300

0.2 0 Cys-25

Cys-100

G-Cys1.0-25

G-Cys1.0-50

F-Cys1.0-25

OPD-25

Cys-50

Cys-200

F-Cys1.0-50

OPD-100

G-OPD1.0-25

50

OPD-50

OPD-200

G-OPD1.0-50

Zone of inhibition (mm)

S. aureus

2

(d)

40

G-Cys1.0-200

F-Cys1.0-100

F-Cys1.0-200

20 10 0

50

1.4

(e)

Cys-300

OPD-300

F-Cys1.0-300

0

20

0

30

0

S.aureus Cys-300 G-Cys 1.0-300 F-Cys 1.0-300 OPD-300 G-OPD1.0-300

1.0 F-OPD1.0-50

10

Concentration (g/ml )

G-OPD1.0-100 G-OPD1.0-200

10

G-OPD1.0 F-OPD1.0

30

1.2

F-OPD1.0-25

8

Cys OPD G-Cys 1.0

25 G-Cys1.0-100

4 6 Time (h)

F-Cys 1.0

0.8

OD600

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

Page 40 of 50

F-OPD1.0-300

0.6

F-OPD1.0-100 F-OPD1.0-200

G-Cys1.0-300 G-OPD1.0-300

F-OPD1.0-300

0.4 0.2 0.0

40


0

2

4 6 Time (h)

8

10

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


Figure 3. Anti-bacterial efficacy evaluation of the synthesized CDs. The photographic images of the zone of inhibition (ZOI) developed during disc diffusion method for different control molecules and CDs were compiled in (a).

The studies were performed at different

concentrations ranging from 25 – 300 µg. The corresponding bar diagram of ZOI for E.coli and S.aureus were given in (b) and (d), respectively. The growth inhibition curve for all the samples were presented in (c) and (e) for E.coli and S.aureus, respectively.

41



Figure 4 (a)

(b)

(c)

(d)

(e)

(f)

(g)

(h) 42


Page 42 of 50

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Figure 4. Confocal microscopy (first three columns) and TEM (last column) studies to evaluate the potential of CDs as anti-bacterial agents. Row (a-d) and (e-h) is for samples treated with E.coli and S.aureus, respectively where, (a & e) G-Cys1.0, (b & f) F-Cys1.0, (c & g) G-OPD1.0 and (d & h) F-OPD1.0.

The first, second and third column of the confocal microscopy is for

propidium iodide channel (PI, Ex. 477 nm, band pass: 560-615 nm), nanoparticle emission channel (for G-Cys1.0 and F-Cys1.0 Ex. 458 nm, band pass: 505-550 nm, blue emission; for GOPD1.0 and F-OPD1.0 Ex. 477 nm, band pass: 530–600 nm, yellow emission) and merged images, respectively. Red arrows indicated membrane disruption area for both the bacteria. Confocal microscopy scale: (a) and (d) 20000 nm and (b-c, e-h) 5000 nm. TEM scale: (a) and (c) 500 nm and (b, d, e-h) 100 nm.

43



Figure 5 150

150 Control 100g/ml 200g/ml 300g/ml 500g/ml 1000g/ml

(a)

125

***

***

100

Control 100g/ml 200g/ml 300g/ml 500g/ml 1000g/ml

(b)

***

100

***

***

75

75

50

50

25

25

*** *** ***

G-Cys1.0

F-Cys1.0

G-OPD1.0

0

F-OPD1.0

F-Cys1.0

G-OPD1.0

F-OPD1.0

1.2

1.4

(d)

G-Cys 1.0-0.325 F-Cys 1.0-1.25 F-Cys 1.0-0.625

0.8

F-Cys 1.0-0.325 F-Cys 1.0-1.25

0.6

G-OPD1.0-0.625 G-OPD1.0-0.325

0.4

F-OPD1.0-1.25 F-OPD1.0-0.625 F-OPD1.0-0.325

0.2 550

600

650

700

0.6 0.4 0.2 0.0

X-

0.0 500

0.8

0

G-Cys 1.0-0.625

1.0

1.0

1.

(c)

Triton X PBS G-Cys 1.0-1.25

Relative % hemolysis

1.2

G-Cys1.0

1. 25 0. 62 0. 5 31 2 1. 5 25 0. 62 0. 5 31 1. 25 25 0. 62 0. 5 31 2 1. 5 25 0. 62 0. 5 31 25

0

10 0 PB G S -C y G s1 - C .0 ys G 1 - C .0 y F- s1 C .0 ys F- 1 C .0 y F- s1 C .0 y G s1 - O .0 G PD -O 1 . P 0 G D -O 1 . 0 F- P D O 1. PD 0 FO 1 .0 PD FO 1 PD.0

% Cell Viability

125

Absorbance

ito

n

Wavelength(nm)

Tr

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

Page 44 of 50

(e) T

P

A1

A2

A3

B1

B2

B3

C1

C2

C3

D1

D2

D3

Figure 5. MTT assay of HeLa cells treated with CDs at different concentrations viz., 100, 200, 300, 500 and 1000 µg/ml, at (a) 24 and (b) 48 h, respectively. *** Indicates statistically significant value of p

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