Antiparasitic Ointment Based on a Biocompatibile Carbon Dot

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Antiparasitic Ointment based on Biocompatibile Carbon Dot Nanocomposite Vijay Bhooshan Kumar, Avishay Dolitzky, Shulamit Michaeli, and Aharon Gedanken ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00213 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 14, 2018

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ACS Applied Nano Materials

Antiparasitic Ointment based on Biocompatibile Carbon Dot Nanocomposite Vijay Bhooshan Kumara, Avishay Dolitzkyb, Shulamit Michaelib,*, and Aharon Gedankena,* (Note: Vijay Bhooshan Kumar and Avishay Dolitzky contributed equally to this work) a

Institute for Nanotechnology and Advanced Materials and Department of Chemistry, Bar-Ilan University, Ramat Gan 5290002, Israel b Institute for Nanotechnology and Advanced Materials and Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel *Corresponding authors: [email protected] (A.G.) and [email protected] (S.M.) Tel: 972-3-5318315; Fax: 972-3-73804053

ABSTRACT Toward the development of emerging drugs with high efficacy, non-toxicity and low drug resistance against Leishmaniasis, this study unravels the potential of carbon dots (CDs) and gallium doped carbon dots (Ga@CDs). These nanoscale materials sizing from 4-7 nm prepared by ultrasonication without catalyst were well dispersed in a commercial ointment. The formulated ointments with CDs and Ga@CDs exhibited higher activity against both Leishmania species, a minimal concentration of 30 µg/ml for CDs/Ga@CDs, compared with a commercial counterpart. CDs were virtually non-toxic as attested by in vitro and in vivo experimental data using mice and healthy cells. The “killing” mechanism could be attributed to the leakage of Na and K whereas lysosomal bursting and depolarization of mitochondria, ion leakage were ruled out. The ointments could be considered as a new class of emerging drugs to combat Leishmaniasis, a deadly disease that still infects several million people worldwide, especially in Asia and South America.

Keywords: CDs, Ga@CDs, ointment, fluorescence, sonochemistry, leishmaniasis.

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1. INTRODUCTION Leishmaniasis is one of the most serious vector-borne human diseases, which is caused by obligate intracellular protozoa of the genus Leishmania via transmission by sand flies.1,2 Over 350 million people are at risk, and 14 million people in 98 countries are infected with skin ulcers, nodules, and a deadly visceral leishmaniasis1 (by the World Health Organization). Chronic, visceral, and acute leishmaniasis are a group of protozoan infections caused by obligate intracellular protozoan parasites to effect skin disease or visceral organ damage. Infected patients require prolonged hospitalization with high mortality from the viscral disease, especially in endemic regions of 90 countries worldwide3,4 where two million new cases emerge annually.3,5. For detecting the Leishmania species

6,7

, epidemiological and clinical findings are necessary

but insufficient for the identification of the causative agents. The most common diagnostic test is based on light microscope examination of an in vitro culture from biopsy or lymph aspirate. Others include Montenegro skin test, serological tests, or molecular tests (such as the polymerase chain reaction).8 Such methods, however, are not very sensitive, time consuming and require the culturing parasites or passage through an experimental animal. Antibodybased tests cannot detect early infections or distinguish between the past and present infections, and cross reactions of antibodies with other pathogens9. Thus, it is of urgency to develop the sensitive, reliable and cost-effective procedure for the early detection of this disease. As newly emerging carbon materials, nanoscale CDs exhibit superior optical properties, biocompatibility, and low cost of preparation.10,11. Therefore, they are advocated for diversified applications such as bioimaging12,13, cell imaging12, Anti-Inflammation14, VisibleLight-Activated Bactericidal15, dye degradation10, chemiluminescence16, solar cells17,18, and gene delivery19. Of notice is the disruption of the iron metabolism of Pseudomonas 2 ACS Paragon Plus Environment

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aeruginosa by gallium ions (Ga3+) 20–22, which is attributed to the similar chemical properties between Ga3+ and Fe3+. They are alike in their charge, ionic radius, electronic configuration, and coordination number. Moreover, the CDs are water soluble and free of toxic heavy metals, such as cadmium or lead.23,24 Recently, CDs and Ga-doped CDs (Ga@CDs have been synthesized from polyethylene glycol by catalyst-free ultrasonication25. In particular, Ga@CDs exhibit antibacterial activity against Pseudomonas aeruginosa26, promote the growth of neuron cells27 and are proven effective in photodynamic therapy28. In this study, our goal is to create fluorescent CDs based ointment against L. donovani, a common protozoan leishmanial parasite as a model with no side effects that might damaged the body of the host, and good solubility. The pristine ointment lack fluorescence properties, which makes it impossible for the researchers to track the location of the oinment in cells or tissues during cell imaging, drug targeting and delivery applications in vivo. The addition of the fluporescing dots makes the oinment detactable. In this context, we report on the biocompatible fluorescing CDs based ointment for anti-parasitic agent. It will enable to trace the NPs in the cells. The cytotoxicity and biocompatibility aspect of CDs/Ga@CDs was also investigated and validated for the human blood cell and mice, in vivo. A plausible parasitic killing mechanism is proposed and supported by pertinent experimental evidence.

2. EXPERIMENTAL METHODS 2.1 Chemicals: PEG-400 (99.98%) and gallium (Ga, 69.7 g/mole, 99.999%) were purchased from Sigma-Aldrich. The cetomacrogol cream ointment was purchased from Blush Pharmacy, Nexcape Pharmaceuticals Ltd., Hertfordshire, UK. 2.2 Synthesis of CDs and Ga@CDs: CDs and Ga@CDs were synthesized as described previously

25,28

. Briefly, a granule of gallium (~140 mg) was inserted into a glass test tube

containing 20 ml of PEG-400. The tip of an ultrasonic Ti-horn transducer (Sonics and Materials Inc., USA, model VCX 750, frequency 20 kHz, 230V AC) was penetrated in the 3 ACS Paragon Plus Environment


solution to ~25 mm above the test tube bottom. Upon the melting of gallium, ultrasonic irradiation was applied for 2.5 h at an amplitude of 70%, enabling the dispersion of the gallium as particles to form a gray suspension. The suspension was separated by centrifugation at 10,000 rpm for 15 min, resulting in a light brown-yellow supernatant with Ga@CDs. The same procedure was followed for the synthesis of carbon dots without gallium. Finally the dialysis bag (3kD) was used for the purification of CDs or Ga@CDs materials. 2.3 Preparation of the CDs/Ga@CDs ointment: The CD suspension obtained by sonicating PEG-400 for 2.5 h was mixed with the ointment (Scheme-1). The CD amount in the PEG-400 was calculated from the sonicated solution absorbance. The CDs/Ga@CDs ointment was prepared by mixing various weight percentages (1, 2, and 5 wt%) of CDs suspended in the PEG-400 solution and cetomacrogol cream with bath sonication for 10 min at 10 °C (icewater bath). Cetomacrogol is an emollient or moisturizer for the treatment dry skin. It also acts as an emulsifier in commercial ointments without any antiparasitic activity as confirmed in this study. 2.4 Analytical techniques: The fluorescence of Ga@CDs was measured using a Varian Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). The CD/Ga@CD ointment nanocomposites were probed by a transmission electron microscope (TEM, JEOL 2100, with an accelerating voltage of 200 kV). The TEM samples were prepared by mixing two drops of a CD/Ga@CD ointment suspension (0.2 ml) with isopropanol (5 ml), followed by sonication for 2 min at room temperature. A few droplets of the mixture were applied to a carbon-coated copper TEM grid and dried under vacuum. Dynamic light scattering (DLS) measurements of the ointment were performed by a ZetaSizer Nano-ZS (Malvern Instruments Ltd., Worcestershire, UK) for size characterization. The Fouriertransform infrared (FTIR) spectra of CDs and the ointment were recorded using a FTIR spectrophotometer (Bruker TENSOR 27, Platinum ATR diamond F) from 4000-400 cm-1. A Bruker D8 Advance (Bruker AXS GmbH, 49, 76187 Karlsruhe, Germany) and a Philips 4 ACS Paragon Plus Environment

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PW1050 X-ray diffractometer (Cu Kα radiation, operating at 40 kV/30 mA with a 0.0019 step size and a 0.5 s step) were used for X-ray diffraction (XRD) measurements. The chemical composition and oxydation state of CDs/Ga@CDs were analyzed by X-ray photoelectron spectroscopy (XPS) using a VG ESCA 3 MK II XPS installation ( eV).

2.5 Cell lines: Parasites. L. donovani promastigotes, L. donovani promastigotes chromosome (Ld1S-GFP) and L. major promastigotes were grown at 270 C in the M199 medium at pH 7.4 with 10% fetal calf serum. THP-1 human leukemia cultured cells (THP-1 cell lines) were grown at 37 0C and 5% CO2 in the RPMI medium with 10% fetal calf serum. 2.6 Antiparasitic assay: L. donovani parasites (2×106) were treated with CDs or Ga@CDs at different concentrations (0, 10, 20, 30, and 40 µg/ml), and incubated for 24 h. Effector cells were washed with phosphate buffer saline (PBS) and stained with propidium iodide (PI). The sample with 10,000 cells was analyzed by fluorescence-activated cell sorting (FACS). The data are expressed as the mean ± structural equation modeling (SEM) of three different experiments. The results were analysed using a one-way analysis of variance (ANOVA) with multiple comparison Bonferroni post hoc analysis. . 2.7 Lysosomal bursting analysis: Leishmanial parasites (106) were treated with 30 µg/ml of CDs for 5 min. The cells were washed with PBS, and the lysosome was stained with a lysotracker (red). Images taken by a Nikon Eclipse 90i (x63) camera were used for counting the number of cells with the lysosome. 2.8 Plate cultures of parasites: Parasites (108) were plated on a semi-agar medium, and cells were treated by CDs/ointment nanocomposites and incubated for 72 h at 27 °C. Proteins were shifted to nitrocellulose membranes and were stained with Ponso. 2.9 Mitochondrial and inductively coupled plasma (ICP) sample preparation: L. donovani (2×107) parasites were incubated with 30 µg/ml of CDs and 150 nM of tetramethylrhodamine methyl ester (TMRM) for 30 min in a serum-free medium. After 5 ACS Paragon Plus Environment


washing twice with PBS, the resulting cells were analyzed by FACS. The same materials were also analyzed by ICP to measure the efflux of Na+ and K+ from the leishmanial parasite treated with the CDs/Ga@CDs@Oi nanocomposites. 2.10 in-vivo toxicity: Laboratory-bred healthy female mice (12 weeks) were maintained under the controlled temperature at 27 ± 2 °C with a 12 h light/dark cycle where food and water were provided. Among 18 BALB/c mice, they were divided into three groups and injected intravenously into the tail vein with 0.4 or 0.8 mg/kg of C-dot NPs, or with PBS as a control. After one week of observation all mice exhibited no clinical signs were identified. For the toxicity of the C-dots, several biochemical and hematological parameters were tested seven days following the injection, focusing on liver and kidney toxicity. All animal experiments were performed in compliance with the Guidelines for the Care and Use of Research Animals established by the Bar-Ilan University Animal Studies Committee. 2.11 Statistical evaluation: The in vivo and in vitro experiments on mice were repeated five replicates, and the results are expressed as the mean ± SD (standard deviation). Data obtained under various stages were examined statistically by the group t-test for independent samples (P ≤ 0.01). Statistical analysis of collected data was performed with the GraphPad Prism software.

3. RESULTS AND DISCUSSION 3.1 Physical and chemical CDs and CDs/ointment nanocomposites: CDs and Ga@CDs, the ointment, and the CDs and Ga@CDs in the ointment, were visualized under daylight. The CDs, Ga@CDs, ointment, CDs@Oi, and Ga@CDs@Oi appeared as light yellow materials (Figure S1a), but under UV light exposure (365 nm), the emission turned to greenish blue (Figure S1b). There was no change in the fluorescence even after the drying of the CD@Oi nanocomposites.


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The TEM results revealed the successful dispersion of CDs and Ga@CDs in the ointment with the sizes ranging 4-8 nm from (Figure 1a), compared to 5-9 nm for Ga@CDs (Figure 1b). The chemical composition of of single particles of Ga@CDs was analysis by HRTEM with EDS spectrum. The EDS spectrum reveals that most of the gallium atoms are inside the CDs (Figure S2a, S2b). The fluorescence emission of the supernatant solution containing CDs/Ga@CDs was monitorred in the 420-510 nm region (the most intensed emission is found centered at 470 nm, see supporting information Figure S3a,b) under different excitation wavelengths (330 up to 490 nm). The pristine ointment shows very negligible fluorescence (Figure S3c), whereas CDs@Oi shows identical emission spectrum to that of the CDs but the fluorescence intensity is reduced (Figure S3d). For the assessment of antiparasitic activity, CDs/Ga@CDs were added to the ointment to form a nanocomposite ointment. After mixing various CD concentrations into the ointment, the TEM image was recorded (Figure 1, panels’ c and d). The d-spacing estimated from the HRTEM and selected area electron diffraction (SAED) patterns 0.21 nm was observed. The micrograph depicted the virtually uniform distribution of CDs and Ga@CDs in the ointment (Figure S4a and S4b), and the size of CDs remained unchanged. Figure 1c, and Figure 1d present the TEM micrograph of CDs and Ga@CD particles, respectively incorporated in the ointment. To know the crystallinity of the CDs and Ga@CDs was analysed by XRD analysis. The CDs and Ga@CDs were dried on hot plate at 150 °C and powders of CDs and Ga@CDs were used for the XRD analysis. A single broad common peak at 2θ=23.1 is observed for both sample which is the typical for graphitic nature of CDs/Ga@CDs. The large XRD reflection intensity of Ga@CDs may be due to the doping of Ga (Figure S5). In a previous reports28 we have explained the presence of Ga in the C-dots on the basis of XPS and Zetapotentials. The details are presented in reference 28 and repeating the assignment is redundent. The doping of Ga and chemical composition of CDs/Ga@CDs was examined by XPS (Figure S6). The XPS measurement, confirming that Ga@C-dots are 7 ACS Paragon Plus Environment


composed of Ga 2p (1118.6 eV Fig. S6d ), Ga 3p (106.4 and 109.5 eV, Fig. S6b), and Ga 3d (20.3 eV, Fig. S6c) C 1s (284.9 eV (Fig. S6f)), and O 1s (532.3 eV, Fig. S6e). Recently Ga@C-dots were also analysed by HRTEM with high resolution energy dispersive spectroscopy. The HRTEM images revealed that both CDs and Ga@CDs were highly crystalline with an estimated d-spacing of 0.21 and 0.22 nm, respectively, in agreement with the literature29. The nanoparticles were monodispersed in water or isopropanol medium as confirmed by DLS. The mean hydrodynamic diameters of the CDs/Ga@CDs composites in the ointment (Figure 2 and Table 1) increased from 500 to 600 nm. The CDs were incorporated in the ointment as their hydroxyl and carboxyl group on the surface can form hydrogen bonding with -C=O of the ointment and/or electrostatic interaction (Figure 3a). The peak at 1748 cm−1 is attributed to the stretching of carbonyl groups (C=O) of the polymer. In the PEG bonding, the peak at 1094 cm-1 is the stretching band of the C–O bond. The peaks at 1650 and 1530 cm-l represent the C=O stretching vibration and –OHbending vibration of alcohol, respectively (Figure 3). Apparently, stable hydrogen bonds were formed as a result of the interaction of the CDs/Ga@CDs surface carboxyl/hydroxyl groups with the ointment CO groups (Figure 3b). 3.2 Parasitic activity of CDs@Oi nanocomposites: Social motility in parasites enable their culturing on semisolid agarose surfaces to form colony-like patterns. This feature was applied to investigate the influence of materials on their growth. In this context, we compared CDs-, Ga@CDs-ointments with the ointment per se. Parasites (108) plated on semisolid agarose surfaces were treated by the CDs- and Ga@CDs- ointments and incubated for 72 h at 27 °C. The Leishmania parasitic proteins were shifted to a nitrocellulose membrane and stained with Ponso. The growth of untreated cells (PBS) and cells treated with the ointment to form colonies was expected whereas CDs exhibited some noticeable effect on the colony formation. (Figure S7). Notice also is the significant anti-parasitic activity of the CDs8 ACS Paragon Plus Environment

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ointment, compared with the pristine ointment (Figure S7a) or CDs alone (Figure S7c). The Ga@CDs ointment also exhibited a higher anti-parasitic activity compared with galliumdoped CDs alone (Figure S7b). The antiparasitic activity of CDs@oi is stronger than either oi or of the CDs. This is because the CDs in the ointment are well dispersed (see Figure 1c), whereas the CDs in the solution are moving and colliding and they aggregate even over a very short times. That is the reason that the activity of CDs in the ointment is more effective. Finally, the incorporation of Ga and CDs in the ointment slightly enhanced the anti-parasitic effect over the CDs-ointment. Considering the long-term cytotoxicity effect of Ga, only the CDs and CDs-ointment were used in the subsequent experiments. 3.2.1 In vitro drug sensitivity assay: 10-30 and 10-40 µg/ml of the CD ointment were established as the required concentration for obliterating Leishmania parasites (Figure 4a, and 4b) An experiment was also conducted to examine the effect of CDs alone on THP-1 cells30,31 using fluorescence microscopy. These cells were differentiated to macrophage cells and used as the host cell for leishmanial parasites by using 12-O-tetradecanoylphorbol-13acetate (PMA). In this case, THP-1 cells (106) were plated in a 24 well plate and treated with PMA, followed by incubation at 37 °C for 48 h to become macrophage cells. The cells were then infected with L. donovani promastigote (GFP) parasites (5×106) and kept for 24 h at 37 °C. After washing the cells for the removal of unreacted parasites, the infected cells were then treated overnight with CD particles at 30 µg/ml. The macrophage nuclei were stained with DAPI (blue), and the results were obtained by ‘live imaging’ microscopy, using a Zeiss Observer Z1 (X40). As shown Figure 5b and 5d, there was a significant reduction in the number of parasites, suggesting that the CDs were able to penetrate the host cell and exhibited a detrimental effect on parasites in the amastigote form of Leishmania. The results shown in this work demonstrate a highly effective nanoparticles as a drug against Leishmaniaparasites. We are using unique CDs which is carbon-based materials comparing to other inorganic 9 ACS Paragon Plus Environment


materials previously described.32 This unique property of our particles makes it a non-toxic and efficient tool to deal with Leishmania parasites. The toxicity of the CDs was studied by comparing untreated cells with cells treated with 40 and 80 µg/ml of CD particles. The toxicity was analyzed by measuring the DNA fragmentation, one of the best markers of apoptosis or necrosis33, at cell cycle events using a flow cytometer. Apoptosis or necrosis is recognized by a "sub G1 peak / hypodiploid peak” (Figure 6). The DNA was stained with PI. Despite at 80 µg/ml of CDs, there was no decrease in the cell population in the sub-G1 phase of the cell cycle (in THP-1 monocytes). Therefore, the CD particles were virtually non-toxic to the monocyte cells compared to the Leishmania parasites. 3.2.2. In-Vivo toxicity: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), released from damaged liver cells to the blood, are markers for liver toxicity. For the evaluation of hematological parameters, ~150 µl of blood were collected in EDTA-coated tubes. For the evaluation of biochemical parameters, ~350 µl of blood was collected in non-coated gel tubes, centrifuged (4000 g, 4 min, RT), and serum separated. Both EDTA-coated tubes and serum samples were transferred to American Medical Laboratories (AML, Herzliya Medical Center, Israel) for further analysis. Additionally, raised bilirubin serum levels, as well as decreased total protein and albumin serum levels, can indicate the liver dysfunction (Table 2). Hematological parameters including blood cell counts are also useful markers for a clinical health condition. Based on the obtained values for such parameters, the injection of CDs NPs at 0.8 mg/kg was virtually nontoxic to the mice. The formulation of the ointment with CDs at low dosage and nontoxicity appears promising as a potential nanomaterial to combat leishmaniasis provided the long-term effect to human is minimal, a subject of further study. The available drugs still have several shortcomings such as adverse effects to human 34,35, species-specific activity,34 or low efficacy35 10 ACS Paragon Plus Environment

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3.3 A proposed mechanism of killing the Leishmania parasite: The CD with many surface functional groups, such as OH, COOH, and C=O, were internalized by Leishmania as reflected by the confocal microscopy data (Video 1), causing the morphological changes in L. donovani. The untreated parasite (without CDs) appeared in their normal shape together with daughter cells, whereas the treated parasite (with CDs) was under stress. Similarly to untreated cells, the cells treated with CDs still appeared in red, an indication that the lyosome was still intact (Figure S8). Next, we examined if the killing activity of the CDs is due to the depolarization of the mitochondrial membrane. Tetramethylrhodamine methyl-ester (TMRM) was used to examine chnages in membrane potential 36. The results indicate that there was no appreciable difference between the treated and untreated cells regarding the mitochondrial membrane potential (Figure S9). In brief, there is a decrease in the mitochondrial membrane potential during the process of apoptosis as TMRM cationic materials enter the cells and accumulate in the negative mitochondrial matrix, depending on the mitochondrial membrane potential.37 Other possibilty is that the positive charge on the surface of CDs play some role in antiparasitic activity. However, such an effect was not observed from the result of the CDs treatment. Finally, the leakage of Na+ and K+ was confirmed by ICP, as reflected by the higher Na+ and K+ concentrations in the treated cells, by ~11 % as summarized in Table 3. Thus it seems that the nanoscale CDs interact with the the cell membrane of Leishmania and can potentially poke holes in the outer membrane causing entrance of ions resulting in cellular imbalance (Figure 7). In addition, the CDs which are small can enter the parasite via the flagellar pocket on route to the endocytic pathway. Upon their penetration, CDs can interact with the endocytic vesicles and generate pores (Figure 7a-c). Consequently, the demage especially to the cell membrane should lead to cytoplasmic leakage and death of the parasite (Figure 7d-e). Ga@CDs and CDs can produce reactive oxygen species (ROS) by photoexcitation28. However, ROS should affect the mitochondrial memebrame potential and



since we obaerved little effect on the mitochondria this effect may not be domininat in the killing mechanims of the parasites by CDs.

4. CONCLUSIONS Synthesized nanoscale CDs and Ga@CDs (4-8 nm) incorporated in the ointment exhibited a comparable anti-parasitic activity against L. donovani and L. major. The Ga undoped CDs were equaly effective against the parasites but displayed no harm to the host cells. Pertinent evidence pointed out the killing mechanism could be attributed to the ion imbalance due to the penetration of the nanomaterials in the cell membrane and interact with other biomolecules and organelles. However, lysosomal bursting and membrane depolarization were ruled out as the cause oft he anti-parasitic activity. Considering their efficacy and non-cytotoxicity, these new materials are warranted for further investigation to assess their potential application against Leishmaniasis and other parasites.

ACKNOWLEDGMENTS The authors are grateful to Ortal Lidor-Shalev for help with the TEM measurements, to Daniel Raichman for Raman measurements, and to Merav Muallem for the HRSEM measurements, carried out at the Department of Chemistry of Bar-Ilan University, Israel. List of abbreviations CDs: carbon-dots DLS: dynamic light scattering PBS: phosphate buffer saline Ga@CDs: Gallium doped carbon-dots Oi: ointment HRTEM; high-resolution transmission-electron microscope.


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Supplementary Information Supplementary data include the optical image, fluorescence, confocal microscope and toxicity data of CDs, CDs@Oi.

Competing interests

None of the authors have any competing interests in the manuscript.



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Video

vijay1.mp4 Video 1. Effects of CDs before and after treatment on Leishmania (release Na+ and K+ ion).



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Tables Table 1: Summary of particle sizes and zeta potentials. Type of material

Size of the particles by DLS (nm)

Size of the particles by TEM (nm)

Zeta potentials (mV)

CDs

~7

~5

-25

Ga@CDs

~8

~6

+19

CD@Oi

~60 and ~500

~60, ~300

+15

Ga@CDs@Oi

~60 and ~500

~60, ~300

+24

Ointment

~70 and ~500

~60, ~300

+20

Table 2: Analyses of the biochemistry and the hematological parameters after seven days following intravenous injection of 0.4 or 0.8 mg/kg of C-dot NPs. Data are expressed as mean ± SD according to the results of a mixed-design ANOVA with repeated measurements and a multiple comparison Bonferroni post hoc analysis. (NA - not applicable). Control

0.4 mg/kg

0.8 mg/kg

units

Mean (SD)

SD

Mean

SD

Mean

SD

Total protein

g/dl

5.2

0.4

5.1

0.2

5.1

0.2

WBC

10^3/µl

7.2

2.1

9.0

1.2

9.9

2.1

RBC

10^6/µl

9.9

0.5

9.9

0.3

10.1

0.2

HGB

g/dl

15.8

0.7

15.9

0.3

16.0

0.6

HCT

%

48.3

4.1

49.9

1.2

50.4

1.4

MCV

fL

48.8

2.1

50.4

0.5

49.9

0.1

MCH

Pg

15.9

0.3

16.1

0.2

15.8

0.2

MCHC

g/dl

32.8

1.6

31.9

0.3

31.7

Neutrophils

%

20.7

4.8

16.2

0.7

16.7

3.8

Lymphocytes

%

71.5

2.6

74.9

3.4

74.4

4.5

Monocytes

%

1.0

0.2

1.1

0.2

1.5

0.1

Eosinophils

%

6.2

4.3

7

3.4

6.6

2.3

Basoohils

%

0.2

0.0

0.3

0.06

0.2

0.1

Platelets

10^3/µl

689.7

261.7

862.3

67.5

771.5

81.4

Creatinine

mg/dl

0.03

N/A

0.07

0.01

0.12

0.1

Urea

mg/dl

47.3

9.7

42.4

10.2

41.4

1.1


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Albumin

g/dl

3.8

0.3

3.8

0.4

3.7

0.1

Total Bilirubin

mg/dl

0.04

0.1

0.1

0.0

0.07

0.0

ALP

IU/L

180.3

54.4

228.5

23.4

209.0

19.2

AST=SGOT

IU/L

177.8

119.9

506.0

296.9

222.5

39.0

ALT=SGPT

IU/L

144.3

99.4

187.7

150.0

153.3

47.8

Na

mmol/L

145.5

1.0

146.0

2.9

146.0

2.8

K

mmol/L

11.4

0.8

11.6

1.2

11.2

0.3

Calc

mg/dl

9.8

0.3

10.1

0.5

9.7

0.3

Phos

mg/dl

7.8

0.9

9.7

2.5

8.1

0.6

Glucose

mg/dl

90.5

16.5

117.5

59.7

92.3

13.9

Cholesterol

mg/dl

93.0

9.6

122.5

46.4

90.8

3.6

Glob

g/dl

1.4

0.1

1.4

0.2

1.5

0.1

Table 3: ICP analysis of leakage ions after treatment with the parasite. Type of materials Untreated cell (treated with PBS only) Treated cell (treated with CDs) PBS + CDs PBS

Na+ ± Std. dev. K+ ± Std. dev. Differences in (ppm) (ppm) Na+ ion = parasite - PBS 11200 ± 80 1070 ± 67 350 ± 57

Differences in K+ ion = parasite - PBS 170 ± 17

12800 ± 150

1200 ± 10

1950 ± 30

300 ± 10

10950 ± 140 10850 ± 150

1000 ± 30 900 ± 50

100 ± 10 0

100 ± 10 0



Figures

Scheme 1. The representative scheme of CDs/Ga@CDs formation by sonication and CDs/Ga@CDs@Oi by vgorous stirring conditions.


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Figure 1. HRETEM images of (a) CDs (inset: lattice image), (b) Ga@CD (inset: lattice image), (c) CDs ointment, and (d) Ga@CDs ointment.



Figure 2. Size distribution of (a) CDs (b) CD@Oi, (c) Ga@CDs@Oi, and (d) ointment.


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Figure 3. (a) FTIR spectra of CDs, ointment, and CDs@Oi nanocomposites, (b) Possible chemical interaction of CDs and Ointment



Figure 4. Parasitic activity of CDs at various concentration (0-45 µg/ml) for (a) L. major and (b) L. donovani. (** p value < 0.005, *** p value < 0.0001)

Figure 5. Effects of CDs on the parasitic growth of (a,b) L. major and (c, d) L. donovani.


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Figure 6. Effects of (a) CDs, (b) Ga@CDs, and (c) Ointment on THP-1 cells cell cycle and cytotoxicity. The percentage of sub-G1, G0/G1, S, and G2/M fractions were analysed using the CellQuest software.

Figure 7. Plausible mechanism involved in antiparasitic action of CDs, (a-c) Interactions of CDs with cell membrane of Leishmania, (b, c) entrance of CDs into the Leishmania (d, e) cell damage of cell membrane and Na+ and + outflow along with cytoplasmic leakage.



TOC graphics Antiparasitic Ointment based on Carbon Dots Nanocomposite


Page 26 of 26

Antiparasitic Ointment Based on a Biocompatibile Carbon Dot

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