Antitubercular activity of ZnO nanoparticles prepared by solution combustion synthesis using lemon juice as bio-fuel

Accepted Manuscript Antitubercular activity of ZnO nanoparticles prepared by solution combustion synthesis using lemon juice as bio-fuel

G.K. Prashanth, P.A. Prashanth, Priyanka Trivedi, Vinita Chaturvedi, B.M. Nagabhushana, S. Ananda, Amani Erra, Y. Tejabhiram PII: DOI: Reference:

S0928-4931(16)31375-3 doi: 10.1016/j.msec.2017.02.093 MSC 7427

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

17 November 2016 6 January 2017 21 February 2017

Please cite this article as: G.K. Prashanth, P.A. Prashanth, Priyanka Trivedi, Vinita Chaturvedi, B.M. Nagabhushana, S. Ananda, Amani Erra, Y. Tejabhiram , Antitubercular activity of ZnO nanoparticles prepared by solution combustion synthesis using lemon juice as bio-fuel. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi: 10.1016/j.msec.2017.02.093

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ACCEPTED MANUSCRIPT Antitubercular activity of ZnO nanoparticles prepared by solution combustion synthesis using lemon juice as bio-fuel

G.K. Prashantha, b, P.A. Prashanthb, c, *, Priyanka Trivedid, Vinita Chaturvedid,**, B.M.

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Department of Chemistry, Sir M. Visvesvaraya Institute of Technology, Bengaluru-562 157,

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Nagabhushanae, S. Anandaf, Amani Errag, Y. Tejabhiramh,

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Department of Chemistry, Sai Vidya Institute of Technology, Bengaluru-560 064, India

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Biochemistry Division, Central Drug Research Institute, CSIR, Lucknow-226031, India e

Department of Chemistry, University of Mysore, Mysuru-560 006

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Department of Chemistry, M. S. Ramaiah Institute of Technology, Bengaluru-560 054, India

Department of Internal Medicine, Presence Saint Joseph hospital, Chicago, IL 60657 USA

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Department of Ophthalmology and Visual Sciences, University of Illinois, Chicago-60612, USA

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Corresponding authors:

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*1. Dr. P. A. Prashanth, Professor & Head, Dept. of Chemistry

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Research and Development Centre, Bharathiar University, Coimbatore-641 046, India

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b.

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India

Sai Vidya Institute of Technology, Rajanukunte, Bengaluru-560 054 [email protected], Ph: 91 9663313591, Fax: 91-80-2846 8193

**2. Dr. Vinita Chaturvedi, Senior Principal Scientist Biochemistry Division, CSIR-Central Drug Research Institute Sector 10, Jankipuram Ext., Sitapur Road, Lucknow-226031

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[email protected], Ph: 0522-2772450, 2772550

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Antitubercular activity of ZnO nanoparticles prepared by solution combustion synthesis using lemon juice as bio-fuel

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G.K. Prashantha, b, P.A. Prashanthb, c, *, Priyanka Trivedid, Vinita Chaturvedid,**, B.M.

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Department of Chemistry, Sir M. Visvesvaraya Institute of Technology, Bengaluru-562 157,

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g.

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Nagabhushanae, S. Anandaf, Y. Tejabhiramg

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Research and Development Centre, Bharathiar University, Coimbatore-641 046, India

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Department of Chemistry, Sai Vidya Institute of Technology, Bengaluru-560 064, India Biochemistry Division, Central Drug Research Institute, CSIR, Lucknow-226031, India e

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Department of Chemistry, M. S. Ramaiah Institute of Technology, Bengaluru-560 054, India

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Department of Chemistry, University of Mysore, Mysuru-560 006 Department of Ophthalmology and Visual Sciences, University of Illinois, Chicago-60612, USA

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Corresponding authors:

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h.

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India

*1. Dr. P. A. Prashanth, Professor & Head, Dept. of Chemistry

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Sai Vidya Institute of Technology, Rajanukunte, Bengaluru-560 054 [email protected], Ph: 91 9663313591, Fax: 91-80-2846 8193

**2. Dr. Vinita Chaturvedi, Senior Principal Scientist Biochemistry Division, CSIR-Central Drug Research Institute Sector 10, Jankipuram Ext., Sitapur Road, Lucknow-226031

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ACCEPTED MANUSCRIPT [email protected], Ph: 0522-2772450, 2772550 Abstract: In this study, we report the synthesis, structural and morphological characteristics of zinc oxide (ZnO) nanoparticles using solution combustion synthesis method where lemon juice was used as the fuel. In vitro anti-tubercular activity of the synthesized ZnO nanoparticles and

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their biocompatibility studies, both in vitro and in vivo were carried out. The synthesized

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nanoparticles showed inhibition of Mycobacterium tuberculosis H37Ra strain at concentrations

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as low as 12.5 µg/mL. In vitro cytotoxicity study performed with normal mammalian cells (L929, 3T3-L1) showed that ZnO nanoparticles are non-toxic with a Selectivity Index (SI) >10.

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Cytotoxicity performed on two human cancer cell lines DU-145 and Calu-6 indicated the antiResults of blood hemolysis

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cancer activity of ZnO nanoparticles at varied concentrations.

indicated the biocompatibility of ZnO nanoparticles. Furthermore, in vivo toxicity studies of ZnO

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nanoparticles conducted on Swiss albino mice (for 14 days as per the OECD 423 guidelines)

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showed no evident toxicity.

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Keywords: ZnO nanoparticles; Anti-tubercular; Hemolysis; MTT assay; Acute toxicity

Word Count for Abstract: 148 Word Count for Manuscript: 3,519 Number of References: 53 Number of Figures: 5 Number of Tables: 2 Number of Supplementary online-only files: 0 4

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1. Introduction:

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Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis (M. tb), an intracellular

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pathogen. The bacteria have been reported to infect the lungs, but can also damage other parts of

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the body including the skeletal and nervous system [1]. Current TB treatment regimen, DOTS (Directly Observed Treatment Short-course), requires patients to follow a combination therapy

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which includes the use of four drugs, namely isoniazid (INH), rifampicin (RMP), pyrazinamide

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(PZA) and ethambutol (EMB) for a period of 2 months (Intensive phase) followed by 4- month treatment with two drugs, INH and RFM (continuation phase) [2]. The primary challenge in the

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treatment is patients’ non-compliance, due to long treatment duration, high dosing frequency,

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and adverse side effects of anti-TB drugs. As a consequence, we have seen the emergence of drug resistant M.tb strains [3,4] which increase the necessity for a much compliant treatment

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regimens.

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Use of inorganic oxides, as opposed to organic antimicrobial agents has a lot of advantages including greater stability, robustness, and longer shelf life. ZnO is an inorganic antimicrobial

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agent which is generally recognized as safe (GRAS) under US-FDA listings for use in human beings and animals (21CFR182.8991). Also, it is an essential inorganic material with multiple applications in cosmetics, pigments and coatings, antimicrobials, spintronics, bio-imaging, drug delivery and catalysis. [5-9]. In the past decade, ZnO nanoparticles (NPs) have received much attention for their implications in anti-virals [10] and anti-cancer therapy [11-15]. Additionally, recent reports have shown that ZnO NPs are effective not only against gram-positive but also

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ACCEPTED MANUSCRIPT gram-negative bacteria [13,15]. Although there have been few reports on zinc based materials in the diagnosis or treatment of TB [16-19], reports on the anti-TB activity of ZnO NPs are meager. Recently,- a study conducted by Bheemanagouda N Patil et al [20] suggested the anti-TB activity of ZnO NPs using microplate alamar alue dye assay (MABA) method. They showed the activity

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to be in the microgram range.

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In this paper, we have demonstrated the Anti-TB activity of ZnO NPs by the agar proportion assay, which is considered as a gold standard as it shows inhibition /killing of bacterial growth.

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ZnO NPs were prepared by solution combustion synthesis (SCS) using a bio-fuel. The present

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study, therefore, involves the synthesis of ZnO NPs by simple, convenient and environmental friendly SCS using lemon juice as a bio- fuel [21-23]. Anti-TB activity of the synthesized ZnO

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NPs was investigated against M. tb H37Ra strain. Additionally, anti-carcinogenic activity

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performed on two human cancer cell lines, e.g. DU-145 (human prostate cell line) and Calu-6 (human pulmonary adenocarcenoma) by 3-[4, 5-dimethylthiazol-2-yl]-2,5- diphenyl tetrazolium

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bromide (MTT) assay, indicated the anticancer capability of ZnO NPs. Further, biocompatibility

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of the ZnO NPs was determined by conducting in vitro cytotoxicity and in vivo toxicity studies. The in vitro cytototoxicity studies were carried out by the MTT assay in the normal L929 mouse

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fibroblast cells, 3T3-L1 cells and hemolysis of sheep RBCs. The in vivo toxicity was evaluated in the normal Swiss mice.

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2. Materials and methods

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2.1 Materials

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Zinc nitrate hexahydrate [Zn(NO3)2.6H2O, AR 99% SD Fine], Dulbecco's Modified Eagle's

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medium [Gibco], Dimethyl sulfoxide [C2H6SO, AR 99% Merck], DPPH [C18H12N5O6, > 90%

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Merck], MTT [C18H16BrN5S, 97.5%, Sigma Aldrich] were procured commercially and fresh

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lemons were purchased from the local market.

All the cell lines used for cytotoxicity testing were procured from the American type culture

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collection (ATCC).

Anti-TB drugs, isoniazid (INH), rifampicin (RFM) and ethambutol (EMB) were purchased from

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Sigma, USA. M. tuberculosis H37Ra (25177) was procured from ATCC. OADC (Oleic acid,

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albumin fraction IV, dextrose and catalase) enrichment was purchased from BD (Becton

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Dickinson), USA. Anticancer drug colchicine was procured from Sigma Aldrich.

2.2 Synthesis of ZnO NPs by solution combustion synthesis (SCS)

Filtered lemon juice (10 mL) was mixed with 4.0 g of Zn(NO3)2.6H2O were taken in 40 mL of double distilled water and dissolved completely under stirring. Here zinc nitrate and lemon juice act as oxidizer and fuel, respectively. The petri dish containing the mixture was placed in a

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ACCEPTED MANUSCRIPT preheated muffle furnace (375 + 10 °C). Within a short while the solution boiled to form a transparent gel followed by rapid combustion of the fuel. The sample was calcined at 600 °C for 3 h.

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2.3 Characterization techniques

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The crystalline phase and crystal structures of ZnO NPs were studied by X-ray diffraction using

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Panalytical X’pert diffractometer with Cu Kα radiation (λ=1.5418 Ǻ) as the source. Surface morphology of the samples was studied by field emission scanning electron microscopy (FE-

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SEM) performed on FEI Quanta FEG 200 - high resolution scanning electron microscope. The shapes and particle size were investigated by high resolution transmission electron microscopy

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(HRTEM) carried out on JEOL 3010 instrument with a UHR pole piece.

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2.4 In vitro anti-tubercular activity evaluation by proportion assay

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The assay developed by Mc Clachy, measures the potential of the test compound to kill (or inhibit) the multiplication of the M. tb [24]. The proportion assay as explained by Mc. Clachy

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was adopted to test the anti-tubercular activity of ZnO NPs [1,24]. ZnO NPs were tested at different concentrations ranging from 1.56 to 50 µg/mL. ZnO NPs were dispersed in dimethyl sulfoxide (DMSO) to make stock solutions (1.00 mg/mL). The working dilutions were also made in DMSO. To 1.90 mL 7H10 agar medium in glass tubes at temperature 45-50 °C (containing 10 % OADC enrichment), 0.1 mL of ZnO dispersion (test) or DMSO (negative control) or Anti-TB drugs isoniazid, rifampicin, ethambutol (positive controls) was added, mixed and allowed to

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ACCEPTED MANUSCRIPT solidify as slants. Bacterial culture, M. tb H37Ra (ATCC 25177) was harvested from Lowenstein-Jensen (L-J) medium and its suspension (1.0 x 107 bacilli/mL) was made in normal saline containing 0.05 % Tween-80. 10 µL of this suspension was inoculated onto each tube and incubated at 37 °C for 4 weeks. The lowest concentration of a compound/drug which produced

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no visible bacterial growth was considered as its minimum inhibitory concentration (MIC).

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2.5 Assessment of anti-carcinogenic activity and cytotoxicity by MTT assay

Cytotoxicity of ZnO NPs was evaluated by using 2, 2-diphenyl-1-picrylhydrazyl hydrate (MTT

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assay) on human prostate carcinoma cell line DU-145 and lung cancer cell line Calu-6 as reported in our earlier studies with slight modifications [15]. DU-145 and Calu-6 (procured from

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ATCC) of 80 % confluent were trypsinized. The viable 50, 000 cells/well were seeded in a 96

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well plate and incubated for 24 h at 37 °C, 5 % CO2 incubator. ZnO NPs from 0-320 µg/mL in Dulbecco’s Modified Eagle’s medium without fetal bromine serum were incubated for 24 h.

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After incubation with ZnO NPs the media was removed from the wells and 100 µL/well of the

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MTT (5 mg/10mL of MTT in 1 × Phosphate buffered saline, the solution was filtered through 0.2 μM filter) working solution was added and incubated for 3 to 4 h. After incubation with MTT

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reagent, the media was removed from the wells and 100 µL of DMSO was added to rapidly solubilize formazan and absorbance was measured at 590 nm. Colchicine was used as positive control in suggested concentrations. Percent of inhibition was calculated as [100 − (As/(Ac) × 100] and cell viability was calculated as [As × 100/Ac] where, As and Ac are the absorbance values of the sample and control respectively.

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ACCEPTED MANUSCRIPT Cytotoxicity of the ZnO NPs was also evaluated towards normal healthy mouse fibroblast cells, L929 (obtained from subcutaneous connective tissues), 3T3-L1 (obtained from embryo), using the same procedure asmentioned above. Here, the compound was considered potentially nontoxic if the IC50 (concentration causing 50% loss in cell viability) was > 10 times its MIC for M.

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tuberculosis H37Ra. The data from this toxicity testing and MIC values were used to calculate a

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selectivity index (SI), the ratio of IC50: MIC.

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2.6 In vitro cytotoxicity by blood hemolysis

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The hemolysis activity test was performed against ZnO NPs following the standard procedure [25]. In brief, to 9.0 mL of the blood sample collected from sheep, 1 mL of 3.8% sodium citrate

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was mixed in order to inhibit the coagulation of blood. The sample was centrifuged at 3000 rpm

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for 5 min. The supernatant having platelet poor plasma was discarded. The pellet containing RBC was suspended in 10 mL of phosphate buffer saline (PBS) of pH 7.4. The cells were

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suspended in PBS to obtain a uniform suspension of cells. ZnO NPs with different

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concentrations (0.25, 0.5, 1.0, 2.5, 5.0, 10.0 mg/mL) were taken in different test tubes. To all the test tubes 2 mL of erythrocyte suspension was added and the test tubes were inverted. The tubes

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were then gently shaken to retain the contact of the blood with ZnO NPs and incubated at 37 °C for 90 min. The samples were centrifuged at 3000 rpm for 5 min to pellet out the RBC cells. The supernatant was then separated and the absorbance was measured at 540 nm against a PBS blank solution. The percentage of hemolytic index was calculated by using the following formula [26].

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ACCEPTED MANUSCRIPT Hemolysis (%) ODtest sample – ODnegative control × 100 ODpositive control – ODnegative control

Where, OD is the optical density value. Triton X-100 and PBS served as positive and negative

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controls respectively.

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2.7 Assessment of in vivo acute toxicity

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Acute toxicity study was carried out as per the Organization for Economic Co-operation and

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Development (OECD) 423 protocols. The procedure carried out was as reported in our earlier studies [15]. The animals used were Swiss albino mice of weight 25.0 + 5.0 g. The animals were

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of either the sex. Their age at the start of the studies was 8-10 weeks. 3 animals/dose levels were

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studied. The acclimatization was one week prior to dosing. The animals were identified by cage number and marking on animals. The route of administration was oral [27-29]. The animals were

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housed in separate cages under controlled conditions of temperature 22 + 2 °C. All animals were

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given standard diet and water regularly.

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Methodology followed was as per the guidelines of OECD 423. In brief, the acute toxic class method set out in this guideline is a stepwise procedure with the use of 3 animals of a single sex per step. Depending on the mortality and or the moribund status of the animals, on average 2-4 steps may be necessary to allow judgment on the acute toxicity of the test substance. The ZnO NPs were administered orally to a group of experimental animals at one of the defined doses. The substance was tested using a stepwise procedure, each step using three animals of a single sex (normally female). Absence or presence of compound-related mortality of the animals dosed 11

ACCEPTED MANUSCRIPT at one step will determine the next step, i.e.; no further testing is needed, dosing of three additional animals, with the same dose and, dosing of three additional animals at the next higher or the next lower dose level. The dose level to be used as the starting dose was selected from one

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of the four fixed levels, 5, 50, 300 and 2000 mg/kg body weight.

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3. Results and discussion

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3.1 Crystal structure and morphological studies

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The PXRD data was analyzed using Origin 8.1 software (Origin Lab Corporation, USA). The XRD pattern of ZnO NPs is presented in Fig.1 (A). The diffraction pattern agrees with the

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standard joint committee on powder diffraction standards (JCPDS) No. 36-1451 corresponding

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to zincite pattern and can be indexed as hexagonal wurtzite type. Absence of other impurity peaks indicates high purity of the synthesized products. The average crystallite size D was

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calculated using the Scherrer equation, D = k λ / β Cos θ, where k is the Scherrer constant, λ is

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the wavelength of the X – ray used (1.54 Ǻ), θ is the Bragg angle and β is the full-width at halfmaxima (FWHM) of the diffraction peaks. The average crystallite size of ZnO NPs was

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calculated to be 19 nm. The FE-SEM micrograph of ZnO shown in Fig.1 (B) reveals that besides the spherical crystals the powder also contains several voids or holes, the reason for which can be attributed to the release of hot gases that escape out of the reaction mixture during combustion [30]. It is through pores of various sizes and shapes that the crystallites are interlinked to one another [30]. The HRTEM image of ZnO NPs is shown in Fig. 1 (C). It indicates that the particles are spherical shaped. The mean particle size by histogram was found to be 33 nm.

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ACCEPTED MANUSCRIPT 3.2 Anti-tubercular activity

Results of anti-tubercular activity of the test sample are shown in the Table 1. ZnO NPs showed complete inhibition/killing of the bacterial growth at 12.50 µg/mL. Media containing only

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DMSO (no drug control) showed bacterial growth whereas those containing anti-TB drugs

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showed complete inhibition/killing of the M. tb bacilli at their respective MICs.

While detailed mechanism of the bioactivity of ZnO is still under discussion, many mechanisms

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have been proposed related to this: (a) One of the possible mechanisms is based on the abrasive

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surface texture of ZnO - binding of ZnO NPs to the bacterial surface is due to electrostatic forces that directly kill bacteria [31], (b) mechanical destruction of the cell membrane caused by

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penetration of the NPs [32], (c) release of Zn2+ ions from the nanoparticles [33] and (d) active

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oxygen generated from the powder [34-38]. Thus the main two probable mechanisms involved in the interaction between NPs and bacteria suggested by several investigations are (1) the

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production of increased levels of ROS, mostly hydroxyl radicals and singlet oxygen [34,36-39]

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growth [34,35].

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and (2) Zinc toxicity of cell membrane by adhesion of ZnO particles which inhibits the bacterial

There are very few reports on the anti-TB activity of NPs against Mycobacterium tuberculosis H37Ra. ZnO NPs initiate a lipid peroxidation reaction subsequently causing DNA damage, glutathione depletion and disruption of membrane morphology, and electron transport chain, which leads to cell apoptosis [40]. The concentration and size are two important factors that may affect cell apoptosis [20]. ZnO NPs may get attached on the surface of the bacterial cell

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ACCEPTED MANUSCRIPT membrane [20] and subsequently enter into the cytoplasm of mycobacterium via endocytosis and the smaller sized nanoparticles penetrate into the bacterial cell membrane which inactivates the enzymes essential for adenosine triphosphate production that leads to the formation of reactive

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oxygen species and eventually bacterial cell apoptosis [41-43].

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3.3 Cytotoxicity towards human cancer cells

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It is evident from MTT assay that ZnO NPs induce dose dependent activity in DU-145 and Calucells. IC50 values for cytotoxicity test of DU-145 and Calu-6 were derived from a nonlinear

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regression analysis (curve fit) based on sigmoid dose response curve (variable) and computed using Graph Pad Prism 5 (Graphpad, San Diego, CA, USA) as shown in Fig. 2 (A-B)

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respectively. IC50 values of ZnO NPs on DU-145 and Calu-6 are presented in Fig. 3. Results

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indicate that the cells investigated in our studies respond differently to ZnO NPs at different concentrations. Cytotoxicity of ZnO NPs was found higher towards Calu-6 (IC50 57.7 µg/mL)

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compared to DU-145 (IC50 44.73 µg/mL) cell lines. The marked cytotoxicity against DU-145

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and Calu-6 cancer cells suggests an exciting potential for ZnO NPs as novel alternatives to cancer chemotherapy. Earlier studies have shown that ZnO NPs induce cytotoxicity in a cell

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specific and proliferation dependent manner by rapidly dividing cancer cells being the most susceptible and quiescent cells being the least sensitive [13,44]. However, the anticancer activity of ZnO NPs, in particular the mechanism of apoptosis in cancer cells due to ZnO NPs is still not clear.

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ACCEPTED MANUSCRIPT Interestingly, IC50 values of ZnO NPs towards normal mouse cells (L929) and 3T3-L1 cell lines as shown in Fig. 2 (C-D) were quite high, i.e. 130.9 µg/mL and 172.1 µg/mL respectively. The NPs showed selectivity index (SI) of 10.47 for L929 cells and 13.768 for 3T3-L1. Results shown as SI indicate that these NPs are non toxic to normal healthy cells.

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Determination of IC50 values for cytotoxicity of colchicine on DU-145, Calu-6, L929 and 3T3-

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L1 is shown in Fig. 2 (E-H) and IC50 values are presented in Fig. 3. The results indicate the

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cytotoxicity of colchicine on both cancerous and normal cells. Further, the results also vindicate that ZnO NPs synthesized in our studies are more toxic to cancer cells DU-145 and Calu-6 and

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less toxic to normal cells L929 and 3T3-L1 cells. It is important to mention that, ZnO NPs

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showed selective anti-cancer activity. Earlier studies by other research groups have shown that ZnO NPs are more toxic to cancer cell and less toxic to normal cells [13, 45-47]. Mohd Bakhori

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et al have shown non toxic level of ZnO nanostructures on L929 cells [45]. The same finding has

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been confirmed by Ieda et al. [46]. They have shown that, ZnO nanostructures exert higher cytotoxicity against cancer HeLa cells (human cervical cancer cells), in comparison to the

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noncancerous cell line L929. Studies also indicate that dose-dependent cytotoxicity of ZnO

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against HL60 (acute myeloblastic leukemia) cells with a CC50 of 52.80 μg/mL, whereas the CC50 value against normal peripheral blood mononuclear cells was 741.82 μg/mL [13]. The regulation

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of oxidation–reduction (redox) reactions is critical to a cell, as it influences metabolic and other signal-transduction pathways. When ROS (Reactive Oxygen Species) generation exceeds the cellular antioxidant defenses, cell damage ensues. Cellular defenses to ROS include antioxidant scavengers, such as ascorbate, glutathione and thioredoxin, and antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase and thioredoxin reductase. The above findings may be understood by the fact that healthy cells can regulate the amount of ROS

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ACCEPTED MANUSCRIPT generated by the ZnO NPs, whereas cancer cells cannot. Therefore, treatment of ZnO NPs showed higher cytotoxicity towards cancer cells resulting in to cell death [47].

ZnO exhibits surface active properties best known for their application in many electronic and

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photoelectrical applications [48,49]. These properties have also been extensively used to prepare

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antimicrobial coatings and anti-cancer agents [50]. The negatively charged surface of ZnO stems

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from the presence of multiple oxygen vacancies which are produced due to electron hole pairing in the presence of an excitation wavelength [51]. These negatively charged oxygen vacancies are

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a result of reactive oxygen species (ROS) released from the surface of ZnO which have been

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linked to their excellent antimicrobial and anti-cancer properties [51,52]. Thessicar et al [53] have previously demonstrated that negatively charged ZnO tetrapod structures (ZnOTs) can be

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used as effective traps for positively charged virion particles present in a medium. They were

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also able to show that the presence of ZnOTs (and ROS released) causes an elevated immune response at the site of application [53]. We believe our ZnO NPs also pursue a similar process

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for the generation of toxicity against cancer cells and M. tuberculosis. The smaller size of our

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ZnO NPs would be able to stick onto the surface of the target followed by endocytosis and subsequently release ROS for the disruption of the bacterial DNA. While these are speculations,

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we believe our hypothesis stands close to those mentioned elsewhere. Fig. 4 shows schematic representation of the possible mechanism of the bio activity of ZnO NPs.

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ACCEPTED MANUSCRIPT 3.4 Blood hemolysis

The results of hemolysis assay are depicted in Fig. 5. Since 5% hemolysis is considered as permissible limit for biomaterials, within the limitations of this study, up to a concentration of

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5.0 mg/mL of ZnO NPs can be taken for hemolysis activity. Therefore ZnO NPs synthesized in

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our present studies are biocompatible in nature up to a concentration of 5.0 mg/mL. These results

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are in good agreement with the literature [15].

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3.5 Acute toxicity

The acute toxicity study was performed for 14 days. General behavior and lethality were

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evaluated to assess the acute toxicity of ZnO NPs. Observations of acute toxicity studies are

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presented in Table 2. As visualized from it, no mortality was observed throughout the dosing schedule of 14 days in all dose level in all groups. Thus LD50 (lethal dose, 50 %) cut off was

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4. Conclusions

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calculated as 2000 mg/kg body weight, as per the methodology adopted.

The present work was conducted for the evaluation of anti-tubercular activity of ZnO NPs prepared by lemon juice fueled solution combustion synthesis. Anti-tubercular activity was observed at a concentration of 12.5 µg/mL of ZnO NPs against M. tb H37Ra. Further, sufficiently higher IC50 values towards L929 cells (130.9 µg/mL) and 3T3-L1 (172.1 µg/mL) indicate that ZnO NPs are non toxic for healthy mammalian (mouse) cells. The selectivity index

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ACCEPTED MANUSCRIPT (IC50/ MIC) with respect to anti-TB activity was found greater than 10 which reflects good therapeutic value of ZnO NPs as anti-TB agent.

IC50 values determined by MTT assay

performed on two cancer cell lines indicated that the NPs do possess moderate anti-carcinogenic activity. Blood hemolysis studies conducted proved the bio compatibility of ZnO NPs at varied

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mice showed that the ZnO NPs caused no systemic toxicity.

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concentrations. No mortality in the acute toxicity studies conducted in out bred Swiss albino

In conclusion, we report that the ZnO NPs carry a significant step towards the development of

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cost effective, biocompatible and environment friendly effective anti-tubercular and anti-cancer

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agents. The anti-TB results observed in our studies are promising and hence further experimentations shall be carried out in ex vivo and in vivo models in order to understand the

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efficacy of ZnO NPs as an anti-tubercular agent.

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Acknowledgements

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The author G.K. Prashanth thanks the management of Sri KET and Dr. M.S. Indira, Principal, Sir MVIT, Bengaluru for the support and encouragement extended towards this project. The authors

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acknowledge Pinnacle Biomedical Research Institute, Bhopal for the facility extended towards the animal studies [the animal experiment was approved by institutional animal ethics committee (IAEC) of PBRI, Bhopal (Reg. No. 1283/PO/c/09/CPCSEA), with the protocol reference No. PBRI/IAEC/13-14/PN-395], Nanotechnology Research Center, SRM University for FE-SEM, IITM for HRTEM measurements.

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ACCEPTED MANUSCRIPT 53. T.E. Antoine, Y.K. Mishra, James Trigilio, Vaibhav Tiwari, Rainer Adelung, Deepak Shukla, Prophylactic, therapeutic and neutralizing effects of zinc oxide tetrapod structures against herpes

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ACCEPTED MANUSCRIPT Figure captions

Fig. 1 (A) PXRD pattern, (B) FE-SEM and (C) HR-TEM image of ZnO NPs

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Fig. 2 Determination of IC50 of ZnO NPs (µg/mL) towards (A) DU-145, (B) Calu-6, (C) L929

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cells, (D) 3T3-L1 and positive control (colchicine) towards (E) DU-145, (F) Calu-6, (G) L929

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cells, (H) 3T3-L1 [data represent mean ± SD (standard deviation)]

Fig. 3 IC50 values of (A) ZnO NPs and (B) positive control (colchicine) in DU-145, Calu-6,

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L929 and 3T3-L1 cells

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Fig. 4 Schematic diagram depicting the possible mechanism of anti-microbial and anti-cancer

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activity of ZnO NPs

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(percent) haemolysis]

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Fig. 5 Hemolysis of blood by ZnO NPs [data show mean ± SD (standard deviation) of %

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Fig. 3 (A-B)

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Fig. 4

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Graphical abstract

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ACCEPTED MANUSCRIPT Table 1. In vitro anti-tubercular activity evaluation of ZnO NPs against Mycobacterium tuberculosis H37Ra.

Test samples

3

RFM*

4

EMB*

5

DMSO

12.50

25.00

++

++

++

-- --

-- --

50.00

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INH*

6.25

-- --

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3.12

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ZnO NPs

1.56

MIC

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Test concentrations (µg/mL)

(No drug-Control)

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S. No.

+ ++ +

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Notes: ++ Visible bacterial growth; -- -- No visible growth.

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*Anti-TB drugs: INH-Isoniazid, RFM-Rifampicin and EMB- Ethambutol.

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(µg/mL) 12.50 0.025 0.20 2.00

ACCEPTED MANUSCRIPT Table 2. Assessment of acute toxicity of ZnO NPs in Female Swiss albino mice.

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5.0

2

50.0

Male

0/3

Female

0/3

Male

0/3

2000.0

0/3 0/3

Female

0/3

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Female

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300.0

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Lethality

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Test Groups (n=3)

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Dose

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Note: NPs were administered orally.

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ACCEPTED MANUSCRIPT Highlights  ZnO nanoparticles (NPs) were prepared solution combustion synthesis using biofuel  ZnO NPs showed inhibition of Mycobacterium tuberculosis H37Ra strain at

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12.5 µg/mL

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 Cytotoxicity of ZnO NPs was proved on DU-145 and Calu-6 cell lines

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 ZnO NPs were proved to be biocompatible by blood hemolysis and MTT assay

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 Acute toxicity studies on SA mice showed no evident toxicity over a 14 days

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period

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