Evaluation of Mixture Magnesium Oxide and Zinc Oxide Nanoparticles against Multi-drug-resistance Mycobacterium tuberculosis by Microplate Alamar Blue Assay

Objective The current study is the first experimental study of which has evaluated the MICs and MBCs of ZnONPs, MgONPs, and mixture MgONPs-ZnONPs against H 37 Rv Mtb and MDR-Mtb.Results The MIC of MgONPs and ZnONPs were 0.195 and 0.468 µg.ml -1 against 10 4 of H 37 Rv Mtb. As well, 0.166 µg.ml -1 of MgONPs-ZnONPs was able to inhibit 10 -4 H 37 Rv Mtb. The MIC of MgONPs against 10 4 concentrations of MDR-Mtb was 12.5 µg.ml -1 . The MIC of MgONPs/ZnONPs against 10 4 concentrations of MDR-Mtb reached to 0.664 µg.ml -1 . Based on the results, the MBC value of ZnONPs increased to 1.875 µg.ml -1 against 10 -4 concentrations of MDR-Mtb. Testing showed that the MBCs of MgONPs/ZnONPs reached to 1.328 µg.ml -1 against 10 4 concentrations of MDR-Mtb. The half maximal inhibitory concentration (IC50) against MDR-TB was 0.779 µg.ml -1 for ZnONPs and 0.883 µg.ml -1 for MgONPs-ZnONPs. The MgONPs-ZnONPs was not toxic to Vero cell lines however ZnONPs could inhibit the Vero and HepG 2 cell lines. We found that ZnONPs and mixture MgONPs-ZnONPs not only have higher bactericide behavior but might have also synergistic effects against MDR-TB.

Today, nanotechnology has attracted medical researchers for over a century and is now with heavy steps used in biomedical sciences [2]. The researchers have found, metallic NPs have shown anti-tubercular properties against Mtb [3]. In fact, there is a focus of interest related to using mixed metallic NPs because of their outstanding potential against 3 MDR-TB [2,4,5].
Studies have shown that in the process pathogenesis of pulmonary tuberculosis, the metallic NPs can penetrate within the calcified granuloma and can attach to Mtb and kill it. The metallic NPs can also eliminate Mtb into the macrophage without any toxicity effect. Therefore, the metallic NPs especially mixed metallic NPs have potentially capable of electrostatic interactions with the Mtb cell membrane [6].

Study design and bacterial isolates
The multidrug resistance strain of Mtb isolated from a 55-year-old man who was proven tuberculosis by imaging, clinical findings, histological and cytological observations, previously. The patient had consumed first-line anti-tubercular treatment for 6 months but the symptoms of TB had reappeared about 7 months after completing the treatment.
Spectrum sample of patients was cultured in LJ medium for 28 days. The susceptibility drug testing was done by the indirect proportion method to determining of MDR-Mtb [7].

Preparing Nanoparticles
In this study, MgONPs (average diameter of 20 nm) and ZnONPs (average diameter of 4 nm) were purchased from Tehran University of Medical Sciences (Tehran, Iran). Before each experiment, MgONPs and ZnONPs were sterilized through heating in an oven at 200 °C for one hour.

Antibiotic susceptibility test
To determine the minimum inhibition concentration (MICs) of NPs, the microplate Alamar blue (MABA) assay is used [8]. Two hundred microliter (200 µl) of sterile deionized water added to outer-perimeter wells of 96-well microplates. Then, 100 ml of 7H9 GC broth was added to each well. 100 µl of MgONPs (12.5 µg.ml -1), ZnONPs (30 µg.ml -1) and MgONPs-ZnONPs (42.5 µg.ml -1) solutions were added to each well, and the serial dilutions were done. A hundred microliter (100 µl) of H37Rv Mtb (Razi serum and vaccine research institute, IRAN) was added to the wells [9]. The plates incubated at 37°C for 5 days. Fifty microliters (50 µl) of a freshly prepared 1:1 mixture of Alamar blue reagent and 10% Tween 80 added to well and reinsulated at 37°C for 24 hours. The blue color in the well interpreted as no growth and a pink color scored as growth. The MICs was defined as the lowest drug concentration which prevented a color change from blue to pink [9].
To determine the minimum bactericidal concentration (MBCs) of NPs against MDR-Mtb (Razi serum and vaccine research institute, IRAN), proportion method was also done. 10 ml of melted löwenstein-Jensen (LJ) agar and 10 ml of NPs were poured and subsequently, serial dilution was performed. On the following day, 100 µl of 10-2 and 10-4 McFarland of bacteria were added and then they are incubated at 37 °C. The colony-forming unit (CFU) of bacteria was counted after 28 days. The atomic force microscopy (AFM) (Institute for color science & technology, IRAN) of mixture MgONPs-ZnONPs fulfilled by commercial AFM system (Nanosurf, Switzerland) [10]. The TEM image was prepared at the Institute of Biochemistry and Biophysics (IBB) of Tehran University.

MTT assay
To MTT assay, Vero and HepG 2 cell lines (Institute for tuberculosis research, the University of Illinois at Chicago, USA) (1×104 cells per well) were seeded in 96-well plates containing 100 µl of DMEM, separately [11]. 100 µl of MgONPs, ZnONPs, and MgONPs-ZnONPs were inoculated to each well. Next, 5 mg MTT per ml added. Cell viability calculated using DMSO treated Vero and HepG 2 as the 100% viable control and measured (A540 of NPs × treated sample/A540 of control) × 100.

Statistical analysis
Statistical analyses were prepared by using of Kruskal-Wallis test and Mann-Whitney U test. The statistical significance threshold was resolute as P-value ≤ 0.05.

Discussion
The current study is the first experimental study of which has evaluated the MICs and MBCs of ZnONPs, MgONPs, and MgONPs-ZnONPs against H 37 Rv Mtb and the MDR-Mtb. In this study, the toxicity effects of the NPs have evaluated, as well. This study reported the 7 MIC of MgONPs against MDR-Mtb was 12.5 µg.ml -1 (Table 1A) reported that ZnONPs has ability to inhibit Mtb at 12.5 µg.ml -1 [13]. We showed that the ZnONPs has the ability to eliminate MDR-TB at 1.875 µg.ml -1 (Table 1A)

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding support
This study was supported by Guilan University of Medical Sciences. This funding source had no role in the design of this study and will not have any role during its execution, analyses, interpretation of the data, or decision to submit results.

Acknowledgement
This research supported by vice chancellor of research and technology, Guilan University of Medical Sciences.
We thank our colleagues from Guilan University of Medical Siences who provided insight and expertise that greatly assisted the research although they may not agree with all of the interpretations/conclusions of this paper. We would also like to show our gratitude to the Dr. Hadi Sedigh Ebrahim-Saraie for sharing their pearls of wisdom with us during the course of this research, and we thank 3 "anonymous" reviewers for their so-called insights. We gratefully acknowledge the dedicated efforts of the investigators and coordinators participated in this study.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.