BioNanoScience https://doi.org/10.1007/s12668-017-0480-5
Biosynthesis of MgO Nanoparticles Using Lactobacillus Sp. and its Activity Against Human Leukemia Cell Lines HL-60 V. Mohanasrinivasan 1 & C. Subathra Devi 1 & Avani Mehra 1 & Suman Prakash 1 & Aditi Agarwal 1 & E. Selvarajan 1,2 & S. Jemimah Naine 1
# Springer Science+Business Media, LLC, part of Springer Nature 2017
Abstract The present study reports a low-cost, eco-friendly, and reproducible microbes Lactobacillus sp. mediated biosynthesis magnesium oxide nanoparticles. The nanoparticles were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and high-resolution transmission electron microscopy (HRTEM). The morphological characteristics were found to be spherical, oval in shape, individual nanoparticles as well as a few aggregates. The XRD shows the crystallographic plane indicating that nanoparticles structure dominantly corresponds crystalline. The biosynthesized magnesium oxide nanoparticles showed corresponding functional peaks. The cytotoxic effects of the magnesium oxide nanoparticles could significantly inhibited HL-60cancer cell lines proliferation in a time and concentration-dependent manner by MTT assay. L. sporogenes mediated magnesium oxide nanoparticles had potential to inhibit the cancerous cells by 60% while L. plantarum mediated nanoparticles found to inhibit the growth by 50%. The biosynthesis of nanoparticles has been proposed as an environmental friendly and cost effective alternative to chemical and physical methods. Hence, this report added the value for the application of magnesium oxide nanoparticles in biomedical and nanotechnology applications with the absence of adverse side effects from nonpathogenic, mesophilic Lactobacillus plantarum and Lactobacillus sporogenes for the nanoparticles. Keywords Lactobacillus sp. . MgO nanoparticles . Anti-cancer activity
1 Introduction Cancer is known to be one of the most fatal diseases, where uncontrolled cell growth is observed. Every year there is an increase in the number of casualties due to certain forms of cancer since in most of the cases a satisfactory therapeutic agent is yet to be discovered. Nanotechnology in recent times has become a crucial branch of science and technology. The past few years have witnessed nanoscience and technology being applied in various fields of research, such as cancer, where nanotechnology can help in the early detection and treatment of cancer [1]. Nanoparticles, due to their small size
* C. Subathra Devi
[email protected] 1
School of Biosciences and Technology, VIT University, Vellore, India
2
Department of Genetic Engineering, School of Bioengineering, SRM University, Kattankulathur, Tamil Nadu, India
and higher surface area have the ability to interact with biomolecules present on both the surface and inner of the body cells [2].The use of metallic nanoparticles like magnesium oxide MgO have long been used as antimicrobial agents since these offer greater surface area in contact with the bacterial cells [3]. It has been reported that the antibacterial activity of MgO nanoparticles is attributed to the production of reactive oxygen species (ROS) which induce lipid peroxidation in bacteria [4]. Even though researchers have now started to focus on the use of MgO nanoparticles in the field of cancer, none have focused on the use of biologically synthesized MgO nanoparticles for the treatment of cancer. The advantages of biological synthesis of nanoparticles over chemical and physical synthesis have been well emphasized in earlier publications. Bacteria, especially thermophilic bacteria, are a better choice for the synthesis of nanoparticles since they have the natural ability to adapt to the extreme changes in their environment [5]. The use of Lactobacillus, a group of mesophilic facultative anaerobic bacteria offer a wide range of advantages, owing to their ability to undergo both oxidation and reduction reactions [6, 7].
BioNanoSci.
Fig. 1 a XRD pattern for L. plantarum mediated nanoparticles. b XRD pattern for L. sporogenes mediated nanoparticles
Therefore the present study aimed at a cost effective and efficient method of MgO nanoparticle synthesis with the potential to inhibit cancerous cell growth.
2 Materials and Methods 2.1 Biosynthesis of MgO Nanoparticles Lactobacillus plantarum VITES07 [8] and Lactobacillus sporogenes were chosen for the biosynthesis of nanoparticles
a
and therefore inoculated in MRS broth and incubated at 37 °C for 24 h. The bacterial cultures were then further diluted with sterilized uninoculated MRS broth in the ratio 1:3. Post dilution, 0.1 M magnesium nitrate [Mg (NO3)2] was added to each diluted culture, followed by drop wise addition of 0.2 M NaOH, to delay the process of transformation. The cultures were then kept in a water bath at 40 °C for 15–20 min for the white colored precipitate to settle down. The cultures were finally incubated undisturbed at room temperature for 10 h [7]. Post 10 h of incubation, the cultures were centrifuged at 5000 RPM for 15 min. The supernatant was carefully
b
100
100
1 4 0 2 .2 5
75
50 1 3 8 4 .8 9
3 4 4 4 .8 7 3 4 1 9 .7 9
25
0 4000
0
3000
2000
1500
1000
500
4000
3 4 4 6 .7 9
25
1 6 4 1 .4 2 1 6 2 9 .8 5
50
5 3 8 .1 4
%T 1 0 8 7 .8 5
1 6 4 1 .4 2
75
5 3 8 .1 4
2 9 2 6 .0 1 2 8 5 4 .6 5
%T
3000
Fig. 2 (a) and (b) depict the FTIR pattern of L. plantarum and L. sporogenes mediated nanoparticles
2000
1500
1000
500
BioNanoSci.
After sonication and stabilization, the samples were prepared by coating on carbon-coated copper grids and air dried before TEM analysis.
2.3 Cytotoxic Potential of Nanoparticles
Fig. 3 TEM micrograph of MgONPs
discarded and the pellet was washed twice with distilled water. The nanoparticles were then carefully dried and obtained in the powder form. The obtained nanoparticles were in the hydroxide form which were converted to oxide form by calcinating the nanoparticles at 300 °C for 4 h [9].
A concentration of 8 mg/mL of nanoparticles was prepared and assessed for cytotoxic potential against human leukemia cell lines HL-60. Approximately 25 μL of cancer cells were grown in a 96-well plate, following which the plate was incubated at 37 °C for 24 h. Post incubation, the cells were washed with 100 μL serum free medium and let to starve for 60 min at 37 °C. The cancer cells were then treated with different volumes of the nanoparticles for 24 h. At the end of 24 h, the medium was aspirated and serum-free medium which also contained 0.5 mg/mL MTT was added to the cells and incubated for 4 h at 37 °C in the presence of CO2. The MTT containing medium was then washed off and the cells were treated with 200 μL phosphate buffered saline (PBS). The obtained crystals were then dissolved by adding 100 μL DMSO. Viable cancer cells reduce MTT, a tetrazolium salt to form a blue colored product, which is measured spectrophotometrically at 570 nm [10]. The average of the viable cells was calculated using Graph pad prism software after carrying out the experiment in triplicates.
2.2 Characterization of Nanoparticles The calcinated nanoparticles were then characterized using Fourier infrared spectroscopy (FTIR), X-Ray diffraction (XRD), and high-resolution transmission electron microscopy (HRTEM). The particle size and nanostructure were studied by high-resolution transmission electron microscopy in a JEOL 3010 (HRTEM), Japan, operating at 200 KeV. Dry powder of particles was suspended in de-ionized water at a concentration of 1 mg/mL and then sonicated at room temperature for 10 min at 40 W to form a homogeneous suspension.
3 Results and Discussion 3.1 XRD Analysis of Nanoparticles Figure 1a and b depicts the XRD patterns for the Lactobacillus plantarum VITES07 mediated as well as Lactobacillus sporogenes mediated nanoparticles, where mostly unidentified peaks have been observed, with the exception of one recognized peak in case of Lactobacillus sporogenes mediated LS 125
100
100
% o f c e ll V ia b ility
% of cell Viability
LP 125
75 50 25 0
75 50 25
control
6
13
25
50
Concentration in g/mL
100
Met-25
0
control
6
13
25
50
100
Met-25
Concentration in g/mL
Fig. 4 (a) and (b) Percentage of cell viability shows the effective nanoparticle concentration toxic to human leukemia cell lines HL-60 (LP L. plantarum, LS L. sporogenes)
BioNanoSci. Fig. 5 Morphological changes in human leukemia cell lines HL-60 after treated with L. plantarum mediated nanoparticles
Control
50µg/mL
nanoparticles. From the obtained XRD patterns, the presence of impurities in the nanoparticles could be inferred. Previously Sundarajan et al. [9] also reported the presence of unidentified peaks in the XRD pattern of MgO nanoparticles calcinated at 300 °C, indicating the presence of impurities. Moreover, the magnesium oxide nanoparticles were synthesized by simple wet chemical method and characterized by XRD analysis and calcinated to 700 °C with 13. 3 nm in size [9]. Phytoassisted synthesis of magnesium oxide nanoparticles with an aqueous extract of Swertia chirayaita showed a cubic phase in XRD pattern with
