Document Type : Original Research Article

Authors

Sri Devaraj Urs Academy of Higher Education and Research

Abstract

Abstract
Green based nanoparticles were synthesized to investigate the cytotoxic effects on human peripheral blood mononuclear cells (hPBMCs). Nanoparticles were made by mixing using aqueous Allamanda cathartica L. latex (3%) and magnesium oxide (1mM) at 370C on shaker for 24 hours. The developed nanoparticles were characterized by Scanning Electron Microscopy, Transmission Electron Microscopy, Fourier Transmission Infra-Red, Zeta potential and X-ray diffraction. in vitro hPBMCs were tested in vitro with different concentrations of synthesized nanoparticles and found maximum concentration at that 100 µg/mL showed about 96% dead cells from trypan blue dye exclusion method. Though controlling the proliferation of cells, the nanoparticles did not exhibit any genotoxicity, indicating that the nanoparticles showed their effect on periphery of the cells, broke them that led to the death of cells. This is the first report of green route development of MgO nanoparticles using the latex of Allamanda cathartica L. and finding their efficacy on hPBMCs. Further analysis is required to confirm their use as therapeutics in leukemia.

Keywords

INTRODUCTION
During the twenty-first century, nanoparticles (NPs) in nanotechnology have gained importance in multidisciplinary fields; broadly, medicine, agriculture, energy science, engineering and so on. The growing interest in preparation of these nanoparticles is mainly valued the environment and other living beings. Use of metals in nanoparticle synthesis is an advantage since these metals are somewhere or the other linked to the body's functions. The various metabolic activities of a living system are controlled by enzymes; their functions are regulated by the cofactors, such as zinc, copper, iron, potassium, calcium and magnesium. Inorganic based metal and metal oxide nanoparticles are in practice with silver, titanium, zinc, copper etc [1] (Jeevanandam et al. 2018). In recent past, the antioxidant properties of NPs, for example, nanomagnesium, nanosilver, nanozinc, nanoselenium and so on have been proved to have oxidation reducing capacity in diabetes, cardiotoxicity, diabetes neuropathy and so on [2] (Heydary et al. 2015). Metal oxides, in spite of having many modes of nanoparticle synthesis, are widely accepted in “green synthesis”, which means, synthesizing nanoparticles with biological sources from microscopic algae to macroscopic plants. Green approach is not only easy but also rapid, cost effective and eco friendly. Several types of plant derived chemicals, called phytochemicals, include proteins, alkaloids, phenolic compounds, tannins, glycosides and polysaccharides participate in nanoparticle synthesis, thus considered as safe. These compounds involve the bio-reduction of metals and their oxides at the time of eco-friendly nanoparticle formation. Green nanotechnology uses and develops nano sized particles by blending metals with plant material. The synthesized particle size is smaller than 100 nanometers. Many previous studies authenticated from the research that the synthesized nanoparticles limit oxidative stress, genotoxicity and apoptotic activities [3] (Kuppusamy et al. 2016). Magnesium oxide (MgO) is widely used metal oxide in non-medical fields (cosmetics, electrical insulation, food packaging industry, desiccation units) [2] (Heydary et al. 2015). It is a thermostable, inexpensive and biologically compatible metal. In healthcare, MgO has antibacterial properties. It is a part of antacid (relieves heartburn), laxative (short term), quick emptying of the bowel (just before surgery), also used as dietary supplement and MgO is recognized as safe/ non-toxic by the U.S. Food and Drug Administration (21CFR184.1431) [4] (Cai et al. 2018). Keeping in mind the uses of MgO in healthcare, an attempt has been made for preparing MgO nanoparticles (MgONPs) by using latex from Allamanda cathartica L., a medicinal plant. In the plant kingdom, only a limited number of plants produce latex and one among them is A. cathartica L., belonging to the family Apocynaceae. Latex is a rich source of glycosides, saponins, alkaloids, tannins and other phytochemicals [5] (Kiranmayee et al. 2020). In addition to other essential phytochemicals, the whole plant of A. cathartica contains plumieride, plumericin, and allamandin. The isolated and identified chemical constituents from leaves are ursolic acid, ß-sitosterol, ß-amyrin, plumericin, sesquiterpenes, and plumieride. Along with the above, stem and bark contain triterpenoids, glucoside, and alkaloids.  Whereas flowers contain kaempferol, quercetin and other flavonoid compounds; roots are rich with triterpenoids, plumieride, alkaloid and glucoside. The whole plant shows ethanopharmacological properties. To name a few, laxative, an antibiotic, treats malaria, jaundice, enlarged spleen, cough, anti-inflammatory, purgative, antioxidant properties. Sap contains antibacterial and anticancer properties [6] (Chandreyi et al. 2019). Hyperglycemia and Mg deficit are common in type 2 diabetes. Glucose and insulin are the key regulators in Mg metabolism [7] (Mario and Ligia 2015). Because of its poor bioavailability, MgO gets putrefied by digestive juices and acids. The best way to escape from this and increase bioavailability is to use MgO nanoparticles, in particular, synthesized by green approach [8] (Patil 2020). As human Peripheral Blood Mononuclear Cells (hPBMCs) are having direct contact with NPs, in the present study MgONPs were made by adopting a green approach. Normal cell and tissue protein denaturation is the side effect of NPs. Based on the medicinal properties of plant latex and the metal, MgO nanoparticles were synthesized by green approach. The synthesized particles were characterized by the standard methods, and tested their genotoxic efficacy on hPBMCs. As far as our knowledge is concerned, this is the first report of using A. cathartica latex and MgO for green route development of nanoparticles and finding their efficacy on hPBMCs.

MATERIALS AND METHODS
The plant, A. cathartica (golden trumpet), growing on Sri Devaraj Urs Academy of Higher Education and Research, Kolar campus has been identified by Dr. Kiranmayee (one of the authors of this paper) and authenticated again by Horticulture college, Kolar. Specimen was submitted to Dr. Madhava Chetty, Department of Botany, Sri Venkateswara University, Tirupati and obtained voucher specimen number as 0109. The trimmed young twigs were gathered at morning hours to collect latex and stored at 4oC till NP synthesis. By following Harbourne’s protocol, phytochemical analyses for alkaloids, flavonoids, saponins, tannins, glycosides, reducing sugars were carried out from 3% latex [9] (Harborne 1973).

Nanoparticle synthesis
Equal proportions of MgO (1mM) and latex (3%) were mixed till latex is completely solubilized in MgO solution and facilitated the conditions (37oC, 135 rpm, 24 h) for nanoparticle development [10] (Banerjee et al. 2014). The suspension was clarified at 10,000 rpm for 10 min and the pellet was stored at 4oC till further use.

Characterization of latex nanoparticles
All the characterization studies were carried out in the department of Microbiology and Cell Biology (MCBL) and Spectroscopy Analytical Test Facility and Materials Research Center (MRC), Indian Institute of Science (IISc), Bangalore, Karnataka, India. From the UV-Visible spectrophotometer (Nanodrop 8000), absorption/particle spectrum was obtained between 200 and 700 nm. For Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) [11] (de F Navarro Schmidt 2006), the particles were centrifuged at 4000 rpm for 20 min. Carefully transferred the dried pellet onto a clean slide and images were captured in a SEM, Quanta (200 FEI). For TEM, metal coated copper grids were filled, dried with 10 µL NPs and images were captured by tecnai G2 Sprit Bio TWIN at a voltage of 120 kV [12] (Strober 2001). The following were applied to get X-ray diffraction (XRD) data: Bragg angle 2θ at 2/min scan rate, Seifert Rayflex 300TT X-ray diffractometer, CuK (λ = 1.542 Å) radiation, 40kV voltage, 30mA current. Zeta potential/ scattered light intensity and particle size distribution were carried out on Horiba Scientific SZ-100. This helped us to measure and confirm the nanoparticle size distribution. Nicolet 6700 Fourier Transform Infrared Radiation (FRIT) was used to get the functional group information by using potassium bromide pellets at a range of 400-1cm to 4000 cm-1. 

hPBMC collection and trypan blue dye exclusion 
Since PBMCs are being isolated from human blood, we obtained ethics clearance (SDUAHER/KRL/Res. Project/128/ 2017-’18) from the institute ethics clearance department. The volunteer's blood was collected to isolate hPBMCs by Ficoll gradient method as per the protocol without any alteration [12] (Strober 2001). After the treatment (Table 1), equal volumes of the cells and 0.4% trypan blue were mixed and left at room temperature (37oC) for 10 min and 10 µL of undisturbed suspension was used to count the viable and non-viable cells in the hemocytometer.
Viability %= Number of cells/Total number of cellsX100
   
Effect of nanoparticles on DNA
By hemocytometer, cells were counted (104) and subjected to DNA fragmentation assay [13] (Pattern and Hirzel 2017). Pelleted cells were lysed in 20 µL of TES buffer (Tris-1M, EDTA-20mM and SDS-1%), added 10 µL each of RNase and proteinase K, incubated at 37oC for 1 h and 50oC for 1 hour and 30 min respectively. Spun down the sample, the entire supernatant was mixed with 6 X DNA loading dye, ran the agarose gel at 80 volts and documented the image.

Statistical analysis
Performed the experiments for three times, their averages were presented to minimize the errors.

RESULTS AND DISCUSSION
Without any complexity, the present study focused on NP synthesis by using easily available A. cathartica latex and MgO. When mixed and incubated equal proportions of 1 mM MgO and 3% aqueous latex, initial colourless solution turned into bottle green colour (Fig. 1), indicating that the phytochemicals of latex have been reduced and became solubilizing agents for the formation of MgO nanoparticles.

Characterization of particles was carried out by UV-Visible spectrophotometry, FTIR, SEM, TEM and Zeta potential 
Metal nanoparticles are good conductors and have plasmon resonance. The size of metal nanoparticles and light absorption are directly proportional to each other [14] (Kelly et al. 2003). This is the most widely used method to find the absorption spectra of nanoparticles, because the bands/peaks are correlated to diameter and the ratio of metal nanoparticles [15] (Philip 2008). Before the efficacy check, the green MgONPs were characterized by spectrophotometry. This gives the peak concentration. The nanoparticle size between 2 and 100 nm is conveniently measured at wavelengths between 300 and 800 [16] (David and Vanden Bout 2002). The absorption peak at 329 nm is the characterization of plasma resonance of synthesized MgONPs (Fig. 2), proven the development of nanoparticles UV-V is peak at 329 nm clarifies the reduction of latex phytochemicals and able to form MgONPs. As shown (Fig. 2), the absorption maximum was noted roughly at 329 nm, which coincided with another study [17] (Renata Dobrucka 2018). Further confirmation was carried out by SEM, TEM, FTIR and X-RD analyses.
FTIR spectroscopy helps to identify the likely biomolecules/phytochemicals responsible for the reduction and capping of the MgONPs. Also, helps to know the possible interaction between metal and the phytochemicals. The spectrum (Fig. 3) was taken between 4000 and 400 cm -1. The broad and strong peak at 3149 cm -1 is a stretching of the O-H group in an alcohol, which is a carboxylic acid [17] (Renata Dobrucka 2018) and a small peak at 2352.23 is due to O=C=O stretch. The results are synchronized with Ahmed et al. [18]  (Halbus Ahmed et al. 2019). 
SEM scans the surface and produces images of any sample that scans. The morphology of MgONPs obtained from SEM is shown in Fig. 4. The scale bar was 10 µm, 20, 50, 100 and 300 µm. The particles were in mass like and formed large clusters as mentioned in the previous study [17]  (Renata Dobrucka 2018).
In TEM, a beam of electron scans the sample, detects the sample’s surface topology and provides the shape and size. In the present study, the shapes of MgONPs noted were spherical, cylindrical, rectangular, hexagonal and round; the measured area, angle, length and diameter of the particles were shown (Fig. 5). About 36 particles were measured and the average particle size was noted as 32.3 nm. The minimum and maximum sizes noted were 10.6 and 61.3 respectively (Table 2).
Zeta potential/ scattered light intensity and particle size distribution were carried out on Horiba Scientific SZ-100. This helped us to measure and confirm the nanoparticle size distribution (Fig. 6).
MgONPs XRD analysis was carried out  and XRD analysis of the synthesized particles helped us to understand the crystal nature. (X-ray tube: Empyrean Cu LFF (9430 033 7300x) DK400339, Filter: Beta-filter Nickel, Detector: PIXcel-1D detector) (Fig. 7). 

Cytotoxicity on hPBMCs
The effect of MgONPs on hPBMCs was tested at 20 µg/mL, 40, 60, 80, and 100 µg/mL concentrations. At 72 h, cell viability percentage was assessed with trypan blue dye exclusion method. Increased concentration showed decreased viable cell percentage. The comparative data among the treatments was shown (Table 3). 
In recent years, nanoparticle based drug delivery systems have attained much attention. These high efficient nanoparticles are being synthesized or fabricated under suitable and ordinary conditions. Because of having large surface area, the synthesized particles have efficient biological interactions in  the in vitro model. Many research findings open up new avenues as NPs can be customized to induce antiproliferative action against cancer cells. They medicate the formation of reactive oxygen species and induce apoptosis and necrosis [19] (Vinardell and Mitjans 2015). NPs have high accessible area to relate to cell and other organelle membranes. Due to this property, membrane leakage happens and cancer cells induce ablation. Therefore, researchers focus on nanoparticle mediated therapeutics in cancers, most often, the NPs-derived anticancer drugs [20] (Behzadi et al. 2018). In the present study an exploration of the latex mediated MgONPs in vitro model by using hPBMCs was done. This will give an overview of understanding the fate of NPs in an in vivo model, when used to control proliferative activity of lymphocytes in leukemia. The potential activity of MgONPs as antimicrobial agents and their usage in drug delivery have been elucidated in many reports, but not been explored on hPBMCs. Many studies reported the possibility of preparing MgONPs from plant and its derivatives. These have proven their antimicrobial activity and wide range of effects on humans. The safe and unsafe use of NPs depends on the size, surface area, conditions used for their formation, concentration etc [21] (Mohsen Safaei et al. 2019). With respect to this, we used different concentrations of MgONPs on hPBMCs and noted the viable and non-viable cells.
The main apprehension about any drug is its distinguishing nature between normal and cancer cells. To keep this in mind, many researchers use green nanoparticles with maximum surface area and having potential antiproliferative effects [21] (Mohsen Safaei et al. 2019). When the particles are internalized into any cell, they act either on cytosol or on nucleus. If they are capable of entering into the nucleus, they may show DNA damage. If this occurs, then the DNA becomes a ladder pattern and subsequently the cell will collapse. To show the resultant action of MgONPs on DNA, we lysed the cells in lysis buffer and loaded in 1% agarose gel (Fig. 8). Unlike other metals [5] (Kiranmayee et al. 2020), the DNA fragmentation pattern indicates that there was neither shearing nor breaking of DNA into fragments. This might indicate that the increased concentration of MgONPs showed decreased percentage of viable cells, signified that the particles did not show any effect on DNA and instead, killed the cells just by bursting them. According to a report by Stoimenov et al., MgONPs exhibited antibacterial activity by bursting the membrane [22] (Peter Stoimenov et al. 2002). Present results are in coordination with Stoimenov, where MgONPs made from latex affected the cell membrane. In other experimental conditions [23, 24] (Ge et al. 2011; Karthikenyan Krishnamoorthy  et al. 2012), cytotoxicity of MgONPs was observed in cancer cells at 200 µg/mL and 300 µg /mL, however, in our experiment, the maximum concentration used was 100 µg/mL with per cent non-viable cells of 96. Though MgONPs have many applications, limited literature is available on its effect on hPBMCs. Reports are available on cytotoxicity of human umbilical vein endothelial cells [20] (Behzadi et al. 2018), human cardiac microvascular endothelial cells [25] (Sun et al. 2011), less toxic effects against human astrocytoma U87 cells [26] (Lai et al. 2008), applied MgO nano-shells in cancer therapy [27]  (Martinez Boubeta et al. 2010). MgONPs have been used as hyperthermia agents and biosensor in liver cancer treatment [28, 29] (Chalkidou et al. 2011; Lei et al. 2012). Machine dependent study on the harmfulness of MgO nanoparticles to cancer cells and MgONPs were recently been applied in nano-cryosurgery in tumor management [30] (Di et al. 2012). All these investigations support the fact that MgONPs show cytotoxic effects on cancer cells not limited to hPBMCs. Based on the preceding in vitro and in vivo studies, NPs show a wide variety of sound effects on humans. For functional execution, the NPs shall have nano range size, larger surface area, period of exposure for NPs synthesis, functional group adherence from the plant source and stability. All these properties specify whether the synthesized NPs have safety feature or not. In review, a few published articles proved their toxic nature and a few showed their positive effects and all these are under the survivelance of concentration and the size of MgONPs. Therefore, in the present study, we tested different concentrations of MgONPs on hPBMCs. Contrary to Ge [23] (Ge et al. 2011), we reported lower concentration (100 µg/mL) promoted the death of cells. Using low concentration was advantageous and coinciding with other studies [26, 31], (Lai 2008 et al.; Hasbullah et al. 2013). Taken together the literature survey and the present study results, green approached MgONPs’s usefulness and harmfulness on hPBMCs depend on phytochemicals of latex, composition of these phytochemicals, particle concentration, size and the target cells [2] (Heydary et al. 2015). Documentations claim that NPs have high adsorption potential to plasma proteins. Since MgONPs are the potential drug delivery candidates, having magnetic resonance image and suitable even at failed thermoregulation situations, exploration is needed to test against human chronic myelogenous leukemia and safety assessment of human serum albumin (HSA) as it contributes the formation of protein corona on the NPs [32] (Lesniak et al. 2012). To conclude from the present research results from latex mediated MgONPs on hPBMCs, these particles have a potential role in antiproliferative activity.

CONCLUSION AND LIMITATION
Allamanda cathartica latex, which can be collected from the trimmed twigs and MgO, which is available at low cost, are good sources of synthesizing nanoparticles on shaker at 370C. The authors used 3% aqueous latex and 1 mM MgO for NP synthesis. The colourless solution turned to bottle green colour indicated the plant latex has been reduced and became a stabilizing agent for MgONPs. The biophysical characterization techniques proved the biosynthesis of MgONPs. On an average, the particle size noted was 32 nm, which is a suitable particle to target the cells to be examined. When using latex MgONPs, an increased percentage of non-viable hPBMC cells were noted at as low as 100 µg/mL. Though nanoparticles did not fragment DNA, particles might stick to the cells, burst open the cells and finally led to cell death. Therefore, low concentration of latex mediated MgONPs can play antiproliferative role on cells. White blood cell number will be high in leukemic condition. Much research is needed to prove that latex mediated MgONPs might be a good source to control leukemic cells. Limitation of the study was to check the effectiveness of these green MgONPs on Leukemia cells. 

ACKNOWLEDGEMENT
Authors acknowledge the department of Cell Biology and Molecular Genetics for providing all the essentials for the successful completion of this work. 

CONFLICTS OF INTEREST/COMPETING INTERESTS
Authors declare no conflict of interest

ETHICS CLEARANCE
No: SDUAHER/KRL/Res. Project/128/ 2017-’18

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