Comparative analysis of transition and post transition metal mediated Allamanda cathartica L latex nanoparticles on human peripheral blood mononuclear cells

2.1.1. Test for alkaloids

2.1.2. Test for flavonoids

2.1.3. Test for saponins

2.1.4. Test for tannins

2.1.5. Test for glycosides

2.1.6. Test for reducing sugars

Equal volumes of 1 mM metal solution (Cu, Zn and Fe) were mixed properly with 3% water soluble latex solution and the mixture was incubated at 37 °C in an incubator shaker (135 rpm) for 24 h. The development of colour was observed and the samples were stored at 4 °C [17].

To check the absorption spectra of the synthesised NPs, UV-visible spectrophotometer was used. Particle spectra were obtained from Nanodrop 8000 between 200 and 700 nm. The gold standard methods for characterisation of NPs are scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The preparation protocol followed [16, 17], with a monor modification. Briefly, 2 ml of green NP was centrifuged at 4000 rpm for 20 min. The dried pellet was scrapped carefully with forceps and placed onto a glass slide. The images were captured in a SEM, Quanta 200 (FEI) and carbon coated copper 300 mesh grids were used for TEM analysis. About 10 μL green metal NPs was placed on metal coated copper grids which were dried under lamp. Tecnai G2 Sprit Bio TWIN with a voltage of 120 kV was used to obtain the images. The X-ray Diffraction (XRD) measurements were recorded on Bragg angle 2θ with 2/min scanning rate. This was done on a spectroscope by using Seifert Rayflex 300TT X-ray diffractometer with CuK (λ = 1.542 Å) radiation that was operated at a voltage of 40 kV and a current of 30 mA with Cu k-α radiation (1.5405 Å). To disclose the involved organic functional groups on the NPs, Fourier Transform Infrared spectroscopy (FTIR) was performed on Nicolet 6700 FTIR. Method involved was potassium bromide pellet and the range of the scan was from 4000 cm⁻¹ to 400 cm⁻¹. SEM, TEM, FTRI and XRD were done in Department of Microbiology and Cell Biology (MCBL), Spectroscopy Analytical Test Facility, and Materials Research Centre (MRC), Indian Institute of Science, Bengaluru.

Central ethics clearance was (SDUAHER/KRL/Res. Project/128/2017-18) obtained from the institute before we start the isolation of hPBMCs from leftover blood (collected in heparin tubes) from Central Diagnostic Laboratory, Sri Devaraj Urs Academy of Higher Education and Research. Without any modification, Ficoll gradient method was followed (1). hPBMCs were allowed to grow in RPMI for 4 h and inoculated with 20, 40, 60, 80 and 100 μg/ml green NPs from 1 mg ml⁻¹ stock. Minimum and maximum concentrations of DMSO, metal and 3% latex were used as controls. To name the controls: only hPBMC, hPBMC + DMSO (20 μL), hPBMC + DMSO (100 μL), hPBMC + 1 mM metal solution (20 μL) hPBMC + 1 mM metal solution (Cu, Zn and Fe) (100 μL), hPBMC + 3% latex (20 μL) hPBMC + 3% latex (100 μL).

Trypan blue (0.4%) in balanced salt solution and hPBMCs suspension were mixed in equal volumes and undisturbed for about 10 min at room temperature. About 10 μl of this suspension was transferred onto a clean slide and the cells were counted in hemocytometer [20].

DNA ladder pattern of hPBMCs was noted by taking 10⁴ cells and lysed with TES buffer (1 M Tris, 20 mM EDTA and 1% SDS) [21]. To the clarified cell pellet, 20 μL of lysis buffer was added and mixed properly, 10 μL RNase was added to the lysed cells, and left for one hour at 37 °C. The temperature was shifted to 50 °C, 10 μL Proteinase K was added and incubated for 1.5 h. The entire volume was loaded in 1% agarose gel with 6X DNA loading dye and ran at 80 to 100 volts. Image was documented by UV-transilluminator.

All the experiments were repeated three times to minimise the error and standard errors were calculated.

Tests for phytochemicals revealed that the aqueous latex has the following phytochemicals: flavonoids (yellow), saponins (frothing), glycosides (red) and reducing sugars (orange red).

Latex NPs were made by mixing equal volumes of 3% aqueous latex with 1 mM transition (copper chloride and zinc oxide) and post-transition (iron sulphate) metal solutions. Overnight incubation developed the colours, viz, dark brown, greenish black and bottle green with iron sulphate, copper chloride and zinc oxide respectively (figure 1). This indicated the excitation of surface plasmon resonance (SRP). Latex phytochemicals are responsible for the formation of NPs [26].

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Figure 2 gives the information about the UV-visible spectrum of NPs wherein, the absorption maximum was perceived at 575, 450 and 370 nm for copper, iron and zinc respectively. A study conducted by [27] showed the same peak pattern for zinc, [28] copper and [29] iron. The light coloured mixtures changed to dark colours, which is indicative of formation of oxide particles by irreversible interaction between phytochemicals and metals.

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The morphology, surface and size details of metal latex NPs were studied by SEM and TEM. The advantage of taking both the microscopies into consideration is that both will provide similar but distinct analysis. TEM images help us to know the size and shape of the synthesised NPs. By TEM, the captured particles were 21; the particle diameter ranged from 17.9 to 59.2 nm. The average size of the particle is 33.5 nm. Besides, the synthesised NPs are mostly round or spherical, a few were aggregated, having a wide-range size dispersal and figure 3 shows the TEM and SEM images of the particles. The results are in accordance with Viet et al [30]. Latex phytochemicals offer flexible control over the size and shape of the NPs. In NP biosynthesis, phytochemicals first reduce the metal ions and stabilise the metals in the form of NPs. So it is thumb of rule that phytochemicals of the latex control the size and shape of NPs. Results showed that the synthesised NPs are mostly round or spherical, hexagonal, aggregated and rectangular below 59, 38.6 and 55 nm for Cu NPs, Zn NPs and Fe NPs respectively (figure 3).

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The shape of the NPs was noted as spherical, rectangular, irregular, angular, hexagonal and aggregated. The NPs diameter ranged from 17.9 to 59.2 nm (Cu NPs); 20.5 to 38.6 nm (Zn NPs) and 29.2 to 55.4 nm (Fe NPs).

In general, FTIR spectroscopy analyses chemical composition of biological and non-biological materials. This analysis provides both qualitative and quantitative measures of material. The compounds present in test sample form a cap to metals thus stabilising the metal NPs. This method helps to identify the likely interactions between the metal NPs and test sample, in our study, the latex. IR spectra are the signatures of amide linkages of proteins and polypeptides. Figure 4 shows the FTIR spectra of the NPs taken between 4000 and 400 cm⁻¹. The present study results reveal different stretches of bonds at different peaks.

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In figure 4, Cu NPs FTIR spectrum, a broad peak at 3175.61 cm⁻¹ presents O-H stretch, strong and broad and compound class is carboxylic acid. Peak at 2922.49 cm^-1 might be carboxylic acid O-H stretch, −C–H stretch medium appearance and compound class is alkane; 2850.15 cm⁻¹ peak might be carboxylic acid O-H stretch, medium –C-H stretch, alkane; peaks at 2359.57 cm⁻¹ and 2337.23 cm⁻¹ are strong, O=C=O stretch carbon dioxide, carboxylic acid O-H stretch; peak at 1731.46 cm⁻¹ might be strong, C=O stretch aldehyde, aldehyde C=O stretch; and 1402.47 cm⁻¹ peak might be strong, C-F stretch fluoro compound. FTIR spectrum of FeNPs is presented in figure 4. Peak at 3272.48 cm⁻¹ is strong, broad, O-H stretch, alcohol/phenol, carboxylic acid; 2922.67 cm⁻¹ peak might be medium C-H stretch alkane/alkyl, carboxylic acid O-H stretch; peak at 2850.64 cm⁻¹ is medium C-H stretch alkane, carboxylic acid O=H stretch; and 2357.30 cm⁻¹ peak might be strong, O=C=O stretch, carbon dioxide. FTIR spectrum of Zn NPs shows peak at 3176.24 cm⁻¹, a strong, broad O-H stretching carboxylic acid; 2922.12 cm⁻¹ peak is a strong, broad N-H stretch amine salt, C-H alkyl stretch; peak at 2849.97 cm⁻¹ might be medium, C-H stretching, alkane and alkyl stretch; 2357.80 cm⁻¹ peak is a strong, O=C=O stretching, carbon dioxide and 1730.53 cm⁻¹ peak is a strong, aldehyde C=O stretch, α, β-unsaturated ester. The results are in agreement with a study conducted by using Euphorbia jatropha latex [27].

To test the effectiveness of the synthesised NPs as nano-carrier antiproliferative drugs, we tested the cytotoxic property of these NPs in terms of DNA damage, hPBMCs were exposed to different controls (positive and negative), and treatments. Cytotoxicity of NPs was carried out on hPBMCs with controls, NPs dose range was between 20 and 100 μg ml⁻¹, replicated three times and the results were expressed as mean ± S.D. The synthesised NPs were made to 1 mg ml⁻¹, the hPBMCs were treated at 20, 40, 60, 80 and 100 μg ml⁻¹, the minimum (20 μg ml^-1) and maximum concentrations (100 μg ml⁻¹) of DMSO, latex and metal were treated and incubated for 72 h. All the controls and treatments were stained with trypan blue to differentiate live and dead cells. In all the experiments, we observed a dose depended response, which means that as the NPs' concentration increased, there is an increased percentage of dead cells and decreased percentage of viable cells. Images were captured using CARL ZEISS microscope (Axion imager A2). Figure 5 represents the percentage of viable and dead cells obtained from respective treatments and controls. The order of antiproliferative activity of the NPs was Cu (86%)>Fe (87%)>Zn (95%). In-depth investigations are needed to find the mode of action of NPs (figure 5).

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Researchers are focusing on interaction between small molecules and DNA. One of the primary intracellular targets of anticancer drugs is the DNA of any cell type and interaction between them causes DNA damage by blocking cell division and leads to cell death [31]. Enthusiastically many researchers interested in finding out the binding properties of NPs to DNA, as new cancer drugs [32, 33]. The qualitative evaluation of NPs is the DNA fragmentation (figure 6).

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Control hPBMCs in A, B and C were free of any treatment, the DNA is still seen in the well, since there is no genotoxicity, indication of undamaged DNA, where as in other lanes, though there is no shearing of DNA, fragmentation was noted. Similar results were noted when MCF-7 cells were treated with Cu NPs at 160 and 320 μg ml⁻¹ doses, but comparatively, the NPs concentration was lesser (maximum 100 μg ml⁻¹) in our study [34].

In this study, equal volumes of 1 mM metal solution and 3% water soluble latex solutions were mixed properly at 37 °C in an incubator shaker (135 rpm) for 24 h to synthesise NPs. The change in colour indicates the formation of NPs. The NPs were characterised by standard methods and their efficacy was tested against hPBMCs. Barar et al in 2015 prepared metal NPs by using anticancer drug, mitoxantrone (MTX) [35]. The maximum particle size by TEM was 35 nm, wherein, the NPs synthesised by us also showed an average particle size of 34.6 nm. The smaller NPs of <10 nm of any metal can be removed quickly from the blood stream whereas larger particles are cleared by mononuclear phagocyte system. In our study we used hPBMCs, wherein if these cells are under uncontrolled condition, when given NP treatment, because of their nanosize, NPs can eliminate the diseased cells quickly from the blood stream to kidney to remove from the body. In other words, particle size and penetration go hand in hand; accumulation with the tumour is higher when smaller NPs interact with tumour. Alone, Cu metal at both the concentrations showed less toxicity to the cells, indicating the safe use of Cu metal in synthesising the NPs. The capping phytochemicals make Cu more effective in controlling the proliferation of hPBMCs, where the NPs can be used to control the division of lymphocytes in leukaemia. The mechanism of action of NPs on hPBMCs and non-effectiveness on normal cells with minimum side effects has to be elucidated. Though the NPs are non-toxic, latex is more effective in making the NPs more stable and biocompatible. Among the used transition and post-transition metals and metal oxides, it is evident that Zn NPs when mixed with Allamanda latex showed 95% effective antiproliferation property. Zinc, the 2^nd most transition metal, is considered to be an essential micronutrient required for the metabolic activities of humans, animals and plants [28]. Zn NPs can also be used for selective destruction of tumour cells and have a great potential in drug delivery applications [36]. ZnO-NPs have also been shown to exhibit strong protein adsorption properties, which can be used to modulate cytotoxicity, metabolism or other cellular responses [37]. Apart from these many applications, ZnO, due to its low toxicity, is listed as 'Generally Recognised as Safe' (GRAS) by the US Food and Drug Administration (21, CFR 182, 8991). In several studies it is evidenced that Zn NPs showed an increased in vitro cytotoxicity in glioma, colon, bone, breast, leukaemias and lymphomas [38–40]. Cancerous cells of lymphatic family when matched with the normal cells were approximately 28 to 35 times more vulnerable to Zn NPs mediated cytotoxicity [39, 41].

It is discussed by Rasmussen et al in 2010 [36] that Zn NPs can be a source of selective destruction of cancer cells and also have unlimited prospects in drug delivery applications. Modulation of cytotoxicity is due to ZnO NP's strong protein adsorption properties [42]. Ravindra et al in 2011 used Calotropis procera latex and ZnO for NPs synthesis and the average particle size was noted between 5 and 40 nm [43] and the maximum NPs in our works were 31 nm. Effect of ZnO NPs at a concentration of 1000 μg ml⁻¹ against PBMCs was determined by Rashmirekha Pati et al, [18] and clarified that ZnO NPs are significantly less, which is 15% reduction in cell viability. In our study, about 95% cells were dead when treated with a highest concentration of only 100 μg ml^-1 [18].