Thymbra spicata leaf extract driven biogenic synthesis of Au/Fe 3 O 4 nanocomposite and its bio-application in the treatment of di ﬀ erent types of leukemia

: Herein, the bio-inspired synthesis of Au nanoparticles (NPs) adorned Thymbra spicata extract functionalized Fe 3 O 4 NPs as a novel magnetic nanocomposite has been demonstrated. The plant phytochemicals act as a nat-ural and non-toxic reductant as well as the in situ stabilizing agent of the NPs. The Au – Thymbra @Fe 3 O 4 composite was characterized over a range of advance physicochemical techniques like fourier transformed infra red (FT-IR) spectroscopy


Introduction
After the advent of nanoscience and technology long back in 1970's, the arena has been gradually enriched and developed with fascinating materials following diverse techniques and methodology [1,2].In modern days, nano-biotechnology is an enthralling subject, a perfect and complementary combination of nanoscience and biotechnology, more precisely, biotechnology-inspired synthesis and bio-application of nanomaterials [3,4].There are plethora of applications of these advanced functional nanomaterials in assorted fields like medicines and pharmaceuticals, drug delivery, bioengineering, agriculture, sensing, electronics, optics, cosmetics, energy and catalysis [4][5][6][7].Now, among the different nanomaterials, nanoparticle (NP) research is currently an interesting and hot topic having remarkable prospects [8,9].Regarding their synthesis, the biomolecular assisted pathway has garnered significant consideration in the last few years, accounting from the importance and necessity of following sustainable pathways in material sciences.In addition, biogenic synthesis involves several advantages like controlled shape and size, well-defined morphology, uniform dispersion, biocompatible, non-toxic, easily scalable and enhanced stability [10][11][12][13][14]. Explicitly, microorganisms (bacteria, virus, fungi, actinomycetes, yeast, etc.), enzymes and phytochemicals (plant extract) mediated biosynthesis of NPs have been the most promising pathway.The phytosynthesis over plant extract is considered as a distinctive, green and eco-friendly route which is free from complex and irk some methods like cell culture, incubation, microbial isolation, maintenance of proper conditions, etc.The synthesis is also quite fast, inexpensive, hassle free and uses water as the only green solvent [3,[15][16][17][18][19][20][21].
They also exhibit very good antimicrobial properties against a wide variety of micro-organisms [25,28].We are particularly interested on the anticancer applications of bio-engineered AuNPs as nanoformulated drug, due to having its unique pharmacokinetics, high surface area to volume ratio, superior drug carrying ability, appliance of minimum dose, biocompatibility and stability in the cell environment [29][30][31][32].Several current literature demonstrated the marvelous anti-cancer properties of bio-functionalized AuNPs against a number of diverse carcinogenic cell lines, both in vitro as well as in vivo, without any undesirable side effects [33][34][35][36][37][38].
Herein, we have demonstrated the bio-application of Au-Thymbra@Fe 3 O 4 nanocomposite toward the apoptosis or regression of proliferation of human leukemia cells.Two kinds of leukemia cells were used in the study, such as acute myeloid leukemia (32D-FLT3-ITD, HL-60/vcr) and acute lymphoblastic leukemia (MOLT-3, TALL-104).Leukemia is one of most lethal and invasive cancers and causes high rate of mortality.These days the nano-formulated biogenic drug like molecules have come up with tremendous potential in order to avoid the prevalent adverse effects encountered in the conventional treatment procedures like surgery, chemotherapy, etc. [34][35][36].In our investigations with Au-Thymbra@Fe 3 O 4 nanocomposite, we observed some excellent results against the said leukemia cancer cells like low cytotoxicity and % of cell viability that could make the material a potent anti-leukemia drug in the future.

Experimental method 2.1 Preparation of Thymbra spicata leaf extract
Cleaned and dried Thymbra spicata leaves (2.0 g) was added over DI H 2 O (50 mL) and gently warmed (70-80°C, 0.5 h) and filtered over Whatman-1 paper.The pale colored filtrate was treated as the Thymbra extract.

Green synthesis of the Au-Thymbra@Fe 3 O 4 NPs
The Fe 3 O 4 NPs were prepared following reported methods by co-precipitation.0.5 g Fe 3 O 4 NPs were sonicated for 0.5 h over 100 mL of DI H 2 O and the precursor solution of Au (1 mM HAuCl 4 , 50 mL) was slowly introduced.After swirling the mixture for 10 min, 10 mL of the Thymbra extract was added into it and heated under stirring (80°C, 1 h).The final composite product (Au-Thymbra@Fe 3 O 4 ) was isolated magnetically, rinsed carefully and dried at 60°C.

Antioxidant activities of Au-Thymbra@ Fe 3 O 4 nanocomposite
In order to determine the antioxidant potential of the final material in terms of DPPH scavenging capacity, equal volume of DPPH solution in MeOH (300 µM, 1 mL) was mixed to diverse concentrations of sample suspensions (1-1,000 µg/mL, 1 mL).Subsequently, these mixtures were vortexed and kept for 0.5 h.The change in color intensity (purple to pale yellow to colorless) was monitored by subjecting them to UV-Vis spectrophotometer at a wavelength of 517 nm.The final % inhibition (% I) or antioxidant capacity was determined using equation (1).
where A 0 and A s are the absorbances of standard and that of samples.

Anti-leukemia properties of Au-Thymbra@Fe 3 O 4 nanocomposite
Cytotoxicity of the Au-Thymbra@Fe 3 O 4 bio-nanocomposite were evaluated against the following cell lines by standard MTT process.I) 32D-FLT3-ITD, Human HL-60/vcr, MOLT-3 and TALL-104 (Leukemic) II) HUVEC (Normal) To carry out this research study, cancer cell lines were cultured in 10% complete culture medium (DMEM) and HUVEC cells in 10% complete culture medium (RPM) containing FBS and penicillin-streptomycin antibiotics in T25 flasks for cell culture.They were kept in a CO 2 incubator (temperature 37°C, 80% humidity, 5% carbon dioxide pressure).MTT test (Sigma, France) was used to investigate the toxicity of NPs.In this method the bluish violet formazan crystals are formed by the action of succinate dehydrogenase enzyme in the cells that are dissolved in solvents like DMSO and isopropanol.Finally, the % of cell viability was determined spectrophotometrically.At first, 96-well plates were prepared for each cell line and 5,000 cells were added to the plate well.Cell counting was performed using trypan-yellow dye and neobar slide.After incubation for a day and ensuring the adhesion of cells to the plate substrate.the bio-nanomaterial was added to each well in different concentrations and incubated for 48 h.Then the MTT solution was added to each well and again transferred to the incubator for 4 h.After draining the extra dye, DMSO solvent (100 µL) was added to them to dissolve the purple   Due to innate magnetism, the nanocomposite particles were recovered magnetically with ease.Thereafter, morphological structure and physicochemical characteristics of the nanocomposite were ascertained by a number of instrumental techniques like fourier transformed infra red (FT-IR) spectroscopy, scanning electron microscopy, energy dispersive X-ray (EDX) spectroscopy, TEM, inductively coupled plasma-optical emission spectroscopy and vibrating sample magnetometer (VSM).Figure 1 depicts the relative FT-IR spectra of pristine ferrite, plant extract and the final Au-Thymbra@Fe 3 O 4 nanocomposite.The unmodified Fe 3 O 4 NP is portrayed by the vibrations appearing at 446 (octahedral bending) and 586 cm −1 (tetrahedral stretching) of Fe-O-Fe bond.The vibration at 635 cm −1 can be ascribed to the ferrite cubic spinel structure.Two significant vibrational bands corresponding  to O-H stretching vibrations of peripheral hydroxyls and intercalated waters are observed at 3,412 and 1,641 cm −1 , respectively (Figure 1a).The respective vibrational peaks of phyto-organofunctions from Thymbra spicata are found at 3,402 cm −1 (O-H stretching), 2,922 cm −1 (C-H stretching), 1,732 cm −1 (C]O stretching), 1,616 cm −1 (C]C stretching), 1,421 cm −1 (C-O stretching) and 1,076 cm −1 (C-O-C stretching), respectively, which in turn justifies the polyol, flavonoid, flavanol, tannin and terpenoid moieties (Figure 1b).Finally, when we look at the FT-IR spectrum of Au-Thymbra@Fe 3 O 4 nanocomposite in Figure 1c, it seems to be a combination of the earlier ones, implying the fruitful association of Thymbra phytomolecules on Fe 3 O 4 NPs.Nevertheless, the immobilization of AuNPs could be substantiated by minor change in peak positions in Figure 1c as compared to Figure 1a and b.
Electron microscopy (TEM and SEM) was explored to find out the surface morphology, shape and size of Au-Thymbra@Fe 3 O 4 nanocomposite particles (Figures 2 and 3).TEM image evidently represents the spherical shaped particles of Fe 3 O 4 and AuNPs of grey and black colors, respectively (Figure 2).Both Au and Fe 3 O 4 NPs are almost homomorphic, but size wise the former is comparably larger.Sizes of the two NPs are in the range of 15-20 and 20-25 nm, respectively.The image embodies an amalgamation of the two NPs.Surface modifications by the plant phytochemicals over Fe 3 O 4 NPs can be anticipated by the homogeneous layer like appearance over them.Their uniform shapes, nanometric sizes and the resulting high surface area definitely afford some synergistic effect on catalytic activity.Figure 3 displays SEM image of the nanocomposite which also authorizes the spherical shaped particles, supporting the TEM data.However, phytomolecular modification over the NPs could not be identified from SEM. Due to preparation of samples manually, the nanocomposite looks to some extent aggregated.
EDX investigation of Au-Thymbra@Fe 3 O 4 nanocomposite was carried out in order to have information on its chemical composition as well as the quantitative estimation of its constitutional elements.The corresponding profile (Figure 4) clearly displays the occurrence of Fe and Au as metallic components.Similarly, C and O represent the non-metallic counterparts.A characteristic signal appearing at 2.2 keV rationalizes the Au NPs.The non-metals appearing in the lower region corresponds to the phytomolecules from Thymbra spicata extract.The EDX results were emphasized furthermore by elemental mapping analysis.As Figure 5 displays, SEM image of the material has been scanned over X-ray and the corresponding result demonstrates the distribution of same metal and non-metal species over the matrix (Figure 5).Markedly, they are well dispersed with an excellent homogeneity, which definitely has a significant impact on its catalytic applications.
For a magnetic cored material, the study of its magnetic properties is indispensable.Thereby, VSM analysis was carried out to study the same for Au-Thymbra@Fe 3 O 4 nanocomposite in an external magnetic field region of −20 kOe to +20 kOe.The output appears as a magnetic hysteresis curve with a saturation magnetization (M s ) value of 41.6 emu/g (Figure 6).Profile of the curve discloses it as a super-paramagnetic material and attracted by magnets even after the loading of non-magnetic AuNPs and the Thymbra biomolecules.

Evaluation of antioxidant capacity of
Au-Thymbra@Fe 3 O 4 nanocomposite A number of reports revealed that the substances that exhibit considerable anti-malignant properties are also very good antioxidant.Hence, the material Au-Thymbra@Fe 3 O 4 under investigation was explored to study its anti-cancer properties as well as the antioxidant ability.The latter was performed following DPPH radical scavenging studies, coupled with spectroscopic analyses.In order of that, a DPPH free radical solution in EtOH was mixed with the antioxidant material suspensions prepared in a range of concentrations (2, 3, 7, 15, 31, 62, 125, 250, 500 and 1,000 μg/mL).Soon after the mixing, the initial purple solution of DPPH radical turned to pale yellow due to partial or complete quenching.The free DPPH solution (purple) abstracts hydrogen or electron from the experimental antioxidant sample and gets paired up (pale yellow).The change in absorption under UV-Vis absorption spectrophotometer is monitored and subsequently the antioxidant capacity is determined in terms of % inhibition, as displayed in Figure 7.The results were compared with the standard BHT molecule.Markedly, the % inhibition capacity of Au-Thymbra@Fe 3 O 4 progressively increases with the increase in sample load.After 250 μg/mL concentration the results were almost parallel with the standard.This is definitely because of the amplified electronic effects of electron rich Thymbra phytomolecules and ferrite support.The corresponding IC 50 value in quenching 50% of initial DPPH molecules was evaluated as 57 μg/mL as compared to 31 μg/mL for BHT (Table 1).

Anti-leukemia effects of
Au-Thymbra@Fe 3 O 4 nanocomposite The nanomaterials that show potential cytotoxic or anticancer activities produce substantial amount of reactive oxygen species (ROS) during its action that has important role to intercede the proliferation of cancer cells or to destroy them specifically without affecting the normal cells via in vitro or in vivo studies.The sharp interfacial edges of the nanomaterial are also believed to cut through the cancer cell walls thus breaking down the cell DNA.The Au-Thymbra@Fe 3 O 4 bio-nanocomposite was engaged in investigating the cytotoxicity against acute myeloid leukemia (32D-FLT3-ITD, HL-60/vcr) and acute lymphoblastic leukemia (MOLT-3, TALL-104) cell lines.The rich polyphenolic content of Thymbra plant was also believed to help in damaging the leukemic cells.They strongly reduce the ATP content of the cell leading to smash up of the affected cell mitochondria.Moreover, AuNPs are capable of producing high concentration of ROS thus augmenting the cytotoxicity.Green synthesis of Au/Fe 3 O 4 nanoparticles  7 During the cytotoxicity studies, the Au-Thymbra@Fe 3 O 4 bionanocomposite was treated in diverse range of concentrations (0-1,000 µg/mL) of the cultured leukemia cells.
After incubation and other processing including the addition of MTT dye and formazan crystal dissolution, the related absorbances were measured over UV-Vis spectrophotometer.The corresponding outcomes have been recorded in Figures 8  and 9 in terms of % of cell viability at diverse concentrations.Manifestly, the % of cell viability of leukemia cell lines gradually reduced with the increasing doses (>7 µg/mL) of the bio-nanomaterial in all the four cell lines.After examining considerable cytotoxic potential of Au-Thymbra@Fe 3 O 4 bionanocomposite against the four leukemic (32D-FLT3-ITD, HL-60/vcr, MOLT-3, TALL-104) cell lines, it was quite essential to study its effect on the normal healthy cells as well.Hence, the related study was performed over the normal HUVEC cell line which displayed no such significant decrease in % of cell viability, as shown in Figure 10.

Conclusion
Herein, we demonstrated a biogenic system for the synthesis of Thymbra spicata plant phytochemicals templated AuNPs decorated over magnetic Fe 3 O 4 NPs.After adsorption over the ferrite surface, the oxygen rich plant phytomolecules assist the biogenic reduction of Au 3+ ions into AuNPs.The phytomolecules additionally stabilize the AuNPs by encapsulation and thus protect from self-aggregation.It   was meticulously analyzed by several advanced physicochemical techniques.SEM and TEM data confirmed the globular shaped particles with their size around 15-25 nm, without manifested agglomerations.The % of cell viability of leukemic cell lines was found to decrease with the increase in the dose of Au-Thymbra@Fe 3 O 4 .The corresponding IC 50 of the Au-Thymbra@Fe 3 O 4 bio-nanocomposite were determined as 125, 150, 133 and 170 µg/mL against the 32D-FLT3-ITD, HL-60/vcr, MOLT-3 and TALL-104 cell lines.The present analysis reveals the Au-Thymbra@Fe 3 O 4 bio-nanocomposite to be anticipated as a prospective chemopreventive and chemotherapeutic drug against leukemia cells.

3
Results and discussion3.1 Analysis of data of Au-Thymbra@Fe 3 O 4 nanocompositeHerein, a sustainable biogenic method for the fabrication of AuNPs over nano-magnetic Fe 3 O 4 surface has been documented employing Thymbra spicata leaf extract as a safe reductant and stabilizer.The stepwise synthesis of Au-Thymbra@Fe 3 O 4 nanocomposite involves the adsorption of Au 3+ ions over plant phytochemical modified Fe 3 O 4 NPs followed by in situ bio-reduction of the ions to AuNPs and stabilization by capping (Scheme 1).

Table 2 :
The IC 50 of Au-Thymbra@Fe 3 O 4 bio-nanocomposite in the antiacute leukemia test

Table 1 :
The IC 50 of Au-Thymbra@Fe 3 O 4 nanocomposite and BHT in the antioxidant test