Sustainable Microwave Assisted Synthesis and Anti-proliferative Response of Starch-Based CNT-IO and CNT-ZO Nanocomposites: A Comparative Study

Starch based carbon nanotubes-iron oxide (CNT-IO) and carbon nanotubes-zirconium oxide (CNT-ZO) nanocomposites (NCs) were fabricated using facile microwave irradiation technique from different plant-based starch sources namely Tapioca sago (TS), Hordeum vulgare (HV) and Eleusina coracona (EC) and metal oxide precursor solutions. The prepared NCs were studied by different characterization techniques such as SEM, TEM, XRD, Raman spectroscopy and FTIR spectroscopy. The Structural morphology of Starch based CNT-IO and CNT-ZO NCs observed like a rolled tubular structures exhibiting strong blue luminescence with the charge transfer between CNT and metal oxide. We have evaluated the anti-proliferative and anti-oxidant activity of starch-based CNT-IO and CNT-ZO NCs using MCF-7 cell line compared with the standard drug camptothecin and DPPH assay. Among the various NCs studied antioxidant activity of CNT-IO and CNT-ZO obtained from starch source Eleusina coracona was effective with IC50 values of 12.3 and 18.2 µg/mL respectively. The results suggest that EC/CNT-IO and EC/CNT-ZO NCs are most efficient in cytotoxicity against MCF-7 cells.


Introduction
Green nanotechnology aids in the synthesis of eco-friendly, safe and commercially viable nano-technology based materials. The main objective of green technology plays a vital role in fabrication of materials that are safer. The electronic, photochemical, chemical and optical properties of metallic, carbon, polymeric and metal oxide based nanoparticles synthesized by green nanotechnology has been received great attention rather than other materials [1]. The combination of nanoparticles by green technology, utilizing both biotechnology and nanotechnology, has gained much importance due to increasing need for the synthesis of customized and eco-friendly materials [2].
The exceptional and enhanced optoelectronic, catalytic, magnetic properties were exhibited by metal oxide nanomaterials in comparison to their counterparts [3][4][5][6]. The noble metal nanoparticles are extensively studied for the freeelectron excitations which exhibits strong optical absorption in the visible region [7]. The novel zirconium and iron oxide metal nanoparticles are under high priority due to their numerous applications [8,9]. These neoteric metal oxide nanoparticles are utilized in bio-labelling, optics (nonlinear), chemical reactions (catalyst), electric batteries to intercalate materials (optical receptors), anti-bacterial applications and selective spectral coating in absorption of solar energy.
It was earlier reported that different inorganic substrates like zeolites and silica were intrigued by carbon based metal nanoparticles, those displayed structure with core shell frames [10][11][12]. These materials were found to have important and effective attributes viz., surface functionalization, photoelectric, physical and chemical characteristics which protect the species from tough unfavorable conditions with increased stability [13,14].
On the other hand, starch is eco-friendly, feasible and sustainable polysaccharide [15,16]. Plant based starch has complex coarse grained structures which are semi crystalline in nature. These crystals range from 1 to 100 mm in diameter [17]. Due to its undesirable characteristics like biodegradable and in-toxic nature, starch is extensively used as drug carrier [18]. Application of metal oxide nanoparticles with starch polymers have attained great recognition for its ability of natural polymer shielding, thus increasing the forces to build up the consistency between different attractive forces acting on nanoparticles [19].
The advantages of microwave energy made them to utilize more in the studies for nanoparticle synthesis. The characteristic feature of homogenous heating is the most advantageous in using the energy source from microwave for producing specific nanoparticles. It was earlier reported that synthesis of iron oxide nanoparticles in compatible with organic materials using microwave method [20]. The synthesized iron oxide nanoparticles using microwave energy were utilized in the application of molecular imaging [21]. Mostly, hydrothermal technique was employed in synthesis of zirconia nanoparticles, often few reported, stated that nanoparticles of zirconia were synthesized using microwave method [22,23].
The present work is focused on the development of new nanomaterials based on metal (Fe and Zr) oxides and carbon, where the soluble starch extracted from Eleusina coracona [EC], Tapioca sago [TS] and Hordium vulgare [HV] were used as the carbon sources. The nanomaterials were synthesized by microwave irradiation, which is very rapid and simple technique. The metal (Fe, Zr) oxide particles generated during the synthesis of nanoparticles were stabilized by the EC, TS and HV starch solution, which acts as carbon source in this technique. The starch solution is mixed with the metal oxide precursor solution and the reaction mass obtained was further processed without the addition of any precipitating agent.
The synthesized materials were confirmed and characterized by XRD, Fourier transform infra-red spectroscopy (FT-IR), FT-Raman, scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV-Vis spectroscopy and photoluminescence spectroscopy. MTT and DPPH assays were performed on MCF-7 cancer cell lines to analyze the cytotoxicity and the anti-oxidant activities of the synthesized NCs, respectively.

Synthesis of CNT-Iron Oxide and CNT-Zirconia Oxide Nanocomposites
The starch sources from Eleusina coracona [EC], Tapioca sago [TS] and Hordium vulgare [HV] were employed in the synthesis of CNT-IO and CNT-ZO NCs with microwave method using anhydrous ferric chloride and Zirconyl oxy chloride as precursors. The seeds of EC, TS and HV purchased from the market, were washed thoroughly with distilled water and dried in sunlight. The completely dried seeds of EC, TS and HV were finely powdered by grinding. These powders were used for the preparation of 5% (w/v) starch solutions, separately. Six beakers with starch solutions (two with TS, two with HV and two with EC) were exposed to 800 W microwave energy for 10 min. Immediately, to the hot starch solutions of EC, TS and HV, added anhydrous FeCl 3 (2 g) to produce CNT-IO NCs and ZrOCl 2 ·8H 2 O (1.168 g) to produce CNT-ZO NCs separately, then exposed the beakers with mixture solutions to 800 W energy for other 10 min. in a microwave oven. The colloidal solutions are formed after the treatment. To the colloidal solutions, added ethanol and washed the solutions several times with distilled water. The washed colloidal solutions were incubated for 12 h for aging. After aging, the solutions were dried at 100 °C for 24 h to obtain NCs. The NC powders produced consists of CNT-iron oxide (IO) and CNT-zirconia oxide (ZO). The illustration scheme for synthesis of CNT-IO and CNT-ZO NCs is shown in Fig. 1.

Characterization
Structural Properties of NCs were thoroughly studied using different characterization techniques. The particle size and crystalline phase of the nanocomposites were studied by X-ray diffraction (XRD) on X'pert Pro X-ray diffractometer (P analytical B.V., Netherlands) using Ni filtered Cu Kα radiation (λ = 1.5406) from 2θ = 0° to 60°. The morphology of nanocomposites were investigated using ZEISS EVO 18 model Scanning Electron Microscopy, operating at 40 kV and a current of 30 mA at a scan rate of 0.388 min −1 . The Transmission Electron Microscopy (TEM) images of the nanocomposites were obtained on a JEOL JSM-6500F with an accelerating voltage of 200 kV to know the morphology. The chemical composition of designed nanocomposites were characterized by FTIR and spectroscopy using a Bruker Alpha FTIR spectrophotometer in order to know the organic moieties present in the sample using KBr pellet method (wavenumber range 800-3600 cm −1 ) at room temperature.

Cell Cultures
Human breast cancer cell line MCF-7 was purchased from NCCS and the cells were maintained in DMEM (Dulbecco's modified Eagles medium), supplemented with 10% FBS, penicillin/streptomycin (0.5 mL −1 ) at 37 °C in 5% CO 2 incubator. Exposure studies with CNT-IO and CNT-ZO NCs were conducted in MCF-7 cells seeded in culture grade flasks (T25 flasks, Tarson Inc., India).

Anti-proliferative Activity of NCs Using MTT Assay
To understand maximum efficacy of NCs, MTT assay was performed to assess cell metabolism, proliferation, and viability [24]. Concisely, cells were seeded at 5 × 10 3 cells density per well and incubated overnight in DMEM supplemented with 10% FBS at 37 °C and 5% CO 2 . Next day, old media was discarded and replaced with 100 µL of fresh media with different concentrations of NCs (5, 10, 25, 50,100 µg/mL) in 96 well plate and incubated for 48 h. Cells were incubated with fresh media with 100 μL of MTT solution (0.5 mg/mL −1 ) for 3 h at 37 °C in 5% CO 2 incubator. Cells with metabolically active mitochondria reduce MTT salt to chromophore formazan crystals which appears as precipitate in the 96 well plates. Camptothecin is used as a standard drug control. Microplate reader is used to read the optical density of solubilized crystals in DMSO at 570 nm [25,26]. Average value of the optical density of the biological replicate standardized using negative control (cell culture medium and cells) and positive control (with drug) is used to determine Cell viability for  each treatment group. All the experiments were performed in triplicates and IC 50 was calculated from the graph thus attained.

Anti-oxidant activity of NCs
1,1-Diphenyl-2-picryl hydrazyl (DPPH) assay was used to study antioxidant property, and potential to scavenge the free radicals [27,28]. Reduction of ethanolic DPPH by a hydrogen donating antioxidant is the basis of the DPPH assay, DPPH is reduced to its non-radical form (DPPH-H

XRD Analysis
The X-ray diffraction analysis of starch-based CNT-IO and CNT-ZO NCs synthesized by microwave method disclosed the crystalline nature of the nanocomposites. In Fig. 2a and b, the crystalline peaks at 2θ = 19.62°, 21.88°, 26.29°, 41.6° were exhibited by the NCs in the X-ray diffraction analysis. The peak at 26.29° corresponding to the graphitic (002) planes and the sharp peak at 41.6° corresponds to (100) planes of CNT. Moreover, improper alignment of CNT causes more defective structure [29][30][31] due to the metal ion incorporation.
The nano sized carbon dots with CNT's were identified by the prominent peaks below 20° and the later increased peak width was observed. In addition to the CNT peaks, in (α) phases, respectively [29,32]. The results of CNT-IO NCs disclosed the reduced crystallinity nature in TS/CNT-IO compared to HV/CNT-IO and EC/CNT-IO i.e., the starch composite of TS/CNT-IO is less crystalline among the tested starch nanocomposites. In the process of conversion of starch to carbon dots and tubes, the formation of inverse spinel structure of iron oxide particles is crucial and is clearly distinguishable. In Fig. 2b, the lattice (111), (200), (220), (311) and (211) planes of the tetragonal phase of ZO [33] produce the peaks at 2θ = 30.5°, 34.62°, 50.58°, 59.9° and 61.9° for CNT-ZO NCs, the peaks of composite ZO are slightly shifted when compared to the peaks of pure ZO synthesized by green technology [22]. The results and observations for CNT-ZO NCs revealed the reduced crystallinity nature in TS/CNT-ZO and HV/CNT-ZO compared to EC/CNT-ZO NCs. Thus the reduced crystallinity made the ZO more amorphous.

UV-Vis Spectra
The UV-Vis spectrum of starch-based CNT-IO and CNT-ZO NCs at 200-1100 nm wavelengths were recorded and depicted in Fig. 3a and b. Prominent absorption bands at 321 nm, 360 nm, 408 nm, 500 nm, 655 nm and 730 nm related to CNT-IO NCs was shown in the UV-Vis spectra in Fig. 3a. The charge transfer between O (2p) orbitals and Fe 3+ (d) orbitals also the transitions in the ligand field of Fe 3+ generated a strong absorption band at 321 nm in the UV-Vis spectrum, whereas, the formation of ferric oxide nanoclusters with α and ϒ phases developed weak absorption bands at 360 nm, 408 nm, 500 nm and 655 nm [34,35]. Moreover, the formation of CNT-IO complex is due to the π → π* transition of π electrons in CNT, which is observed as a peak at 730 nm and is also confirmed by XRD analysis [36].
In Fig. 3b, the UV-Vis spectra showed that the CNT-ZO NCs has a prominent absorption band at 212 nm, 290 nm, 300-400 nm and 650-720 nm. The tetragonal Zirconia developed a characteristic blue peak, which is shifted from the bulk Zirconia to create a strong absorption band at 212 nm. Further, the charge transfer from ligand to metal between O (2p) and Zr 4+ (d) orbitals results in generation of absorption peak at 290 nm. Weak and broad absorption bands are observed at 300-400 nm and 650-720 nm which corresponds to Zr-O transitions and the formation of CNT-ZO complex due to π → π* transition of CNT π electrons, respectively [36].

FTIR Analysis
The FTIR analysis revealed information about crystal morphology, nature of surface hydroxyl groups for starch based CNT-IO and CNT-ZO NCs ( Fig. 4a and b). Fiure 4a corresponds to CNT-IO NCs and Fig. 4b corresponds to CNT-ZO NCs. In both Figs. 4a and b the characteristic FTIR peaks at 3416 cm −1 , 1650 cm −1 , 1470 cm −1 , 1034 cm −1 is due to the -OH stretching, -OH bending vibration, -CH stretching and -CO stretching with respect to the carbon phase of starch solutions [36][37][38][39][40]. The additional peak at 584 cm −1 in Fig. 4a confirms the Fe-O Stretching of deposited IO in the carbon nanostructure [37] and the peaks at 2926 cm −1 , 600-800 cm −1 in Fig. 4b confirms the CH stretching, Zr-O bonds in the carbon nanostructure [38].

FT-Raman Analysis
The Raman analysis is studied for starch-based CNT-IO and CNT-ZO NCs, shown in Fig. 5a and b. In XRD and FTIR analysis, the characteristic peaks of carbon material in the composites are not clearly defined, hence the Raman spectra reveals the nature of carbon component present in the composites. In both the Fig. 5a and b, the corresponding Raman shift at ~ 1350 cm −1 and ~ 1580 cm −1 are characteristic D and G bands of carbon materials present in both the composites respectively [41][42][43][44].

Band Gap Energy
It is very essential to evaluate the material band gap for the nanomaterial semiconductors. The distance between the conduction band and valence band of electrons is said to be band gap. It represents the minimum energy required for the electron in the valence band to excite to conduction band to participate in conduction. Thus the energy required is known as band gap energy. Insulators possess large (> 4 eV) band gap energy whereas it is low for semiconductors (< 3 eV). The calculated band gap energies of CNT-IO and CNT-ZO NCs are tabulated in Table 1. The band gap energies were calculated using the following formula mentioned below  where E bg corresponds to the band-gap energy and λ to the wavelength (nm) absorbed by the material.

Photoluminescence Spectra
The luminescence of pristine IO and ZO nanoparticles in the visible and UV regions were reported in our earlier papers [22,32]. The intrinsic defects generated during the nanoparticle synthesis produces emission peaks in the visible region; moreover, the excitonic recombination generates an emission in the UV region. The surface passivation of the defects in the nanoparticle results in varied PL responses by the CNT matrix embedded with pristine IO and ZO nanoparticles. The increased luminescent properties exhibited by the defects in the CNT-metal oxide structures were studied experimentally and theoretically [46,47]. Depending on the method of synthesis, defects in the surface and the morphology of the metal-oxide (M-O), the emissions of the M-O may be reduced or intensified. The high quality IO and ZO dots synthesized by green technology exhibited characteristic broad yellow-green, orange red emission (large defects, ionized oxygen vacancy) with a strong near band edge emission [22,32]. On the other hand, in Fig. 6a and b, the pl spectra recorded for CNT-IO and CNT-ZO NCs at 360 nm excitation wavelength showed the quenching effect showing lower order broad visible range emissions at 450 nm extending its tail to longer wavelength compared to sharp bands in pristine IO and ZO nanoparticles. These characteristic emissions may result due to reduction in the visible luminescence or replenishing the defects in the nanoparticles with CNT or the pristine IO and ZO transfers the photoexcited charges to CNT empty state orbitals. The enhanced reduction in the luminescence was more in CNT-IO and CNT-ZO NCs than pristine metal (Fe, Zr) oxide nanoparticles. In the process of IO and ZO nanoparticles synthesis with CNT network exhibited a strong blue emission and slightly green emission, generally produced by the concentration of CNTs and oxygen vacancies. In this synthesis, may be the sharp edges of pristine metal (Fe, Zr) oxide nanoparticles are connected to the CNT network.

SEM and TEM
Surface morphology and chemical composition of starchbased CNT-IO and CNT-ZO NCs were analyzed by scanning electron microscope equipped with energy-dispersive X-ray spectroscopy is shown in Fig. 7a and b. Present studies shows that IO and ZO particles with CNT frame work are observed as perfect dispersed spherical balls and rods under SEM analysis. The structures are of irregular morphology, may be due to slight agglomeration of particles. EDAX analysis shows signal characteristics of Fe and O, Zr and O with incorporation of C without any impurity shown in Fig. 7e and f. TEM is used to investigate the microstructure of starchbased CNT-IO and CNT-ZO NCs. Due to preferred heterogeneous nucleation and interfacial interactions, many IO and ZO nanocrystals on the CNTs are densely deposited. The CNT has a rolled tube-like structure that can be seen clearly. The CNT plays a pivotal role in allowing IO and ZO nanoparticles to disperse on its surface, TEM image ( Fig. 7c  and d), which depicts the lattice of both starch based CNT-IO and CNT-ZO NCs, reveals the close interfacial contact between CNT-IO and CNT-ZO.

Anti-proliferative Activity
Carbon nanotubes are known as modern tools in nano-biotechnology for their unique properties, have great potential applications in suitable substrates, cell growth in tissue engineering, biomedical devices, and as vectors for gene transfection [48,49]. Daxiang Cui et al. [50] who reported that the SWCNTs effect on HEK293 cells, cells were arrested in G 1 phase after 25 μg/mL exposure of SWCNTs in the medium, and this resulted in drastic decrement in the cell number in the S phase. Also Bottini et al. [51] showed the cytotoxicity effect of MWCNT carboxylic on T lymphocyte and T leukemia cells, which led to 50% cell death after 24 h, with the use of TB. Tian et al. [52] showed that the effect of SWCNTs on cell viability of human keratinocytes at the concentration of 100 μg/mL gave 79, 50, and 31% viability after days one, three, and five, respectively, which correlates to our results. Another study has also reported functionalized CNT (hydroxylic (-OH) and carboxylic (-COOH)) reduced cell viability to 33% with use of the MTT assay [53,54].
In the present study, MCF-7 (human breast cancer cell lines), were employed in assessing the cytotoxicity of starchbased CNT-IO and CNT-ZO NCs. Cytotoxic studies of pristine IO and ZO nanoparticles on MCF-7 cell lines were reported earlier in our previous papers [22,32] showed good cell viability with IC 50 values of 87.2 µg/mL and 59.70 µg/ mL. IO and ZO nanoparticles with CNT conjugation dramatically reduced cytotoxicity compared to pristine IO and ZO nanoparticles, may be related to decreasing probability in accessibility of Fe/Zr source with cells. The blocking of the entrance of CNT or the bonding sites of IO and ZO (generation of ROS) resulted in reduced toxicity. CNTs plays a crucial role in release of intracellular dissolved metal ions (that generate ROS) into cytosol, reach nucleus and results in the death of the cell. Moreover, tracking studies revealed that the nucleus of MCF-7 cells contains CNT-IO and CNT-ZO NCs suggesting the effective deliver of target compounds into cytosol and nucleus of the MCF-7 cells.
In conclusion, the results indicated dose dependent cellular mitochondrial activity CNT-IO and CNT-ZO NCs shown in Fig. 8a Table 2 might be due to the starting material is a bio material capped with metal ions inducing greater potential towards cancer cell death, opens a new avenue in biomedical applications.

Antioxidant Activity
The radical scavenging activity of starch-based CNT-IO and CNT-ZO NCs synthesized by microwave technique are depicted in Fig. 9a and b. The DPPH solution colour changed from deep violet to pale yellow gradually in presence of starchbased CNT-IO and CNT-ZO NCs. The low peak intensity of DPPH can be clearly visible from UV-Vis absorption curves. The free radical scavenging percentage was calculated from the absorbance value decreased at 517 nm. The radical scavenging activity remains almost same with increase in the concentration of starch-based CNT-IO and CNT-ZO NCs that compared with Ascorbic acid as standard antioxidant. The IC 50 values are shown in Table 3.

Conclusions
Sustainable starch-based CNT-IO and CNT-ZO NCs were synthesized by simple and rapid microwave irradiation technique. The structural morphology of starch-based CNT-IO and CNT-ZO NCs were rolled tube like structures exhibiting strong blue luminescence with the charge transfer between CNT and Metal oxide. Moreover, the enhanced antiproliferative and antioxidant activity has been observed for the synthesized NCs. Hence, in the forthcoming years, starch based CNT-IO and CNT-ZO NCs by green technology using microwave technique may serve as cost effective and ecofriendly method for the synthesis and production of luminescent starch based carbon/metal (Fe, Zr) oxide composites in larger scale and acts as promising materials in bio imaging by tuning its luminescent properties.