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Capping of Nanoparticles: An Alternative Approach for Reducing Nanoparticle Toxicity in Plants

The present era has witnessed a new dawn in technology innovations with the entry and use of nanomaterials in the industries and in the products used and to be used in day-to-day life creating a huge possibility of ending up in the food chain. Several studies in the past has highlighted toxicity of nanomaterials due to their size. However, we cannot stop technological advancements provided by the nanomaterials fulfilling human needs but can find a solution to the toxicity of the nanomaterials for a better future and safe environment. In this study, we propose capping of nanomaterials to reduce the toxicity without compromising their functionality. Capping of the nanomaterials is used to passivate nanomaterials but the same capping also helps in the reduction of surface reactivity leading to low toxicity. We studied phytotoxicity in the presence of one of the most extensively used metal nanoparticles (copper nanoparticles) on Eleusine corcana G. (finger millet) and Paspalum scrobiculatum L. (Kodo millet). Copper nanoparticles were synthesized by the hydrometallurgical methods. Ethylenediaminetetraacetic acid (EDTA) was used to cap the nanoparticles during the synthesis. In vitro studies results showed that the toxicity of copper nanoparticles is significantly reduced after capping. Anti-bacterial activity studies showed no change in efficacy of copper nanoparticles after capping. This study highlights the use of capping to reduce the toxicity of nanomaterials without sacrificing their required applicability.


Introduction
We are living in the era of nanotechnology, and with the development of new characterization and fabrication technologies, new nanotechnology-based products are constantly developing. Recent advances in semiconductors, polymers, metallurgy etc 1-2 has enabled the technology to reach to the masses which widespread applications to human race (Liu et al 2018,). Lots of different fabrication methods are developed to synthesize copper nanoparticles [18][19][20] . However, we choose an industrial scale synthesis method generally used to extract copper from copper sulphate waste, making the study a greener effort for sustainable environment.
In this report, we propose a widely used capping of nanomaterials method (used to enhance stability of nanomaterials) to reduce environmental toxicity. Copper nanoparticles with and without capping are synthesized by hydrometallurgical methods. The effect of nanoparticles of the growth of millets are studied in-vitro. Anti-bacterial activity of nanoparticles were studied to see effect of capping on the applicability of nanomaterials.

Particle size measurement
Transmission electron microscopy (TEM) was used to determine average particle size of synthesized copper nanoparticles (Cu NPs). Fig. 1 (a-b) is showing TEM images of un-coated and coated CuNPs. ImageJ software tool was used to measure particle size. It is clear from the micrographs that the particle size of both coated and uncoated copper particles is in the nanometer range. The average particle size of uncoated and coated CuNPs is ~ 8 nm and ~11 nm, respectively. A little higher particle size could be due to capping layers on the surface of the CuNPs. Agglomerated particles are visible in the micrograph of coated CuNPs because of the presence of EDTA capping layers. Where, λ is wavelength of the X-rays used, βtot is full width at half maximum of the peak, K is a constant (K = 0.95 used for near circular samples) ,and θ is corresponding Bragg's angle. The crystallite size is calculated by size component (Kλ/L) which is equal to the intercept on the y-axis (βtot cosθ). The approximated crystallite size is ~10 nm. The results of XRD also suggest nanocrystalline CuNPs as observed in TEM.

Influence of nanoparticles on plant morphology
Germination percentage of PR-202 was almost equivalent to that of control for all treatments while it was reduced for GPUK-3 ( Table 2). The findings are like the earlier studies where different metal nanoparticles drastically reduced the germination percentage in wide variety of plants as of iron on rye grass, barley and flax 26 , cerium oxide on maize, tomato, cucumber and soybean 27 and zinc oxide on corn 28 . Germination index was measured by calculating both the total and mean germination rate of seeds after specific time duration. Germination index was decreased in Kodo millet and more significantly upon uncoated nanoparticles exposure. The plausible reason could be that nanoparticles may be accumulated in the actively growing tissues especially in meristem, disrupting the regular growth mechanism and allowing the cells to choose alternate growth pathways to mitigate the toxicity effects causing decrease in . We did not observed this difference in Finger millet and the reason is not known to us at the moment. The Fig. 3 (a-b) shows length measurements of in vitro grown finger millet and Kodo millet, respectively. In the case of Kodo millet, significant reduction in shoot and root length after uncoated CuNPs treatment as shown in Fig. 6 like what we observed in Finger millet. Here, the growth was reduced in the plantlets after coated CuNPs treatment too. The relative growth was higher in the plantlets treated by coated

Enzyme assay
The immediate molecular response by any plant species under stress is the production of reactive oxygen species which in turn execute the signal transduction pathways by acting as secondary messengers to relay the information further to trigger the defense mechanism of the plant 32 . Different cell organelles like mitochondria, peroxisome, etc. detoxify these ROS by the outburst of antioxidant enzymes like superoxide dismutase, catalase, peroxidase 33 .
Normally it has been reported several times that introduction of heavy metal ions or particles like silver and gold (above certain concentration) increase the enzymatic activity like POX and CAT activity in the test plants due to increment in the ROC in the tissues [references]. But here, reduction in POX activity was seen in both the cases and more pronounced in the case of coated CuNPs (Fig 7). This is due to combine effect of polymer capping of nanoparticles which reduced the speed of oxidation of metal particles into ions causing less ROC in the tissues and lesser concentration of metal nanoparticles in the medium. Copper nanoparticles coated with polymers are stabilized and released slowly thereby reducing their participation in redox reactions. The uptake and incorporation of nanoparticles into the plant cells have already been discussed by many researchers 36-39 . CuNPs is reported to elevate the levels of catalase in roots of lettuce exposed to NPs hydroponically ( Trujiilo-Reyes et al 2014) and it was found that in roots of lettuce exposed to Cu/CuO NPs, the activity of catalase (CAT) increased, while that of ascorbate peroxidase (APX) decreased.

Chlorophyll content
Copper element plays important role in metabolism of macromolecules as well is directly involved in

Conclusions
Polymer capping has minimized the toxic behavior of copper nanomaterials to an extent in food plants.
The results show a new way of reducing the toxicity caused by applied nanomaterials in the environment.