Synthesis, Characterization and Safety Assessment of Zinc, Manganese and Copper Nanoparticles for Incorporation in Broiler Chicken Feed

Zinc, manganese and copper are important trace minerals in poultry nutrition. In birds, zinc is necessary for proper feathering, growth, skeletal development, skin quality, reproduction and disease resistance through boosting the immune system (Naz et al., 2016). Similarly, manganese is required for embryonic development, weight gain, bone growth, blood sugar homeostasis, immunological function, cellular energy, blood coagulation, cellular defense against free radicals and reproduction (Olgun, 2017). Copper is also regarded as a critical trace element in poultry diet and it aids in the creation of connective tissues like collagen and elastin, as well as the development of the nervous system through dopamine synthesis Mroczek-Sosnowska et al. (2013).
       
Traditionally, trace minerals have been supplemented in poultry diets using inorganic salts such as zinc oxide, manganese oxide or sulphate and copper sulphate. However, in recent years there are increased concerns about the usage of inorganic trace mineral salts due to their poor bioavailability and potential to cause mineral antagonism, which impairs absorption of minerals and increases environment pollution.Minerals, particularly trace minerals, have a limited bioavailability in the bodies of animals (Star et al., 2012). Trace element nanoparticles can minimise mineral antagonism in the gut, resulting in the increased absorption and less excretion in the environment.
       
Further, these may also help to improve the immunological responses and digestive efficiency of birds, resulting in increased feed efficiency (Marappan et al., 2017).
       
Nanoparticles can be produced through different techniques, which can be broadly classified into top-down method and bottom-up method. Top-down method is also called as physical method whereas; bottom-up method is also called as chemical method. Top-down method involves breaking down of a bulk material into nano-sized particles using cutting, grinding and etching techniques, i.e., nano materials are produced from larger entities without atomic-level control (Umer et al., 2012). Ball milling is the simplest mechanical top down (destructive) method of synthesis of nanoparticles.
       
Nanomaterial toxicity is strongly influenced by factors such as particle size and surface area (Nel et al., 2009). Because each type of nanoparticle has its own unique physicochemical properties, the harmful effects on cells may differ. Some harmful effects are irreversible and permanent, resulting in cell death, whereas others are reversible if nanoparticle exposure is eliminated, allowing cells to proliferate normally. Hence, this study was carried out to synthesize, characterize and evaluate the cytotoxicity effect of nano forms of zinc, manganese and copper.
 

Synthesis of nano forms of zinc, manganese and copper
 
Nano forms of zinc, manganese and copper were synthesized through physical method at the Department of Animal Nutrition, Madras Veterinary College, Chennai during 2021. Ball milling was the technique employed for the synthesis. The procedure of synthesis of nano forms of zinc, manganese and copper was optimized by method development. Nano form of zinc was synthesized by grinding feed grade zinc oxide for eight hours in planetary ball mill (Retsch PM 100) @ 250 cycles/min using fifty numbers of zirconium balls with 5 mm diameter in 50 ml capacity zirconium jar.
       
Nano form of manganese was synthesized by grinding feed grade manganese sulphate for four hours in planetary ball mill (Retsch PM 100) @ 250 cycles/min using fifty numbers of zirconium balls with 5 mm diameter in 50 ml capacity zirconium jar.
       
Nano form of copper was synthesized by grinding feed grade copper sulphate for two hours in planetary ball mill (Retsch PM 100) @ 250 cycles/min using fifty numbers of zirconium balls with 5 mm diameter in 50 ml capacity zirconium jar.
 
Characterization of nano forms of zinc, manganese and copper
 
The properties of the materials are likely to be far different from the bulk materials, when the particle size is reduced to nano size. In the present study, Transmission Electron Microscopy, Particle Size Analyzer, X Ray Diffractometer, Fourier Transform Infra-red (FTIR) spectroscopy and Inductively Coupled Plasma Mass Spectrometry were used to analyze the properties of synthesized nanoparticles like particle size, shape, zeta potential and elemental concentration. The nanoparticle yield was calculated by weighing the final product and comparing it to the precursor used.
 
In vitro cytotoxicity assay
 
In vitro cytotoxicity assay was carried out in vero cell line (African green monkey kidney cell line) to ensure the safety of nano particle source of zinc, manganese and copper as per the method of Mosmann, (1983). Serially diluted nano particles were incubated in ninety-six well plates for 72 hours containing vero cell line at the concentration of 106 cells/ml with solvent following standard protocol. The concentrations of nano zinc selected were 20, 40, 60, 80, 100, 250, 500, 800 µg/ml. Nano manganese was added at the concentrations of 25, 50, 75, 100, 250, 500, 750, 1000 µg/ml.Similarly, nanocopper was added at the concentrations of 5, 10, 15, 20, 25, 50, 100, 150 µg/ml.  The level of concentrations for each nano form of mineral was fixed considering their standard recommended incorporation level (BIS, 2007) in poultry feed. The samples were run in triplicates.
       
At the end of incubation, colorimetric method measured the reduction of yellow 3- (4, 5 dimethythiazol-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase of live cell. Mean was calculated from the triplicate readings of OD values for each sample. The absorbance value of blank was subtracted from all the samples in order to derive corrected absorbance values. From the corrected absorbance values, calculation was done using the following formula:

Synthesis of nano forms of zinc, manganese and copper
 
Nano particle source of zinc, manganese and copper were successfully synthesized through physical method using planetary ball mill. Zinc oxide, manganese sulphate and copper sulphate were used as sources to produce zinc, manganese and copper nanoparticles respectively. Optimum conditions (such as grinding jar size, sample volume, size and number of grinding balls used, speed of rotation in rpm and duration of grinding) required for preparation of nano forms of zinc, manganese and copper were standardized. The major advantages of mechanical milling are simpler to operate, low cost of production of nanoparticles and the possibility to produce it in large scale (McCormick and Froes, 1998).
 
Characterization of nano forms of zinc, manganese and copper
 
The product yield, particle size, shape, zeta potential and elemental concentration of the prepared nano particles are presented in Table 1. The recovery percentage of nano particle source of zinc, manganese and copper synthesized through ball milling method were 95.75, 95.50 and 96.25 respectively.


       
The particle size assessed through both particle size analyser and Transmission Electron Microscopy confirmed that the synthesized nano forms of zinc, manganese and copper were less than 100 nm. The TEM image of the synthesized nano forms of zinc, manganese and copper are presented in Fig 1, Fig 2 and Fig 3 respectively. Mean size assessed through TEM were 80.64 nm, 20.33 nm and 12.98 nm for synthesized nano forms of zinc, manganese and copper respectively. Whereas, mean size assessed through particle size analyser were 18.20 nm, 16.90 nm and 70.90 nm for synthesized nano zinc, manganese and copper respectively.






       
The X-Ray diffraction (XRD) pattern of synthesized nano particle source of zinc, manganese and copper are presented in Fig 4, Fig 5 and Fig 6 respectively. The data recorded were analyzed using Jade 6.0 software. X-Ray diffraction pattern confirms that the synthesized nano particle source of zinc, manganese and copper were free of impurities as it does not contain any characteristic XRD peaks other than their respective element peak and the samples are nano in nature.






       
FTIR spectroscopy is used to estimate the type of the functional groups and their involvement during bio reduction. The FTIR spectrum of synthesized nano particle source of zinc revealed well-defined peaks at around 552 cm^-1 and 1163 cm^-1. The FTIR spectrum of synthesized nano particle source of mangane serevealed well-defined peaks at around 625 cm^-1 and 3137 cm^-1. The FTIR spectrum of synthesized nano particle source of copperr evealed well-defined peaks at around 565 cm^-1 and 3104 cm^-1.In the present study, the unique finger printing for zinc, manganese and copper were evident in the respective nano particle source of zinc, manganese and copper respectively.The observed FTIR results confirmed that synthesized zinc, manganese and copper nanoparticles were without any significant impurities.
       
The elemental concentrations were analyzed by using Inductively Coupled Plasma Mass Spectrometry (ICPMS). The results revealed that nano zinc contains 76.57% elemental zinc, nano manganese contains 34.04% elemental manganese and nano copper contains 21.52% copper. The elemental concentration of the synthesized nano particle sources of zinc, manganese and copper were almost same as that of the original mega particle source from which they were produced. This indicates no loss in zinc, manganese and copper as a result of ball milling.
       
The shape of the synthesized nanoparticles were analysed through TEM. The shapes of nano manganese and copper obtained in this study were spherical. Spherical shape of nano particles has an advantage of not causing injury while being transported across the tissue (Xu et al., 2012). Thus nano particle source of manganese and copper produced will not cause damage to cells / tissues on oral administration. Though the shape of nano zinc synthesized was hexagonal, it was reported that hexagonal shaped zinc oxide nanoparticles are safe to administer orally in poultry Mohd Yusof et al. (2021).   
       
Nanoparticles with zeta potential values more than +25 mV or less than -25 mV are known to be extremely stable. Due to Van Der Waal inter-particle interactions, dispersions with a low zeta potential value will eventually coalesce (Nanocomposix, 2012). In our study it was observed that the zeta potential (mV) of the synthesized zinc, manganese and copper nanoparticles were -33.50, -29.90 and -25.10 respectively. Thus the zinc, manganese and copper nanoparticles synthesized in our study could be of good stability.
       
Concurring with this study, nano particles of zinc, manganese and copper having similar size and shape were produced by other researchers. Geetha et al., (2016) revealed that the TEM images of ZnO nanoparticles confirmed that the particles were hexagonal in shape. The average particle size was found to be 50-100 nm. Cherian et al., (2016) reported that the SEM images of manganese nanoparticles were found to be spherical in shape with the diameter range of 40.5 to 70 nm. In FTIR a strong absorption peak was found at 625 cm^-1 which is similar to our result. Sufeesh et al., (2019) synthesized copper nanoparticles through physical method using ball mill. The synthesized copper nanoparticles were spherical in shape with the average particle size of 14.67 nm and zeta potential of -12.3 mV. The elemental concentration of copper in the synthesized nanoparticle was found to be 23.26%.
 
In vitro cytotoxicity assay
 
The results of MTT assay on per cent cell viability for synthesized nano forms of zinc, manganese and copper are presented in Table 2. A value above 75 per cent indicates good cell viability. Nano-zinc showed good cell viability up to the concentration 250 µg/ml. The per cent cell viability for nano zinc at 250 µg/ml concentration was 75.57%. This safe concentration level of 250 µg/ml is equivalent to 250 mg/ kg which is more than 3 folds of recommended dose of 80 ppm zinc in broiler chicken diet.


       
Nano-manganese showed good cell viability up to the concentration 500 µg/ml. The per cent cell viability for nano-manganese at 500 µg/ml concentration was 75.85%. This safe concentration level of 500 µg/ml is equivalent to 500 mg/kg which is 5 folds of recommended dose of 100 ppm manganese in broiler chicken diet. Nano-copper showed good cell viability up to the concentration 50 µg/ml. The per cent cell viability for nano-copper at 50 µg/ ml concentration was 76.24%. This safe concentration level of 50 µg/ml is equivalent to 50 mg/ kg which is more than 4 folds of recommended dose of 12 ppm copper in broiler chicken diet.
       
Inorganic minerals are relatively inert in their bulk form. However, cytotoxicity assay of any nano particle source is essential, as the particle size decreases, toxicity increases. Nano particles interact with proteins and enzymes within cells and they can interfere with the antioxidant defense mechanism leading to formation of reactive oxygen species, which further causes inflammatory response and destruction of the mitochondria finally leading to apoptosis or necrosis Scharand et al. (2010). 
       
In a cytotoxicity study, Saranyaet al. (2017) studied the in vitro cytotoxic effects of green synthesized ZnO nanoparticles in Vero, PK 15 and MDBK cell lines. The ZnO nanoparticles revealed better cell viability at lower concentration (10 μg/100 ìl) in all type of cells (Vero, PK 15 and MDBK cells). Similar results were reported by Tabassum et al. (2021) who observed the in vitro cytotoxic effects of manganese nanoparticles in human breast cancer cell line. The results showed that there is significant dose- dependent inhibitory effect of manganese nanoparticles. When the concentration of manganese nanoparticles increased from 25 to 125 µg/ml, the cell viability decreased from 94% to 61 %. Sufeesh et al., (2019) studied the in-vitro cytotoxic effects of copper nanoparticles in vero cell lines. The results of the study revealed that copper nanoparticles showed good cell viability at 100 μg/ ml concentration. Hence, in this study, the results obtained revealed that safest inclusion level of nano zinc, manganese and copper in broiler chicken feed is up to 250 ppm, 500 ppm and 50 ppm respectively.  
 
 

Zinc, manganese and copper nano particles would be successfully and effectively synthesized through physical method using planetary ball mill and the same could be characterized by several techniques as followed in this study for its quantity, size, shape, stability and purity.
       
The result of the present study indicated that nano forms of zinc manganese and copper had all the characteristics of nano particle. Based on the result of in vitro cytotoxicity assay we would say that nano forms of zinc, manganese and copper are non-toxic at our inclusion level. Hence, the synthesized zinc, manganese and copper nanoparticles could be used as a feed supplement for broiler chicken at the dosage of 80 ppm, 100 ppm and 12 ppm respectively as per the BIS specifications.
 

The authors are very much grateful to the authorities of Tamil Nadu Veterinary and Animal Sciences University Chennai for providing necessary facilities to carry out this research study.
 

None.