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Pitfalls and Challenges in Nanotoxicology: A case of Cobalt Ferrite (CoFe2O4) Nano-composites Farooq Ahmad, and Ying Zhou Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00377 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on January 25, 2017

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Chemical Research in Toxicology

Pitfalls and Challenges in Nanotoxicology: A case of Cobalt Ferrite (CoFe2O4) Nano-composites

Farooq Ahmad †, ǁ*, Ying Zhou †, ‡ * †

College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310032,

China ǁ

State Key Laboratory of Metal Matrix Composites, School of Material Science and Engineering,

Shanghai Jiao Tong University, Shanghai, China ‡

Research Center of Analysis and Measurement, Zhejiang University of Technology, Hangzhou,

China *Co-Corresponding and co- first author:

Farooq Ahmad and Ying Zhou contributed equally in this Manuscript and will be considered as co-first author and co-corresponding author as well.

*Farooq Ahmad College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310032, Zhejiang, China State Key Laboratory of Metal Matrix Composites, School of Material Science and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China Tel.: +86 57188320666 Email: [email protected]

*Ying Zhou

ACS Paragon Plus Environment


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Research Center of Analysis and Measurement, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou 310032, Zhejiang Province, China. Tel.: +86 571 88320568 E-mail: [email protected]

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TOC Graphic

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Abstract Nanotechnology is developing at a rapid pace with promises of the brilliant socio-economic future. The apprehensions of vivid future involvement with nanotechnology make the nano objects ubiquitous in the macroscopic world of humans. Nanotechnology helps us to visualize the new mysterious horizons in engineering, sophisticated electronics, environmental remediation, bio-sensing and nanomedicine. In all these hotspots, cobalt ferrite (CoFe) nanoparticles (NPs) are the outstanding contestant because of its astonishing controllable physicochemical and magnetic properties with easiness of synthesis method. The extensive use of CoFe NPs may easily penetrate into the human body unintentionally by ingestion, inhalation, adsorption etc. and intentionally instilled into the human body during the biomedical diagnostics and treatment. After being housed into the human body, it might induce oxidative stress, cytotoxicity, genotoxicity, inflammation, apoptosis and developmental, metabolic and hormonal abnormalities. In this review, we compiled the toxicity knowledge of CoFe NPs aimed to provide the safety usage of this breed of nanomaterials. Key words: Nano cobalt ferrite, Oxidative stress, Genotoxicity, Apoptosis, Malformation, Toxicity

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Introduction In the new millennium, innovations in technology boost the modernized human society with improved health and sanitation conditions. Modern technological innovations are also closely allied with encroachment in nanotechnology, which are transforming the current and future with improved attributes of nano-technological products. Although, the spacious use because of endless benefits, the nanotechnology is also packed with negative side effects for human society and health. 1-3 Among nanomaterials (NMs), magnetic nanoparticles (MNPs) are one of the very vital and extensively exploited sub category of NMs. MNMs are usually multi-component, characteristically enclosed with nano-scale magnetic constituents to trigger the response to an external magnetic field. Thus far, with the rapid development in nanotechnology, novel magnetic nanomaterials with combination of diverse range of materials such as liquid crystals, gels, self healing polymers and silica, carbon or organo-metallic frameworks have emerged.

4-11

This

enables the utilization of magnetic force for the control of properties and flow of the liquids, corresponds to plentiful practical applications. 12 Recently, MNPs are overwhelmingly used in sophisticated nano-based medicine, electronics, environmental remediation, biosensing, medical imaging, improved drug and gene delivery, nanoprobes, catalyst and optical devices.

13-24

Above all, in nanomedicine, MNPs are used in

cellular therapy, tissue repair, biosensors, drug delivery, magnetic resonance imaging, magnetic fluid hyperthermia and solar cells.

25-31

All these applications oblige elevated magnetization

values of NPs, size of less than 100 nm with homogeneous physical and chemical properties. Furthermore, it is indispensible to comprehend the biological as well as environmental fate and

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32


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Magnetic Nanoparticles Magnetic NPs 70%

Iron oxide NPs 24%

CoFe NPs 6%

Cobalt ferrite Nanoparticles Synthesis 79% Catalyst 2%

Toxicity 3%

Sensor 2%

Electrocnic s 1% Medicine 13%

Fig.1 Scientific papers published concerning the synthesis and toxicity studies of magnetic and cobalt ferrite nanoparticles (data from ISI web of science as searched on October. 08, 2016) prospective toxicity of MNPs for their rumbling appliances in nano-tech based rapid point of care like in diabetics, pregnancy testing, electrolyte and blood analysis, cardiac marker, cancer, drug abuse and infectious disease diagnostics and lot more.

16, 27, 28, 33

For example, magnetic

nanofluids effectively reduce tumor progression in the cat mammary glands. 34 In the last decade, magnetite (Fe3O4) was the most investigated MNPs because of easier to synthesis and having much control over their physicochemical properties. 26

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Cobalt-ferrite (CoFe) NPs belongs to the crystal family of spinal ferrites (MFe2O4) and are the most prevalently exploited NPs in nanomedicine.

35

These NPs are considered superlative to be

exploit in nanotech, because of comparatively larger coercivity (~5 kOe), permeability, reasonable magnetic saturation (~80emu), chemical stability, mechanical hardness, high electromagnetic and magneto-optic performance atomic level.

32, 38

25, 36, 37

and tunable magnetic properties at the

Therefore, CoFe is a talented contestant material for high-density magnetic

recordings, ferrofluids, drug delivery,39 magnetic resonance imaging (MRI), cancer treatment and in magneto-optical devices. 14, 25, 36, 40 Up to October 08, 2016, more than 19800 studies on iron oxide NPs, which the number of papers published on cobalt ferrite NPs were more than 1700 and only 37 papers had discussed the biological effects of CoFe NPs had been published (Fig.1). This number is far more less as their main area of application is in the medicine as illustrated above. Since, all above mentioned features make CoFe NPs outstanding and remarkable candidates for biomedical applications, but the release of higher amounts of cobalt and iron, agglomeration/aggregation and reduced surface functionality by diverse collection of coating materials (bio-macromolecules) and higher toxicity limits their use in biomedical applications. The good news is that these hitches can be conquered by surface fashioning of CoFe NPs with attuned, non-hazardous, and water-stable/dispersing material.38 Coating of Oleylamine over the CoFe NPs significantly reduces their toxicity in healthy cells (MRC5 and dental MSCS) as compared to the cancerous cells (HeLa and A549). 41 Broad spectrum applications of CoFe NPs ranges from cancer therapy, drug delivery, magnetic recording material and in transducers necessitate the fabrication of mono-dispersed and homogenous physicochemical properties. 39, 42

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Fig. 2 Shows physicochemical properties, synthesis method and possible applications of Cobalt ferrite nano-composites. Synthesis methods play very important role in undermining the toxicity of NPs as it controls the cation distribution, physicochemical properties and presence of residual reagents. CoFe NPs can (Fig.3) be manufactured by four different ways, i-e.; co-precipitation method, conventional hydrothermal, micro emulsion and sol-gel method. But the most used method is co-precipitation method, because of its simplicity, use of less hazardous chemicals with stable mono-dispersed suspension, uniform size distribution and better magnetic properties for safe use in nanomedicine.43 The details of the different synthesis routes and their respective advantages and 7


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disadvantages are out of the scope of the review, but the readers may find different methods of synthesis in elsewhere. 44-57 Doping of CoFe NPs with some other metal oxides (e.g., Zn, Ni, Cu, Mn or Cr) significantly enhance the magnetic, chemical, hyperthermia and antibacterial characteristics.25, 43 The reason for improved antibacterial property lies in the fact that doping results in the CoFe NPs with irregular shape with sharp edges are more lethal than the round shaped counterparts. Doping with copper yields the most lethal CoFe NPs composites, but again it also depends on the specie of test model. 25 Folic acid (FA) fabrications of CoFe NPs generate mono-dispersed with enhanced peroxidase activity of NPs. Such FA fabricated CoFe NPs can be exploited in tagging HeLa cells (folate receptors) from NIH-3T3 cells (without foliate receptors).35 The FA fabrication also enhances the targeted drug delivery potential of CoFe NPs. Alcantara et al (2011) reported the attachment of methotrexate and doxorubicin (anti-cancer drugs) with FA conjugated CoFe NPs by simple organic coupling reaction, with superior cytotoxicity and apoptosis in HeLa cells. 58

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Fig. 3 Characterization of Co-Fe NPs by TEM images prepared by co-precipitation method. (A) Bright field image, (B) dark field image, (C-D) high resolution images, inset- magnification of single particle. Reprinted from Limor Horev-Azaria, Giovanni Baldi, Delila Beno, Daniel Bonacchi, Ute Golla-Schindler, James C. Kirkpatrick, Susanne Kolle, Robert Landsiedel, Oded Maimon, Patrice N. Marche, Jessica Ponti, Roni Romano, François Rossi, Dieter Sommer, Chiara Uboldi, Ronald E. Unger, and, C. V., and Korenstein, R. (2013) Predictive Toxicology of cobalt ferrite nanoparticles: comparative in-vitro study of different cellular models using methods

of

knowledge

discovery

from

data.

Part.

Fibre.

Toxicol.

10,

(https://particleandfibretoxicology.biomedcentral.com/articles/10.1186/1743-8977-10-32).32 Toxicity of nano-CoFe Toxicity of nano-CoFe on respiratory system

9


1-17.

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During the course of fabrication, circulation, exploitation and recycling, nano-CoFe is easy to spread into the air. Therefore, industrial or commercial CoFe NPs might be fall into one of habitual constituent of interior or exterior environment. As inhalation (after injection) was the most familiar route of CoFe NPs to enter human body, there was also a possibility that nanoCoFe did impairment to respiratory system. Respiratory tract and intravenous system became the primary target organ system for the inhaled as well as biomedical used NPs, respectively. So there were a lot of studies that paid attention to this significant problem.

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Mechanographic

investigation of guinea pig airways evidenced that powdered nano-CoFe with size of 5-20 nm (0.5mg/ml) meticulously compromised the functionality of normal contractile job of trachea and major bronchi, measured by Musson-1M ultrasonic nebulizer (OJS Altay Instrumental Factory Rotor) at the rate of 30-min per day for 4 days. Mechanical function disability of the respiratory system advocated the both in vitro and in vivo inhalation of powdered nano-CoFe abruptly augment the contractile amplitude of the air passage ways which put an immense force on the delicate respiratory membranes terrorizing the bursting of them. This results in dwindled surface area and abridged body oxygen leading to hypoxia condition. This hypoxia condition if sustains longer than norm escorts to cell death in different parts of the body. In addition, presence of nano-CoFe elicits the disproportionate discharge of histamine and adrenergic salbutamol.

60

But

this study did not provide any information regarding the coating material and zeta-potential of used CoFe NPs. Moreover, the exposure concentration (0.5mg/ml) used in this study was even higher than to be used for MRI imaging,61, 62 which make results ambiguous and redundant. In in vitro respiratory toxicity after exposure for 24-72 hours of pristine CoFe NPs (10 nm) at 23.5282.5 µg/ml dosage, was measured by the Alamar blue, MTT and neutral red assays. These assays clearly presents that CoFe NPs cause severe oxidative stress to the respiratory system in 10



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the order; lung slices from rat>NCIH441 cells>A549 lung cells. 45The results also confirmed that sensitivity to different NPs also varies among different types of cell lines. 32, 63 This indicates that nano-CoFe not only disrupt the normal functionality of respiratory system by compromising the alveolar membranes mediated by oxidative stress and inflammation but can also reach into the different vital parts of the body e.g., liver by circulatory system. Toxicity of nano-CoFe on Skin The route of exposure is very important in predicting the possible toxicity of NPs and skin is one of the major portals of entry for NPs into the human and animal bodies. Disease states such as atopic and contact dermatitis, acne, seborrheic dermatitis, inflamed skin and psoriasis can make the skin more permeable. In addition, simple acts such as shaving or injuries such as sunburns, cuts, or scrapes can increase cutaneous permeability.

64-66

NPs in undergarments or skin care

products may enter into the female reproductive tract and incite alterations of the reproductive lining. Thereafter, they may circulate to the entire body through the circulatory system of the animals or humans. 64 Well there are also evidences that NPs may not give harm to the skin but may penetrate and accumulate into the stratum corneum by photohydrolysis, which in turn increases the risk of higher circulatory metal ions (M+n) in the blood and other system organs such as kidney and liver.

67

A recent in vitro study contradicted the above report and revealed

that bare CoFe NPs with size of ~27nm when exposed to human keratinocytes cells (HaCaT) at 0.5,1, 2 and 4 mg/mL concentration caused the toxicity in a dose dependent way. Prussian blue, MTT and apoptosis assays showed very moderate toxicity at 2mg/mL, while the metabolic analysis showed that even at the moderate toxic concentration of 2mg/mL; CoFe NPs induces the oxidative stress by elevation in cellular alanine, taurine, ethanol and succinate levels and

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decrease in isoleucine in human keratinocytes cells (HaCaT). The elevation in the alanine level is connected with apoptosis.68 While the decrease in isoleucine indicates the cellular energy perturbations incited by CoFe NPs exposure. The leucine and isoleucine are branched-chained and most abundant essential amino acids. Among essential amino acids, leucine is involved in protein synthesis and degradation, leptin secretion, energy-balance regulation, and so on. Valine, leucine, and isoleucine biosynthesis pathway can supply the TCA cycle with various anaplerotic substrates including α-ketoisocaproate, which can be further metabolized to acetyl coenzyme A and acetoacetate. 68, 69 Fe3O4 NPs with size of 65nm induce toxicity in human dermal fibroblasts (HDF) and cells of the squamous tumor cell line (SCL-1) , oxidative stress was build up inside the cells by the excessive generation of reactive oxygen species (ROS) leading to the instigation of lipid peroxidation process.

70

In another study, polygonal smooth surfaced Fe3O4 NPs with

size of 25nm at concentrations of 25-100µg/mL induces dose dependent cytotoxicity and apoptosis in skin epithelium A431 cells after 24 hours of exposure. Dose dependent oxidative stress was evident by the depletion of glutathione and elevation of ROS and lipid peroxidation. 71 Interaction of nano-CoFe with U87MG cells evidenced by synchrotron radiation X-ray fluorescence illustrated the significant reduction in the viability of these cells. In fact, these NPs were also accumulated into the cytoplasm near the perinuclear region and compromised the nuclear membrane.

72

Red-allotrope selenium NPs (rSeNPs) with size of 123.5±33.6 nm

exposed to head and neck squamous cell carcinoma (HNSCC) and human dermal fibroblast (HDF) cells at concentrations ranging from 0.01 to 100 µg rSeNP/mL media, decrease the viability of HNSCC and HDF cells with severe inculcation of apoptosis. These rSeNPs also have higher tendency to accumulate near the lysosomal and mitochondrial region of HNSCC and HDF 12



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cell lines and believed to persuade the possible energy perturbations leading to the apoptosis.73 Exposure of HaCaT cells to 10µg/ml of tannic acid modified 13 and 33 nm AgNPs led to a significant rise in the production of ROS in comparison to control and the activation of phosphorylated ERK pathway.74 This is the very good indicator of not only the cellular stress but also perturbed the cellular viability and proliferation (decrease in Ki-67 percentage).74, 75 Therefore, NPs not only use the skin as a transport route to enter into the body but also provoke oxidative stress (by excessive ROS generation), apoptosis and isoleucine based energy imbalances. This also leads to the imbalance of Ca+2 transports, alteration in metabolism and excretory function of normal skin. These all dreadful things may also (1) leads to the increased chances of skin damage with and without photocatalysis, (2) makes the skin pores wide open providing the gateway of entry for the various ambient pathogens and contaminants into the body, from where they may transport to the different parts of the body and foster the cultivation of complex diseases. Toxicity of nano-CoFe on liver and kidney Once the NPs housed into the animal body either intentionally or unintentionally, they would be distributed into the whole body through the circulatory system. Liver is the vital organ of the body involved in the detoxification and production of digestive biochemicals, which will be the core distribution site for CoFe NPs. In addition, nano-CoFe can reside for longer duration inside the liver and cause inflammation leading to disturbances in liver functionality. In vivo (whole animal, mice) after intravenous injection and in vitro (Hep3B cells) study reported that rhodamine B isothiocyanate coated with silica shell nano-CoFe exhibit substantial liver accumulation. In mice, the CoFe NPs were accumulated 50 times more than the normal one and the target site is the vesicle like spaces in cytoplasm.76 MTT assay confirmed, nano-CoFe 13


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divulged ruthless cytotoxic effects mediated by oxidative and metabolic perturbations directly dependent on dosages and treatment times in liver. In addition to oxidative and metabolic stress, carcinogenesis and inflammation and growth arrest in liver cells were very prominent phenomena's observed after nano-CoFe exposure. As the liver is the focal clearing house of the animal body, so the compromised functionality of the liver results in perturbed metabolic activity and body become the waste house for harmful metabolites. Furthermore, the hemotoxylin and eosin staining revealed that CoFe NPs causes deformation of nuclei in the viable cells.

77

CoFe

NPs also induce extraordinary cytotoxicity and oxidative stress in HepG2 cell-line (liver), MDCK cell-line (kidney) and Caco-2/ TC7 cell-line (intestine). Caco-2/ TC7 and MDCK celllines are more vulnerable to CoFe NPs as compare to HepG2 cell-line (liver), indicating the compromised major cleaning route of toxicants out of body. 32 In in vitro accumulation of nanoCoFe in cancer cells (MiaPaCa2) of human liver illustrates moderate toxicity on the basis of their viability and proliferation index, however disparity in response to nano-CoFe were incredibly diverse from cell to cell lines of same and different organisms belongs to diverse intricate system organization.78 The fate of liver accumulated CoFe NPs was further assessed in invertebrate terrestrial isopod (Porcellio scaber) model, the results showed those gastric juices of Porcellio scaber biodegrade the CoFe NPs into Co+2 and Fe+3 ions. In addition Co+2 ions accumulated in comparatively high concentration inside the hepato-pancreas compromising the structure and normal functionality, also released Co+2 ions are substantially more toxic than Fe+3 and CoFe NPs. What is more, the accumulation of Co+2 ions are also the main precursor of genotoxicity.79 Another study revealed that in vivo investigation of polyol coated zinc doped cobalt ferrite (Zn0.8Co0.2Fe2O4) NPs with size of 8nm in New Zealand rabbit showed excessive accumulation in liver and kidney leads to inflammation with 50% increase in WBCs, granuloma and multiple 14



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congestions. These toxic effects comes from the NPs itself and not from the release of metal ions,80 which is clear contradiction with the study of Novak et al.79 But the drawback of this study is, they did not observe the dissolution behavior of (Zn0.8Co0.2Fe2O4) NPs inside the specific cells that is why we do not know the actual reason of contradiction with the previous study. Zirconia nanoparticles (ZrO2NPs) with 20nm size at dosage of 0.1 mg/ml leads to increase in creatinine content, congestion and destruction of the glomerular capsule space in wistar rats.81 From what we have listed above, nano-CoFe not only accumulate into the vital detoxifying organs of the body but also cause oxidative stress, cancer and complete destruction of the internal structure of liver as well as kidney. This may also leads to inhibit the excretion of harmful toxins and metabolites from the body and accumulates in higher concentrations among various organs and sites of the body at dangerously excessive levels causing cell deaths, joints degeneration and impairment etc.. Toxicity of nano-CoFe on reproductive system and embryo development The adsorption leading to internalization of CoFe NPs into cells or organs results in the biodistribution and biodegradation through circulatory system and biological enzymes respectively. Internalization and accumulation of CoFe NPs inside the cells induces physicomechanical destruction of sub-cellular organelles and to delicate biological membranes. In vitro acute toxicity (5 day) profiling of nano-CoFe to embryonic stem cells (ES-D3) illustrates pristine nano-CoFe were relatively more toxic compared to Gold (Au) or silane coated CoFe NPs and cause cytotoxicity leading to abnormal cellular differentiation process. 82 The mechanism of endocytosis of nano-CoFe in Chinese hamster ovary was corresponded by macropinocytosis (membrane ruffle) and clathrin mediated routes in a time dependent way. CoFe NPs occupied almost all the intracellular space of Chinese hamster ovary cells within 24 hours of exposure. In 15


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addition, no excretion of nano-CoFe was observed even after 24 hours of accumulation. This illustrates accumulation of nano-CoFe leads to the impairment of excretory role of hamster ovary cells either by blocked or destructive excretory system.83 Accumulation of nano-CoFe into human ovary cancer cell lines (A2780) exhibited severe cytotoxicity leading to death of cells. In addition, exposure of nano-CoFe at higher concentration illustrates the decreased clonogenicity and epithelial cell adhesion,78 with most probable cytotoxicity driving forces involved were disturbed cellular iron balance, perturbations in multiple cellular signaling pathways, DNA damage, oxidative stress, mechanical destruction of cytoskeleton with accompanying changes in gene expression.84 Recently it is also reported that polyol coated zinc doped cobalt ferrite (Zn0.8Co0.2Fe2O4) NPs also reduces the viability of Human Umbilical Vein Endothelial Cells (HUVECs) and hence leads to premature abortion.

80

Spermine coated cobalt ferrite NPs also

proved effective in curing the human breast cancer MCF-7 cell line compared to the Fe3O4 NPs, it is because of the higher hyperthermia and coercivity induced by the combining the magnetic constituent (Co) with Fe3O4.85 Meanwhile, recent studies revealed that in vivo exposure of CoFeNPs induce developmental malformations like arrested development, absence of head and late eyes spot development, finfold abnormality and tail flexure with absence of tail extension, cardiac malformation/ pericardial edema, yolk sac edema and spinal cord abnormality in developing Zebrafish embryos in a time (96 and 168 hpf) and concentration dependent way. CoFe NPs also put mechanical pressures by adhering to the external chorion, blocking the air passage ways posing sever hypoxia condition. In addition, CoFe NPs cause serious oxidative stress by excessive ROS generation compromising the antioxidant defense systems and aggravation of apoptosis. These developmental malformations and oxidative stressors were further corresponds to thyroid endocrine disruptions and sever DNA damage.13, 86 Moreover, 16



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nano-CoFe potential of inducing oxidative stress, accumulation, alter the cell signaling, DNA damage in the reproductive system leads to abnormal development of fetus, premature abortions, genetic mutations with a possible transfer to the next generations leads to the impairment of the organ systems and improper development, and poses the more serious threats to the life of mother during pregnancy. Toxicity of nano-CoFe on central nervous system The significance of studying the harmful effects of NPs on nervous system can be understood by the fact that brain is the most crucial organ of the body involved in regulation, communication and homeostasis in response to internal and external changes, neuron and glial cells, organized in specialized structures, cooperate to allow the articulated functions provided by the brain.

30

Recently, the CoFe NPs are extensively utilized in in vivo use, so there is a pressing need to know about the long and short term exposure effects of these NPs on the nervous system, but unfortunately this information is still lacking. The NPs may also enter the animal neural network through skin (commercial exposure of CoFe NPs) and most probably by circulatory system (intravenous injection) trans-synaptic transport and by crossing the blood brain barrier (BBB).87 However, the neural toxicological effects can be predicted from analogous studies of particulate matter (PM) for long and short term exposure.

88

For example, the well studied oxidative stress

paradigm is the root of various diseases like hyperglycemia, cardio vascular disease, cancer, neuro-degeneration and pulmonary disorder etc.88-91 It is also well known that metal ions (Fe+3, Co+2, Zn+2, Mn+2 etc.) accumulation leads to multiple neurodegenerative diseases including Parkinson and Alzheimer disease.91-93 The deregulation of metals in the body leads to the overloading of metal ions in the brain or other body parts consequently results in impairment of nervous system.94 Iron has traditionally thought to cross the blood-brain barrier (BBB) by 17


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multiple

routes

like

transferrin-mediated

route

through

the

capillary

endothelium,

circumventricular or olfactory nerves. Additionally, ferrtin is also act as an auxiliary protein for iron transport.

95

It has also reported that iron and cobalt could generate the ROS within brain

synapses and interfere with transduction of neurotransmitters96-98 as well as compromised the normal functioning of neuron protection proteins e.g., prions and melatonin, causing the sever hypoxia in hippocampus.

91, 93

It is also reported that nano-CoFe can cross the sensitive neural

membrane and accumulate inside the SH-SY5Y neural cells instigating the necrosis process.99 Even though nano-CoFe have the potential to induce the damage to the CNS but still there are lake of studies explaining the mechanisms of toxicity induced by this breed of NPs. There is a natural fact that brain is highly vulnerable to oxidative stress and requires metal ions (Fe+3, Co+2) in a balanced amount for its high respiratory activity, as well as generation of numerous neurotransmitters and myelin development. All these activities make the brain highly vulnerable to the slight imbalances in oxidation equilibrium and lead to the neurodegeneration and associated disorders. 88 Toxicity of CoFe NPs on Immune system Peripheral blood system is also very important for healthy animal body as it protects the body from foreign pathogens. If the peripheral blood system stopped working or damaged by any means directly shortens the life span of that animal. Nano-CoFe (~5.6 nm) showed significant (p

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