Molecular and immunological toxic effects of nanoparticles

Accepted Manuscript Title: Molecular and immunological toxic effects of nanoparticles Authors: Rajan Kumar Pandey, Vijay Kumar Prajapati PII: DOI: Reference:

S0141-8130(17)33394-9 https://doi.org/10.1016/j.ijbiomac.2017.09.110 BIOMAC 8287

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International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

5-9-2017 21-9-2017 27-9-2017

Please cite this article as: Rajan Kumar Pandey, Vijay Kumar Prajapati, Molecular and immunological toxic effects of nanoparticles, International Journal of Biological Macromolecules https://doi.org/10.1016/j.ijbiomac.2017.09.110 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Molecular and immunological toxic effects of nanoparticles Running Title: Nanoparticle-mediated immunotoxicity

Rajan Kumar Pandey, Vijay Kumar Prajapati*

Rajan Kumar Pandey: Department of Biochemistry, School of Life Sciences, Central University of Rajasthan, Kishangarh, 305817, Ajmer, Rajasthan, India *Vijay

Kumar Prajapati: Department of Biochemistry, School of Life Sciences, Central University of Rajasthan, Kishangarh, 305817, Ajmer, Rajasthan, India Word Count: 11,864 Corresponding author Vijay Kumar Prajapati [email protected] [email protected] Abstract Nanoparticles have emerged as a boon for the public health applications such as drug delivery, diagnostic, and imaging. Biodegradable and non-bio degradable nanoparticles have been used at a large scale level to increase the efficiency of the biomedical process at the cellular, animal and human level. Exponential use of nanoparticles reinforces the adverse immunological changes at the human health level. Physical and chemical properties of nanoparticles often lead to a variety of immunotoxic effects such as activation of stressrelated genes, membrane disruption, and release of pro-inflammatory cytokines. Delivered nanoparticles in animal or human interact with various components of the immune system such as lymphocytes, macrophages, neutrophils etc. Nanoparticles delivered above the threshold level damages the cellular physiology by the generation of reactive oxygen and nitrogen species. This review article represents the potential of nanoparticles in the field of

nanomedicine and provides the critical evidence which leads to develop immunotoxicity in living cells and organisms by altering immunological responses.

Keywords- Immunotoxicity, ROS generation, nanoparticles, DNA damage

1. Introduction The past decades have witnessed exponential growth in the development and production of the engineered nanoparticle, worldwide; due to their astonishing physiochemical properties [1-6]. In this era, nanotechnology broadens the opportunity for inventors, producers, and consumers of almost all sectors due to their activity at the molecular level. Nanoparticles are engineered structures with at least one dimension of less than 100 nm with distinctive properties. Nanoparticles have shown widespread applications including pharmaceuticals, electronics, coating, cosmetics, and Photonics. The engineered nanostructures type may be a nanotube, nanowire, nanocrystal, spherical and dendritic aggregated nanomaterial, quantum dots and metal nanoparticles etc. [7]. As compared to the bulk material, nanoparticle shows higher surface to volume ratio, results in an enhanced contact area with their surroundings and specially designed nanoparticles can be used for the immunomodulatory purposes to serve specific functions like anti-inflammatory, vaccine adjuvant and immunosuppressive drugs [8]. But, due to the novel physical and chemical properties, nanoparticle needs a proper evaluation of their possible toxic effect on human health. In 1982, first, specific toxicological research was conducted to check the toxicity associated with the nanoparticle [9]. Afterward, there has been a 25 times increase in research publication of toxicological research from 2005 to 2016 (PubMed, 09/2017). Immunotoxicity, induced by the nanoparticles can be evaluated by measuring the level of proinflammatory cytokines and in most of the cases, high level of

cytokines are associated with the low therapeutic efficacy, adverse reactions, and toxicity upon nanoparticle treatment [8]. The level of toxicity of nanoparticles is highly dependent on their size and dosage of administration, the expression level of receptors specific to them, their internalization mechanism, properties of the target cell system, the charge on their surface, surface coating and their cellular uptake [10]. However, the surface charge of a nanoparticle has shown their important role in complement activation. The negatively charged liposome has shown their capability to increase the complement activation and complement-activation related pseudoallergy. A highly negatively charged nanoparticle has shown to increase both hemolysis and complement activation in vitro. But the increasing hydroxylation of the negatively charged nanoparticle surface leads to the increasing complement activation [11-13]. Recently, Tirtaatmadja et al. reported that variation in the plasma protein concentration leads to the substantial changes in the composition of hard corona that adsorbed on the surface of various nanoparticles that ultimately affects the release of inflammatory cytokines like interleukin-8 (IL-8) and tumor necrosis factor (TNFα) in 10 % plasma concentration, but not at higher concentrations [14]. Some of the nanoparticles have also been proven to be carcinogenic in nature which provides another limitation to their utilization in industrial and biological aspects. Such damaging effects are resultant of reactive oxygen species that damage at the DNA level, mutations, apoptosis, cell cycle inhibition, enhanced

secretion

of

cytokines

and

chemokines,

inflammatory

responses,

immunosuppression and reduced viability of the major cell types involved in innate and adaptive immune system. Due to such major drawbacks, there should be a modification in nanoparticle-based applications to decrease the extent of damage being caused to human health and their surrounding environment. The different nanoparticle included in this review for their immunotoxicity presentation are alumina, cadmium, carbon black, carbon nanotube, cobalt chrome alloy, fullerene, gold, graphene, iron oxide, nanoceria, nickel, platinum, silica,

silver, titanium dioxide and zinc oxide nanoparticles (Fig. 1). In this review, we have explored the current scenario of the potential immunological toxicity induced by different nanoparticles.

2. Molecular and immunological toxicity induced by nanoparticles 2.1. Alumina nanoparticles Aluminium is the third most abundant element in the earth’s crust and is widely used in a number of large-scale industries. It used as a component of many alloys because of its ability to modify the physical properties. Alumina nanoparticles, an oxidized form of aluminium oxide (chemical name: Aluminium oxide) have been widely utilized for their application in the cosmetics and ceramics industry, domestic and biomedical products such as bone implants [15]. These particles formed an essential component in artillery and wear-resistant coatings on propeller shafts of ships. Their use in drug delivery systems increases the solubility of the same and they were also utilized to enhance the specific impulse per weight of composite propellants of solid rocket fuels [16, 17]. They also have shown great potential as drug delivery systems. But, the extent and type of toxicity they give rise to, is a field which needs to be explored more [7]. Alumina nanoparticles have been shown to cause substantial neurotoxicity in ICR (Institute of Cancer Research) strained mice. Experiments have shown that surface chemical characteristics play a major role in determining the extent of toxic effects that these nanoparticles cause. Most of the damage may be due to the generation of reactive oxygen species and induction of caspase-3 gene. Necrosis and mitochondrial impairment were considered as the main reasons behind the diminished and altered neuro-behavioral functions

[18]. A significant increase in the reactive oxygen species levels and activity of antioxidant enzymes indicated generation of oxidative stress in red blood cells (RBCs), brain and liver. Their presence in brain augmented the levels of dopamine and norepinephrine, indicating that the nanoparticles were strongly neurotoxic. It has been observed that alumina nanoparticles reduced the splenic levels of zinc and iron. Further, they affected the functioning of macrophages and suppress the level of α-Naphthyl acetate esterase (ANAE) within the cells and also the production of interleukin like IL-2 and cytokines such as TNF- α [19]. Reduced viability and altered morphology were the major effects of nano alumina on HBMEC (Human Brain Microvascular Endothelial Cells) cultures. As a result, apoptotic body formation, cell shrinkage, and mitochondrial dysfunction increased in a dose-dependent manner. In vitro, as well as in vivo experiments demonstrated that F-actin expression along with many other tight junction proteins showed great diminution [20]. Intra-peritoneal administration of nanoscaled aluminium oxide suspension and non-nanoscaled aluminium oxide suspension of varying concentrations gave rise to inflammatory conditions in the brain. It is being assumed that the nanoparticles successfully crossed the blood-brain barrier which ultimately leads to glial activation in the brain. Previous in vitro study has shown that the generation of reactive oxygen species was induced in glial cell line BV-2 by alumina nanoparticles [21]. Aluminium compounds such as aluminium hydroxide and aluminium phosphate are often used as an adjuvant for human and animal vaccines. The use of aluminium adjuvant enables efficient uptake of antigen particles and help in mounting of a strong immune response by activation various components (complement system, macrophages etc.) 2.2. Cadmium nanoparticles Cadmium-based quantum dots (QDs) is one of the most extensively used nanoparticles in nanobiotechnology research. Their high photostability and high photoluminescence quantum

yield put them ahead of another conventional bio-probes. QDs combine two most essential properties of probes, i.e. resistance to photo-degradation and can be constructed in very small hydrodynamic size in such a way that diffusion and the behavior of the molecule may not affect [22]. These particles fall under an important class of fluorophores for biological, biomedical and bio-sensing based applications [23]. Their unique photophysical and photochemical properties such as high brightness, superior photostability, tunable emission spectra and high specific bio-activity makes them suitable to be utilized as stable fluorescent probes in various fields of biomedical research [23]. They were also used to improve biological imaging at the intracellular level for the detection of cancer at various stages. The most popular forms of QDs that are commercially available, constitutes of cadmiumtellurium (CdTe) or cadmium-selenium (CdSe) cores. Experiment on oysters (Crassostrea gigas), a sentinel species, has shown certain physiological changes when exposed to cadmium nanoparticles. The phagocytic capacity of hemocytes was diminished and resulted in immunosuppression [24]. In mussels (Mytilus edulis), it was reported that cadmium quantum dots (CdQDs) severely affects hemocytes viability as compared to dissolved cadmium inducing major immunodeficiency. It was also reported via discriminant function analysis that the mussel could distinguish between the toxicity of dissolved cadmium and quantum dots. In trout fishes, macrophages were more significantly affected when exposed to dissolved cadmium without affecting the phagocytic ability much. Also, lymphocyte transformation was more affected by dissolving cadmium rather than CdQD. As compared to CdQD, which failed to bring about any changes in mouse macrophages, dissolved Cd greatly reduced their viability without affecting their phagocytic capacity. These QDs were also able to affect T-lymphocyte transformation. Additionally, different levels and types of toxicity were observed in different forms of cadmium administered to a human being. The toxicity of CdQD and dissolved Cd was studied in

humans using blood samples and found that monocyte viability was severely decreased in both the cases along with lymphocyte transformation, thus resulting in immunodeficiency. It was also observed that toxicity induced by both QDs and dissolved Cd-elicited similar responses [25]. Another researcher reported that when cadmium quantum dots were administered to three animals, namely Mytilus edulis, Oncorhynchus mykiss and Elliptio complanata into three categories which were filtered, permeate (materials that passed through the ultrafilter membrane) and retentate (materials retained on the ultrafilter membrane). It was observed that the host animals reacted differently to the three different fractions containing nanoparticles of different sizes. While the small fraction of permeate often did not elicit any response, nanoparticles of the permeate fraction behaved similarly to dissolved cadmium. On the contrary, the larger fragments elicited a strong response that reduced over time. Amongst the three organisms, Mytius edulis showed the highest susceptibility to QDs compared to the other two. The immune system of freshwater species was found to be less affected than marine water species (Mytilus edulis). It might be due to negative charge cancellation and aggregation of the nanoparticles [26]. Recently, it was reported that macrophages treated with 1.25 and 2.5 nM concentrations of QDs for a time duration of 4h, resulting in increased intracellular reactive oxygen species (ROS) levels that lead to apoptotic events within the cells. Treatment of RAW264.7 macrophages with the aforementioned concentration of QDs leads to decrease in phagocytic activity at the 24hr post-treatment while unpredictably increases at 4-6 hrs [27]. 2.3.Carbon black nanoparticles Carbon black nanoparticles (CBNPs) are a gaseous particulate, released in the form of diesel exhaust from the locomotive vehicles. These nanoparticles have been known for their toxicological effects in humans and other organisms [28]. Carbon black finds its application as a high-volume industrial chemical being used in various industrial procedures such as the

production of rubber, tyre, paint, toner, and printing ink. These particles can be considered as a model substance present in diesel soot responsible for air pollution as well as different diseases. Printex 90, having an average primary particle size of 14nm [28] and large surface area per mass unit, is the commercial name of CBNPs that is composed of pure carbon with very low concentrations of impurities such as polycyclic aromatic hydrocarbons (PAH) and endotoxins. In human monocyte cell line (THP-1 cells), the effect of micro and nanocarbon black particles was studied. Nano size carbon black particles (100- 800 𝜇g/ml) proved to be more toxic in a concentration dependent manner than micro sized particles (400 and 800 𝜇g/ml). Nano sized particles decreased the viability of cells up to 41%, while micro sized particles reduced the same up to 56% [29]. Along with a decrease in cell viability, elevated levels of pro-inflammatory cytokines (TNF-) and chemokines were also observed [30]. Analysis of gene expression showed that monocyte chemo attractant protein-1 (MCP-1) coding gene was highly expressed but the phagocytic capacity of cells were affected after the nanoparticles administration [29]. Carbon black nanoparticles were also given to ovalbumin sensitized splenic leukocytes derived from transgenic mice. They acted as adjuvants in enhancing the Th2 associated cytokine pathways by increasing the genes for Th2 cytokines such as IL-4, and IL-10 [31]. Although cell division was suppressed, cell viability remains unaffected while cytokines and chemokine production was suppressed in the cells being treated with carbon black nanoparticle only. . Exposure of sperm cells to carbon black nanoparticles affected its fertilization with egg and even development of an embryo in purple sea urchin, Paracentrous lividus. Nanoparticles attached to the sperm surfaces and hindered various signaling pathways occurring in early embryonic development. On gastrula stage, the embryo was seen to have many abnormalities. In pluteus stage, embryos showed skeletal deformities; even larval shape

and size were affected. Reduction in cholinesterase and propionyl cholinesterase activity was also detected in fertilized embryos [32]. After the intranasal administration of carbon black nanoparticle to the pregnant mice, no deaths were observed. The male newborns showed a slight increase in weight, inflammatory response and increase in immune-phenotype. An increase in the number of T-lymphocytes and B-lymphocytes were also reported [33]. Haemocytes of blue mussel treated with carbon black nanoparticles at a concentration of 5 and 10 µg/ml; showed a reduction in the mitochondrial number and loss of mitochondrial membrane potential in a dose-dependent manner. Other responses noted were extracellular lysosomal release at different concentrations (1, 5, and 10 µg/ml) of the carbon black nanoparticle accompanied by the generation of nitrogen oxide and reactive oxygen species. Inflammatory pathways consisting of MAPKs (mitogen-activated protein kinases) and JNKs (Jun N-terminal Kinases) were also highly active [34]. The addition of carbon black nanoparticles resulted in decreased cell viability to the fetal bovine serum. Cells in a serumfree culture medium easily took in the nanoparticles. Also, serum proteins hindered in cytotoxicity of the nanoparticles by adsorbing on their surface [35]. Size acts as a major determining factor of inflammatory response with small size carbon black nanoparticle eliciting the maximum secretion of granulocyte macrophage colony-stimulating factor. Carbon black nanoparticles have been reported to induce cytotoxicity by generating oxidative stress when administered to bronchial epithelial cells [36]. 2.4. Carbon nanotubes Carbon nanotubes (CNTs) are being produced at a rate of hundreds of tons annually to utilize their physiochemical properties. General types of these nanoparticles include single-wall carbon nanotubes (SWCNTs), double-wall carbon nanotubes (DWCNTs), and multi-wall carbon nanotubes (MWCNTs). CNTs are one of the components in electronic devices, protective clothing, sports equipment, medical devices and drug delivery vehicle. Their

characteristics fiber-like geometry and durability are shared with asbestos [37]. The unique physiochemical properties of SWCNTs include small size, large surface area, and high reactivity which forms the basis of their potential application in high-strength materials, reinforced rods, quantum wires, mechanical memory, sensors, electronic devices, and water treatment [38]. The biomedical applications of SWCNTs consist of drug delivery systems, cancer diagnosis, imaging, and treatment. When it comes to drug delivery, one-dimensional structure and ultra-high surface area of these nanoparticles enhances their drug delivery capacity and allowing their passive diffusion inside the cell or endocytosis [39]. The specialty of these nanocarriers is that they prevent tissue level toxicity and embolism condition due to blockage of blood vessels and delivers the drug to the target site in our body via injectable routes [40]. It has come to light that MWCNTs cause disruption of the immune system both in vitro and in vivo. In vitro, MWCNTs exposure to mature human monocyte-derived macrophage (HMM) cells gain entry both actively and passively into the cell. Significant toxicity and diminished cell viability via necrosis were observed in a dose-dependent manner in the case of unpurified as well as purified MWCNTs [41-43]. An experiment conducted with MWCNTs using murine macrophages (J774.1) showed decreased cell viability as the concentration of nanoparticle and time of exposure increased. The nanoparticles interact with macrophage receptor with collagenous structure present on the plasma membrane, a murine scavenger receptor and subsequently lead to its rupturing [44]. In vitro, studies conducted on human bronchial epithelial cell line BEAS-2B showed well-dispersed MWCNTs. The addition, MWCNTs resulted in increasing cytokines in a dose-dependent manner and phosphorylation of a signal transduction cascade component ERK1 of RAS, RAF, MEK, ERF pathway which is very crucial for cell adhesion, cell cycle, cell survival etc. [45]. Other factors that are phosphorylated were p38 (A component of the MAPK family, which gets

selectively activated under the effect of inflammatory cytokines and various other environmental stresses) and HSP 27 i.e. heat shock protein, which serves as a chaperone protein, antioxidant and also functions to inhibit apoptosis [46]. Migration inhibitory factor (MIF), which often acted as a proinflammatory cytokine and participated in immune response was also phosphorylated in larger concentration. Another factor, NF-B that has a role in the expression of cytokines and another pro-inflammatory related gene, showed increased phosphorylation [47]. In similar experiments conducted on the mesothelial cell line, it has been seen that SWCNTs do result in oxidative stress generation in the cell. As a result of this, many constituents of cellular stress pathways are activated such as histone 2AX phosphorylation, activation of PARP-1, AP-1, NF-B, p38 etc. The damage caused consisting of DNA damage and cell death in a dose-dependent manner [48]. Trouts exposed to carbon nanotubes showed gill irritation, mucus secretion, and elevated ventilation rates. The increase in edema was also observed in secondary lamellae and hyperplasia in primary lamellae with changing mucocyte morphology. Also, hemoglobin and hematocrit content went down while Na+ K+-ATPase activity was seen to be slightly elevated. Some reduction in Cu and Zn ion concentrations in the gills and brain were observed according to the dose of the carbon nanoparticle administered. However, it was unclear whether the nanoparticle affects bronchial Cu and Zn transport. Thiobarbituric acid reactive substances (TBARS) often generated as by-products of lipid peroxidation can be used to assess the damage caused by ROS, thus indirectly measuring the ROS content. The decrease in TBARS content in various tissues like brain, gills, intestine, and liver was observed whereas glutathione level was unaffected. Cell morphology was affected and apoptotic bodies were clearly visible. Many cells were observed in the early stage of nuclear necrosis. Ventral surface of cerebellum exhibited

swellings. Aggressive nature in fishes was observed and was explicit at highest nanoparticle concentration. Contradictory to what was observed in an earlier experiment, higher metal concentration in brain lowered fishes’ defensive abilities and a lot of aggressive behavior was detected which requires more studies to be conducted on this topic. Similar to the observation of an earlier experiment, in which granuloma formation in the lungs of rodents was monitored upon exposure to carbon nanotubes; tumor formations in fish liver were also observed on prolonged exposure [49]. However, fishes exposed to high concentration of nanoparticles, showed swelling of blood vessels of the cerebellum (ventral surface), thus indicating that nanoparticles might affect the blood-brain barrier and cardiovascular system in fishes. A large accumulation of nanoparticles was observed in intestines with some regions showing inflammation and degradation of the intestinal mucosa [50]. Male C57Bl/6 mice were found to be immunosuppressed after being given 1mg/m3 of carbon nanotubes via inhalation for 6 hours per day for 14 consecutive days resulting in the activation of cyclooxygenase enzymes in the spleen due to the signals received from the lungs. Broncho-alveolar lavage fluid (BALF) proteins extracted from mice that were exposed to MWCNTs successfully suppressed T-cell dependent antibody response in naive spleen cells [51]. By changing the surface characteristics of the MWCNTs, their binding capacity to various receptors can be altered thus lessening their toxicity, as was clear from an experiment conducted in vivo (mice) and in vitro (macrophages). Also, the accumulation of MWCNTs mostly occurred in lungs, liver, and spleen after 24 hrs. when administered by injection into a tail vein. Pulmonary inflammation was considerably higher on the first day after administration. Accumulation in lungs was very prominent which activated alveolar macrophages and strongly stimulated inflammatory response [52]. It was found that purified carboxylated multi-walled carbon nanotubes on intravenous injection in mice showed varied results. After uptake by mononuclear phagocytic organs, the particles which had formed

agglomerates were retained in the lungs while those which remained in suspension formed fewer aggregates and were easily eliminated out of the host system. The agglomerates were also responsible for a wide range of inflammatory responses [53]. Double-walled carbon nanotubes (DWCNT) were shown to be recognized by Toll-like receptors, leading to the release of pro-IL-1β. Caspases are a family of cysteine proteases having a very important role in programmed cell death i.e., apoptosis. Inflammatory caspases such as caspases-1 and -5 get activated due to the formation of inflammatory complexes known as inflammasome which results in activation of pro-inflammatory cytokines such as IL-1 [54]. Also Nlrp3, an inflammasome, belonging to the NOD-like receptor (NLR subfamily of pathogen recognition receptor), gets activated when DWCNTs was engulfed by monocytes hence triggering an immune response [55]. Studies have shown that some hydrophobic regions of Toll-Like Receptors were able to bind to small-sized carbon nanostructures (5,5 armchair SWCNTs containing 11 carbon atom layers). Through this experiment, it was also seen that CNTs induces an excessive expression of specific cytokines and chemokines [56]. Dispersity is a critical factor for assessing the extent of immunotoxicity of a nanoparticle. An experiment conducted on female BALB/c mice and its macrophage cell line demonstrated that nanoparticles which have a number of carboxyl groups on its surface displayed more dispersity due to enhanced solubility. As a result, ROS production decreased thereby resulting in reduced cytotoxicity [57]. Bivalent mollusks, when exposed to nanoparticles showed that nanoparticles were taken up by the gills, which were then directed to intestines and their intracellular uptake lead to ROS generation and lysosome rupturing [58]. 2.5. Fullerene Nanoparticles

The fullerene (C60) finds its wide applications in biomedical research due to its anti-HIV activities, scavenging of reactive oxygen species and free radicals. It has also shown apposite role in antitumor effects, anti-aging treatments, degeneration of articular cartilage and treatment of secondary progressive multiple sclerosis [59]. Fullerenol, a derivative of fullerene [C60(OH)X] can inhibit allergic responses and prevent mitochondrial dysfunction in cellular models of Parkinson’s disease. Structurally C60 is icosahedral which resembles a soccer ball with 60 vertices and 32 faces (12 pentagonal and 20 hexagonal) composed entirely of carbon atoms [59]. In an experiment conducted on the IL-2-dependent T lymphoblastoid WE17/10 cell line, C-60 nanoparticles have demonstrated good biocompatibility. Once activated by the receptor, T-lymphocyte activation leads to secretion of IL-2. IL-2 acts to stimulate growth, proliferation and further differentiation of T-cells. C-60 nanoparticles have shown less dispersion and also did not elicit IL-2 dependent proliferation. Low reactivity of the nanoparticles also leads to its lessened intracellular uptake. Due to their hydrophobic nature, C-60 nanoparticles mostly formed aggregates and increases the proliferation of CD4+ T cells due to the low amount of ROS generation. Another thing to be noted is that they did not affect CD25 expression [60]. Female C57BL/6 mice suffering from hypersensitivity showed suppression of symptoms after being intravenously given fullerene nanoparticles. Interleukin release (IL-4, IL-6, IL-17) occurring in a mouse suffering from DTH (delayed type hypersensitivity) also showed attenuation after being administered to nano-C-60, while no effect was seen with interferon- (IFN-). IL-17 suppression also resulted in up-regulation of T-regulatory cells. Cytokine promoting cell death (TNF-) showed an increase after the introduction of C-60 nanoparticle in these mice [61]. Human keratinocyte (HaCaT) and human lung carcinoma cell line (A549) did not show any toxic effects on being exposed to C-60 fullerene nanoparticle except for

suppression of cell proliferation. The significant effect of nanoparticles on the cells was the generation of ROS [62]. Human peripheral blood mononuclear cells (PBMCs) were used to study the effect of C60 fullerene in apoptosis condition induced by oxidative stress. Both TNF- and dRib (reducing sugar) were used to introduce apoptotic conditions in the cell. But the radical scavenging activity enabled fullerene to successfully inhibit apoptosis in both the cell types. However, in the case of TNF- induced apoptosis, carboxy-fullerene prevented apoptosis more efficiently by preventing mitochondrial membrane depolarization. The mechanism behind this is thought to be an enzyme inhibiting activity of carboxy fullerene [63]. C60(OH)20 nanoparticles has also garnered interest due to their antitumor effect on immune cells. After administration in serum samples, secretion of Th1 cytokine (IL-2, IFN-γ, and TNF-α) was increased along with an increase in the production of Th1. Alternatively, secretion of Th2 cytokine i.e., IL-4, IL-5, and IL-6 was decreased while the viability of immune cells remained unaffected. By synthesizing TNF-, the fullerene nanoparticles helped in the elimination of tumor cells [64]. In Daphnia pulex a water flea, administration of fullerene resulted in oxidative stress. The release of toxicity biomarkers namely glutathioneS-transferase and catalase was also observed. In oysters (Crassostrea virginica), a lot of harmful effects were observed after administration of fullerene. Lysosome rich hepatopancreatic tissue displayed accumulation of fullerene nanoparticles that resulted in lysosomal destabilization without any increase in lipid peroxidation levels. C-60 nanoparticles effect on developing embryos showed disastrous results [65]. Experiments were conducted to assess fullerene nanoparticle cytotoxicity on Mytilus hemocytes. Extracellular lysozyme activity was found to be elevated in a dose-dependent manner. ROS and NO (Nitric oxide) generation showed a similar trend after cells were treated with fullerene nanoparticle. Also, phosphorylation of p38 MAPK showed an initial increase which then gradually

declined. Apart from induction of mild inflammatory responses, these nanoparticles did not bring about any significant cytotoxicity in hemocytes [66]. Stronger effects of membrane destabilization in lysosomes were reported in mussel (Mytilus galloprovincialis) digestive glands after treating with the C-60 nanoparticle. Accumulation of lysosomal lipofuscin was also observed. Data showed that the catalase activity was not much affected by nanoparticles, while Glutathione transferase (GST) activity was stimulated a bit. The nanoparticle can lead to toxicity by inducing oxidative stress in the mussel digestive gland [66]. C60 nanoparticles resulted in a strong inflammatory response in the lungs of mice due to increased production of interleukins such as IL-1, IL-6, and TNF-α. Also increased secretion of Th1 cytokines [IL-12 and IFN-γ] in BALF was observed. In comparison to MHC class I, MHC class II has shown increased gene expression along with an increased number of T-cells. BAL cells also showed an arrest in the G1 stage of the cell cycle and lung tissue displayed the overall great extent of pathological damage [67]. Fish embryos were not affected after being given hydroxylated fullerene. Although the number of monocytes, lymphocytes, thrombocytes, and neutrophils remained unaffected, the functionality of neutrophils was slightly reduced. Also, IL-11 and myeloperoxidase were secreted more due to upregulation of their respective genes whereas a reduction was observed in the case of elastase-2 [68]. 2.6. Gold nanoparticles Gold nanoparticles (AuNPs) possess a huge potential to be used as inert carriers for medical purposes and are also widely used in plasmon-based labeling and imaging, optical and electrochemical sensing, therapeutic treatment and diagnostics for various diseases such as cancer, Alzheimer’s disease, hepatitis, arthritis, diabetes etc [69]. They are also being used as a drug delivery vector and in photo-thermal therapy. Gold nanoparticles can be distinguished

from bulk gold in their physical appearance as in they appear wine red in solution which is entirely different from the yellow color of bulk gold [18]. The type of immune response, a particular nanoparticle generates depends on a lot of factors. One of them is its surface nature that determines its interactions with various components of the immune system. Associating nucleotide material with the nanoparticle to alter its surface interactions is one such way. This association provides many unique properties of the conjugate particle such as increased uptake by the cells, more stability inside the cells and resistance against the activity of nucleases. It is considered that the negative charge present on the surface coupled with localized high salt concentration is the reason behind the conjugate’s resistance to degradative enzymes [70]. It has been reported that the class of receptors that recognize viral nuclei are responsible for activating interferon production, thus mounting an intense immune response. However, in an experiment conducted on mouse macrophage cell line RAW-264 shown that gold nanoparticles coated with oligonucleotides, resulted in almost one-fourth decrease in immune response [71]. Also, it indicates that by modifying the surface properties of the gold nanoparticle we could lessen the immune response. Experiments conducted on murine macrophage cell line have shown that gold nanoparticles successfully reduced activation of Toll-like Receptors-9 receptors, which are known to be activated by CpG-ODN. The synthetic analog of CpG DNA known as CpGODN (CpG oligo deoxynucleotides) binds to HMGB1 (High Mobility Group Protein-1). Then, it interacts with TLR9 receptors present in the cell (endolysosomes) to activate the release of various interleukins (IL-6, IL-12) and TNF- [72]. It was also found out that further pathways down the line that were activated by TLR9 (p38, JNK, and ERK) showed reduced activation. It is still under consideration that cathepsin activation resulting from gold nanoparticles resulted in degradation of TLR9. Also after phagocytosis of gold nanoparticles

by macrophages, the secretion of pro-inflammatory cytokines such as IL-6, IL-12p4 was reduced significantly [73]. Dendritic cells generated from bone marrow of C57BL/6 mice did not show signs of cytotoxicity when incubated with gold nanoparticles. While after the excessive accumulation of nanoparticles in dendritic cells, some morphological modifications were seen. Induced secretion of cytokines such as IL-6, IL12p70, IL-1 etc. has been also seen. Experiments conducted using polyethylene glycosylated gold nanoparticles on RAW264.7 cells have shown that LPS (lipopolysaccharides) activated macrophage cells displayed faster internalization of the nanoparticle. Apart from that, these cells also showed greater saturation of the nanoparticles within 24 hrs. as compared to unstimulated cells and showed high levels of IL-6, NO, and iNOS production, known to be pro-inflammatory mediators [74]. Insignificant cytotoxicity was observed in murine macrophage cell line after exposing the cells to gold nanoparticles. Maximum accumulation of the nanoparticles occurred in intracellular vacuoles. In human melanoma cell line MV3 and BLM, gold nanoparticles of 1.4nm diameter were found to be highly toxic; while in human dermal fibroblast cells, the nanoparticles decreased cell motility and adhesion capacity. In HeLa cell line smaller nanoparticles (1.4nm) were found to be more toxic as compared to the bigger nanoparticle (15nm). Similar results were obtained when SK-Mel-28, an endothelial cell line; L929, a fibroblast cell line; and j774A, a phagocytic cell line were exposed to the gold nanoparticle of size 1.4nm. Glutathione-coated gold nanoparticles in BALB/c mice displayed higher retention in tumor tissues and quicker clearance in normal tissues pointing out them as unique therapeutic agents for tumor detection. These nanoparticles also showed reduced retention in the reticuloendothelial system as compared to conventional dyes, which are currently used for the same purpose. Drosophila melanogaster showed high levels of generalized toxicity after ingestion of citrate-capped gold nanoparticles. The flies showed a reduction in their lifespan, a decreased fertility (in both male and female flies), and signs of

DNA damage in gastrointestinal tissue and extremely high expression of Hsp-70 (Heat Shock Protein). Uniform distribution of the nanoparticles was seen in reproductive and enteric tissue [75]. 2.7. Graphene nanoparticles Graphene and its derivatives are one of the most attractive nanoparticles for their application in electronic devices, molecular assembly, chemical sensing and environmental remediation [76]. This wide range of its applications is majorly due to its unique physiochemical properties such as high surface area, extraordinary electrical and thermal conductivity and strong mechanical strength. Graphene is basically single atom thick, two-dimensional sheets of carbon atoms that are having a hexagonal arrangement and sp2 hybridization. It serves as the building block for various other materials such as graphite, large fullerenes, and carbon nanotubes (CNTs). Graphene oxide (GO) is the best possible graphene derivative that has been utilized for its biological and biochemical applications which are mainly because of their unique properties like high solubility in water and other polar solvents, and rich oxygencontaining functional groups [77]. During the chemotherapy of cancer patients, graphene is used for the delivery of anti-cancer drugs to cancerous cells. Graphene oxide has shown tremendous potential for its utilization in the field of bioapplications. The injection of graphene oxide nanoparticles directly to the lungs had shown disastrous results. Graphene oxide could have acted as electron donor resulting in an elevated mitochondrial respiration rate thus producing more ROS as a by-product. However, it was found that increased dispersion of graphene along with the minimized oxidized condition could actually result in less toxicity and more compatibility inside the cells [78]. On human lung cell line BEAS-2B, cytotoxicity and apoptosis were observed after injection of graphene oxide nanoparticles. Oxidized graphene nanoribbons, water solubilized in PEG-DSPE (1,2-

distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)]),

using

various assays were used for assessment of toxicity in different cell types. Out of all the cell lines tested (HeLa, NIH-3T3, MCF-7, SKBR3), HeLa cell lines showed the highest level of susceptibility [79]. Erythrocytes, when exposed to graphene oxide nanoparticles have shown

hemolytic activity whereas graphene sheets have been proved to be more tolerable. It can be concluded that not only particle size but the oxide content of graphene oxide nanoparticles along with their response to their immediate environment are important parameters in determining their toxicological impact [80]. Contradictory observations were obtained, when the toxicity of graphene nanoparticles was studied in human alveolar epithelial cell line A549. It was observed that the cells did not take up nanoparticles thus no effect was seen on membrane integrity and viability of the cells. At low concentrations of the nanoparticle, no viable loss was observed. Induction of oxidative stress and a slight rise in cell death was observed in increasing the nanoparticle dose [81]. Experiments conducted on human peripheral blood T lymphocytes and human serum albumin (HSA) has given great insight into the toxicity of graphene oxide nanoparticles. Pristine graphene oxide (p-GO) and functionalized graphene oxide-containing GO-COOH and GO- poly ethylenimine (PEI) were used to assess the toxicity. In human serum albumin proteins, GO-COOH binds easily to the proteins without bringing about many alterations in structure. P-GO severely affects binding capacity and functionality of HSA while; GO-PEI altered the structure of HSA and rendering it functionless. For T-lymphocytes, p-GO and GO-COOH cause dose-dependent toxicity in the form of DNA damage, lessening of cell viability and ultimately leading to apoptosis without affecting their functionality. The fact where their effects differ is that p-GO leads to ROS generation whereas GO-COOH does not. Similar to the effect on HSA proteins, PEI causes high damage to T-lymphocytes by causing DNA and membrane damage thus suppressing their activity to a huge extent [82].

BALB/c mice sensitized with ovalbumin were used to study the exposure of PEG-GO. With a single administration of PEG-GO, no reduction in body weight or spleen index was observed. While, the production of OVA-specific immunoglobulin IgM, IgG1, and IgG2a continued in the same manner, the production of specific IgE was severely affected. Also, notable secretion of IFN-γ and interleukin-4 from the splenocytes was reported. In comparison, a mitogen concanavalin (sensitized mice after being administered with PEGGO) did not show any alteration in function. It can be inferred that dividing the capacity of Tcells under the influence of a mitogen remained unaffected [82]. It has been shown that GO is capable of complement activation. C3 is a part of complement activation cascade and its cleavage results in the formation of membrane attack complexes followed by generation of inflammatory responses (due to the anaphylotoxic nature of C3a fragment resulting from cleavage). The C-terminal arginine is lost from this fragment resulting in the formation of C3a (des-Arg). While unmodified graphene oxide nanoparticle results in C3 cleavage leading to activation of proteins involved in inflammatory responses. It's PEGylation results in selective and increased adsorbing properties of nanoparticle towards serum proteins. This reduces activation of C3a/C3a (des-Arg) by preventing its attachment to designated receptor thus hindering its activation. Studies also showed that nano-GO-PEG are capable of attenuating the immune response elicited by another nanoparticle [83]. Evidence shows that compared to graphene oxide nanoparticles, polyvinylpyrrolidone (PVP) coated GO have less toxic effects on dendritic cells (DCs), Tlymphocytes and macrophages. A difference was observed in the form of reduced differentiation and maturation of dendritic cells. The secretion of interferon and interleukins remained the same in the presence of both PVP-GO and GO nanoparticles. PVP-coated GO after being administered reduced the apoptosis of T-lymphocytes and even lead to increasing the activity of macrophages [84].

2.8. Iron oxide nanoparticles Iron oxide (magnetite), due to its magnetic properties, is potentially important in biomedical applications such as diagnostic imaging, cell labeling, site-directed drug delivery, anti-cancer hyperthermia therapy, gene delivery, tissue repair, cell separation and magnetic resonance imaging [85, 86]. These nanoparticles are also being utilized for diagnosis and treatment of cancer by antibody targeted therapy [87]. Due to their unique physiochemical properties, they display a high potential to increase the efficacy of anticancer drugs and reverse multidrug resistance. Experimental evidence has shown that after being injected into humans, liver and spleen are the major organs of deposition of iron oxide nanoparticles with uniform distribution in other organs such as brain, heart, lungs, stomach, small intestine, kidneys and even bone marrow [88]. Iron oxide magnetic nanoparticles (MNP’s) coated with polyethyleneimine (PEI) and polyethylene glycol (PEG) were used in various cell lines such as SH-SY5Y, U937 cells, and MCF7 cells, to study the immunotoxicity occurring in these cells. The purpose of coating the nanoparticles was to provide varying degrees of charges to these particles. The U937 cell line showed the lowest level of MNP uptake while PEI-coated MNPs showed a maximum reduction in viability in MCF7 cells. However, MNP-PEI-PEG showed approximately 5% viability reduction in all the three cell lines. Also in all three cell lines, MNPEI nanoparticles showed a maximum generation of ROS whereas MNP-PEI-PEG did not cause much increase in ROS level. Small pits were observed in cell membranes of all cells administered with MNP thus demonstrating a different pattern of cellular response [89]. In mouse, injection into trachea elicited numerous responses. While the level of glutathione decreased, concentrations of many pro-inflammatory cytokines such as interleukins (IL-1, IL2, IL-4, IL-5, IL-6, IL-12), TNF-α, and transforming growth factor (TGF-β) increased in the

BAL fluid along with expression of proinflammatory related genes. Also, the number of IgE, B cell, and CD8+ T cells showed an increase in number. A type of inflammation in the form of microgranuloma in alveolar space was observed which is a result of oxidative stress generated due to the production of ROS [90]. In a similar experiment conducted on mice, it was found that with an injection of iron oxide nanoparticle, the proliferation of lymphocytes increased. Increase in T-cell number was also seen and the production of both Th1 (IL-2, INF-γ) and Th2 cytokines (IL-4, IL-5, IL-10) was strongly influenced [91]. However, in another experiment conducted where mice were administrated with intravenous iron oxide nanoparticle gave quite different results. The mice showed marked suppression of T-cell mediated immunity. Also, Th1 and Th2 immune systems showed differential response to these nanoparticles [92]. In mice splenocytes primed with ovalbumin, on exposure to iron oxide nanoparticle showed a reduction in Th2 immune response. Iron oxide nanoparticles are also known to suppress delayed-type hypersensitivity (DTH) in mice with a reduction in functionality of macrophages and Th1 cells. Even, the expression of pro-inflammatory cytokines such as IL-6 and TNF-α was reduced [93, 94]. In an experiment conducted with dextran-stabilized iron oxide nanoparticles, it was seen that iron oxide nanoparticles caused suppression of mitogen phytohemagglutinin (PHA) induced proliferation and cytokine synthesis. When directly administered to the spleen of rats, these nanoparticles have shown induced proliferation of lymphocytes [95]. In human macrophages, the ultrasmall superparamagnetic iron oxide (USPIO) induced apoptosis occurred with the production of ROS and activation of c-Jun N-terminal kinase (JNK) pathway but without any significant production of TNF [96]. In a similarly conducted experiment in murine macrophage cell line-RAW 264.7 cells, the nanoparticles accumulated in membrane-bound organelle and reduced their phagocytic ability [97]. 2.9. Nanoceria nanoparticles

Nanoceria (CeO2) NPs are attractive agents that are being utilized in pharmaceutical industry, nanotherapeutics, polishing agents, UV-absorbing compounds in sunscreen and UVscattering agents in non-irritating lipsticks. Nanoceria displays catalytic properties due to which they are being utilized as a catalyst in the petroleum refining industry; as additives, to promote the process of combustion of diesel fuels; as sub-catalysts, for the cleaning of automobile exhaust and as electrolytes in solid oxide fuel cells [98]. The unique characteristics of these nanoparticles include their smaller size (1-100nm) which enables them to diffuse easily inside the cell without being aggregated and their increased surface function, which makes them hydrophilic and prevents them from getting conjugated with other ligands or compounds while delivering, targeting, and imaging of certain compounds [99]. Also, the interchangeable variability between two valences of cerium ( Ce3+ and Ce4+) present in nanoceria makes them scavenger molecule for both positively and negatively charged particles. In recent studies, nanoceria has been proved to be an ideal agent for the treatment of glaucoma and retinal diseases and for non-surgical treatment of cataract [99]. The ability of nanoceria to reduce reactive oxygen species has sparked a lot of interest in developing them as an effective tool. They have been proven to be anti-inflammatory in nature as they devour the ROS species that are the main reason behind inflammation as seen in experiments conducted on murine macrophages. Another fact that has come to light is that nanoceria particles are able to suppress innate immunity of sea urchins [100]. The accumulation of nanoparticles inside the body depends upon the route by which they are being administered. The surface of the nanoparticle plays a major role in determining its interaction. Nanoceria is having positive zeta potential; interact better with proteins (in terms of adsorption) as compared to those nanoparticles having negative zeta potential. An experiment conducted on CD1 mice has shown that after intraperitoneal and intravascular administration of nanoparticle, the spleen is the organ where maximum accumulation is seen

followed by liver. The spleen is the main vascular organ and liver is the organ where maximum detoxification takes place. Therefore, it is natural that these organs show maximum deposition of nanoceria. A small amount was also detected in kidney and lungs. However, significant deposition in the lungs is also seen after oral administration [101]. Other experiments conducted on mice of strain C57BL/6 have shown that oropharyngeal administration of nanoceria resulted in inflammation via activation of the mast cells and increase the secretion of a number of cytokines and chemokines such as TNF- and IL-6. Even, osteopontin level seemed to be on the rise [102]. Observations have also depicted a rise in IL-12 and a decrease in NO content. This high amount of toxicity in the form of lung damage and inflammation was observed when Sprague-Dawley rats were administered by cerium oxide nanoparticles [103]. Usual inflammation and increased level of IL- was found to be in female Wistar rats administered with the nanoparticle intra-tracheal. Mild cytotoxicity was observed with acute and chronic neutrophil response in the lungs [104]. When Wistar rats were made to inhale nanoceria, acute cytotoxicity was observed mainly due to oxidative stress. The chronic inflammatory response was seen with increased levels of interleukin (IL-1, IL-6), TNF- and Malondialdehyde. However, glutathione (GSH) content was decreased [105]. Cerium oxide nanoparticles had already garnered a lot of interest due to their ability to destroy ROS. Experimentally, it has been observed that after giving customized cerium oxide nanoparticles (CeNp) to C57BL/6 mice having experimental autoimmune encephalomyelitis (EAE), which is a murine model of multiple sclerosis, a lot of positive response has been received. In line with the antioxidant activity of nanoparticles, a significant decrease in ROS levels in brain cells has been observed. Upon administration, the symptoms that were visible showed the reduced severity and better preservation of motor functions. Cardiac progenitor cells (CPCs) are precursors of all differentiated cells within a specific germ layer. Nanoceria easily enters the cells and get accumulated in the form of

small clusters but do not cause any harm to the cells. The morphology, growth and differentiating ability of the cells remains unaffected after the administration of nanoparticle. These nanoparticles not only reduces ROS content but also stopped further ROS generation [106].

2.10.

Nanoshells nanoparticles

Nanoshells are nanoparticle consisting of a dielectric core surrounded by a thin metallic shell having remarkable optical properties. The most common nanoshells are gold-nanoshell which consisting of a core of silica nanoparticle surrounded by a layer of nanoscaled gold shell. This spherical gold nanoshells core has a diameter of 120nm while the overall diameter is 155nm [107]. It is the plasmon-derived optical resonance properties which are responsible for the optical properties of these nanoshells. The inner and outer surfaces associated plasmon mix together and hybridized to give lower energy state. These unique optical properties of the gold nanoshells made its application in various fields like bio-imaging, photothermal based cancer therapy, biosensing, fluorescent enhancement, surface enhanced Raman and infrared absorption spectroscopies [108]. Nanoshells are right now being utilized as a part of a clinical investigation of the removal of solid tumors in head and neck and prostate cancer. It has also shown its capability to release the antisense DNA oligonucleotide conjugated to complex and become a light-mediated nonviral vector for gene therapy. In spite of the aforementioned key application of gold nanoshells, scientific studies have been conducted to check the toxicity and immunological consequences associated with its application. The preclinical studies have shown the accumulation of gold nanoshells in tumor followed by their clearance from the bloodstream by the organs of reticuloendothelial system. While macrophage cells in the spleen and liver are the major site where long-term retention

of gold nanoshells was evident with little clearance. There were no any toxicological pieces of evidence obtained from these studies. Based on ISO testing standard, the in vitro toxicity screening of gold nanoshells has shown none of the toxicity on the genotoxicity, hemocompatibility, cytotoxicity, pyrogenicity and tissue tolerance parameters. To check the in vivo safety profile and acute systemic toxicity, beagle dogs were administered with single dose gold nanoshells for a time duration of 28 days, none of the sign and symptom of infusion-related reactions were obtained during and after the infusion [107]. 2.11. Nickel nanoparticles Nickel nanoparticles fall under the category of metallic nanoparticles and display characteristics like a high level of surface energy, high magnetism, low melting point, high surface area, and low burning point. These properties allow them to be used as catalysts and sensor in electronic devices and in biomedical applications that include diagnosis of cancer and its therapy. Also, their use in the form of nanorings as memory cells has been explored [109]. Metallic nickel nanoparticles have been reported to show their utility as electrode materials in multilayer ceramic capacitors (MLCC). Nickel and copper alloys have been extensively used in controlled magnetic hyperthermia applications. The use of nanoparticles in medicinal biology has completely revolutionized the whole field. With a huge range of applications, it has become necessary to study their adverse effects on the host. Owing to their extremely small size, it is very easy for them to enter in the body and reach the target organ. Nickel oxide nanoparticles have been found to affect the immune system by increasing the secretion of IFN- and IL-4. Due to oxidative stress, GSH (glutathione) level decreases whereas ROS level increases thereby resulting in cell death via apoptosis [110, 111]. Some experimental evidence is there to show that nickel nanoparticles lead to oxidative stress by activating an apoptotic cascade and are more toxic as compared to nickel microparticles. SIRT-1, a NAD-dependent deacetylase regulates apoptosis by

deacetylation of genes (p53, FOXO, Ku70) playing important roles in aging, energy metabolism, oxidative stress etc. [112]. SIRT-1 inhibits p53 by deacetylating it however due to suppression of SIRT-1, this gene is no longer suppressed and hence apoptosis follows. It might be possible that inhibition of this SIRT-1 by nickel nanoparticles triggers apoptosis [113]. 2.12. Platinum nanoparticles Platinum group elements (PGEs) are one of the components in environmental pollutants and hazardous to human health as well. They are emitted into the environment as vehicle exhaust catalysts thereby contaminating natural resources and leading to their bioaccumulation in flora and fauna via different pathways. PGEs mainly include platinum (Pt), palladium (Pd) and Rhodium (Rh) as nanoparticles which come under the category of potent allergens and sensitizers. Their toxicity is relatively lesser than silver nanoparticles and is known to have antioxidant properties that reduce the production of ROS. This quality of Pt NPs makes them suitable for their use in anti-aging formulations in the cosmetic industry. Experiments conducted on peripheral blood mononuclear cells (PBMC) have shown that platinum nanoparticles do induce certain harmful effects on the immune system. The chlorinated ammonium Pt compounds are the most immunotoxic and have shown the strongest signs of toxicity in the experiment conducted [114]. In cervical cancer cell line (SiHa), platinum nanoparticles led to chromosome condensation and nuclear fragmentation, ultimately leading to cell death [115]. Due to adsorption of proteins on the surface of nanoparticles, nanoparticle-protein corona (NP-PC) are formed which play an important role in determining other interactions of the nanoparticle. Due to the formation of nanoparticles protein complexes, the uptake of Fe-Pt nanoparticles was reduced in HeLa cell line. Plasma proteins have shown a certain amount of reactivity towards the Fe-Pt nanoparticles [116].

2.13. Silica nanoparticles Silica nanoparticles are among the highly utilized NPs in the nanotechnology industry. Their occurrence has been observed in various forms, namely crystalline, simple amorphous and mesoporous. Mesoporous silica (MPS), a highly suitable nanomaterial, synthesized by supramolecular polymer templating methods, is a synthetically modified colloidal silica which has a pore size of the order 2-50 nm and has huge potential in biological applications like, drug labeling, gene or protein labeling, bio-separation etc. [117]. MPS nanoparticles are considered as good drug and nucleic acid delivery systems due to their well-defined structure. Amorphous silica nanoparticles have noncrystalline structure and are being utilized in many industrial and biomedical applications. Some of such applications include their use in pharmaceutical drug, glass, electronic appliances and hydrophobic anticancer drugs [118]. These nanoparticles have a huge number of applications because they can be synthesized in different sizes along with surface modifications. Some of the industrial applications include their use in sunscreens, toothpaste, paints, food, and animal feed additives while biomedical applications consist of drug delivery systems, cancer diagnosis, and its therapy [119]. Silica nanoparticles also form dye-doped nanoparticle when doped with thousands of dye molecules in a single nanoparticle. These particles are attractive luminescent nanomaterial for the production of the strong fluorescent signal [120]. The unique physiochemical properties of these nanoparticles make them suitable for their use in many industries and make their interactions with the biological system so specific, which can often lead to negative regulation of the immune system. MPS causes less cell death by apoptosis as compared to colloidal silica nanoparticle. Colloidal silica nanoparticles were more successful in eliciting vigorous responses such as activation of mediators of inflammatory signaling, induction of pro-inflammatory cytokines and induction of hypersensitivity. The reason behind better biocompatibility of MPS can be attributed to their

large porosity, large internal volume and the high surface area which confers them low cytotoxicity, in vivo [117]. Researchers studying the effect of silica nanoparticles on stem cell differentiation observed that smaller sized nanoparticles (10-30 nm) have a concentration based inhibitory effect on cell differentiation. On the other hand, large sized particles of size 80-400 nm had no effect on the cell growth and function [121]. Experimental evidence proves that silica nanoparticles have caused oxidative stress in various types of cell lines, such as Beas-2B, pulmonary, dermal fibroblasts and tumor cells of the lungs, colon etc. A single insertion of silica nanoparticle is even sufficient to elicit a large number of immune responses. In response of silica nanoparticles, the concentration of NK (natural killer) cells, cytotoxic T-cells increases along with increased pro-inflammatory cytokine secretion. Many genes related to various inflammatory responses were found to be up-regulated when cells were exposed to silica nanoparticles [122]. The action of silica nanoparticle varies according to the charge and size. Smaller particles have a larger surface area thus enabling better interaction with the biomolecules in vivo. Another experiment has shown that upon exposure to the silica nanoparticles of 30nm size, macrophages secrete a large amount of IL-1 which ultimately leads to cell death [123]. Immunosuppression was the major change that was observed through the suppression of proliferation of lymphocytes. The activity of NK cells and production of inflammatory cytokines was suppressed during silica nanoparticle exposure. Another important fact that came to light from the same experiment is that negatively charged silica nanoparticles might cause higher cytotoxicity as compared to the positively charged. The less amount of NO was released and synthesis of cytokines such as IL-1β, TNF-α, IL-12p70, and IFN- was repressed which resulted in stronger immunosuppression [124]. 2.14. Silver nanoparticles

Silver nanoparticles are the most commonly utilized nanoparticles in the industry because of their antimicrobial, optical, catalytic and disinfectant properties. These particles are being used in various consumer products such as cosmetics, clothing, household products, room sprays, food products, medical imaging and biosensing applications. Some of the commercial applications include their use in surgical instruments, water purification products, and paints, which increase the risk of their exposure to the environment [125]. Their ability to generate surface plasmon resonance (SPR) makes them suitable for their use in chemical and molecular sensing. Apart from above applications, the silver nanoparticles are being used in bedding, washing machines, water purification systems, toothpaste, shampoo and rinse, nipples and nursing bottles, fabrics, deodorants, filters, cooking utensils, toys, and humidifiers. Recently, it was also reported that collagen coated silver nanoparticles in collagen hydrogel have shown notable anti-infective properties at significantly lower concentrations against Staphylococcus epidermidis, S. aureus, E. coli and P. aeruginosa. It has also shown a reduced IL-6 level, TIMP1, and CCL24 when implanted subcutaneously in mice with only 0.2μM AgNPs [126]. Besides the useful antimicrobial and anti-inflammatory properties of silver nanoparticle, their intense yellow coloration makes a hindrance for them to become useful in bioengineered corneal replacement.

With a large number of applications in a wide variety of fields, silver nanoparticles have been under extensive study with a focus on their toxic effects. Experimentally, it has been reported that liver and spleen are the main organs where deposition of silver nanoparticles takes place when taken orally. While brain and testes were the two organs in which silver was retained for the longest duration. In comparative studies, it has been found that silver nitrate particles were taken in at the faster rate as compared to silver nanoparticles. Noteworthy, it has been also observed that oral exposure to silver nanoparticles was failed to elicit any significant non-specific immune responses [127]. In vitro studies indicate the

cytotoxic nature of silver nanoparticles by their effect on cellular metabolism. In vivo studies conducted on rats through intravenous administration resulted in an increase in both IgM and IgE levels, when the mouse was given both 20nm and 100nm silver nanoparticles. The T cell, B cell and NK cell populations of the spleen have shown a remarkable increase in number, due to which a large increase in weight of the spleen was observed. NK cell activity was almost reduced to zero as a result of silver nanoparticles. Lymph nodes and the Kupfer cells (specialized macrophages in the liver) have shown no signs of inflammation even in the presence of silver nanoparticles. Further, the effects seen as induction of these nanoparticles are decreasing the production of IFN- and IL-10. The decrease in the concentration of IL-10, IL-6, and TNF- indicates that B-cell activity has been affected in response to silver nanoparticles [128]. The ionic content of the silver nanoparticle affects its cytotoxicity. However, it still remains to be clear whether toxicity is due to ions being released from nanoparticles or due to the nanoparticles themselves. It has been shown that after oral administration, toxicity is related to increasing concentrations of silver ions. Another study presents the T-cell dependent antibody response with respect to keyhole limpet hemocyanin (KLH) indicating the severe impact on the immune system. The functional immune system gets severely impaired due to a reduction in KLH specific IgG thus resulting in overall immunosuppression [129]. Silver nanoparticles have also been shown to affect the blood-brain barrier system. After exposure to silver nanoparticles, pro-inflammatory cytokines such as TNF, IL-1B, and PGE2 were secreted in a large amount due to increased permeability of rat brain microvessel endothelial cells (rBMEC). Size-dependent perforations were observed in rBEMC monolayers on exposure to silver nanoparticles along with the release of pro-inflammatory mediators. Also, another important factor that came to light was that smaller nanoparticles

(25 nm) elicited more response as compared to larger nanoparticles (40-80 nm) and cause more damage such as cerebral microvascular damage. The release of these inflammatory substances affects the microvascular permeability resulting in entry of substances that may result in severe neurotoxicity [130].

2.15. Titanium dioxide nanoparticles Titanium dioxide nanoparticles (TiO2–NP) have a wide range of applications in the manufacturing of paints, paper, floor laminates and food colors (used in chewing gums and other sweet products) and cosmetics. Their use as a pigment in various products is major because of properties like brightness, high refractive index and the ability to resist discoloration [131]. Their ability to block the penetration of UV rays in the skin makes them suitable for their use in sunscreens. These nanoparticles also exhibit photocatalytic properties due which they are being used in wastewaters and as environmental disinfectants. Their biomedical applications include their utility in various pharmaceuticals, medical devices, and treatment of many diseases. There are basically two forms of TiO2–NP that is important for its industrial use. These two forms are rutile (more common) and anatase, out of which the latter one displays higher photocatalytic activity and toxic effects. The commercial TiO2 generally comes with an outer coat of either inorganic or organic substances which are used to enhance its surface properties [132]. Titanium dioxide, apart from its numerous uses in the biomedical field, has also raised questions regarding its potential as an immunotoxic compound. In RAW264.7 cells and BEAS-2B cells, the significant toxicity of titanium dioxide was observed. TiO2 elicited higher toxicity, especially at a concentration greater than 100g/ml. The range of damage caused includes generation of ROS, mitochondrial depolarization, leakage of the plasma

membrane and even cell death. The small size of the particle plays a significant role in cytotoxicity due to the larger specific area and therefore can absorb more biomolecules [133]. The introduction of these nanoparticles triggers the systemic immune system. Spleen and other lymphoid organs were the main targets where deposition of the nanoparticles had taken place. The proliferation of B-cells and T-cells are the main outcome of intra-tracheal administration of titanium dioxide nanoparticles. Also, natural killer cells showed increased activity. Other than this, no significant changes were observed in the secretion of Th1 and Th2 cytokines [134]. When titanium dioxide was administered intragastrically at a lower concentration, not much damage was observed in the splenic tissues; however, at higher concentration, tissue damage was evident. ROS content was greatly increased due to lipid peroxidation. p38, JNK, NF-κB, Nrf-2, and HO-1 genes were up-regulated to tackle with oxidative stress induced in the cells. Damage to a lot of extents was caused due to the introduction of nanoparticles which resulted in the form of lesions and inflammation, leading to lymph node proliferation [135, 136]. Earlier studies have shown that TiO2 proved to be highly toxic, especially in heart, lung, kidney, liver etc. of the injected mice. Spleen, where maximum deposition had taken place showed signs of lesion formation. TiO2 was seen to blocking the blood vessels leading to blood clogging in the pulmonary vascular system that resulting in thrombosis. Hepatic tissue was also found to be damaged by necrosis and fibrosis, due to the presence of the nanoparticle. Even in the lungs, alveolar septal thickening was observed [137]. Titanium dioxide nanoparticles didn’t show much of cytotoxicity in human peripheral blood mononuclear cells. Apart from mild damage to DNA, the cells were not much affected by the presence of TiO2 nanoparticles. Titanium dioxide did not produce many significant changes in human peripheral blood lymphocytes. The expression of adhesion molecules on the surface of the lymphocytes

remained unaffected and no oxidative burst was observed. However, a slight increase in the proliferative capacity of T-lymphocytes was noted [138]. A modular immune in vitro construct (MIMIC) is a system which can be used to study both innate and adaptive immunity. The main components of MIMIC are (1) peripheral tissue equivalent (PTE), which is composed of blood vein endothelial cells, and (2) antigen presenting monocyte-derived dendritic cells (DCs). These DCs act as a link between innate and adaptive immune responses. These two systems in combination act as a yardstick that can be used to measure the immune response of the body against any intruding particle. Proinflammatory cytokines such as interleukins (IL-1α, IL-1β, IL-6, and IL-8), IFN-γ, and TNFα showed elevated levels. Immunosuppressive cytokine (IL-10) production also increased. ROS was also generated which caused lots of further damage. Nanoparticles might cause the activation of DCs along with an increase in expression of DC-specific maturation marker like CD83. T cells also showed increased proliferation. In human myelomonocytic cell line THP-1, the effect of the addition of TiO2 nanoparticles leads to the generation of ROS and an increase in the concentration of kynurenine. However, the tryptophan levels remained as it is [139]. Microglia-mediated neurodegeneration was studied in vitro using titanium dioxide nanoparticles, which was observed to stimulate NO production and MCP production. MCP stands for monocyte chemoattractant protein-1 which is one of the key chemokines that control migration and infiltration of monocytes/macrophages. NF-B is a transcriptional regulator of genes mainly responsible for inflammation. In the presence of TiO2 nanoparticles, its binding capacity to inflammation-related genes increased manifold and more pro-inflammatory mediators were formed. TiO2 is more potent in causing the synthesis of the inflammation-inducing cytokines such as TNF-, IL-1 and IL-6 [140]. In cœlomocytes of Eisenia fetida, lower levels of cytotoxicity were observed. Titanium dioxide nanoparticles did not affect the phagocytic capacity. Only the fetidin and metallothionein

mRNA expression increased and expression of coelomic cytolytic factor mRNA decreased. Superoxide dismutase, catalase, and glutathione-S-transferase, which act as markers for oxidative stress, remained unaltered [141]. Exposure of fishes to TiO2 during early embryogenesis led to a significant alteration in the expression of genes involved in kinase activity, circadian rhythm, immune response etc. In Danio rerio, alteration of gene expression was observed. Changes in function of fish neutrophils, increased expression of IL-11, macrophage stimulating factor 1 neutrophil cytosolic factor 2 was also observed. Most of the nanoparticle accumulation was found to occur in kidney and spleen [68, 142]. Also, intra-tracheal administration of TiO2 nanoparticles in mice led to rising in immunotoxic responses. At a dosage of 0.5 mg/Kg of nano TiO2, pulmonary emphysema was observed. Lymphocytes and macrophages were seen in aggregations and alveolar septa seemed disrupted. At higher concentration (4 mg/Kg of Nano TiO2), accumulation of lymphocytes and macrophages was observed along with inflammation. Terminal bronchioles had collapsed with the thickening of the outer wall. At further higher concentration (32 mg/Kg of nano TiO2) still, greater damage was observed. Extensive nanoparticle deposition was observed in the gaps and alveolar cavity of the lungs with the alveolar septa badly disrupted. Administration of TiO2 micro-particles at a concentration of 32 mg/kg reveals infiltration of inflammatory cells in the lungs. Lamellar bodies which are present in type II cells were observed to be damaged. It was evident that organelle dissolution, endoplasmic reticulum expansion, and other such damages were greater at higher concentration of TiO2 nanoparticle. But, the level of cytokines IFN- and IL-4 was not much altered. T-bet mRNA showed a decrease in expression whereas GATA-3 levels showed a dose-dependent increase. However, not much difference was observed in GATA-3 and T-bet levels in mice that were administered 32 mg/kg of either nano TiO2 or micro TiO2 [143]. The chronic inflammatory

response was seen in mice after intratracheal instillation of titanium nanoparticles and granuloma formation was also observed. Th1 cytokines such as IL-1, IL-6, TNF-, along with Th2 cytokines (IL-4, IL-5, IL-10) showed a dose-dependent increase. The population of B cells increased 9 times in spleen and blood. Even IgE concentration was elevated in serum and BAL fluid. Also, the genes related to antigen presentation and induction of immune cells showed an increase in expression [144]. Toxicity is evident in mice that were given titanium dioxide nanoparticles. Reduced body weight and extensive liver damage are mainly reported. Pathologically, liver tissue shows infiltration by macrophages,

hepatocyte

necrosis,

and macrophage accumulation.

Mitochondrial swelling has been observed along with a collapse of the nuclear membrane in hepatocytes ultimately leading to apoptosis. Both the Th1 and Th2 type of cytokine level increases, especially the IL-4 mediated pathways get activated [145]. 2.16. Zinc oxide nanoparticles Zinc oxide nanoparticles (ZnO NPs) fall under the category of most widely applied NPs in various industries nowadays. The antimicrobial property of ZnO NPs enables their use in sunscreens, cosmetics, coatings, caulks and adhesives. Some other applications of ZnO NPs include their use in electrical appliances, plastics, cement, ceramics, food, glass, rubber, pigments, sealants, toothpaste, textile, in the degradation and complete mineralization process of environmental pollutants due to its photocatalytic property [146]. ZnO NPs are being used in such a wide range of industrial and consumer products because of some special properties like transparency, high isoelectric point, biocompatibility and photocatalytic efficiency [147]. These NPs have been proven for their potential in the treatment of cancer and may be some autoimmune diseases due to their toxic properties against specific disease-causing cells [148]. Their other medical application focuses on the consideration for fabrication material of nerve guidance channels for curing nerve injuries. Due to their porous structure and high surface

area, they can be considered as good drug carriers and one such example is the loading of doxorubicin hydrochloride in mesoporous ZnO. Zinc oxide nanoparticles have been under study for a long time due to their huge potential in therapeutics, drug delivery, cancer treatment and a lot of other aspects of medicinal biology. Adverse effects of these particles are slowly coming to light due to extensive research being done on them. After entering inside the body, they are easy to access in various vital organs like heart, lungs, liver etc. and the severe damage caused by them. ZnO nanoparticles have shown cytotoxic effects on macrophages, an important component of the human immune system. These nanoparticles are inflammatory as they increase the release of various cytokines and chemokines. The toxicity of ZnO nanoparticles increases with a decrease in the size of the particles and increase in time of exposure [29]. One of the major reasons for their high level of damage is because ZnO nanoparticles lead to the release of ROS and increased expression of genes that lead to programmed cell death. One such case has been studied in human liver cells where the mitochondrial membrane potential gets reduced due to very high concentration of ROS. Hence, the cell cycle gets arrested at S or G2 phase. Also, the concentration of pro-apoptotic proteins increases as compared to antiapoptotic proteins and thus cell undergoes apoptosis [149]. It has been experimentally found out that positively charged ZnO nanoparticles are more detrimental as compared to negatively charged nanoparticles. The reason being positively charged ZnO nanoparticles interact effectively with negatively charged acidic acid present at the exterior of macrophages [150]. It has been experimentally validated that ZnO nanoparticle cytotoxicity is the size and dosage-dependent, the underlying fact being the release of Zn2+ ions. In the experiment carried out on a specific phagocytic cell line, it was found out that ZnO, when taken up by specific endosomal compartments, induced mitochondrial injury and pro-inflammatory cytokines. Accumulation of Zn2+, which came from ZnO nanoparticles supplied in the media,

in these compartments led to many adverse effects such as oxidative cell injury, intracellular release of Ca2+, depolarization of mitochondria, organellar clumping and ultimately cell death [151]. We noticed experimental evidence suggesting that ZnO nanoparticles are the reason behind pulmonary disorders. Not just ROS release, but these nanoparticles may stimulate the further release of Zn2+ from metallothioneins promoting further cell injury. These nanoparticles can deplete the ATP reserve of the cell by blocking essential glycolytic enzymes. Thus, it can be concluded that ZnO is responsible for the cytotoxicity by blocking various mitochondrial and lysosomal pathways. 3. Development of novel nanoparticle-based immunotherapeutics Nanoparticles (NPs) have shown an incredible potential to be used as cutting-edge delivery systems for malignancy immunotherapy. The past decade has witnessed the use of the nanosized carrier for vaccine antigen delivery. These carriers include liposome, nanosphere, and polymeric nanoparticle. These nano-carriers have the ability to independently work as an adjuvant and stabilizing the vaccine antigen. Nanomedicine-based frameworks have been shown to promote the professional antigen presenting cells mediated capture and presentation of tumor-associated antigens leading to a wide-ranging, precise and long-lasting immune response. One of the greater advancement of nanoparticles mediated immunotherapy has

shown in cancer. Nanoparticles have shown to improve the biodistribution of cancer drugs by increasing their circulation in bloodstream followed by cellular accumulation due to their enhanced permeability and retention property. The literature survey reveals that nanoparticles have been used as a vehicle for the coordinated delivery of tumor antigens to the professional antigen presenting cells mainly dendritic cells. A polymeric complex consisting of antigenloaded on PLGA-PEG-based nanoparticles has shown to deliver the breast cancer antigen to the dendritic cells followed by improved T-cell recognition [152]. The recent approach of direct Tcell targeting by nanoparticle coat packed with pMHC and co-stimulatory ligands emerges as an

attractive alternative approach. Presently, nanoparticle-based allergen delivery system has gotten much interest as potential adjuvants for allergen immunotherapy as a part of allergy vaccine. Magnetic nanoparticles also have shown a wide variety of application in the medical field. Magnetic particles are generally nontoxic, injectable and can accumulate in the cells easily. Magnetic particles have the ability to react with magnetic force and this application makes it suitable for use in drug targeting and bioseparation including cell sorting. 4. Conclusion Due to the unique physicochemical properties of nanoparticles, they have been utilized to a huge extent in many industrial and medical applications. Their ability to cause toxicity in living organisms marks the limitation to their use. Many types of research have been conducted to show the level of immunotoxicity at molecular and cellular level. Our review concludes that titanium dioxide is responsible for the highest level of toxicity at both immunological levels followed by silica, silver, carbon nanotubes and many other such nanoparticles. The major toxic effects at the immune level include reduced the viability of cell lineage involved in innate and adaptive immune responses, increased secretion of cytokines and chemokines, induced inflammatory responses and immunosuppression (Table1). However, there are several approaches were introduced to lessen the toxicity linked with the nanoparticle. Intralipid was used to diminish the toxicity accompanying by the Ptcontaining anti-cancer nano drugs in spleen, kidney, and liver and it leads to the enhanced bioavailability of the nano-drug. In US and Germany, researchers have introduced a ‘safe-bydesign’ approach to lessen the in vivo toxicity associated with ZnO nanoparticles by doping with iron. Metal nanoparticle coating with PEG is another way to stop protein adsorption on the nanoparticle surface and thus reducing the toxicity. In this review, we have updated the information related with nanoparticles and discussed the adverse changes caused by nanoparticles at the immunological level. This article highlights the toxicity being caused by

nanoparticles based applications in living organisms and opens a new door for further advancement and research in the same field to overcome the above limitations.

Conflict of interest: The Authors have declared no competing interest

Funding: University Grant Commission (UGC), grant No. 30-66/2014-BSR and Science and Engineering Research Board (SERB) grant No. YSS/2015/000716

Acknowledgement RKP is thankful to Department of Science and Technology for providing INSPIRE fellowship. VKP is thankful to UGC and SERB for providing start-up grant.

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Figure 1: Immunotoxic effects induced by nanoparticle. Interaction of nanoparticles with immune cells induces the reduction in viability of immune cells involved in innate and adaptive immune responses, increasing secretion of chemokines and cytokines favoring inflammatory response and immunosuppression

Table 1: Immunotoxicity shown by different nanoparticles

S. No. 1.

2.

Nanoparticl e Alumina

Cadmium

IC50

Size

µg/mL (cell line)

(nm)

866 (291.9-2596) 10-50 (THP1) [154] [153] ----------2-10 [156]

3.

Carbon black

4.

Carbon nanotubes

5.

Fullerene

Administration method

Tested organism

Peripheral

ICR female mice

[155]

[155]

Soluble form [157]

Marine mussel Mytilus galloprovincialis [157] ICR male mice

55.4 (THP1)

14

Intranasal

[153] 15.3 (NR8383)

[28] 0.3

[33] Intratracheal

[158] 1.74 (V79)

[159] 0.7

[160] Inhalation

[161]

[162]

[33] Male mice

and

female

B6C3F1/N mice and Wistar Han rats [162]

6.

Gold

110 (CHO22)

5-400

----

----

58.4 ( MØ J774), 50-70 24.7 (MØ [164] peritoneal)

Water exposure

Zebrafish

[165]

[165]

23.8±1.1 (HepG2), 10-150 18.75±2.1 (MCF-7), 12.5±1.7 (HeLa), [167] 6.4±2.3 (Jurkat) [166]

Intravenous

ICR male mice

[160]

[160]

1058 (374.8-2984) 5,7,18 (THP1) [168] [153]

Intravascular and CD1 mice [101], intraperitoneal C57BL/6 mice [101], [102], Oropharengeal Wistar rats [105]. [102],

[163] 7.

8.

9.

Graphene

Iron oxide

Nanoceria

Inhalation [105].

10.

Nickel

79.46 (33.33-189.4) 3-11 (THP1) [169] [153]

11.

Platinum

57 (A431), (MCF-7)

Water Solution

Male Wistar rats

65 5-7, 8-12 [171]

-

-

[170] 12.

13.

Silica

Silver

14.

Titanium dioxide

15.

Zinc oxide

a) 20nm silica: 20, 50, 80 118.2±7.3 [172] (PC12), 80.2±6.4 (HEK293) b) 50nm silica: 320.7±9.8 (PC12), 140.3±9.6 (HEK293) c) 80nm silica: 380.7±10.5 (PC12), 309.2±11.3 (HEK293) [172] 10.6 (HT-1080), 1-100 11.6 (A431) [172], [174] 19.33 (13.8-27.09) (THP1) [153] a) Stoichiometric: 3-710 432 (103.2[175] 1089) (THP1) b) Nonstoichiometric: 845.2 (233.73056) (THP1) [153] 21.94 (MEF)

20-45

Intraperitoneal

BALB/c mice

[173]

[173]

Intravenous

Wistar rats

[128]

[128]

Inhalation [176]

Dark [176]

agouti

Oral administration C57BL/6 mice [124] [124]

rat