Mohammad Zaki Ahmad1�†, Basel A. Abdel-Wahab2�3, Afroze Alam4, Sobiya Zafar5, Javed Ahmad6, Farhan Jalees Ahmad5, Patrick Midoux7, Chantal Pichon7, and Sohail Akhter7�8�∗�†

1Department of Pharmaceutics, College of Pharmacy, Najran University, 15 791, Kingdom of Saudi Arabia 2Department of Pharmacology, College of Medicine, Assiut University, Assiut, 71515, Egypt 3Department of Pharmacology, College of Pharmacy, Najran University, Najran, Saudi Arabia

4Faculty of Pharmaceutical Sciences, Shoolini University, Solan 173229, Himachal Pradesh, India 5Nanomedicine Research Lab, Faculty of Pharmacy, Jamia Hamdard 110062, New Delhi, India

6Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Raebareli 229010, Uttar Pradesh, India

7Nucleic Acids Transfer by Non Viral Methods, Centre de Biophysique Moléculaire, CNRS UPR4301, Orléans, France 8LE STUDIUM® Loire Valley Institute for Advanced Studies, Centre-Val de Loire Region, 45000, France

Nano-sized inorganic materials as inorganic nanoparticles (iNPs) are widely used for diagnostics, drug delivery and theranostics purposes at preclinical and clinical for various biomedical appli- cations intended for human health care. Per se, iNPs offer distinctive features compared to their organic counterparts while developing imaging agents, nano-drug carriers and theranostics in many aspects. Yet, transition from laboratory to clinic for iNPs restricted or slow by the concern of toxicities around them in vivo and in vitro applications. Researchers have shown that iNPs are potentially useful from a biomedical perspective but can also give rise to unexpected and haz- ardous toxicities to human health in short and/or in long term exposure. iNPs can potentially cause hostile effect on cells, genetic materials, tissues, organs and proteins due to their compositions, high energy state and other unique physiochemical properties at nano-scale. The Pharmacoki- netic/pharmacodynamics and interaction of iNPs with biological system are largely dependent on the physicochemical properties of iNPs, for instance size, shape, electric charge, chemical composi- tion, surface structure, solubility and aggregation behaviour. Herein we discussed in detail about the toxicity issues (in-vitro and in-vivo) associated with the exposure of iNPs in biomedical applications.

Keywords: Biomedical Nanotoxicology, Inorganic Nanotoxicity, Iron Oxide Nanoparticle, Graphene Nanotoxicity, Gold Nanoparticle, Upconverting Nanoparticles.

CONTENTS 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7873 2. Toxicity of Inorganic Nanoparticles . . . . . . . . . . . . . . . . . . . . . . 7877

2.1. Iron Oxide Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . 7877 2.2. Quantum Dots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7879

3. Gold Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7884 4. Upconverting Nanoparticle . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7885

4.1. Graphene Nanocomposites . . . . . . . . . . . . . . . . . . . . . . . . . 7886 4.2. Carbon Nanotubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7890 4.3. Silica Nanoparticles (SNPs) . . . . . . . . . . . . . . . . . . . . . . . . 7891

∗Author to whom correspondence should be addressed. †These two authors contributed equally to this work.

5. Mechanism of Nanotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7892 6. Reactive Oxygen Species and Free Radical . . . . . . . . . . . . . . . . 7892

6.1. Disruption of the Cytoskeleton Structure . . . . . . . . . . . . . . 7892 6.2. Genotoxicity and Alteration of

Signalling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7893 6.3. Inflammation Mediated Nanotoxicity . . . . . . . . . . . . . . . . . 7893

7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7894 References and Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7894

1. INTRODUCTION Nanoparticles (NPs) are a transitional state of matter some- where between bulk and molecular materials, as a result,

J. Nanosci. Nanotechnol. 2016, Vol. 16, No. 8 1533-4880/2016/16/7873/025 doi:10.1166/jnn.2016.13032 7873

Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application Ahmad et al.

Mohammad Zaki Ahmad is currently a Faculty in Department of Pharmaceutics, College of Pharmacy, Najran University, Saudi Arabia. He instructs pharmaceutics courses to Pharm.D. students. He did major in Pharmaceutics from Dibrugarh University Assam, India. Prior to joining academic, he worked as Junior Research Fellow under University Grant Commission. His research focused primarily in the area of novel drug delivery, drug targeting and nanoparticles as drug carrier.

Basel A. Abdel-Wahab is an associate professor of Basic and Cinical Pharmacology and Toxicology, College of Medicine, Assiut University, Assiut, Egypt. He is also working as faculty in Department of Pharmacology and Toxicology, College of Pharmacy, Najran University, Saudi Arabia. He obtained his Ph.D. in Pharmacology and Toxicology from Moscow Medical Academy (I.M. Schenove), Moscow, Russia. His research focused pri- marily in the area of neuropharmacology and Toxicology, Drug targeting and novel drug delivery systems for targeting drugs to the CNS.

Afroze Alam is specialized in Pharmaceutical Chemistry at M.Pharma level and did his Ph.D. in Pharmaceutical/Medicinal and Phytochemistry. He is working as assistant pro- fessor at Faculty of Pharmaceutical Sciences, Shoolini University, Solan, India. His main area of work is drug design and the natural product chemistry of anticancer, antibacterial, and anti-diabetic molecules. Moreover, his research interest include nanobiomedicines. He published more than 10 papers in internationally recognized high impact biomedical and chemistry journals.

Sobiya Zafar did her graduation (2012) and post-graduation (2014) in Pharmaceutical Sciences from Department of Pharmaceutics, Jamia Hamdard, New Delhi, India. Presently, she is working as a Ph.D. Research fellow at Nanomedicine research lab, Department of Pharmaceutics, Jamia Hamdard, New Delhi, India. Her research work has focus predom- inantly on the co-delivery of chemotherapeutic agents with herbal drugs through nanofor- mulation approach for the chemotherapy of solid tumors.

Javed Ahmad is currently working as a Pharmaceutics faculty at Department of Pharma- ceutics, National Institute of Pharmaceutical Education and Research (NIPER), Raebareli 229010, Uttar Pradesh, India. He is a professional Pharmacy graduate; did his M.Pharm and Ph.D. in Pharmaceutical Science (specilization_Pharmaceutics). In his Ph.D., he was awarded with senior fellowship from Council of Scientific and Industrial Research (CSIR), India. His research interests involve drug delivery and targeting, nanoparticulates and vesicular systems, nanotoxicology and pharmaceutical analysis. Dr. Javed has published more than 10 papers including book chapters in peer-reviewed journals of high impact.

7874 J. Nanosci. Nanotechnol. 16, 7873–7897, 2016

Ahmad et al. Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application

Farhan Jalees Ahmad is an Associate Professor at Faculty of Pharmacy, Hamdard Uni- versity, New Delhi, India and Director of Nanomedicine research Lab in the same insti- tution. His work interest is multi-disciplinary research focus on development of oral and parenteral controlled drug delivery system, drug targeting and novel nanotechnologies for medical applications. He received his Ph.D. in Pharmaceutical Sciences (Pharmaceu- tics) from Hamdard University. Currently, he is the President of Indian Pharmaceutical Association (Delhi) and also serving as the director of food technology program at Ham- dard University. After his Ph.D., He worked as Research Scientist in Ranbaxy Research Labs for 6 years before coming into the academic research. So far, Dr. Ahmad success- fully accomplished 28 projects from government and industry related to nano-therapeutics, bioavailable delivery of herbal drugs, product development, scale-up, technology transfer

and validation. He is credited with 2 US and 6 Indian patents. Moreover, Dr. Ahmad published 6 books and more than 200 research and review papers in peered reviewed journals. He is member of the editorial (advisory) board of a variety of scientific journals.

Patrick Midoux is research Director at INSERM, France. His research activities are cen- tered around gene and mRNA delivery with a special emphasis on the design and con- struction of synthetic vectors. He was among the pioneers for the use pH-sensitive peptide as well as histidine residues on peptide, cationic polymers and lipids to favor endosomal escape of plasmid DNA. The design and insertion NFKB motifs in plasmid DNA and discovery of a peptide mediating its migration on microtubules have greatly contributed to optimize intracellular trafficking of plasmid DNA. Patrick Midoux greatly contributes to the development of mRNA-base vaccines. Patrick Midoux has published 125 papers and 10 patents filed. He is co-founder of the Polytheragene Company.

Chantal Pichon is a full Professor in Molecular and Cell Biology at the University of Orléans. She is currently the head of the Institute of Life Sciences and Chemistry for Life at the University of Orléans. Chantal Pichon has completed a Ph.D. in Cellular Biology and Microbiology (1991) at the University of Aix-Marseille before spending 2 years at the AFRC (Cambridge, UK) as post-doc fellow. She has been appointed as an assistant professor in 1993 and full professor in 2005 at the University of Orléans. She is per- forming her research activities at the Center for Molecular Biophysics of CNRS (Orléans, France) where she is coordinating the department of Cell Biology and New therapeutic targets (4 teams) and co-leads the team “Nucleic acids transfer by non-viral methods” with Dr. Patrick Midoux (INSERM Research Director, Deputy Director of CBM). The main focus of the team is the development of chemical-based vectors for DNA, RNA (messen-

ger RNA, replicons) and siRNA. The team is the pioneer of histidine-based nanomedicines for cellular transfection and has developed novel strategies to improve uptake by chemical targeting and/or ultrasound trigger, the nuclear import and the cytosolic diffusion. In parallel with those investigations, the team is also involved in establishing specific cell tools and the identification of new targets for cancer and musculoskeletal tissue regeneration. Chantal Pichon has a track-record of 107 peer-reviewed publications and 20 grants including regional grants (ARD2020, APR), academic funding (ANR), european grants (integrated project, REGPOT) and private companies funding.

Sohail Akhter is currently working as Le Studium research fellow for CBM-Centre de Biophysique Moléculaire (CBM)-CNRS/University of Orléans, UPR4301, Orléans, France. Presently, Dr. Akhtar’s work has focused on the development and optimization of non- viral nano-carriers (for example-lipidic and polymeric nanoparticles) for selective intra- cellular of mRNA intended for therapeutic vaccination. Earlier, Dr. Akhter was working as Senior research fellow at U.S. Food and Drug Administration (US-FDA)/The Center for Drug Evaluation and Research (CDER)/DPQR, USA and had also served as research associate at the Department Pharmaceutics, Utrecht Institute of pharmaceutical sciences, Utrecht University, Netherlands. He received team excellence award-2015; U.S. Food and Drug Administration (US-FDA)/CDER/DPQR for the research on novel non-destructive chemometric method and PAT tools. In the year 2013, he was awarded by European

foundation for clinical nanomedicine (CLINAM) and Nanomedicine European technology platform fellowship-2013 for the lecture in The European Summit for Clinical Nanomedicine, 2013 (6th CLINAM 2013) at Basel, Switzerland.

J. Nanosci. Nanotechnol. 16, 7873–7897, 2016 7875

Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application Ahmad et al.

Dr. Akhter is a professional Pharmacy graduate; did his M.Pharm and Ph.D. in Pharmaceutical Science (speciliza- tion_Pharmaceutics_Nanomedicine). In his Ph.D., he was awarded with senior fellowships from Council of Scientific and Industrial Research (CSIR), Department of Biotechnology (DBT) and University Grant Commission (UGC) of GOVT. India. His research interests involve application of bio-materials in drug delivery and targeting, development and charac- terization of nanoparticulates and vesicular systems, biopharmaceutics, nanotoxicology and bioanalysis. Dr. Akhter has published more than 45 papers including book chapters in peer-reviewed journals of high impact. He is professionally associated with many pharmaceutical and biomedical associations. He is member of the editorial (advisory) board of a variety of scientific journals.

NPs have a large surface area to volume ratio i.e., relatively large functional area, which leads to an amendment in bio- logical activity compared to the parent bulk materials.1�2

In the last two decades, the use of Inorganic nanoparticles (iNPs) in investigational and clinical settings has levitated exponentially due to their extensive range of biomedical applications, targeted drug delivery, cell imaging and cell tracking.3 Due to the inimitable nanocharacteristics and exceptional physicochemical, electrical, mechanical, optical and thermal properties, NPs are unified into all provinces of our life through their applications.3 NPs pro- vide talented multifunctional platforms that suffice the potential necessities of medical diagnosis and therapy. With the fast build-up of progressive knowledge on the distin- guishing properties and exceptional utilities of nanomate- rials, application of NPs in the field of medical has shown inordinate auspicious possibilities of Nanomedicine (NM) as theranostic and diagnostic agent. Nowadays NM is a gratifying hotspot in the investigational field of medicines. The current biomedical application of NPs comprises the nanoparticle-targeted drug delivery, biomedical imaging and as a theranostic agent.3–5 The altered physicochemi- cal properties of iNPs play critical roles in the outstanding performance of NM.6–9 For example (i) nanosizing of drug or formulating of drug molecules as NPs leads to staged changes in physiological properties as compared to their bulk or micronized form.9

(ii) NPs straightforwardly enter into the tissue and cells, permitting the researchers to perceive the histopathological and molecular state throughout the disease treatment or diagnosis with NM.10

(iii) Another important aspect of NPs is their large sur- face area to volume ratio which makes the NPs appropriate for various surface modifications to achieve long circu- lation time, better biocompatibility and desired targeting potential.11

(iv) Multifunctionality of iNPs at one-platform is the key to realize the vision of multi-mode imaging, theranostic and personalized therapy.8–10

Although iNPs holds novel therapeutic functions with high efficacy but their toxicity apprehension is an essential issue in biomedical applications. In this context, nanotox- icity refers to the knack of the particles at nano-scale to adversely interfere with the normal tissues or cellular structures and their development along with the normal body physiology.2�11 Over the past decade, iNPs have

extensively been explored in R&D as diagnostics, ther- anostics and drug carriers in various diseases including different form of cancers. This growing trend of apply- ing iNPs in animal and clinical studies raised the concern of having exposure of such active materials. Therefore, incidence of subsequent iNPs interactions necessitate a more profound knowledge of iNPs’ effects on cells.12 The toxic effect of iNPs interactions with biological systems can be jaggedly alienated into two groups. The first group includes the intense delivery of chemical agents recog- nized to be toxic. The second group involves the noxious effects of comparatively benevolent materials derived from the size-related disruption of biological structures.13 Fur- ther, particle-related issues, the administered dose, route of administration and extent of tissue distribution appear important parameters in iNPs toxicity. After the adminis- tration of iNPs in the body for biomedical applications, via any route, interactions between iNPs and the biolog- ical systems (such as proteins, cells, tissues and organs) are unavoidable till the iNPs reached to its effective sites. The distribution of the administrated iNPs in human body and associated toxicity in particular tissue/organ has been illustrated in Figure 1. Inside the systemic circulation iNPs interact with various serum proteins and form a protein layer on the surface of iNPs called as protein corona.14

This protein corona can influence the biocompatibility and therapeutic efficacy of iNPs.15 With the bloodstream, iNPs are subjected to bio-distribution depending on the physio- logical properties of iNPs and anatomical feature of vas- culature in organ and tissue.16 In some organ and tissue (e.g., Spleen and Liver), iNPs undergoes size-dependent tissue-specific extravasation.16 Subsequently, these iNPS are co-opted into cells and execute their intended in vivo activity (diagnostic and/or therapeutic). After the biodis- tribution, iNPs undergoes the metabolic processes by cytochrome P4509�17 and after that iNPs can be excreted from body via kidney or faeces.10 The pharmacokinetics of the administrated iNPs and their toxicity that might ascend has been illustrated in Figure 2. Nevertheless in some cases, parts of the iNPs are retained in the body due to the incomplete excretion and consequently they may interfere with normal functions of the tissue and organs and induce nanotoxicity inside the body (Table I). Here, in this review we extensively discussed the in-vitro and in-vivo toxicity of inorganic nanoparticles which are widely investigated in various biomedical application.

7876 J. Nanosci. Nanotechnol. 16, 7873–7897, 2016

Ahmad et al. Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application

Figure 1. Different route of iNPs exposure to the body and vital organs that may get affected with different conditions from broad biodistribution of iNPs.

2. TOXICITY OF INORGANIC NANOPARTICLES

2.1. Iron Oxide Nanoparticles In the presence of moist air iron (Fe) reacts with atmo- spheric oxygen (O2� to form different types of iron oxide (e.g., Magnetite, Haematite, Wustite, Maghemite).

Among all these, Magnetite (Fe3O4� nanoparticles have been extensively investigated for many years in hopes of using them in biomedical research.3�18 Owing to decent biocompatibility and enhanced investigative performance, magnetic iron oxide nanoparticles (mIONPs) have been established as a protuberant magnetic resonance imaging

J. Nanosci. Nanotechnol. 16, 7873–7897, 2016 7877

Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application Ahmad et al.

Figure 2. In-vivo fate as ADME (Absorption, Distribution, Metabolism and Elimination) of iNPs; Pharmacokinetics and possible in-vivo nanotoxicity of inorganic nanomaterials at each metabolic steps.

(MRI) agent.9 Superparamagnetic iron oxide nanoparti- cles (SPIONs) consist of an iron oxide core coated with carbohydrate; SPIONs are usually less toxic and hence- forth proposed as an alternative MRI contrast agent.2�9

Till now about 10–15 iron oxide nanoparticles have been approved or are at the different clinical phase as a contrast agent (e.g., GastroMark®, Ferumoxsi, Feridex®, Resovist®, Combidex®, Feraheme® etc.).9 SPIONs have also found wide application in research and development as nanomedicine in many therapy as drug carrier, ther- anostics, diagnostics.19 Considering its more exposure to the human and the environment due to increased produc- tion and increased used, significant number of studies have been carried out to investigate its safely, biodistribution and pharmacokinetics. Biodistribution study of SPIONS (Ferumoxtran-10®) with five different animal models (Monkey, Dog, Rabbit, Rat and Mice) shows that SPIONS were taken up by liver, spleen and lymph node within 24 hr and experienced progressive metabolism. More- over, dose dependent toxicity was observed with repeated injection.20 Further in an important clinical trial (Phase III), 152 patients were injected with Ferumoxtran-10�R�

for the diagnosis of lymphatic metastasis. Ferumoxtran- 10�R� demonstrated reasonable performances with rare occurrence of common side effect (Urticaria, Vomit- ing, Headache, Back pain, and Vasodilatation etc.).21

Under certain circumstances, SPIONS may get exposed to biological system and result in undesired side effect due to degradation of surface coating of particles.21 The intra- tracheally implanted Fe2O3 nanoparticles goes to systemic circulation after crossing the alveolar-capillary barrier and selectively taken up by spleen, kidney and testicles. Plasma t1/2 of Fe2O3 nanoparticles was found to be 22.8 days and lung clearance was 3.06 �g/day.22 Thus systemic toxic- ity may also arise from long term exposure of inhaled Fe2O3 nanoparticles. Size dependent toxicity of Fe2O3 nanoparticles was observed in male Sprague Dawley rats. More severe lung injury related to oxidative stress was caused due to nanosized particles than submicron sized particles as well as lung fibrosis was triggered by Fe2O3 nanoparticles phagocytised with alveolar macrophages.22

Furthermore, Fe2O3 nanoparticles delay the coagulation process by prolonging the prothrombin time and acti- vated partial thromboplastin time.22 Similar pulmonary

7878 J. Nanosci. Nanotechnol. 16, 7873–7897, 2016

Ahmad et al. Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application

fibrosis was observed in adult male Wistar rats after intratracheally administering mIONPs at a single dose of 5 mg/kg.23 After a single i.v injection of �-Fe2O3 nanopar- ticles to rats, 75% of administered �-Fe2O3 nanoparticles were excreted via urine within 72 h. However, remain- ing �-Fe2O3 nanoparticles (25%) causes inflammation in the vital organ of the body (kidney, lungs, and liver).24

Similarly, Feng et al. (2011) reported that injected Fe2O3 nanoparticle induces metabolic abnormalities in spleen and kidney of rats.25 Some investigators reported that mIONPs can cross the blood-brain barrier (BBB) and may cause CNS toxicity.9 Wang et al. (2007) studied the neurological toxicity of Fe2O3 nanoparticles in rat. Intranasally admin- istered mIONPs can reach to brain via olfactory path- way and causes fatty degeneration of neuron in CA3 area of hippocampus.26 mIONPs were mostly deposited in the striatum, hippocampus and olfactory bulb region of the brain27 and induces size-dependent oxidative stress-related nerve cell damages.28 Irregular arrangement of neuronal cell, cellular swelling and damage of neuronal integrity was also observed in the brain of exposed rat.27 mIONPs with different surface coatings shows a different toxic consequence on primary cortical neurons.9�29 For exam- ple, mIONPs coated with polydimethylamine impaired the integrity of cell membrane and potentiated cellular death while mIONPs coated with aminosilane showed lower toxicity at higher concentration and lonely affected the metabolic activity only.9�29 Similarly, mIONPs coated with dextran partially altered the viability of cell higher concentrations.29 These finding suggest that mIONPs can bypass the BBB and induces neurotoxicity in brain.

mIONPs can penetrate the placental barrier and induces the reproductive and developmental toxicity in pregnant mice.30 Associated with the reproductive and develop- mental toxicity of iNPs, the expected genotoxicity of mIONPs are highly concerned. At higher concentration (50 �g/mL) mIONPs induces significant genotoxic effects in human bronchial epithelial cells BEAS-2B.31 Consistent with above report, similar reproductive and developmental toxicity in zebrafish was reported by Ref. [32]. Exposure of zebrafish with mIONPs results in mortality, hatching delay and malformation.32 Similarly, positively charged SPIONs showed dose-dependent genotoxicity in L-929 fibroblast.33 Incubation of human aortic endothelial cells (HAECs) with mIONPs results in localization of NP into cells. This localization of mIONPs results in mitochondrial disruption, cytoplasmic vacuolation and cell death. Fur- thermore, they upsurge the expression and secretion of the inflammatory factors ICAM-1 and IL-8.19 Consistent with the above results, internalization of mNIOP with cell line PC12 results in reduced cell viability and nerve growth factor in dose dependent manner.34 However, it may be noted that coating of mIONPs with dextran or PVA renders the mIONPs almost non-toxic, though they were mostly absorbed by the secluded brain-derived endothelial and

microglial cells.35 Thus, it may be concluded that neuro- toxicity associated with mIONPs me be linked with surface modification of NPs. Redox state of iron in NPs also influences the toxic-

ity of particles. For example, redox state of iron in NPs intensely modifies the cellular uptake of mIONPs and ini- tiates the oxidative DNA damage.36 Similarly, no toxicity of �-Fe2O3 NPs were observed in Escherichia coli while ferrous containing NPs were extremely cytotoxic.37 It may be due to production of reactive oxygen species (ROS) by interaction of oxygen with Fe+2.9�37 Further it was also reported that, nanoparticles based on Fe3O4 and FeO (zero valent iron) are easily oxidized in physiological or biologi- cal media due to their high sensitivity towards oxidation.38

2.2. Quantum Dots Quantum dots (QDs) are semiconductor nanocrystals.39�40

First generation QDs include CdSe, CdS, CdTe and PbS.41

Initially, QDs have been explored as fluorescent mark- ers to study the complexity of biological interactions at the cellular level, in organisms, such as, bacteria, ani- mals, plants and humans. QDs offer good quality con- trast imaging with high sensitivity even for the deeper tissues. Moreover, being used as tissue-specific vascu- lar marker, blood vessels and lymph nodes imaging are also possible with QDS.41�42 In recent time, its thera- nostic potential has been extensively investigated for the complex disease therapy including cancer. First-generation QD-based drug delivery has innate toxicity due to release of Cd and lead. This noxiousness of first generation QDs leads to the development of second generation QDs (InAs/InP/ZnSe), which are free from Cd.40 Solubility of QDs was enhanced by the addition of organic coating (polyethylene glycol, 12-pentacosadiynoic acid). Further, targeting efficiency of QDs was enhanced by conjugat- ing the biomolecules with surface coated QDs.2 Owing to high biocompatibility, long t1/2 and better photochemical firmness, QDs are extensively used in biomedical field of diagnosis and treatment.9�43 However, little information is available regarding the disposition and biodistribution of QDs and their consequences on health of human as well as animal. The toxicity of cadmium (Cd) and selenium (Se) are well known.44 Therefore, this propinquity of QDs and interaction with physiological system demands the toxico- logical consideration of QDs. Toxicity of QDs most likely arises due to liberation of

metal ions from the core–shell.44 For example, cytotoxic effect of CD–Se QDs was reported by Ref. [45] due to surface oxidation and release of free Cd2+. Exposure to air intensified the toxicity of Cd–Se due to augmented oxida- tion and fast release of free Cd2+.44 Similarly Karabanovas et al. reported the liberation and toxicity of Cd+2 from orally administered Cd–Se QDs.46

The kidney and liver are the main target organs for QDs toxicity because of the accumulation of QDs within these

J. Nanosci. Nanotechnol. 16, 7873–7897, 2016 7879

Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application Ahmad et al.

Table I. Toxicity induced by inorganic nanoparticles (iNPs) in research and development—in-vitro studies.

Concentration Exposure Nanoparticles Coated/uncoated of iNPs time Cell line Major outcomes Reference

Iron oxide nanoparticles

—Functionalized with 3,4-dihydroxy- phenylethylamine hydrochloride

1, 10, 100 �g/ml 72 h —Immortalized human umbilical vascular endothelial cells (HUVEC, Ea.hy 926 clone

—Cell death within 24 hours of exposure through oxidative stress

[1]

0.02, 0.2, 2 mol Fe/L

—BV2 microglial cells —Induction of cells proliferation, phagocytosis and generation of ROS and NO

[2]

—Polydimethylamine —Aminosilane —-Dextran

1, 5, 10% v/v of iNPs solution

24 h —Neuronal cultures —Polydimethylamine functionalized nanoparticles induce cell death at all concentrations

—Aminosilane coated particles affected metabolic activity only at higher concentrations

—Dextran-coated nanoparticles partially altered viability at higher concentrations

[3]

10, 25, 50, 250 �g/ml

24 h —SV40-transformed BEAS-2B

—Human lung fibroblasts (IMR-90)

—Cyto- and genotoxicity—Intracellular generation of reactive oxygen

—Cytotoxicity and DNA damage

[4]

– Zebra fish culture (Daniorerio)

—Developmental toxicity in these embryos, causing mortality, hatching delay, and malformation

[5]

—Coated with tetraethyl Orthosilicate (TEOS), (3-aminopropyl) trimethoxysilane (APTMS), TEOS-APTMS, or citrate

100–1000 ppm 24 h —L-929 fibroblasts —Affect the cell viability and DNA stability of L-929 fibroblastic cells in a dose-dependent manner

[6]

2, 20, 100 �g/ml 6, 12, 24 h

—HAECs —U937 cells

—A significant increase in nitric oxide (NO) production

—Endothelial dysfunction

[7]

0.15, 1.5, 15 mM 2, 4, 6 days

—PC12 cells —Dose-dependent diminishing viability and capacity of cells to extend neurites in response to their putative biological cue, i.e., nerve growth factor

[8]

Polyvinyl alcohols (PVAs) (PVA-SPIONs)

11.3 �g Fe/ml —Brain-derived endothelial EC2

—Murine N9 —N11 microglial

—No inflammatory activation [9]

Dextran 1–100 �g/ml 24 h Human lymphoblastoid cell line (MCL-5)

—Oxidative DNA damage [10]

7–700 mg/L 0.33 h —Escherichia coli —Oxidative stress [11]

—Albumin �-Fe2O3-3.4 mg; X/XO-5 Mm/ 2.1 mu/mL

—Xanthine/Xanthine Oxidase

System

—Production of highly reactive hydroxyl radicals

[12]

—L-glutamic acid (Glu) 0, 8, 16, 32, 64, 128, 240, 400, 480, 560, 640, 800 �g/ml

36 h —Chinese Hamster Lung (CHL) cells

—Production of ROS. Reactive —Depletion of glutathione

depletion and inactivation of some antioxidant enzymes (glutathione reductase, superoxide dismutase)

[13]

7880 J. Nanosci. Nanotechnol. 16, 7873–7897, 2016

Ahmad et al. Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application

Table I. Continued.

Concentration Exposure Nanoparticles Coated/uncoated of iNPs time Cell line Major outcomes Reference

2, 20, 100 �g/ml

6, 12, 24 h

—Human umbilical endothelial cells (ECV304 cells)

—The loss of mitochondria membrane potential

—The apoptotic chromatin condensation in the nucleus

[14]

—Citric acid —Dextran

0.1–20 mM 1, 6, 24 h

—Human umbilical vein endothelial cells (HUVECs)

—Disruption of cytoskeletal structure —Disorganized actin fibre and tubulin

networks

[15]

– 0–1 mg/ml —Porcine aortic endothelial cells

—Disruption of actin cytoskeleton —Altered endothelial cell morphology

and mechanics

[16]

QDs PEG-coated silanized QDs

8 or 80 nM 48 h —Lung fibroblasts (IMR-90)

—Skin fibroblasts (HSF-42)

—Down-regulation of genes involved in controlling the M-phase progression of mitosis, spindle formation, and cytokinesis

–

[17]

—CdSe-core 0.0625, 0.25, 1 mg/ml

—Primary hepatocytes

—Liberation of free Cd2+ ions due to deterioration of the CdSe lattice

—Acutely toxic to cell

[18]

—Coated individually with polyethylene glycol, amines, and carboxylic

Acids

5, 10, 20 nM 24, 48 h

—Mouse —Bovine corneal

stromal cells

—Damage to corneal epithelium barrier —Influence the viability of corneal

stromal cell

[19]

—Naked QDs 1, 5, 10 �g/ml 24 h —Human breast cancer cells (MCF-7)

—Damage to the plasma membrane, mitochondrion, and nucleus

[20]

—Mercaptosuccinic acid (MSA)-capped CdTe QDs

0.1–100 �g/ml 24 h —Human umbilical vein ECs (HUVECs)

—Oxidative stress —Mitochondrial network fragmentation —Disruption of mitochondrial

membrane potential —Endothelial toxicity and induction of

endothelial apoptosis

[21]

—Human breast cancer cells (MCF-7)

—Activation and translocation of p53 with subsequent upregulation of downstream targets Puma and Noxa

—Decreased cell viability

[22]

—Albumin conjugates 0.01–100 �g/ml 24 h —Rat pheochromocytoma cells (PC12)

—Size dependent cytotoxicity —Chromatin condensation and

membrane blebbing

[23]

Gold nanoparticle

24 h —CFP-LC3 and Lamp1-RFP stable cell lines

—Accumulated in lysosomes Impairment of lysosome degradation

capacity —Blockade of autophagy flux

[24]

—Triphenylphosphine —Connective tissue fibroblasts, epithelial cells, macrophages, and melanoma cells line

—Size dependent cellular toxicity [25]

1 nM 72 h —MRC-5 human lung fibroblasts

—Upregulation of autophagy proteins, microtubule-associated protein 1 light chain 3 (MAP-LC3) and autophagy gene 7 (ATG 7)

—Oxidative stress and lipid peroxidation

[26]

0.1, 0.5, 1 nM 24, 48, 72 h

—MRC-5 human foetal lung fibroblast cell line

—DNA damage and down regulation of DNA repair genes

[27]

J. Nanosci. Nanotechnol. 16, 7873–7897, 2016 7881

Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application Ahmad et al.

Table I. Continued.

Concentration Exposure Nanoparticles Coated/uncoated of iNPs time Cell line Major outcomes Reference

13 nm- 0–189 �g/ml

45 nm- 0–26 �g/ml

3, 6 days —Human dermal fibroblasts

—Diminished expression of extracellular matrix (ECM) proteins, collagen and fibronectin

—Decreased decreases in motility of cell

—Disruption of cytoskeleton

[28]

0.5 mM 4, 24, 48 h

—Dendritic cells —Perturbation of the immune response

—Altered secretion of cytokines

[29]

—Positive, neutral and negatively charged

0, 10, 25, 50, 100 �g/ml

24 h —Human keratinocyte cell line (HaCaT)

—Cell morphology was disrupted by all three NPs

—Increased nuclear localization of p53 and caspase-3 expression with charged GNPs

[30]

Upconverting nanoparticle

1–5000 �g/ml 24 h —HeLa cell —Caenorhabditis elegans

—Very low toxic [31]

Graphene nanoparticles

Pharyngeal aspiration 1, 5, 10 �g/cm2 24 h —THP-1 cells —Expression of IL-1� [32]

—Functionalized with Polyethylene glycol (PEG), polyethylene glycol-polypropylene glycol-polyethylene glycol (Pluronic P123), and sodium deoxycholate (DOC)

3.125, 6.25, 12.5, 25.0, 50.0, 100.0 �g/ml

48 h —Mice fibroblast cells (line L929)

—Toxicity depends on the type of dispersant and concentration of the nanomaterials in the suspensions

[33]

—Pristine graphene —Carboxyl functionalized hydrophilic graphene

0–300 �g/ml 24 h —Vero cells —Pristine graphene accumulates on cell membrane causing high oxidative stress leading to apoptosis

—Carboxyl functionalized internalized by the cells without causing any toxicity

[34]

5–100 �g/ml 1–5 days —Human fibroblast cells —Dose dependent toxicity —Chronic toxicity under high dose —Higher dose leads to conglomeration of airways and death due to suffocation

—Lung granuloma formation —Decreased cell adhesion, inducing cell apoptosis, entering into lysosomes, mitochondrion, endoplasm, and cell nucleus

[35]

20, 100 �g/ml 4, 6 h —A549 (adenocarcinomic human alveolar basal epithelial)

—Concentration-dependent cytotoxicity

—Physical damage to the cell membrane

[36]

Carbon nanotubes

SWCNTs 50 �g/ml 24, 48, 72, 96 h

—A549 cells —Strong cytotoxic effect within the MTT assay

[37]

−OH functionalized and pristine MWCNTs

1–40 �g/ml 2, 4, 24 h —A549 cells —Earlier genotoxic effect and direct DNA damage

—Induction of Apoptosis –

[38]

MWCNTs with different concentration of iron impurities

5, 10, 30, 60 �g/ml

24, 48, 72 h

—Rat pheochromocytoma (PC12)

—Reduced cell viability and increased cytoskeletal disruption

—Diminish the ability to form mature neurites

—Adversely influence the neuronal dopaminergic phenotype

[39]

7882 J. Nanosci. Nanotechnol. 16, 7873–7897, 2016

Ahmad et al. Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application

Table I. Continued.

Concentration Exposure Nanoparticles Coated/uncoated of iNPs time Cell line Major outcomes Reference

—SWNTs —MWCNTs

SWNTs- 0–226 �g/cm2

MWCNTs- 0–22.60 �g/cm2

6 h —Alveolar macrophage —Necrosis and degeneration of macrophages

[40]

—SWCNTs 2, 4, 8 �g/ml 48 h —Human bronchial epithelial cells

—Human fibroblasts

Increased NO production —Decreased cell viability

[41]

Silica nanoparticles

50, 100, 200, 200 �g/ml

48 h —Human lung epithelial cells (A549 cells)

—Dose-dependent cytotoxicity —Generation of ROS, and membrane lipid peroxidation

[42]

—Plain (unlabeled) and FITC-labeled

25 �g/ml of 0.07, 0.5, 5 �m particles

24, 48 h —HEp-2 cells (human epithelial) and RPMI 2650 cells (epithelial, human nasal septum

—Nucleoplasmic protein aggregation and impaired nuclear function

[43]

—Fish cell lines —Size-, time-, temperature-, and dose-dependent as well as tissue-specific toxicity

[44]

24 h —EAHY926 cell line —Cytotoxic damage —Release of lactate dehydrogenase —Decrease in cell survival

[45]

0.1–1000 �g/ml 24 h —Mouse epidermal Langerhans cell line XS52

—Size dependent cytotoxicity [46]

Amine modified 100, 259, 500 �g/ml 24 h —A549 cells —RAW 264.7

—Surface charge dependent toxicity —Damage to plasma membrane —Haemolytic activity

[47]

25, 75, 100, 200, 400, 500 �g/ml

4 h —HaCaT cells —Decreased cell viability —Damaged cell membrane —Depletion of glutathione —Generation of ROS

[48]

50, 100, 200 �g/ml 20 h —Human bronchial epithelial cell line (BEAS-2B)

—Increased phosphorylation of NF-�B p65 and MAP-kinases p38 and JUN-N-terminal protein kinase pathways (JNK)

—Increases in membrane-bound transforming growth factor-� (TGF-�) release and EGFR phosphorylation

[49]

—Human keratinocyte cell line (HaCaT)

—Increased production of IL-6 —Induction of skin inflammatory disease

[50]

2.5–60 �g/ml 12, 24, 48 h

—Human SK-N-SH —Mouse neuro2a (N2a) neuroblastoma cells

—Impaired morphology of cell —Diminished of dendrite-like process

—Decreased the cell viability —Induced cellular apoptosis —Increased deposit of intracellular �-amyloid 1-42 (A�(1-42)) and enhanced phosphorylation of tau at Ser262 and Ser396, two specific pathological hallmarks of Alzheimer’s disease

[51]

25–200 �g/ml 4–96 h —Embryo of zebra fish —Decreased hatching rate of zebrafish embryos

—Increased mortality and cell death —Embryonic malformations, including pericardial edema, yolk sac edema, tail and head malformation

[52]

J. Nanosci. Nanotechnol. 16, 7873–7897, 2016 7883

Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application Ahmad et al.

vital organs.9�47 For example, biodistribution study of sur- face coated QDs reveals that 8.6% of the infused dose of agglomerated QDs persists in hepatic tissue and it was major entity inducing the toxic effects in vivo.10 Similarly consistent with the above report48 reported that intravenous administration of Cd–Se QDs results in accumulation of NPs in liver and spleen of mice.48 Intraperitoneal injection of Captopril-conjugated QDs in mice results in the accu- mulation of QDs inside the kidney, spleen, liver and brain where toxicity of QDs may arise.49 After two years post injection of QDs injected into Balb/c and nude mice, flu- orescence of QDs was still observed in the spleen, lymph nodes and liver.50 This finding suggests that complete elim- ination of QDs from the body may be considerably more problematic than expected.50 Sadaf et al. (2012) reported the significant renal injury of mice after the intravenous administration of QDs. A noticeable rise in the blood urea, nitrogen and creatinine concentration in mice indi- cates the risk of QDs in nephrotoxicity.51 After 48 h of intraperitoneal injection in mice, Cd–Se QDs shows exten- sive biodistribution in many organs with the maximum in the liver. The retention of QDs in the liver induces hepatic damage supplemented with modifications to hepatic lob- ules and the generation of reactive ROS.52 QDs can cross the placental barrier and induces toxicity in the fetus. For example, Cd–S/Cd–Te QDs toxicity was reported in preg- nant mice and fetus due to transfer of QDs from mother to fetus.53 Toxicity of hematological system was observed after the intravenous injection of QDs coated with carboxyl group and amine group in mice. The injected QDs were capable to activate the coagulation cascade and induce pul- monary vascular thrombosis.54 Moreover, exposure of QDs (Cd–Se) to the eyes leads to corneal damage.55

For better understanding of in-vivo nanotoxicity, it is important to study the in-vitro toxicity of QDs. 24 h expo- sure of MCF-7 cells with Cd–Te QDs resulted in dose dependent chromatin aggregation, shrinkage and deformed nuclei.56 Consistent with the above report, exposure of human umbilical vein endothelial cells with Cd–Te QDs also leads to dose dependent reduction in cell viability.57

Similarly, incubation of bovine corneal stromal cells with Cd–Se/Zn–S QDs results in declined cell viability by 50% in 48 h.55 Potential genotoxicity of Cd–Te QDs was reported in human breast cancer cells (MCF-7).58 Treat- ment of MCF-7 with QDs induces global hypoacetyla- tion with alteration in DNA helix, implying a global epigenomic response.58 After the incubation of N9 murine microglial cell line, the larger Cd–Te was uniformly dis- tributed in the cytoplasm whereas smaller Cd–Te was exclusively taken up by nuclear compartment of N9 cells.59

Further it was observed that smaller size QDs severely damage the chromosomal network.59 Likewise, Nabiev et al. (2007) reported the size-dependent nuclear toxicity of QDs in human macrophages.60 Small sized QDs actively distributed inside the nucleus and damaged nucleoli and

nuclear histones.60 Qu et al. (2011) reported the long- term toxicity of QDs on reproduction of Caenorhabditis elegans.61 Similarly, Hoshino et al. (2004) reported the specific localization of mercaptoundecanoic acid coated QDs in the nucleus and subsequent mutilation of DNA.62

3. GOLD NANOPARTICLES Due to enhanced surface area at the nano scale with gold nanoparticles (GNPs), the light absorption and scattering efficiency of GNPs may increase up to 4 to 5 times as compared to their macro counterpart. This feature makes them particularly attractive tools for biological imaging, detection, theranostics, and drug therapy.4�5�63 Further- more, GNPs in specific shapes (e.g., rods, stars, cages and nano-shells) display localized surface plasmon reso- nant (SPR) property that can cause hyperthermia and it strongly favour their applicability in oncology.4�5 Once GNPs are sufficiently accrued at the site of target, it may result in irreversible localized thermal cellular destruction and imaging by the irradiation of the light of an appropri- ate wavelength (NIR range) due to SPR.4�5 These outstand- ing properties of GNPs make them exceptional theranostic agents for diagnosis and treatment of tumour in deeper tissues.4�5

As a chemical, gold is noble metal and chemically inert, thus GNPs were expected to be nontoxic at cellular level.12

Nevertheless, GNPs display tremendously altered proper- ties with respect to gold compound, so it is essential to consider the safety/toxicity issue of gold nanovectors at molecular as well as vital organ level.4�5 For example, at nanosize (1–4 nm) GNPs can penetrate the nuclear mem- brane and bind with DNA.12 Since the electronegativity of gold is 2.54, it can be easily attracted by DNA grooves and may lead to potential genotoxicity.12 Therefore, it is nec- essary to consider the genotoxicity and cytotoxic profile of GNPs. Study conducted by Jong et al. (2008) revealed that after 24 hr of injection, GNPs with the size range of 1 nm–10 nm shows extensive tissue distribution in brain, liver, spleen, blood, kidney, heart, testis, lungs, and thymus whereas the larger GNPs (50 nm–100 nm) were only con- fined in the liver, spleen and blood.64 Chen et al. (2009) reported the size dependent toxicity and lethality of GNPs in mice. GNPs with the size range 50 to 100 nm did not showed any significant toxicity, however GNPs ranging from 1 nm to 37 nm induced severe toxicity.65 Histopatho- logical examination of major organ indicates the damaged integrity of lungs, increase of Kupffer cells in the liver and diffusion of white pulp in the liver.65 Surface modification of GNPs with immunogenic peptides results in augmented toxicity of GNPs.65 Cho et al. (2009) reported that single intravenous injection of PEGylated GNPs in mice induced acute inflammation and apoptosis in the liver.66 Further- more, it was also found that PEG coated GNPs accumu- lated inside the macrophages of spleen and Kupffer cells of liver.66 Intranasal administration of GNPs in rats results in

7884 J. Nanosci. Nanotechnol. 16, 7873–7897, 2016

Ahmad et al. Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application

accumulation of NPs inside the olfactory bulb and translo- cation of GNPs from lungs to other vital organ. Accu- mulated GNPs down regulates the expression of genes and decreases the phosphatidylserine 36:1 species in the lungs.67 Similarly, single intravenous injection of GNPs in rats results in accumulation of GNPs in spleen and liver.68 Accumulation of GNPs in liver results in altered in-vivo expression of gene related to lipid metabolism and detoxification of xenobiotic and cell cycle.68 Size depen- dent biodistribution of citrate-coated GNPs was reported by Sonavane et al.69 Upon intravenous administration of citrate-coated GNPs of various sizes (15, 50, 100 and 200 nm) in mice showed that maximum amount of admin- istered dose in liver, lung, spleen, kidney, brain, heart and stomach was found for 15 nm citrate coated GNPs.69

Similarly Hilyer et al. (2001) reported the size dependent biodistribution of smaller GNPs (4, 10, 28, 58 nm) in mice. After oral administration, the smallest GNPs (4 nm) were found in the brain, lung, spleen and kidney, while the largest (58 nm) GNPs were confined only in gas- trointestinal tract.70 Effect of GNPs on embryonic and fetal development in pregnant Wistar rats was reported by Wolterbeek et al.71 After the intravenous administration of GNPs, it was found that GNPs were accumulated in the spleen, liver, kidney, thymus, brain and heart. GNPs were also detected in uterus and placenta but no GNPs could cross the blood-placenta barrier.71 Toxicological effect of GNPs in mice via three different routes (oral, intravenous, intra peritoneal) was reported by Zhang et al.72 Among all the routes, orally administrated GNPs caused significant decrease in blood count, spleen index and body weight. Oral and intra peritoneal routes induced maximum toxicity and intravenous (tail vein) the minimum.72

Dose dependent biodistribution and toxic effect of citrate-coated GNPs in mice was reported by Lasagna-Reeves et al.73 Repeated intraperitoneal injec- tion (for 8 days) of citrate-coated GNPs in mice showed that highest fraction of administered dose was accumu- lated inside the liver followed by kidney and spleen.73

Moreover, it was also reported that citrate coated GNPs could cross the blood brain barrier and accumulate in the brain.73 Size dependent nephrotoxicity of GNPs in rat was reported by Abdelhalim et al. (2011a).74 Further, it was also found that GNPs altered the integrity of renal tubules with cloudy inflammation and vacuolar degeneration of nephron74 and induced toxicity in cardiac tissue.75 Oxida- tive stress-induced DNA impairment and loss of cell via- bility along with the diminished expression in antioxidant enzymes and the appearance of lipid peroxidation were reported in the brain of male Wister rats after intraperi- toneal injection of GNPs (20 �g/kg body weight).76

GNPs also show the size-dependent cellular uptake. For example, GNPs with the size range of 50 nm are more susceptible for cellular uptake, followed by 25 nm and 10 nm GNPs.77 Further, it was also found that

internalization of GNPs induces size dependent lysosome degradation.77 Consistent with the above results, cell line study with GNPs of different size (ranging from 0.8 nm to 15 nm) showed that IC50 for the GNPs with the size of 1.4 nm ranged from 30 to 56 �m, while GNPs with 15 nm in size were nontoxic.78 Similarly, incubation of GNPs with MRC-5 (representing human lung fibroblast) resulted in oxidative stress damage and lipid peroxidation79

and oxidative DNA damage.80 Accumulation of GNPs in human dermal fibroblast leads to slow growth of cell and diminished expression of extracellular matrix protein.81

Further, it was also observed that vacuoles formation dis- rupt the cytoskeleton structure and inhibits cell contraction and motility.81 Incubation of GNPs with dendritic cells results in alteration of immune system due to modulation in the secretion of immune-related cytokines after the cel- lular uptake of GNPs.82 The surface charge also influences the toxicity of GNPs.9 For example incubation of charged and neutral GNPs with human keratinocytes demon- strated that LD50 for charged and neutral GNPs were 10 �g/mL, and 25 �g/mL respectively.83 Compared to neutral GNPs, a decreased mitochondrial membrane poten- tial was reported in incubated cell with charged GNPs.83

4. UPCONVERTING NANOPARTICLE The term upconversion is a process in which low energy near infrared radiation is converted to higher energy (UV/visible light) radiation by multi-photon near infra- red (NIR) absorption and subsequent emission of rays of shorter wave length.84 Following the photo excitation with NIR, upconverting nanoparticles (UCNPs) emitting light of shorter wave length.39 UCNPs show superior chemi- cal, physical and photostability and unlike QDs, they do not blink.39�85 These unique properties of UCNP makes them potential candidate for biological imaging and in other biomedical application ranging from cellular labeling to photodynamic therapy.9�86�87 Although there are great interests in biomedical applications of UCNPs, however a better understanding of toxicological profiles of UCNPs in biomedical application is instantly needed to know the benefit to risk ratios. So far, only few toxicological data are available for

UCNPs. Biocompatibility and tissue distribution study of polyacrylic acid (PAA)-coated UCNPs in mice was reported by Xiong et al. (2010). PAA-coated UCNPs primarily accumulated in the spleen and liver, and the all exposed organ were normal in treated group. Fur- ther, it was also found that biochemical and histopatho- logical analysis does not indicate any toxicity in mice treated with PAA-coated UCNPs for more than 4 month.88

Rufaihah et al. (2008) reported that intravenous adminis- tration of polyethylenimine-coated upconversion fluores- cent nanocrystal in healthy rat results in accumulation of UCNPs in lung, spleen, kidney and blood.89 However, within 24 hours of post injection, declined level of UCNPs

J. Nanosci. Nanotechnol. 16, 7873–7897, 2016 7885

Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application Ahmad et al.

was noted in the entire vital organ. Furthermore, no any nanocrystal was observed at 7th day of post injection.89

Similarly Yin et al. (2012) reported that UCNPs does not show any systemic toxicity in mice.90 Chen et al. (2011) investigated the toxic effect of UCNPs in C. elegans. They reported that UCNPs did not produce noticeable toxic effect in the worms.91 Similarly Zhou et al. (2011) reported the understated toxicity of UCNPs in egg produc- tion, egg viability, and growth rate of N2 hermaphrodites’ C. elegance.92

4.1. Graphene Nanocomposites Graphene is a thin two-dimensional carbon nanomaterial possessing excellent electronic, thermal and mechanical properties that has attracted intense interest in diverse areas such as nanoelectronics, solar energy harvesting, biolog- ics. Graphene oxide (GO) serve a nanocargos to deliver the hydrophobic drugs, nucleic acids, and other molecules intracellularly for therapeutic and bioimaging purposes. However, the therapeutic potential of any nanomaterial for biological application is determined critically by its biocompatibility.93�94

The properties of graphene and their nanocompos- ites is an important factor for producing toxicity. It includes surface area, layer number, lateral dimension, surface chemistry, and purity. Surface area plays a sig- nificant role in the biological interaction of graphenes. Smaller nanoparticles (<10 nm) have a greater surface area and their atoms are exposed in a significant fraction on their surface.95 Monolayer graphene has a larger sur- face area and its every atom lies on the surface.96 The surface area of graphene decreases as the layer number increases. The higher surface areas of the graphene family increase the propensity of surface reactions including reac- tive oxygen species (ROS) production, antioxidant deac- tivation, or ROS quenching.97 The lateral dimensions of graphene mainly affect cellular uptake, renal clearance, and blood-barrier transport. In the last decades, much lit- erature evidenced that the toxicity of graphene and their nanomaterials was based on the generation of ROS. Nor- mally, cellular homeostasis process always maintains the ROS production and elimination at equal rate or balanced by antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase.98–100 Graphene nano- composites directly interact with cells or gets accumulated on the cells and further cause overproduction of ROS, which may lead to apoptosis or cause alteration of polyun- saturated fatty acids, lipids and protein denaturation in the cell membrane to cause skin disorders and along with this damage the genetic material also.101 Moreover, graphene nanomaterials also possess hydrophobic surface, and thus they directly interact with membrane lipids to produce tox- icity. Substantial literature reported that ROS generation takes place in a concentration and time-dependent man- ner. Overproduction creates oxidative stress by the admin- istration of graphene. Schinwald et al. (2012) reported

that large non-functionalized graphene with the lateral size of 25 �m caused local inflammation followed by the formation of mesothelial granuloma. Graphene con- tains 1–10 layers with 5 64 ±4 56 �m thickness.102 Upon intrapleural injection at the dose of 50 �g in C57BL6 mice for upto 7 days, a granuloma and inflammatory reac- tion was observed. When graphene with 1.2–5.0 nm in thickness was administered through intratracheal injection at a dose of 50 �g in C57BL/6 mice for 8–12 weeks, inflammation was observed in the lungs.102 Functional- ization of graphene is performed by oxidation, reduction, polymers, drug, and targeting molecules, to enhance solu- bility and biocompatibility. Yang et al. (2011) found that by functionalization with PEG and its administration into mice at a dose of 20 mg/kg, no any significant toxicity was observed.103 Another researcher reported that PEGy- lated graphene oxide (GO) did not produce agglomeration. Wojtomiszak et al. (2012) found that when functional- ization of GO was done with PEG, PEG-polypropylene, sodium deoxycholate and glycol-PEG, the highest biocom- patibility was observed with PEG on L9C29, and toxicity depended on the type of functional materials and con- centration of nanomaterials.104 Other researchers reported that PEG-conjugated GO administered at the dose of 80 mg/mL on human cervical cancer cells showed no sig- nificant toxicity. Another study was executed on human cervical cancer cells (HeLa) with the use of magnetic- based PAA and FMA-conjugated graphene. This conju- gated nano-system did not produce any significant amount of ROS and no toxicity was found.105�106 Sasidharan et al. (2011) reported that carboxyl group-conjugated graphene produced toxicity on monkey renal cells.94 Conjugation of GO with dextran improved stability and further studies on HeLa cells found minimum toxicity.107 Wang et al. (2011) reported that GO caused apoptosis in the human lung epithelial cells.108 Hu et al. (2011) reported physical dam- age of the cell membrane by interaction with GO.93 Wang et al. demonstrated on Kung Ming mice and inject by intravenously administering GO with the dose of 0.1, 0.25 and 0.4 mg. At 0.4 mg dose, granuloma formation was observed in the lungs, kidney, liver and spleen.108 More- over, it was not filtered by the kidney.108 Another similar study reported by Zhang et al. (2011) on Kung Ming mice with the use of radiolabelled GO revealed higher uptake in the lungs. Furthermore, at the dose of 10 mg/kg, toxic signs were observed, such as pulmonary oedama, inflam- mation and granuloma formation in the lungs of mice.109

Another study reported by Duch et al. (2011) observed that administration of the pristine graphene, GO and pluronic- functionalized graphene into the lungs of mice resulted in severe lung injury via the formation of ROS.110 The impor- tant toxicity reported for graphene and their nanocompos- ites along with their possible mechanism are summarized in Tables I and II. In summary, we can say that the biodistribution and pharmacokinetics is directly related to

7886 J. Nanosci. Nanotechnol. 16, 7873–7897, 2016

Ahmad et al. Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application

Table II. Toxicity induced by inorganic nanoparticles (iNPs) in research and development—in-vivo studies.

Route of

Coating/carrier administration Dose Cell line/Animal Major outcomes References

Iron oxide nanoparticles

—Ferumoxtran-10 —Ultrasmall superparamagnetic iron oxide nanoparticles

i.v. —Mice, —Rats, —Rabbits, —Dogs —Monkeys

—Toxicity was observed only at very high exposure levels

—Teratogenic in rats and rabbits

[53]

—Ferumoxtran-10 i.v. Fe-2.6 mg/kg bw —Human volunteer —Safe and effective and facilitated improved diagnostic performance

[54/53]

—Radaiolabeled59 Fe —Intratracheally 4 mg/rat —Male Sprague-Dawley rats

—Systemic accumulation and lung retention

—Potential lung and systemic cumulative toxicity

[55/54]

– —Intratracheally Low dose- 0.8 mg/kg bw

High dose- 20 mg/kg bw

—Male Sprague-Dawley rats

—A very weak pulmonary fibrosis

—Moderate cytotoxic effect

[56/55]

—Functionalized with 3,4-dihydroxy- phenylethylamine hydrochloride

—Fluorescein isothiocyanate (FITC) chromophore

i.v. 0.8 mg/kg —Wistar rats —Perivascular inflammation [1]

—Dextran coated USPIO

i.v. 25 mmol/L Fe —Rats —The disturbance and impairments of hepatic, renal and splenic functions

[57]

– —Intranasal 40 mg/kg bw —Mice Fatty degeneration of neuron in the CA3 area of hippocampus

[58]

– —Intranasal —CD-ICR male mice —Pathological alteration in olfactory bulb, hippocampus and striatum, and caused microglial proliferation and activation

[2]

– —Intranasal —Mice —Oxidative stress —Neurodendron degeneration,

[59]

Dimercaptosuccinic acid (DMSA)

—Intraperitoneally 50 mg/kg 100 mg/kg 200 mg/kg 300 mg/kg

—Pregnant mice —Presence of NPs in foetal liver and placenta

—Significant decrease in the infant’s growth

—Significantly decrease in spermatogonia, spermatocytes, spermatids and mature sperm of infant

[60]

Polyvinyl alcohols (PVAs) (PVA-SPIONs)

—Parenteral 3 mg Fe/kg —Mice —Well tolerated by mice [9]

QDs —Modified with hydroxyl group

i.v. 5 nmol/mouse —Male ICR mice —Aggregated in hepatic tissue and it was difficult to clear

[61]

—(ZnS coated QDs) Oral —Rat —Toxic due to liberation of liberation of Cd(2+)

[62]

i.v. 40 pmol —Mice —Very long half-life —Deposited in spleen, liver, and kidney

–

[63]

J. Nanosci. Nanotechnol. 16, 7873–7897, 2016 7887

Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application Ahmad et al.

Table II. Continued.

Route of Coating/carrier administration Dose Cell line/Animal Major outcomes References

—Captopril- conjugated QDs

i.p. 0.68 mg containing 50 nmol Cd

—Male ICR mice —Deposited in the inside the blood vessels of the liver, kidney and brain

[64]

—Coated with PEG-5000

i.v. —Balb/c and nude mice

—Persist and retain fluorescence for up to two-years in vivo, with significantly blue-shifted emission

[65]

—Silica coated CdTe i.v. 5 nmol/mouse Mice —Increased concentration of blood urea nitrogen (BUN) and creatinine (CREA)

—Mild nephrotoxicity

[66]

—CdSe core i.p. —Mice —Liver dysfunction —Morphological alternation to the hepatic lobules and increased oxidative stress

[67]

—Naked Qds- —Silica shell —Polyethylene glycol

i.v. 20, 50, 86, 125 �g Cd

—Pregnant mice —Cross the placental barrier [68]

—Carboxyl surface coated QDs—Amine surface coated QDs

i.v. —Mice —Vascular thrombosis in the pulmonary circulation with carboxyl-QDs

–

[69]

—Coated individually with polyethylene glycol, amines, and carboxylic

Acids

—Intrastromal injection

—Mouse —Damage to corneal epithelium barrier [19]

Gold nanoparticles

– i.v. —Rat —Size dependent tissue distribution [70]

Naked GNPs i.p. 8 mg/kg/week —BALB/C mice —Size dependent toxicity —Camel-like back and crooked spine —Increase of Kupffer cells in the liver —loss of structural integrity in the lungs

—Diffusion of white pulp in the spleen

[71]

—Coated with PEG (MW 5000)

i.v. 0.17, 0.85, 4.26 mg/kg /bw

—Male BALB/c mice

—Accumulate in the liver and spleen —Acute inflammation and apoptosis in the liver

[72]

i.v. 0.01 mg/kg —Rat —Accumulated in the liver, spleen, kidney, and lungs

—Alter the gene expression of target organ

[73]

i.v. 1 g/kg —Mice —Size dependent bio-distribution and toxicity

—Accumulated in liver, lung and spleen —Can cross blood brain barrier

[74]

i.v. 45 �g/ml —Pregnant Wistar rats

—Accumulated in liver and spleen, lungs, kidneys, thymus and heart, brain and also in the uterus and placentas

[75]

—Citrate coated —Oral —i.p. i.v.

137.5– 2200 �g/kg

—Male ICR mice —Dose dependent toxicity —High dose induces loss in body weight, red blood cells, and haematocrit

—The oral and intraperitoneal routes showed the highest toxicity, and the tail vein injection showed the lowest toxicity

[76]

7888 J. Nanosci. Nanotechnol. 16, 7873–7897, 2016

Ahmad et al. Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application

Table II. Continued.

Route of Coating/carrier administration Dose Cell line/Animal Major outcomes References

—i.p. 40, 200, 400 �g/kg/day for 8 days

—Male C57/BL6 mice

—Dose dependent biodistribution —Cross the blood-brain barrier and

accumulate in the neural tissue

[77]

—i.p. 50 �L and 100 �L of 10, 20, 50 nm GNPs for 3 or 7 days

—Male Wistar-Kyoto rats

—Renal tubular alterations —Atrophy and necrosis in renal cells

[78]

—i.p. 100 �L of 10, 20, 50 nm GNPs for 3 or 7 days

—Male Wistar-Kyoto rats

—Congested heart muscle with prominent dilated blood vessels, scattered and extravasations of red blood cells

–

[79]

—i.p. 20 �g/kg bw of 20 nm GNPs for 3 days

—Male Wister rats

—Oxidative stress —Impairment of the antioxidant

enzyme glutathione peroxidase in rat brain

Generation of 8-hydroxydeoxyguanosine (8OHdG), caspase-3 and heat shock protein70 (Hsp70), and IFN-�, which may lead to inflammation and DNA damage/cell death

[80]

—Citrate coated 0–0.8 mg/mL —Humandermal- fibroblasts

—Alteration of Cell growth, and protein synthesis

[81]

Upconverting NPs

Polyacrylic acid-coated

i.v. 15 mg/kg —Mice -

—No overt toxicity [82]

Graphene NPs —Pharyngeal aspiration

50 �g/mouse —THP-1 cells —Inflammation in lung and the pleural space

Expression of MIP-1�, MCP-1, MIP-2, IL-8, and IL-1� BAL

[32]

PEG i.v. 20 mg/kg —Mice —Accumulate in the reticuloendothelial system (RES) including liver and spleen

[83]

—Dextran functionalized

Graphene —Radio labelled with 125I

i.v. 20 mg/kg Balb/c mice —Accumulates in the reticuloendothelial system including liver and spleen

[84]

i.v. Low dose-0.1 mg Midle dose-0.25 mg

High dose-0.4 mg (for 1, 7, 30 day exposure)

—Mice —Dose dependent toxicity —Chronic toxicity under high dose —Higher dose leads to

conglomeration of airways and death due to suffocation

—Lung granuloma formation

[35]

Radio labelled with 188Re

i.v. 20 mg/kg —Sprague– Dawley rats

—Long-time retention—Inflammation cell infiltration

—Pulmonary oedema and granuloma formation

[85]

—Pegylated —Intratracheal 50 �g/mouse —C57BL/6 mice —Severe and persistent lung injury [86] Carbon

nanotubes —SWCNTs —Intratracheal —ApoE-/-mice

—C57 mice —Inflammation and DNA damage [87]

—SWCNTs —MWCNTs —Ground CNTs

—Injected directly into lungs

0.5, 2, 5 mg/animal

—Female Sprague– Dawley rats

—Pulmonary lesion —Alveolitis —Induction of inflammatory and

fibrotic responses —Over production of TNF-�

[88]

J. Nanosci. Nanotechnol. 16, 7873–7897, 2016 7889

Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application Ahmad et al.

Table II. Continued.

Route of Coating/carrier administration Dose Cell line/Animal Major outcomes References

—SWCNTs —Intratracheal Low dose-2 mg/ml

High dose-10 mg/ml (50 �l)

—Mice —Dose-dependent epithelioid granulomas in lungs

–

[89]

—SWCNTs —Intratracheal 0.5 mg —Male ICR mice —Release of proinflammatory cytokines,

—Chronic pulmonary inflammation and granuloma formation

[90]

—SWCNTs —Intrapharyngeal instillation

10–40 �g/mouse —C57BL/6 mice —ApoE−/− mice

—Oxidative insults, in lung, aorta, and heart tissue

—Provokes pulmonary toxicity —Mitochondrial oxidative modifications and accelerated atheroma formation

[91]

—SWCNTs —Mouse pharyngeal aspiration

40 �g/mouse —Female C57BL/6 mice

—Decline of antioxidants (GSH, ascorbate) as well as accumulation of lipid peroxidation products

[92]

—Functionalized SWCNTs with Diethylentriamine- pentaacetic (DTPA)

i.v. 60 �g and 400 �g in 200 �l of phosphate buffer saline

—BALB/c mice —Rapid blood clearance —Significantly improved toxicity profile compared with their non-functionalized counterparts

[93]

Silica nanoparticle

i.v. 10 mg/kg bw —Mice —Accumulated in lungs, liver and spleen

—Causes Hepatotoxicity

[94]

Fruorescence dye-labelled

i.v. 50 mg/kg —Mice —Trapped by macrophages in the spleen and liver

—Severe inflammatory response

[95]

—Oral —Mice —Histopathological alterations in kidney, lung, testis, liver, spleen, and stomach

—Significant changes in albumin, cholesterol, triglyceride, total protein, urea, HDL, and LDL

—Altered activity of alkaline phosphatase and aspartate aminotransferase

[96]

—i.v. 20 mg/kg every 3 day for 13 day

—Mice —Alter the maturation process of sperm in the epididymis

[97]

the colloidal properties, hydrophilicity, biodegradation and non-biodegradation parameters of this inorganic nanoma- terial, and their manipulation is very crucial for reducing the toxicity of this important nanomaterial.

4.2. Carbon Nanotubes Carbon nanotubes (CNTs) are nanosized tiny tubes of about 1–3 nanometers in diameter and hundreds to thou- sands of nanometers long.111 CNTs are grouped into two classes depending on the number of layers; sin- gle wall carbon nanotubes (SWCNTs) and multiwall car- bon nanotubes (MWCNTs), displaying enormous potential for applications in electronics, optics, materials science,

nanotechnology, biology, and medicine.112 CNTs have been widely explored in cancer therapeutic and imaging. Hong et al., performed molecular imaging with SWC- NTs and evaluated combination of Gd3 and functionalized SWCNTs, which when applied to MRI achieved high res- olution and good tissue penetration.113 CNTs have been used to detect cancer biomarkers, such as, P type car- bon nanotubes for prostate specific antigen in prostate cancer, carbon nanomaterial for carcinoma antigen-125 (CA-125).114�115 SWCNTs were developed for delivery of doxorubicin to colon cancer, cisplatin to squamous carcinoma.116�117 CNTs, have emerged as one of the most attractive approaches for neurological applications. CNTs have shown evidence of their strong mechanical properties,

7890 J. Nanosci. Nanotechnol. 16, 7873–7897, 2016

Ahmad et al. Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application

electrical conductive capacity, and morphological similar- ity to neurites. Furthermore, the structural features and dimensions of CNTs were also found to be similar to many elements of the neural machinery, enhancing molecular level interaction and hence better control over the physio- logical activity and neuronal information processing.118

Various studies shows that toxic effect of CNTs depends on the properties of nanotubes such as single wall (SWCNTs) or multiple wall (MWCNTs), surface area, size and size distribution, functionalization, cellular uptake, degree of aggregation, dispersibility and presence of metallic impurities.111�119–121 The outcome of various tox- icological studies on CNTs has become the hot topic of debate as some investigators have reported that CNTs are cytotoxic to the primates and other mammalian cells. For example, Ursini et al. (2012) reported that non- functionalized MWCNTs shows higher cyto-genotoxicity of human lungs’ epithelial cells as compared to func- tionalized MWCNTs.122 Similarly, efficient dispersibility of CNTs could suppress the immunotoxicity of CNTs.123

Impurities present in CNTs are usually transition metal; these are used as a catalyst during the CNTs synthesis. For example inhibitory effects of iron impurities present with CNTs were demonstrated by Meng et al.124 They reported that contaminated CNTs exhibit higher neural damage of PC12 cells in comparison to non-contaminated CNTs.124

Jacoben et al. (2009) evaluated the toxic and inflammatory potential of three carbonaceous particles; CB, fullerenes C60 (C60) and single walled carbon nanotubes (SWCNT) as well as gold particles and quantum dots (QDs) in apolipoprotein E knockout mice (ApoE−/−).125 Signifi- cant increases in level of Il-6, Mip-2 and Mcp-1 mRNA were found in lung tissue after instillation of SWCNT, CB and QDs at 3 h and 24 h. There was statistically significant increase in DNA damage as well as neutrophil fractions in BAL cells, and protein level in BAL fluid. However, much weaker inflammatory responses were caused by Gold and C60 particles.125 Muller et al. (2005) reported that administration of CNTs in Sprague-Dawley rats induces overproduction of TNF-� by macrophages, form- ing parenchymal granulomas.126 Lam et al. (2004) reported that CNTs induces interstitial granulomas and pulmonary injuries in a dose-dependent manner.127 Similarly, intra- tracheal instillation of SWCNT into male ICR mice induces alveolar macrophage activation, various chronic inflammatory responses, and severe pulmonary granuloma formation.128 Jia et al. (2005) reported that exposure of SWCNTs results in profound cytotoxicity in alveolar macrophage (AM) after a 6-h exposure in vitro.129 Sin- gle intrapharyngeal instillation of SWCNTs induces acti- vation of heme oxygenase-1 (HO-1), a marker of oxidative insults, in lung, aorta, and heart tissue in HO-1 reporter transgenic mice.130 Stoker et al. (2008) demonstrated the incubation of normal bronchial epithelial cells and normal human fibroblasts with SWCNTs. They reported the over

production of nitrous oxide and decreased cell viability after exposure to different concentrations of SWCNTs.131

Exposure of mice to SWCNTs induces an unnatural robust pulmonary inflammatory response causing an early onset of fibrosis, which is followed by oxidative stress and depletion of antioxidant.132

4.3. Silica Nanoparticles (SNPs) Wide-spread use of SNPs in non-medical field is already established (e.g., in cosmetics, polishing, varnished and in food stuffs).2�133 Very recently, SNPs have been intro- duced in the field of medical.2 Owing to the easy surface modification silica is widely used for coating materials, in biomedical field.134 SNPs have been used in cancer ther- apy, drug delivery and DNA delivery.135�136 SNPs with sev- eral unique properties such as, hydrophilic surface favoring protracted circulation, excellent biocompatibility, versatile silane chemistry for surface functionalization, low cost of NP production, and ease of large-scale synthesis, make them amenable for biomedical applications.137

Despite the potential benefits, there is a concern that exposure to certain types of SNPs may lead to adverse health effects. At a moderate dose i.e., <20 �g/mL are considered as safe.2�138 However, at higher dose (>20 �g/mL) SNPs show dose dependent cytotoxicity on A549 cells.139�140 Additionally, SNPs exhibit size- dependent cytotoxicity with smaller diameter being more toxic than bigger,141�142 this is due to the fact that cel- lular uptake is more efficient with small particle which correlates with an increased Cytotoxicity.2�143 For exam- ple, size-dependent intracellular localization and cyto- toxicity of silica particles, using the mouse epidermal Langerhans cell line XS52 was reported by Nabeshi et al. (2010). On treatment with silica particles of diameters 70, 300, and 1000 nm, cellular uptake and cytotoxic- ity increased with reduction in particle size.144 Impact of SNPs design on cellular toxicity and haemolytic activ- ity was evaluated by Yu et al. (2011). They reported that surface charge and pore size govern cellular toxic- ity of SNPs. Hemolysis assay showed that the haemolytic activity was porosity and geometry-dependent for bare SNPs and surface-charge-dependent for amine-modified SNPs.145 Interaction of SNPs with HaCaT cells was stud- ied by Liang et al.146 They reported that SNPs internalized into HaCaT cells and localized in the cytoplasm, lyso- somes and autophagosomes. Decreased cell viability and damaged cell membrane integrity showed the cytotoxicity of SNPs.146

SNPs accumulate mainly in lungs, liver, and spleen.134�147 After intravenous administration of SNPs in mice, it is taken up by macrophages, and could poten- tially cause liver injury.147 Similarly, Hassankhani et al. (2014) reported that oral administration of SNPs causes significant changes in albumin, cholesterol, triglyceride, total protein, urea, HDL, and LDL as well as in alka- line phosphatase and aspartate aminotransferase activity.

J. Nanosci. Nanotechnol. 16, 7873–7897, 2016 7891

Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application Ahmad et al.

In addition, histopathological examinations demonstrated that SNPs have toxic effects on various tissues includ- ing liver, kidney, lung, and testis.148 After the exposure of human bronchial epithelial BEAS-2B cells to SNPs increased phosphorylation of NF-�B p65 and MAP- kinases p38 and JUN-N-terminal protein kinase pathways was observed.149 Increases in membrane-bound trans- forming growth factor-� (TGF-�) release and EGFR phosphorylation were also observed on SNPs exposure, and pre-treatment with inhibitors decreased the libera- tion of IL-6 and CXCL8.149 Similarly, Nagakura et al. (2014) reported that SNPs induces the production of IL-6 production in human keratinocytes leading to the induction of skin inflammatory diseases.150 Yang et al. (2014) investigated the cellular uptake of SNPs in human SK-N-SH and mouse neuro2a (N2a) neuroblastoma cells. It was found that the treatment of SNPs impaired the morphology of SK-N-SH and N2a cells at different concentrations.151 Moreover, two specific pathological hallmarks of Alzheimer’s disease (AD) that increased deposition of intracellular �-amyloid 1-42 (A�(1-42)) as well as increased phosphorylation of tau at Ser262 and Ser396, were observed in both cell lines.151 It has been also reported that SNPs influence the maturation process of sperm in the epididymis by causing oxidative stress and damage to the mitochondrial structure, resulting in energy metabolism dysfunction in mice.152 Exposure of zebra fish embryos with SNPs caused embryonic malformations, including pericardial edema, yolk sac edema, tail and head malformation.153 The outcomes of various iNPs treatment in in-vitro and in-vivo study conducted to investigate the effect of these nanomedicines for associated toxicity are summarized in Tables I and II respectively.

5. MECHANISM OF NANOTOXICITY Irrespective of the fundamental differences between the different types iNPs, the nano factor itself is severely responsible for the toxic effect of iNPs.12 For instance, as the size of the iNPs approaches the size of biomolecules (proteins, DNA), the iNPs can reach to the site where larger molecules cannot access (for example in mitochon- dria and nucleus) or it can cross the blood placental barrier.12 The high surface to volume ratio of iNPs ampli- fies the surface accessible for interaction of cellular com- ponents with iNPs.12 Because of these common features for iNPs, it is anticipated that iNPs exert parallel mecha- nisms by which they disturb the cellular homeostasis.

6. REACTIVE OXYGEN SPECIES AND FREE RADICAL

Nanosize of the Particle can lead to the formation of elec- tronic states in the NPs.13 The electronic states present in nanoparticles have impending potential to undergo elec- tron transfer reactions and results in generation of ROS

and can directly disturb the biological electron transfer reactions.13 The most important molecular mechanism for in vivo cellular nanotoxicity is the induction of oxida- tive stress induced by reactive oxygen species (ROS) and free radical.154�155 Free radicals can cause mutilation to the biomolecules through lipid peroxidation, destabilization of protein, and damage to the DNA helix. Further, oxida- tive stress enhances the inflammatory process via upregu- lation of NF-�B, kinase and activator protein involved in inflammation.155 The NPs are treated as a foreign material by the cells and as a reaction to these foreign elements, production of ROS is quite common.12 The large surface to volume ratio of iNPs and its reactive surface are suit- able for substantial oxidizing potential. iNPs can gener- ate ROS as a consequence of contact with lysosomes or interaction of cellular organelles (mitochondria, nucleus, endoplasmic reticulum etc). ROS can also be generated by iNPs by interacting with surface receptor and activa- tion of intracellular signalling pathway or by interaction with redox active protein (NADPH oxidase and Xanthine oxidase).5�12 Iron oxide nanoparticles generate extremely reactive OH− radicals that mediate the toxicity of NPs.156

For example, Zhang et al. (2012) reported the production of ROS in Chinese hamster lung cells due to L-glutamic acid-coated Fe2O3 nanoparticles.

157 Further, glutathione depletion and inactivation of some anti-oxidant enzymes (glutathione reductase, superoxide dismutase) was also observed.157 Similarly, Zhu et al. (2010) reported the loss of membrane potential from mitochondrial surface and apoptotic condensation of chromatin in human umbilical endothelial cells due to iron oxide nanoparticles.158 Like- wise, incubation of MCF-7 cells with Cd–Te QDs results in production of reactive oxygen species and consequent damage of mitochondrial membrane.56 Furthermore, addi- tion of antioxidant effectively protects the cell against QDs induced ROS.56 Liu et al. (2011) reported that QDs is responsible for the production of ROS in mice hep- atocytes with significant increase in hepatotoxic agent, malondialdehyde.52 Oxidative stress related nanotoxicity was also observed with GNPs. For example, incubation of GNPs with human lung fibroblast results in oxidative dam- age and lipid peroxidation of cells.79 Similarly, Tedesco et al. (2008) reported the oxidative stress induced by GNPs in aquatic organism.159

6.1. Disruption of the Cytoskeleton Structure The consequences of iNPs on cytoskeleton network have only recently gained more consideration. Moreover, as the cytoskeleton plays an important role in many intracellu- lar signalling pathways, therefore it is important to inves- tigate whether the iNPs induced cytoskeletal disruption leads to secondary effects. Most commonly, disruption of the cell membranes has been observed as a function of dye diffusion or cytosolic enzyme leakage assays.13 As we know that iNPs has physical dimension of nanometric

7892 J. Nanosci. Nanotechnol. 16, 7873–7897, 2016

Ahmad et al. Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application

range, intracellular volume occupied by iNPs may lead to alteration in cytoskeleton network. For example interac- tion of iNPs with blood components can lead to haemol- ysis and thrombosis.155 In another study it was reported that mIONPs impaired the adhesion of cytoskeleton net- work with extracellular matrix and also disrupted the tubu- lin network and actin fibres of human umbilical vein.160

Consistent with the above report, Buyukhatipoglu et al. (2010) reported that mIONPs leads to cell elongation and increase in actin stress fibre formation.161 Similarly, GNPs showed contrary effect on the cytoskeleton network of A549 human carcinoma lung cells162 and actin fibril of human dermal fibroblast.162�163

6.2. Genotoxicity and Alteration of Signalling Pathway

As discussed earlier, iNPs can disturb the balance of cel- lular homeostasis and thereby alter the intracellular sig- nalling pathways (for example altered gene expression and protein or cascade of genotoxicity due to high level of ROS). Till now only little investigation has been reported for the effect of iNPs on gene and protein expression. Here, we mainly discuss on reported altered gene expres- sion as a result of DNA damaging effect of iNPs (Fig. 3).

To induce genotoxicity, iNPs interact not only with DNA but also with protein involved in replication, tran- scription, or repair of DNA. For example, Baweja et al. (2011) reported that C60 fullerene binds with topoiso- merase II � and inhibits enzymes activity.164 Similarly, Gupta et al. (2011) reported that C60 fullerene inter- act with protein involved in the DNA mismatch repair pathway.165 iNPs can disturb the cell cycle by Interaction with centrioles and mitotic spindle apparatus, leading to deletion or addition of chromosome in daughter cells.166

For instance, long term exposure of iNPs led to abnormal

(a) (b)

Figure 3. (a) Diagrammatic representation of possible intra-cellular uptake and access of iNPs to the nucleus. (b) Schematic overview of the possible consequences of iNPs-induced gene/DNA damage.

alignment and segregation of chromosome and multipo- lar spindle formation.167 Further, it was also demonstrated that iNPs can influence the activity of protein kinase and disturb the regulation of cell cycle (DNA replication and cell division).167

iNPs can generate ROS and free radical that may cause oxidative DNA damage and oxidised base lesions and DNA strand break. DNA base lesion may result in muta- tion and can be carcinogenic.168 Free ions (Fe+2, Ag+, Mn+2 etc.) released from iNPs may be involved in the production of ROS.166�169 For example, Asharani et al. (2008) reported that silver nanoparticle (AgNP) react with hydrogen peroxide and lead to formation of Ag+.170 AgNP interact with mitochondrial function and disrupt the mito- chondrial respiratory chain that leads to production of ROS, which can result in damage to DNA.170

6.3. Inflammation Mediated Nanotoxicity Carbon nanoparticles, such as, graphenes and CNTs have been shown to cause the induction of inflammation in different cells including epidermal keratinocytes, alveolar and bronchial epithelial cells, and cultured monocyte-macrophage cells.171�172 A mechanistic study was conducted, to explain the generation of inflamma- tory responses upon exposure to carbon nanoparticles. It was suggested through the computational model that the Toll-like receptors recognize the carbon nanoparticles as pathogens which trigger innate immune responses and further lead to the secretion of few inflammatory protein mediators including interleukins and chemokines.173

Stoker et al. (2008) developed 5%, 10%, 20% SWCNTs and analysed the production of nitric oxide (NO), an inflammatory marker and cytotoxic response (MTT assay) of the cell layers, upon exposure to different concentra- tions of SWCNTs. NO production dramatically increased

J. Nanosci. Nanotechnol. 16, 7873–7897, 2016 7893

Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application Ahmad et al.

with the increase in SWCNTs concentration indicating inflammatory response in the airway epithelial cells. The fibroblasts also showed mild inflammatory and cytotoxic response.131 Oxidative stress is also linked to inflammation reciprocally, as it leads to the release of pro-inflammatory mediators through the pro-inflammatory cascades such as NF-�B (Nuclear Factor-�B), phosphoinositide 3-kinase (PI3-K) pathways and mitogen-activated protein kinase (MAPK).174 Liu and Sun, reported ROS generation in endothelial cells on exposure to silica nanoparticles, which lead to the induction of apoptosis via JNK/p53 dependent mitochondrial pathways. Furthermore, at high concentrations, the silica nanoparticles also caused the activation of NF-kB due to generation of oxidative stress in the endothelial cells, which resulted in the upregu- lation of CD62E, CD54, IL-6, IL-8, TF and MCP-1.175

Nagakura et al., showed the involvement of purinergic sig- nalling in skin inflammatory diseases. Exposure to silica nanoparticles (SNP30) increased the IL-6 production in human keratinocyte cell line, HaCaT. A dose and time dependent effect on IL-6 production was examined and it was observed that the IL-6 production was increased on increasing the dose from 5 �g/cm2 to 50 �g/cm2. Furthermore, IL-6 production increased in a time depen- dent manner for upto 24 h. The study demonstrated that P2Y11 receptor mediates SNP30-induced IL-6 production in the cells, confirming ATP-P2Y11 purinergic signalling, as a common pathway inducing the skin inflammatory diseases.150 Treatment with titanium dioxide nanoparticles, lead to the toxicity in human bronchial epithelial cell line, mediated by the production of IL-8 via the p38 MAPK and/or ERK pathway.176

7. CONCLUSION iNPs have already found a wide range of biomedical appli- cation including but not limited to drug carriers, imag- ing agents and theranostics. Their unique physicochemical properties are credited for their applicability in modulat- ing the drug efficacy. In unison, published reports clearly indicate that iNPs showing toxicity in in-vitro and in-vivo studies. With the extensive study in nanoparticles tox- icology, at the present, we have an understanding that the degree of toxicity of iNPs varies with the physical characteristics, surface coating, dose and their adminis- tration route. Moreover, it’s also identified that designing of right engineered iNPs by selection of size, modifi- cation of surface properties, improving of solubilisation and organ/cellular specific targeting permit the creation of safer and effective iNPs as nanomedicines. Nonetheless, the published reports have lack of corroboration in much extent and also have significant degree of inadequateness. It is therefore, a careful toxicity testing at cooperative level is required prior stabilising the iNPs in clinical biomedical nanotechnology.

Conflict of Interest None.

References and Notes 1. A. M. Schrand, M. F. Rahman, S. M. Hussain, J. J. Schlager,

D. A. Smith, and A. F. Syed, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2, 544 (2010).

2. L. Yildirimer, N. T. Thanh, M. Loizidou, and A. M. Seifalian, Nano Today 6, 585 (2011).

3. M. Z. Ahmad, S. Akhter, G. K. Jain, M. Rahman, S. A. Pathan, F. J. Ahmad, and R. K. Khar, Expert Opin. Drug Deliv. 7, 927 (2010).

4. M. Z. Ahmad, S. Akhter, Z. Rahman, S. Akhter, M. Anwar, N. Mallik, and F. J. Ahmad, J. Pharm. Pharmacol. 65, 634 (2013).

5. S. Akhter, M. Z. Ahmad, F. J. Ahmad, G. Storm, and R. J. Kok, Expert Opin. Drug Deliv. 9, 1225 (2012).

6. X.-J. Liang, H. Meng, Y. Wang, H. He, J. Meng, J. Lu, P. C. Wang, Y. Zhao, X. Gao, and B. Sun, Proc. Natl. Acad. Sci. 107, 7449 (2010).

7. E. C. Cho, C. Glaus, J. Chen, M. J. Welch, and Y. Xia, Trends Mol. Med. 16, 561 (2010).

8. S.-G. Kang, G. Zhou, P. Yang, Y. Liu, B. Sun, T. Huynh, H. Meng, L. Zhao, G. Xing, and C. Chen, Proc. Natl. Acad. Sci. 109, 15431 (2012).

9. J. Li, X. Chang, X. Chen, Z. Gu, F. Zhao, Z. Chai, and Y. Zhao, Biotechnol. Adv. 32, 727 (2014).

10. Z. Chen, H. Chen, H. Meng, G. Xing, X. Gao, B. Sun, X. Shi, H. Yuan, C. Zhang, and R. Liu, Toxicol. Appl. Pharmacol. 230, 364 (2008).

11. J. D. Byrne, T. Betancourt, and L. Brannon-Peppas, Adv. Drug Del. Rev. 60, 1615 (2008).

12. S. J. Soenen, P. Rivera-Gil, J.-M. Montenegro, W. J. Parak, S. C. De Smedt, and K. Braeckmans, Nano Today 6, 446 (2011).

13. M. M. B. Holl, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 1, 353 (2009).

14. S. Tenzer, D. Docter, S. Rosfa, A. Wlodarski, J. R. Kuharev, A. Rekik, S. K. Knauer, C. Bantz, T. Nawroth, and C. Bier, ACS Nano 5, 7155 (2011).

15. P. Aggarwal, J. B. Hall, C. B. McLeland, M. A. Dobrovolskaia, and S. E. McNeil, Adv. Drug Del. Rev. 61, 428 (2009).

16. B. Wang, X. He, Z. Zhang, Y. Zhao, and W. Feng, Acc. Chem. Res. 46, 761 (2012).

17. E. Fröhlich, T. Kueznik, C. Samberger, E. Roblegg, C. Wrighton, and T. R. Pieber, Toxicol. Appl. Pharmacol. 242, 326 (2010).

18. W. H. Suh, K. S. Suslick, G. D. Stucky, and Y.-H. Suh, Prog. Neurobiol. 87, 133 (2009).

19. M.-T. Zhu, B. Wang, Y. Wang, L. Yuan, H.-J. Wang, M. Wang, H. Ouyang, Z.-F. Chai, W.-Y. Feng, and Y.-L. Zhao, Toxicol. Lett. 203, 162 (2011).

20. P. Bourrinet, H. H. Bengele, B. Bonnemain, A. Dencausse, J.-M. Idee, P. M. Jacobs, and J. M. Lewis, Invest. Radiol. 41, 313 (2006).

21. Y. Anzai, C. W. Piccoli, E. K. Outwater, W. Stanford, D. A. Bluemke, P. Nurenberg, S. Saini, K. R. Maravilla, D. E. Feldman, and U. P. Schmiedl, Radiology 228, 777 (2003).

22. M.-T. Zhu, W.-Y. Feng, B. Wang, T.-C. Wang, Y.-Q. Gu, M. Wang, Y. Wang, H. Ouyang, Y.-L. Zhao, and Z.-F. Chai, Toxicology 247, 102 (2008).

23. B. Szalay, E. Tátrai, G. Nyírő, T. Vezér, and G. Dura, J. Appl. Toxicol. 32, 446 (2012).

24. A. Hanini, A. Schmitt, K. Kacem, F. Chau, S. Ammar, and J. Gavard, Int. J. Nanomedicine 6, 787 (2011).

25. J. Feng, H. Liu, K. K. Bhakoo, L. Lu, and Z. Chen, Biomaterials 32, 6558 (2011).

26. B. Wang, W. Y. Feng, M. Wang, J. W. Shi, F. Zhang, H. Ouyang, Y. L. Zhao, Z. F. Chai, Y. Y. Huang, and Y. N. Xie, Biol. Trace Elem. Res. 118, 233 (2007).

7894 J. Nanosci. Nanotechnol. 16, 7873–7897, 2016

Ahmad et al. Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application

27. Y. Wang, B. Wang, M.-T. Zhu, M. Li, H.-J. Wang, M. Wang, H. Ouyang, Z.-F. Chai, W.-Y. Feng, and Y.-L. Zhao, Toxicol. Lett. 205, 26 (2011).

28. B. Wang, W. Feng, M. Zhu, Y. Wang, M. Wang, Y. Gu, H. Ouyang, H. Wang, M. Li, and Y. Zhao, J. Nanopart. Res. 11, 41 (2009).

29. C. J. Rivet, Y. Yuan, D.-A. Borca-Tasciuc, and R. J. Gilbert, Chem. Res. Toxicol. 25, 153 (2011).

30. A. Noori, K. Parivar, M. Modaresi, M. Messripour, M. H. Yousefi, and G. R. Amiri, Afr. J. Biotechnol. 10, 1221 (2013).

31. K. Bhattacharya, E. Hoffmann, R. F. Schins, J. Boertz, E.-M. Prantl, G. M. Alink, H. J. Byrne, T. A. Kuhlbusch, Q. Rahman, and H. Wiggers, Toxicol. Sci. 126, 173 (2012).

32. X. Zhu, S. Tian, and Z. Cai, PLoS One 7, e46286 (2012). 33. S. C. Hong, J. H. Lee, J. Lee, H. Y. Kim, J. Y. Park, J. Cho, J. Lee,

and D.-W. Han, Int. J. Nanomedicine 6, 3219 (2011). 34. T. R. Pisanic, J. D. Blackwell, V. I. Shubayev, R. R. Fiñones, and

S. Jin, Biomaterials 28, 2572 (2007). 35. F. Cengelli, D. Maysinger, F. Tschudi-Monnet, X. Montet,

C. Corot, A. Petri-Fink, H. Hofmann, and L. Juillerat-Jeanneret, J. Pharmacol. Exp. Ther. 318, 108 (2006).

36. N. Singh, G. J. Jenkins, B. C. Nelson, B. J. Marquis, T. G. Maffeis, A. P. Brown, P. M. Williams, C. J. Wright, and S. H. Doak, Bio- materials 33, 163 (2012).

37. M. Auffan, W. Achouak, J. Rose, M.-A. Roncato, C. Chanéac, D. T. Waite, A. Masion, J. C. Woicik, M. R. Wiesner, and J.-Y. Bottero, Environ. Sci. Technol. 42, 6730 (2008).

38. W.-X. Zhang, J. Nanopart. Res. 5, 323 (2003). 39. S. Akhter, M. Z. Ahmad, A. Singh, I. Ahmad, M. Rahman,

M. Anwar, G. Kumar Jain, F. J. Ahmad, and R. Krishen Khar, Curr. Pharm. Des. 17, 1834 (2011).

40. M. Rahman, M. Z. Ahmad, I. Kazmi, S. Akhter, M. Afzal, G. Gupta, F. J. Ahmed, and F. Anwar, Expert Opin. Drug Deliv. 9, 367 (2012).

41. V. Karabanovas, E. Zakarevicius, A. Sukackaite, G. Streckyte, and R. Rotomskis, Photochemical and Photobiological Sciences 7, 725 (2008).

42. S. Kim, Y. T. Lim, E. G. Soltesz, A. M. De Grand, J. Lee, A. Nakayama, J. A. Parker, T. Mihaljevic, R. G. Laurence, and D. M. Dor, Nat. Biotechnol. 22, 93 (2004).

43. T. M. Samir, M. M. Mansour, S. C. Kazmierczak, and H. M. Azzazy, Nanomedicine 7, 1755 (2012).

44. T. Zhang, J. L. Stilwell, D. Gerion, L. Ding, O. Elboudwarej, P. A. Cooke, J. W. Gray, A. P. Alivisatos, and F. F. Chen, Nano Lett. 6, 800 (2006).

45. A. M. Derfus, W. C. Chan, and S. N. Bhatia, Nano Lett. 4, 11 (2004).

46. V. Karabanovas, E. Zakarevicius, A. Sukackaite, G. Streckyte, and R. Rotomskis, Photochem. Photobiol. Sci. 7, 725 (2008).

47. R. S. Yang, L. W. Chang, J.-P. Wu, M.-H. Tsai, H.-J. Wang, Y.-C. Kuo, T.-K. Yeh, C. S. Yang, and P. Lin, Environ. Health Perspect. 115, 1339 (2007).

48. M. E. Åkerman, W. C. Chan, P. Laakkonen, S. N. Bhatia, and E. Ruoslahti, Proc. Natl. Acad. Sci. 99, 12617 (2002).

49. S. Kato, K. Itoh, T. Yaoi, T. Tozawa, Y. Yoshikawa, H. Yasui, N. Kanamura, A. Hoshino, N. Manabe, and K. Yamamoto, Nanotechnology 21, 335103 (2010).

50. J. A. Fitzpatrick, S. K. Andreko, L. A. Ernst, A. S. Waggoner, B. Ballou, and M. P. Bruchez, Nano Lett. 9, 2736 (2009).

51. A. Sadaf, B. Zeshan, Z. Wang, R. Zhang, S. Xu, C. Wang, and Y. Cui, J. Nanosci. Nanotechnol. 12, 8287 (2012).

52. W. Liu, S. Zhang, L. Wang, C. Qu, C. Zhang, L. Hong, L. Yuan, Z. Huang, Z. Wang, and S. Liu, PLoS One 6, e24406 (2011).

53. M. Chu, Q. Wu, H. Yang, R. Yuan, S. Hou, Y. Yang, Y. Zou, S. Xu, K. Xu, and A. Ji, Small 6, 670 (2010).

54. J. Geys, A. Nemmar, E. Verbeken, E. Smolders, M. Ratoi, M. F. Hoylaerts, B. Nemery, and P. Hoet, Environ. Health Perspect. 116, 1607 (2008).

55. T.-R. Kuo, C.-F. Lee, S.-J. Lin, C.-Y. Dong, C.-C. Chen, and H.-Y. Tan, Chem. Res. Toxicol. 24, 253 (2011).

56. J. Lovriæ, S. J. Cho, F. M. Winnik, and D. Maysinger, Chem. Biol. 12, 1227 (2005).

57. M. Yan, Y. Zhang, K. Xu, T. Fu, H. Qin, and X. Zheng, Toxicology 282, 94 (2011).

58. A. O. Choi, S. E. Brown, M. Szyf, and D. Maysinger, J. Mol. Med. 86, 291 (2008).

59. J. Lovriæ, H. S. Bazzi, Y. Cuie, G. R. Fortin, F. M. Winnik, and D. Maysinger, J. Mol. Med. 83, 377 (2005).

60. I. Nabiev, S. Mitchell, A. Davies, Y. Williams, D. Kelleher, R. Moore, Y. K. Gun’ko, S. Byrne, Y. P. Rakovich, and J. F. Donegan, Nano Lett. 7, 3452 (2007).

61. Y. Qu, W. Li, Y. Zhou, X. Liu, L. Zhang, L. Wang, Y.-F. Li, A. Iida, Z. Tang, and Y. Zhao, Nano Lett. 11, 3174 (2011).

62. A. Hoshino, K. Fujioka, T. Oku, M. Suga, Y. F. Sasaki, T. Ohta, M. Yasuhara, K. Suzuki, and K. Yamamoto, Nano Lett. 4, 2163 (2004).

63. C. R. Patra, R. Bhattacharya, D. Mukhopadhyay, and P. Mukherjee, Adv. Drug Del. Rev. 62, 346 (2010).

64. W. H. D. Jong, W. I. Hagens, P. Krystek, M. C. Burger, A. J. Sips, and R. E. Geertsma, Biomaterials 29, 1912 (2008).

65. Y.-S. Chen, Y.-C. Hung, I. Liau, and G. S. Huang, Nanoscale Res. Lett. 4, 858 (2009).

66. W.-S. Cho, M. Cho, J. Jeong, M. Choi, H.-Y. Cho, B. S. Han, S. H. Kim, H. O. Kim, Y. T. Lim, and B. H. Chung, Toxicol. Appl. Pharmacol. 236, 16 (2009).

67. L. E. Yu, L.-Y. L. Yung, C.-N. Ong, Y.-L. Tan, K. Suresh Balasubramaniam, D. Hartono, G. Shui, M. R. Wenk, and W.-Y. Ong, Nanotoxicology 1, 235 (2007).

68. S. K. Balasubramanian, J. Jittiwat, J. Manikandan, C.-N. Ong, E. Y. Liya, and W.-Y. Ong, Biomaterials 31, 2034 (2010).

69. G. Sonavane, K. Tomoda, and K. Makino, Colloids Surf. B. Biointerfaces 66, 274 (2008).

70. J. F. Hillyer and R. M. Albrecht, J. Pharm. Sci. 90, 1927 (2001). 71. A. Wolterbeek, L. van der Horst-Groeneveld, M. Tegelenbosch-

Schouten, A. Koster-Ammerlaan, P. Bode, and B. Wolterbeek, Reprod. Toxicol. 30, 247 (2010).

72. X.-D. Zhang, H.-Y. Wu, D. Wu, Y.-Y. Wang, J.-H. Chang, Z.-B. Zhai, A.-M. Meng, P.-X. Liu, L.-A. Zhang, and F.-Y. Fan, Int. J. Nanomedicine 5, 771 (2010).

73. C. Lasagna-Reeves, D. Gonzalez-Romero, M. Barria, I. Olmedo, A. Clos, V. S. Ramanujam, A. Urayama, L. Vergara, M. J. Kogan, and C. Soto, Biochem. Biophys. Res. Commun. 393, 649 (2010).

74. M. Abdelhalim and B. M. Jarrar, Lipids Health Dis. 10, 163 (2011). 75. M. Abdelhalim, Lipids Health Dis. 10, 205 (2011). 76. N. J. Siddiqi, M. Abdelhalim, A. K. El-Ansary, A. S. Alhomida,

and W. Ong, J. Neuroinflammation 9, 123 (2012). 77. X. Ma, Y. Wu, S. Jin, Y. Tian, X. Zhang, Y. Zhao, L. Yu, and X.-J.

Liang, ACS Nano 5, 8629 (2011). 78. Y. Pan, S. Neuss, A. Leifert, M. Fischler, F. Wen, U. Simon,

G. Schmid, W. Brandau, and W. Jahnen-Dechent, Small 3, 1941 (2007).

79. J. J. Li, D. Hartono, C.-N. Ong, B.-H. Bay, and L.-Y. L. Yung, Biomaterials 31, 5996 (2010).

80. J. J. Li, L. Zou, D. Hartono, C. N. Ong, B. H. Bay, and L. Y. L. Yung, Adv. Mater. 20, 138 (2008).

81. T. Mironava, M. Hadjiargyrou, M. Simon, V. Jurukovski, and M. H. Rafailovich, Nanotoxicology 4, 120 (2010).

82. C. L. Villiers, H. Freitas, R. Couderc, M.-B. Villiers, and P. N. Marche, J. Nanopart. Res. 12, 55 (2010).

83. N. M. Schaeublin, L. K. Braydich-Stolle, A. M. Schrand, J. M. Miller, J. Hutchison, J. J. Schlager, and S. M. Hussain, Nanoscale 3, 410 (2011).

84. F. Auzel, Chem. Rev. 104, 139 (2004). 85. H. S. Mader, P. Kele, S. M. Saleh, and O. S. Wolfbeis, Curr. Opin.

Chem. Biol. 14, 582 (2010).

J. Nanosci. Nanotechnol. 16, 7873–7897, 2016 7895

Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application Ahmad et al.

86. C. Wang, H. Tao, L. Cheng, and Z. Liu, Biomaterials 32, 6145 (2011).

87. A. Xia, Y. Gao, J. Zhou, C. Li, T. Yang, D. Wu, L. Wu, and F. Li, Biomaterials 32, 7200 (2011).

88. L. Xiong, T. Yang, Y. Yang, C. Xu, and F. Li, Biomaterials 31, 7078 (2010).

89. A. Rufaihah, E. Sim, L. Ye, and N. Chen, Biocompatibility study of PEI-NaYF4: Yb, Er upconversion nanoparticles, 4th Kuala Lumpur International Conference on Biomedical Engineering 2008, Kuala Lumpur, Malaysia, Springer (2008), p. 82.

90. W. Yin, L. Zhao, L. Zhou, Z. Gu, X. Liu, G. Tian, S. Jin, L. Yan, W. Ren, and G. Xing, Chem. Eur. J. 18, 9239 (2012).

91. J. Chen, C. Guo, M. Wang, L. Huang, L. Wang, C. Mi, J. Li, X. Fang, C. Mao, and S. Xu, J. Mater. Chem. 21, 2632 (2011).

92. J.-C. Zhou, Z.-L. Yang, W. Dong, R.-J. Tang, L.-D. Sun, and C.-H. Yan, Biomaterials 32, 9059 (2011).

93. W. Hu, C. Peng, M. Lv, X. Li, Y. Zhang, N. Chen, C. Fan, and Q. Huang, ACS Nano 5, 3693 (2011).

94. A. Sasidharan, L. Panchakarla, P. Chandran, D. Menon, S. Nair, C. Rao, and M. Koyakutty, Nanoscale 3, 2461 (2011).

95. A. E. Nel, L. Mädler, D. Velegol, T. Xia, E. M. Hoek, P. Somasundaran, F. Klaessig, V. Castranova, and M. Thompson, Nat. Mater. 8, 543 (2009).

96. J. Wörle-Knirsch, K. Pulskamp, and H. Krug, Nano Lett. 6, 1261 (2006).

97. I. Fenoglio, M. Tomatis, D. Lison, J. Muller, A. Fonseca, J. B. Nagy, and B. Fubini, Free Radical Biol. Med. 40, 1227 (2006).

98. V. C. Sanchez, A. Jachak, R. H. Hurt, and A. B. Kane, Chem. Res. Toxicol. 25, 15 (2011).

99. G. Oberdörster, E. Oberdörster, and J. Oberdörster, Environ. Health Perspect. 113, 823 (2005).

100. V. Stone, H. Johnston, and R. P. Schins, Crit. Rev. Toxicol. 39, 613 (2009).

101. P. Nguyen and V. Berry, J. Phys. Chem. Lett. 3, 1024 (2012). 102. A. Schinwald, F. A. Murphy, A. Jones, W. MacNee, and

K. Donaldson, ACS Nano 6, 736 (2012). 103. K. Yang, J. Wan, S. Zhang, Y. Zhang, S.-T. Lee, and Z. Liu, ACS

Nano 5, 516 (2010). 104. M. Wojtoniszak, X. Chen, R. J. Kalenczuk, A. Wajda, J. Łapczuk,

M. Kurzewski, M. Drozdzik, P. K. Chu, and E. Borowiak-Palen, Colloids Surf. B. Biointerfaces 89, 79 (2012).

105. J. T. Robinson, S. M. Tabakman, Y. Liang, H. Wang, H. S. Casalongue, D. Vinh, and H. Dai, J. Am. Chem. Soc. 133, 6825 (2011).

106. G. Gollavelli and Y.-C. Ling, Biomaterials 33, 2532 (2012). 107. S. Zhang, K. Yang, L. Feng, and Z. Liu, Carbon N Y 49, 4040

(2011). 108. K. Wang, J. Ruan, H. Song, J. Zhang, Y. Wo, S. Guo, and D. Cui,

Nanoscale Res. Lett. 6, 1 (2011). 109. X. Zhang, J. Yin, C. Peng, W. Hu, Z. Zhu, W. Li, C. Fan, and

Q. Huang, Carbon N Y 49, 986 (2011). 110. M. C. Duch, G. S. Budinger, Y. T. Liang, S. Soberanes, D. Urich,

S. E. Chiarella, L. A. Campochiaro, A. Gonzalez, N. S. Chandel, and M. C. Hersam, Nano Lett. 11, 5201 (2011).

111. R. Kumar, M. Dhanawat, S. Kumar, B. N. Singh, J. K. Pandit, and V. R. Sinha, Recent. Pat. Drug Deliv. Formul. 8, 12 (2014).

112. Y. Liu, Y. Zhao, B. Sun, and C. Chen, Acc. Chem. Res. 46, 702 (2012).

113. H. Hong, T. Gao, and W. Cai, Nano Today 4, 252 (2009). 114. C. Li, M. Curreli, H. Lin, B. Lei, F. Ishikawa, R. Datar, R. J. Cote,

M. E. Thompson, and C. Zhou, J. Am. Chem. Soc. 127, 12484 (2005).

115. C. Ou, R. Yuan, Y. Chai, M. Tang, R. Chai, and X. He, Anal. Chim. Acta 603, 205 (2007).

116. D. Goldstein, T. Nassar, G. Lambert, J. Kadouche, and S. Benita, J. Controlled Release 108, 418 (2005).

117. S. Dhar, Z. Liu, J. Thomale, H. Dai, and S. J. Lippard, J. Am. Chem. Soc. 130, 11467 (2008).

118. A. Nunes, K. Al-Jamal, T. Nakajima, M. Hariz, and K. Kostarelos, Arch. Toxicol. 86, 1009 (2012).

119. R. Singh, D. Pantarotto, L. Lacerda, G. Pastorin, C. Klumpp, M. Prato, A. Bianco, and K. Kostarelos, Proc. Natl. Acad. Sci. U S A 103, 3357 (2006).

120. A. Eatemadi, H. Daraee, H. Karimkhanloo, M. Kouhi, N. Zarghami, A. Akbarzadeh, M. Abasi, Y. Hanifehpour, and S. W. Joo, Nanoscale Res. Lett. 9, 1 (2014).

121. J. Kayat, V. Gajbhiye, R. K. Tekade, and N. K. Jain, Nanomed. Nanotechnol. Biol. Med. 7, 40 (2011).

122. C. L. Ursini, D. Cavallo, A. M. Fresegna, A. Ciervo, R. Maiello, G. Buresti, S. Casciardi, F. Tombolini, S. Bellucci, and S. Iavicoli, Toxicol. In Vitro 26, 831 (2012).

123. M. Rahman, M. Ahmad, J. Ahmad, J. Firdous, F. Ahmad, G. Mushtaq, M. Kamal, and S. Akhter, Curr. Drug Metab. 16, 397 (2015).

124. L. Meng, A. Jiang, R. Chen, C.-z. Li, L. Wang, Y. Qu, P. Wang, Y. Zhao, and C. Chen, Toxicology 313, 49 (2013).

125. N. R. Jacobsen, P. Moller, K. A. Jensen, U. Vogel, O. Ladefoged, S. Loft, and H. Wallin, Part Fibre Toxicol. 6, 2 (2009).

126. J. Muller, F. Huaux, N. Moreau, P. Misson, J.-F. Heilier, M. Delos, M. Arras, A. Fonseca, J. B. Nagy, and D. Lison, Toxicol. Appl. Pharmacol. 207, 221 (2005).

127. C.-W. Lam, J. T. James, R. McCluskey, and R. L. Hunter, Toxicol. Sci. 77, 126 (2004).

128. C.-C. Chou, H.-Y. Hsiao, Q.-S. Hong, C.-H. Chen, Y.-W. Peng, H.-W. Chen, and P.-C. Yang, Nano Lett. 8, 437 (2008).

129. G. Jia, H. Wang, L. Yan, X. Wang, R. Pei, T. Yan, Y. Zhao, and X. Guo, Environ. Sci. Technol. 39, 1378 (2005).

130. Z. Li, T. Hulderman, R. Salmen, R. Chapman, S. S. Leonard, S.-H. Young, A. Shvedova, M. I. Luster, and P. P. Simeonova, Environ. Health Perspect. 115, 377 (2007).

131. E. Stoker, F. Purser, S. Kwon, Y.-B. Park, and J. S. Lee, Interna- tional Journal of Toxicology 27, 441 (2008).

132. A. A. Shvedova, E. R. Kisin, A. R. Murray, O. Gorelik, S. Arepalli, V. Castranova, S.-H. Young, F. Gao, Y. Y. Tyurina, and T. D. Oury, Toxicol. Appl. Pharmacol. 221, 339 (2007).

133. M. J. Akhtar, M. Ahamed, S. Kumar, H. Siddiqui, G. Patil, M. Ashquin, and I. Ahmad, Toxicology 276, 95 (2010).

134. M. Cho, W.-S. Cho, M. Choi, S. J. Kim, B. S. Han, S. H. Kim, H. O. Kim, Y. Y. Sheen, and J. Jeong, Toxicol. Lett. 189, 177 (2009).

135. J. Lu, M. Liong, Z. Li, J. I. Zink, and F. Tamanoi, Small 6, 1794 (2010).

136. C. Hom, J. Lu, and F. Tamanoi, J. Mater. Chem. 19, 6308 (2009). 137. L. Tang and J. Cheng, Nano Today 8, 290 (2013). 138. H. Yang, C. Liu, D. Yang, H. Zhang, and Z. Xi, J. Appl. Toxicol.

29, 69 (2009). 139. M. Chen and A. von Mikecz, Exp. Cell Res. 305, 51 (2005). 140. D. Lison, L. C. Thomassen, V. Rabolli, L. Gonzalez, D. Napierska,

J. W. Seo, M. Kirsch-Volders, P. Hoet, C. E. Kirschhock, and J. A. Martens, Toxicol. Sci. 104, 155 (2008).

141. N. T. Vo, M. R. Bufalino, K. D. Hartlen, V. Kitaev, and L. E. Lee, In Vitro Cellular and Developmental Biology-Animal 50, 427 (2014).

142. D. Napierska, L. C. Thomassen, V. Rabolli, D. Lison, L. Gonzalez, M. Kirsch-Volders, J. A. Martens, and P. H. Hoet, Small 5, 846 (2009).

143. Y.-H. Park, J. N. Kim, S. H. Jeong, J. E. Choi, S.-H. Lee, B. H. Choi, J. P. Lee, K. H. Sohn, K. L. Park, and M.-K. Kim, Toxicology 267, 178 (2010).

144. H. Nabeshi, T. Yoshikawa, K. Matsuyama, Y. Nakazato, A. Arimori, M. Isobe, S. Tochigi, S. Kondoh, T. Hirai, and T. Akase, Die Pharmazie-An International Journal of Pharmaceu- tical Sciences 65, 199 (2010).

7896 J. Nanosci. Nanotechnol. 16, 7873–7897, 2016

Ahmad et al. Toxicity of Inorganic Nanoparticles Used in Targeted Drug Delivery and Other Biomedical Application

145. T. Yu, A. Malugin, and H. Ghandehari, ACS Nano 5, 5717 (2011).

146. H. Liang, C. Jin, Y. Tang, F. Wang, C. Ma, and Y. Yang, J. Appl. Toxicol. 34, 367 (2014).

147. G. Xie, J. Sun, G. Zhong, L. Shi, and D. Zhang, Arch. Toxicol. 84, 183 (2010).

148. R. Hassankhani, M. Esmaeillou, A. A. Tehrani, K. Nasirzadeh, F. Khadir, and H. Maadi, Environ. Sci. Pollut. R 22, 1127 (2015).

149. T. Skuland, J. Øvrevik, M. Låg, P. Schwarze, and M. Refsnes, Toxicol. Appl. Pharmacol. 279, 76 (2014).

150. C. Nagakura, Y. Negishi, M. Tsukimoto, S. Itou, T. Kondo, K. Takeda, and S. Kojima, Toxicology 322, 61 (2014).

151. X. Yang, C. E. He, J. Li, H. Chen, Q. Ma, X. Sui, S. Tian, M. Ying, Q. Zhang, and Y. Luo, Toxicol. Lett. 229, 240 (2014).

152. Y. Xu, N. Wang, Y. Yu, Y. Li, Y.-B. Li, Y.-B. Yu, X.-Q. Zhou, and Z.-W. Sun, PloS One 9, e101572 (2014).

153. J. Duan, Y. Yu, H. Shi, L. Tian, C. Guo, P. Huang, X. Zhou, S. Peng, and Z. Sun, PloS One 8, e74606 (2013).

154. S. Lanone and J. Boczkowski, Curr. Mol. Med. 6, 651 (2006). 155. K. L. Aillon, Y. Xie, N. El-Gendy, C. J. Berkland, and M. L.

Forrest, Adv. Drug Del. Rev. 61, 457 (2009). 156. M. A. Voinov, J. O. S. Pagán, E. Morrison, T. I. Smirnova, and

A. I. Smirnov, J. Am. Chem. Soc. 133, 35 (2010). 157. T. Zhang, L. Qian, M. Tang, Y. Xue, L. Kong, S. Zhang, and Y. Pu,

J. Nanosci. Nanotechnol. 12, 2866 (2012). 158. M.-T. Zhu, Y. Wang, W.-Y. Feng, B. Wang, M. Wang, H. Ouyang,

and Z.-F. Chai, J. Nanosci. Nanotechnol. 10, 8584 (2010). 159. S. Tedesco, H. Doyle, G. Redmond, and D. Sheehan, Mar. Environ.

Res. 66, 131 (2008). 160. X. Wu, Y. Tan, H. Mao, and M. Zhang, Int. J. Nanomedicine 5, 385

(2010). 161. K. Buyukhatipoglu and A. M. Clyne, J. Biomed. Mater. Res. A

96, 186 (2011).

162. H. K. Patra, S. Banerjee, U. Chaudhuri, P. Lahiri, and A. K. Dasgupta, Nanomed. Nanotechnol. Biol. Med. 3, 111 (2007).

163. S. Akhter, I. Ahmad, M. Z. Ahmad, F. Ramazani, A. Singh, Z. Rahman, F. J. Ahmad, G. Storm, and R. J. Kok, Nanomedicines as Cancer Therapeutics: Current Status. Curr. Cancer Drug Tar- gets 13, 362 (2013).

164. J. Ahmad, S. Akhter, M. Rizwanullah, S. Amin, M. Rahman, M. Z. Ahmad, M. A. Rizvi, M. A. Kamal, and F. J. Ahmad, Nanotechnol. Sci. Appl. 8, 55 (2015).

165. S. K. Gupta, L. Baweja, D. Gurbani, A. K. Pandey, and A. Dhawan, Journal of Biomedical Nanotechnology 7, 179 (2011).

166. Z. Magdolenova, A. Collins, A. Kumar, A. Dhawan, V. Stone, and M. Dusinska, Nanotoxicology 8, 233 (2014).

167. S. Huang, P. J. Chueh, Y.-W. Lin, T.-S. Shih, and S.-M. Chuang, Toxicol. Appl. Pharmacol. 241, 182 (2009).

168. M. S. Cooke, M. D. Evans, M. Dizdaroglu, and J. Lunec, The FASEB Journal 17, 1195 (2003).

169. M. Kruszewski, K. Brzoska, G. Brunborg, N. Asare, M.-G. Dobrzynska, M. Dusinska, L. Fjellsbø, A. Georgantzopoulou, J. Gromadzka, and A. C. Gutleb, Advances in Molecular Toxicology 5, 179 (2011).

170. P. AshaRani, G. Low Kah Mun, M. P. Hande, and S. Valiyaveettil, ACS Nano 3, 279 (2008).

171. R. Baktur, H. Patel, and S. Kwon, Toxicology In Vitro 25, 1153 (2011).

172. J. G. Rouse, J. Yang, A. R. Barron, and N. A. Monteiro-Riviere, Toxicology In Vitro 20, 1313 (2006).

173. M. Turabekova, B. Rasulev, M. Theodore, J. Jackman, D. Leszczynska, and J. Leszczynski, Nanoscale 6, 3488 (2014).

174. A. Manke, L. Wang, and Y. Rojanasakul, BioMed. Research Inter- national 2013, 15 (2013).

175. X. Liu and J. Sun, Biomaterials 31, 8198 (2010). 176. E.-J. Park, J. Yi, K.-H. Chung, D.-Y. Ryu, J. Choi, and K. Park,

Toxicol. Lett. 180, 222 (2008).

Received: 30 December 2015. Accepted: 24 March 2016.

J. Nanosci. Nanotechnol. 16, 7873–7897, 2016 7897

View publication statsView publication stats

https://www.researchgate.net/publication/305732091