Biochemistry and Biophysics Reports 13 (2018) 63–72

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Biochemistry and Biophysics Reports

journal homepage: www.elsevier.com/locate/bbrep

Comprehensive cytotoxicity studies of superparamagnetic iron oxide T

nanoparticles
Rakesh M. Patila,c, Nanasaheb D. Thoratb, Prajkta B. Shetec, Poonam A. Bedged,
⁎
Shambala Gavdec, Meghnad G. Joshid, Syed A.M. Tofailb, Raghvendra A. Boharac,d,e,
a
Directorate of Forensic Science Laboratory, Govt. of Maharashtra Kalina, Mumbai, India
b
Material and Surface Science Institute, Bernal Institute, University of Limerick, Ireland
c
Centre for Interdisciplinary Research, D.Y.Patil University, Kolhapur, India
d
Department of Stem Cells and Regenerative Medicine, D.Y.Patil University, Kolhapur, India
e
Research and Innovations for Comprehensive Health (RICH), Cell D.Y.Patil University, Kolhapur, India

A R T I C L E I N F O A B S T R A C T

Keywords: Recently lots of efforts have been taken to develop superparamagnetic iron oxide nanoparticles (SPIONs) for
SPIONs biomedical applications. So it is utmost necessary to have in depth knowledge of the toxicity occurred by this
Toxicity material. This article is designed in such way that it covers all the associated toxicity issues of SPIONs. It mainly
Cellular alteration emphasis on toxicity occurred at different levels including cellular alterations in the form of damage to nucleic
Biomedical applications
acids due to oxidative stress and altered cellular response. In addition focus is been devoted for in vitro and in
vivo toxicity of SPIONs, so that a better therapeutics can be designed. At the end the time dependent nature of
toxicity and its ultimate faith inside the body is being discussed.

1. Introduction these studies include a wide range of nanoparticle concentrations and

exposure time [11,12].
Superparamagnetic iron oxide nanoparticles (SPIONs) have been In addition, various researchers used different cell lines with
found promising candidate in nanobiotechnology for wide range of varying culturing conditions which made things more difficult, as direct
applications such as magnetic separation, drug delivery, magnetic re- comparisons between the available studies and their own results are not
sonance imaging (MRI) and magnetic hyperthermia (MH) [1–4]. Most validated. It is to be note that while working on SPIONs, the reported
importantly the site-specific drug and diagnostics agent delivery by toxicity taken into consideration includes, inflammation, diminished
using SPIONs is the most exciting applications in cancer theranostics mitochondrial activity, the cellular stress mediated generation of re-
[5,6]. The wide ranges of potential bio-applications of SPIONs are in- active oxygen species (ROS) and chromosome condensation [13–18].
fluenced by its physical, chemical, and magnetic properties along with This article is designed in such way that it covers all the associated
its shape and size. The toxicity of SPIONs towards normal cells are toxicity issues of SPIONs. SPIONs are manufactured in higher quantities
hindering its successful implication as therapeutic agent. High degree of in order to meet the demands for rapidly growing field of nanomedicine
nonspecific binding to cell components and biological fluids by SPIONs for biomedical applications. But exposure to human body and eco-
as well as colloidal instability of SPIONs during their delivery into system needs to address. This review mainly aims to collect the tox-
biological media are the main cause of the toxicity [7]. The response of icological in vitro and in vivo data along with major adverse effects of
these particles to living system both in terms of acute and chronic SPIONs [19]
toxicity is main concern in terms of clinical activity [8]. Moreover the
degradation and it's accumulation inside the body of this nanoparticles 2. Why toxicity study of SPIONs?
following administration is very important point of study. Currently the
most trusted and easiest approach to study the In vitro cytotoxicity SPIONs are the most preferred candidate in biomedical applications
studies of nanoparticle is by using different cell lines varying their in- for diagnostics and therapeutics. Many in vivo toxicity appliances of
cubation times and evaluating by colorimetric assays [9,10]. This ap- SPIONs are needed in most of biomedical applications. Hence it is im-
proach has gained lots of publicity. However, the main drawbacks of portant to study the overall toxicity associated with them. SPIONs are

⁎
Correspondence to: D.Y.Patil University, Kolhapur, India.
E-mail address: [email protected] (R.A. Bohara).

https://doi.org/10.1016/j.bbrep.2017.12.002
Received 14 July 2017; Received in revised form 7 December 2017; Accepted 11 December 2017
Available online 08 January 2018
2405-5808/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
R.M. Patil et al. Biochemistry and Biophysics Reports 13 (2018) 63–72

very small in size, comparable with the biomolecules. Such a small size
can cause sequestration of these moieties into various body systems and
can interfere with their normal functioning. They might cross blood-
brain barrier and damage neural functions, also can cross nuclear
membrane and cause mutations. The bare SPIONs have very low solu-
bility which can lead to agglomeration which can obstruct blood vessels
[11].
SPION are coated with a suitable biocompatible material for in-
crease in stability, water dispersibility and biocompatibility.

3. In vitro toxicity studies of SPIONs

In order to confirm the toxicity, different assays are available. Each

assay is based on some different principle, for more accurate results it is
recommended to carry multiple assay for same samples. Some of the
widely used assay are lactate dehydrogenases assay (LDH), Fig. 1. Schematic representation of possible mechanism of SPIONs interaction and
Sulphorhodamine B (SRB) assay, protein assay, neutral red, and 3-(4,5- SPIONs-induced toxicity at cellular level.
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
from the cell. The cellular antioxidant enzymes and other redox mole-
3.1. In vitro assays for cytotoxicity studies of SPIONs cules take care of excessive ROS and counterbalance ROS generated in
the cell [32].
MTT assay is a widely accepted, non-radioactive, colorimetric based
assay [20,21]. MTT is derivative of a tetrazolium salt, which is con-
verted into purple formazan insoluble complex by enzyme within the 3.2. Mechanism associated with in vitro toxicity of SPIONs
mitochondrial dehydrogenases [22]. Recent reports suggest that that
reduction of MTT can also be facilitated by NADH or NADPH within the The most beautiful features of SPIONs is they can be easily attracted
cells and also outside of mitochondria [22]. Therefore further mod- and manipulated by using external magnetic field and in addition the
ification of the initial protocol by Mossmann was proposed [23,24] in superparamagnetic properties, enables them to work as magnetic
order to increase the repeatability and the sensitivity of the assay. Only switches. In addition the least toxic effect shown on human body has
active mitochondria contain these enzymes; therefore, the reaction only attracted researcher to explore this system for maximum biomedical
occurs in living cells [25]. applications [33,34].
The neutral red uptake assay is based on the ability of viable cells to Fig. 1 represents the possible mechanism of SPIONs interaction with
incorporate and bind the supra vital dye neutral red. This assay is cell and toxicity at cellular level. The figure suggests that SPION can
widely used cytotoxicity assay used for biomedical and environmental interact with cell by different mechanisms. The prominent one are, a)
applications. The principle behind this is the weak cationic dye pene- passive diffusion b) Receptor mediated endocytosis c) clatharin medi-
trates cell membranes by the mechanism of nonionic passive diffusion ated endocytosis d) and caveoline mediated endocytosis. After entering
and concentrates in the lysosomes. inside the cell SPION are degraded by enzymes present in lysosomes
The dye binds to lysosomal matrix by electrostatic interaction, and breaks the assembly to form ions. This Fe + 2 ions generates re-
which is then extracted from the viable cells by using an acidified active oxygen species (ROS) by altering mitochondrial and other or-
ethanol solution, and the absorbance of the solubilized dye is quantified ganelle functions and induction of cell signalling pathways which leads
using a spectrophotometer [26]. to activation of inflammatory tells [35,36]. Possible mechanism of
Another important assay commonly used is, LDH leakage assay SPIONs interaction and SPIONs-induced toxicity at cellular level is
which is based on the measurement of lactate dehydrogenase activity in shown in Fig. 1.
the extracellular medium. The silent features like reliability, speed, and
simple evaluation are the major strengths of this assay [27]. 3.2.1. SPION associated plasma membrane toxicity
The most widely used assay for viability study is the trypan blue. The SPION also shows toxicity by damaging the plasma membrane
The assay is simple method of determining cellular viability [28]. In and proteins. In addition to induction of cell signalling pathways,
this the cells are sedimented onto slides and fixed in a mixture of trypan SPION can stimulates the redox reactions and up regulate plasma
blue and paraformaldehyde. The nonviable cells a stain with dark blue membrane proteins which results in the generation of cellular stress and
color, whereas viable cells exclude the dye [29]. The major concern ultimately cell death [37,38].
with trypan blue assay is its difficulty to interpret because of staining It is observed that the toxicity assay based upon mitochondrial
artefacts. functionality (e.g., MTT and XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfo-
A number of techniques for detecting DNA damage (e.g. micro- phenyl)-2H-tetrazolium-5-carboxanilide)), which are based upon re-
nuclei, mutations, structural chromosomal aberrations) have been used ductase enzyme may show large errors [39]. The reason behind this is
to identify substances with genotoxic activity. The comet assay, also the redox active surface of SPIONs could widely impact electron flow
known as single-cell gel electrophoresis (SCGE), is so named because and change the mitochondrial functionality [40–42]. The study done by
damaged cells form a comet-shaped pattern after electrophoresis. It is a Jeng and Swanson [16] showed that SPIONs had a major effect upon
sensitive method to measure genotoxicity and cytotoxicity of chemical mitochondrial function and maximum concentration tested was
and physical agents. The comet assay has also been used to analyse the ([Fe] ≈ 2.5 mM) at this concentration there was statistically significant
capacity of cellular DNA repair [30]. change in the mitochondrial function. In another study done by Au
Continues metabolic process produces reactive oxygen species et al. [40] similar results were observed and the authors have con-
(ROS) such as superoxide and hydrogen peroxide. ROS generation is cluded that SPION alters mitochondrial function as well as decreased
normally counterbalanced by the action of antioxidant enzymes and cell viability.
other redox molecules. However, higher levels of ROS can lead to cel- The study lead by the Stroh et al. [14] confirmed that citrate-coated
lular injury and may damage biomolecules such as DNA, lipids and SPIONs results in a substantial increase in protein oxidation and oxi-
proteins [31]. This excess reactive oxygen species should be eliminated dative stress [14]. The study also concluded that iron was the source to

64
 Table 1
A brief account of in vitro toxicity of SPIONs (bare as well as coated) on different cell types using different cytotoxic assays Adapted from Ref. [58].

a b
Organ Cell type Coating material on SPIONs Assay used Concentration of Exposure time Observation Refs.
R.M. Patil et al.

SPIONs (h)

CNS astrocytes (human Nerve cells) – MTS and LDH 10 μg/mL 6 significantly (p < 0.01) increased MTS production revealed [40]
alteration in mitochondrial function
Schwann cell Dextran dyes (PI) up to 4 mg/mL 48 No change in cell viability [59]
Glioma tetramethyla mmonium11- 0.1–100 μg/mL 24 concentration dependent toxicity [60]
aminoundecanoate
GL261 (mouse brain) Dextran MTT 1–200 μg/mL 24 Higher toxicity was exhibited as compared to bare one [61]

Liver BRL 3A (rat) MTT 0–250 μg/mL 24 h concentration dependent and 50% decrease in viability at 250 μg/ [12]
mL
BRL 3A (rat) LDH 0–250 μg/mL 24 toxic effect at 250 μg/mL was reported [15]
HepG2 (human) Baavi-b USPIO MTT no indication of cytotoxicity [62]
HepG2 (human) amino-surface MTT 0.03 μg/mL to 3 mg/ 5 days LD50 of Gal-ASPIO-278 = 1500 μg/mL [63]
mL
HepG2 (human) amine-surface Cytochrome C 0.03–3000 μg/mL 4 h to 5 days The toxicity is associated with the zeta potential of NPs [63]
SMMC-7721 (human hepatocellular) Chitosan MTT 0–123.52 μg/mL Bare MNPs showed decreased cell viability as compared to coated [64]
one

Pancreas human islet Dextran dyes (PI) 280 μg/mL viability of labelled islets were similar to the control islets [65]
Kidney Cos-7 (monkey) MTT 0.2–23.05 Mm no toxicity detected [66]
Skin dermal fibroblasts (human) PEG, insulin MTT 0–1 mg/mL 24 h 25–50% decrease in viability for bare particles (250 μg/mL); [67,68]
99% viability for PEG-coated (1 mg/mL)
2
HEK Dextran MTT, alamar blue 0–26 μg/cm 24 h Size depended toxicity has been seen. 20 nm particles had shown [69]
a decrease in cell viability, while the 15 and 50 nm particles were
not cytotoxic.
Murine epidermal cells (JB6 P+) Dextran MTT, alamar blue 0–26 μg/cm2 24 h activation of AP-1, 5% reduction in cell viability at the highest [69]

65
dose evaluated (26 μg/cm2)
dermal fibroblasts (human) sodium oleate MTT 0–1000 μg/mL 24 h bare SPIONs shown disrupted cytoskeleton [42]
Lactoferrin or ceruloplasmin coated SPIONs attached to the cell
membrane
hTERT-BJ1 (human) dextran and albumin- derivatized dyes (BrdU) 0.05 mg/mL 24–72 h Albumin-coated particles shown more cell viability as compared [70]
to bare and dextran coated
L929 (mouse) PVA dyes (crystal 800 mM 72 h confirmed the presence of gas vesicles inside Cells [71,72]
violet)
L929 (mouse) PVA MTT 0.2 mM 24 h morphology and size dependent toxicity [73]
L929 (mouse) PEGF and PVA MTT 0.4–1.6 M 24–72 h morphology and size dependent toxicity [71,74]
L929 (mouse) PEGF dyes (NR) 800 mM 24–72 h concentration@800 mM did not change the cell shapes notably [74]
and cells appeared not to be damaged
L929 (mouse) PAA MTT 48 h No observable toxicity was found [55]
L929 (mouse) Chitosan MTT 48 h No observable toxicity was found [20]
L929 (mouse) Chitosan/Glutar-aldehyde MTT 24 h No observable toxicity was found [75]
L929 (mouse) Oleic acid/betain HCl MTT 24 h No observable toxicity was found [76]
3T3 (mouse) MTT 0–30 ppm 72 h no significant difference in the toxicity [77]
HS68 (human foreskin) ethylene glycol MTT 1 mg/mL 24 h no significant difference in the viability of Cells [78]
Melanoma (human) PVA and vinyl alcohol/vinyl amine MTT 12, 61, and 123 μg/mL 2 and 24 h polymer alone (was more toxic than polymer-coated SPIONs; [79]
copolymer
SK-MEL-37 (human melanoma) DMSA, citric acid or lauric acid MTT up to 840 μg/mL cell viability decreased in a dose-dependent manner [80]
HaCaT MTT 0.01–100 mg/mL 24 h cell viability decreased in a dose-dependent manner [81]

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Biochemistry and Biophysics Reports 13 (2018) 63–72
 Table 1 (continued)

a b
Organ Cell type Coating material on SPIONs Assay used Concentration of Exposure time Observation Refs.
R.M. Patil et al.

SPIONs (h)

Blood J774 (murine) Tween 80 MTT 25–500 μg/mL 1–6 h Enhanced ROS generation, leading to cell injury and death; [82]
concentration- and time- dependent damage
macrophages(human) dextran MTS and dyes 100 μg/mL 7 days 20% of macrophages were viable after 7 days [83]
(BrdU)
Mouse WST-1 higher degree of necrosis due to rod shaped [84]
macrophage cells (RAW264.7) LDH Fe2O3was in correlation with both the higher degree of membrane [84]
damage and ROS Production
human monocyte macrophage dextran MTT and NBT 1 and 10 mg/mL up to 14 days only mildly toxic at the highest applied dosage (i.e., particle [85]
concentration of 10 mg/mL)
K562 (human leukemia) Tetraheptyl- ammonium MTT 2.5 μg/mL 72 h inhibition rate = 46% for the cell system incubated with Fe3O4- [86]
PLA
K562 and K562/A02 (human ADM conjugated MTT 20 μg/mL to 5 mg/mL 48 h cell proliferation significantly (P < 0.001) [87]
leukemia)
T lymphocyte cell line (rat) scAbCD3 Tetrazolium 0.15 μg 48 h no detectable toxicity [88]

Muscles A10 (rat) polylactide Redox 10–50 μg/mL 72 h no detectable toxicity [89]
Lung A549 (human) dyes (TB) and ROS up to 80 μg/mL 18 h no or low toxicity [90]
A549 (human) Comet up to 80 μg/mL 4h oxidative DNA lesions in cultured A549 cells after exposure to [90]
40 μg/mL and 80 μg/mL SPIONs
A549 (human) silica MTT 4 mg/mL IC50 = 4 mg/mL [91]
H441 (human) PEI MTT 90 μg/mL 24–48 h Toxicity of tested complexes was acceptable (cell [92]
viability > 80%)
LLC (mouse) poly(TMSM A-r- PEGMA) MTT 1–100 μg/105 cells 12 h no indication of toxicity [93]

Mesenchyma MSC (human) PLL MTT 1–43 days long-term viability, and apoptotic indices were unaffected [94]

66
MSC (human) comet 50–250 μg/mL 24–72 H Did not affect the apoptosis [95]
rMSC (rat) and MSC (human) PDMA Tetrazolium 15 μg γ-Fe2O3/mL 24 h The viability with coating was more as compare to bare [96]
rMSC (rat) HEDP MTS 25, 50, and 100 μg 48 h Concentration dependent toxicity [97]
iron/mL

Heart BAECs redox 90 μg/mL 24 h cell viability was not adversely affected by internalized SPIONs; [98]
Breast B16/DNS and B16/phOx (mouse DNS hapten covalently attached to ATP 100 mM 48 h DNS-CLIO was nontoxic to B16/DNS (DNS receptor positive) and [99]
breast) CLIO B16/phOx (control receptor positive) cells
MCF-7 (human breast) dextrane and phosphatidyl choline/ MTT 100 μg/mL 1–3 days presence of SPIONs in culture medium led to [100]
cholesterol alterations in mitochondria ultrastructural
organization and decrease of oxygen uptake by
mitochondria in sensitive and anticancer drugs resistant cells
H184B5F5/M10, SKBR3 (normal MTS 0.1, 1, 10, and 100 μM 72 h no observable change in cell viability [101]
breast), MB157, and T47D (human
breast cancer)
B16F10 (mouse breast) CMC XTT 1–5 mM 24 h after 48 h, cell viability was reduced (81%) at [102]
concentrations > 1 mM

Prostate glands PC3 (human prostate) TCL-SPIONs MTT 0.1 mg/mL 48 h cytotoxicity was comparable to free Dox [103]
Cervix HeLa (human cervical) PLL MTT 1–43 days long-term viability, growth rate, and apoptotic [94]
indices of the labelled cells were unaffected by
the endosomal incorporation of SPIONs
HeLa (human cervical) dextran, amino-dextran, heparin, MTT 0.05–0.5 mg/mL 24 h viability of cell culture was not significantly Affected [104]
and dimer-captosuccinic acid
KB (human carcinoma) PAMAM and G3 XTT 0–80 mg/mL 4 days dendrimer-stabilized SPIONs did not display cytotoxicity to KB [64]
cells in the predetermined concentration range

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R.M. Patil et al. Biochemistry and Biophysics Reports 13 (2018) 63–72

DNS, mouse melanoma cells with DNS receptor positive; B16/phOx, mouse melanoma cells with control receptor positive; SMMC-7721, human hepatocellular carcinoma cells; PC3, human prostate cancer cells; A2780, human ovarian cancer cells;

Abbreviation of coatings: Baavi-bUSPIO, (Avidin-coated baculoviral vectors-biotinylated ultra-small superparamagnetic iron oxide nanoparticles); PEG, poly(ethylene glycol); PEGF, poly(ethylene glycol-co-fumarate); PLL, poly(L-lysine); PVA,
cells; Cos-7, obtained by immortalizing a CV-1 cell line derived from kidney cells of the African green monkey; OCTY, mouse kidney cells; J774, murine macrophage cells; Schwann, principal glia of the peripheral nervous system; BAECs, bovine

linked iron oxide; PDMA, poly(N,Ndimethylacrylamide); scAbCD3, nonviral gene delivery agent bearing CD3 single chain antibody. c Abbreviation of toxicity methods: MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide); MTS,
Abbreviations of cell types: hTERT-BJ1, Infinity Telomerase Immortalized primary human fibroblasts; L929, mouse fibroblast cell; rodent 3T3, Swiss mouse fibroblast cells; HS68, human foreskin fibroblast cells; HEK, normal human epidermal
keratinocytes; A549, human lung adenocarcinoma epithelial cells; H441, human lung adenocarcinoma epithelial cells; BRL 3A, rat liver cells; HepG2, human liver hepatocellular cells; MSCs, mesenchymal stem cells; rMSCs: rat mesenchymal stem

aortic endothelial cells; HeLa, cervical cancer cells; LLC, mouse Lewis lung carcinoma; GL261, mouse brain tumor cells; K562, human immortalized myelogenousleukemia cells; k562/A02, human leukemiacells; B16, mouse melanoma cells; B16/

oxypoly(ethylene glycol)-oligo(aspartic acid)); WSC, water-soluble chitosan; LA, linoleic acid; PAMAM, dendrimer-stabilized (carboxyl-functionalized poly(amidoamine); G3, dendrimers of generation 3; CMC, carboxymethylCurdlan; CLIO, cross-

(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); XTT, (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide); BrdU, bromodeoxyuridine; LDH, lactate dehydrogenase; ATP, ade-
poly(vinyl alcohol); PEI, polyethyleneimine; ADM, adriamycin; TCL-SPIONs, thermally cross-linked SPIONs; DMSA, meso-2,3-dimercaptosuccinic acid; HEDP, 1-hydroxyethylidene-1.1-bisphosphonic acid; PAA, poly(acrylic acid); MPEG, meth-
MCF-7, human breast cancer cells; SK-MEL-37, human melanoma cells; KB, human epithelial carcinoma cells; H184B5F5/M10, normal breast epithelial cells; B16F10, SKBR3, MB157, and T47D, three types of breast cancer cells; MSTO-211H,
Refs. generate the reactive oxygen species (ROS). This was supported by a

[77]
[85]
dramatic reduction in these levels of ROS via co-administration of an
iron chelator.
not toxic at particle concentration of 1 mg/mL and mildly toxic at Van den Bos et al. [43] also reported a study in which he used
dextran coated SPIONs in dose-dependent manner. It was observed that
there was increase in lipid peroxidation with simultaneous increase in
dose [43]. The key factor for generation of ROS was ferritin which was
reported in rat synaptosomes and which lead to neurodegeneration in
particle concentration of 10 mg/mL after 72 h

vivo [44].
It is also observed that surface coating has particular effect at the
same time the length of a coating can play a significant role and it is
seen that it bear a negative correlation with toxicity [17]. At the same
Concentration depended toxicity

time longer tails coated SPION may undergo degradation into shorter
tails within the intracellular environment and cause toxicity.
The SPIONs being in nanometre size can easily enter into the nu-
clear membrane and may cause damage to DNA and which may results
in generation of ROS. In addition the released ROS further causes da-
mage to nucleic acid and at high concentration may lead to breaking of
Observation

hydrogen bonding in DNA structure.

Damage or injury to cytoskeletal structure is very important area of
research. The toxicity created by SPION needs to confirm, as these fi-
laments are essential element in maintaining cellular and structural
Exposure time

morphology. The study suggests that high doses of SPION lead to in-
24, 48, 72 h

terference with the actin cytoskeleton resulting in decreased cell pro-

liferation [45]. The study done by Soenen et al. clearly shown that
72 h
(h)

human lung mesothelioma cells; HMMs, human malignant mesothelioma cells; HaCaT, human keratinocyte cells; A10, rat aortic smooth muscle cells.

SPION encapsulated in liposomes also called magntoliposomes shown

direct effect on actin cytoskeleton architecture and which leads to
formation of focal adhesion complexes and cell has shown decreased
Concentration of

proliferation ability. The study also reveals that the effect was reversal
and took 7 days to return to normal [45]. Disruption of a cytoskeleton
0–10 mg/mL
0–30 ppm

protein, tubulin, and dynamic cortical meshwork of F-actin are some

SPIONs

other reported effects of SPION [46–48]. Resovit is commercially

available MRI agent formulated with carboxy dextran coated SPION.
When pancreatic islet cells labelled with Resovist, there was increasing
expression in insulin levels [49]. In another study of Resovist on me-
senchymal stem cells showed amplified cellular growth and cell cycle
Assay used

progression. This was accompanied by alterations in the expression of

cell cycle regulatory proteins [50].
MTT
MTT

nosine triphosphate; NBT, Nitrobluetetrazolium; WST, water-soluble tetrazolium; PI, propidium iodide.

Primary human fibroblasts (hTERT-BJ1) cell line shown increase in

cell proliferation in response to transferrin-coated SPIONs [46].
Recent invitro studies have shown the effect of SPION on macro-
Coating material on SPIONs

phages. The study revealed that there was change in cellular behaviours
with cytokine expression. In addition there was increased expression of
IL-1, 4, and 10, TNF- α and inhibition of tumor necrosis factor-α (TNF-
α) which suggest the potential effect on immuno modulatory cap-
abilities [51–53].
Our group has also studied rigorously on in vitro cytotoxicity as-
dextran

sociated with different ferrite and other MNPs such as Fe3O4, CoFe2O4,
Ni-ZnFe2O4, ZnFe2O4 nanoparticles with different coating materials
b

using MTT and trypan blue assays on different cell lines, both cancerous
and normal cell lines [54–57].
Table 1: A brief account of in vitro toxicity of SPIONs (bare as well
as coated) on different cell types using different cytotoxic assays is
discussed in detail.
MSTO-211H (human)

4. In vivo toxicity studies of SPIONs

HMMs (human)

4.1. Mechanism associated with in vivo toxicity of SPIONs

Cell type

The SPIONs are aggregated in a particular tissue by using a magnet

a

for maximum effects for therapy or diagnosis application, which can

Table 1 (continued)

leads to high concentrations in that area [105]. Now this may lead to
high levels of free Fe ions in the exposed tissue which may lead to
cellular damage which can lead to or have a significant impact on fu-
Cancer
Organ

ture generations if the fidelity of the genome in germ cells is not

b
a

maintained [106–108]. It also to be note that iron has been associated

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R.M. Patil et al. Biochemistry and Biophysics Reports 13 (2018) 63–72

with cancer different researchers has explained various mechanisms for proteins [124]. Spindle cell sarcoma and pleomorphic sarcoma in rats
these effects [109,110]. was reported after I/M exposure of iron-dextran complex [125]. Ex-
The physical and chemical characteristics of SPIONs are considered pression of hepcidin was observed in iron-overload in vivo [126–128].
as crucial factors to determine pharmacokinetics, toxicity and bio dis-
tribution of magnetic nanoparticles [57]. Till date very few studies are 5. Fate of SPIONs
available on humans which can discuss the detail property of SPION.
One such study is done on Ferumoxtran-10, which is a dextran-coated In the literature, most of work was carried out to study the toxic
USPIO (ultra-small SPIONs). It has seen that this NPs have shown to effects of SPIONs but a very less data was available on the final desti-
induce the transient effects including urticaria, diarrhoea and nausea nation of SPIONs after exposure in vitro or after administration in vivo.
[111,112]. The same system when it was exposed as commercial con- It is a prime importance to study the clearance or use of SPIONs after
trast agent in living system, adverse events from USPIO were reversible exposure to body for a particular therapy application such as in drug
and diminish with the time [113]. delivery, MRI and hyperthermia.
Chertok et al. [114] checked the possibility of SPIONs as a drug
delivery vehicle for magnetic targeting of brain tumors. Animals were 5.1. Fate of SPIONs in vitro
intravenously injected with nanoparticles (12 mg Fe/kg), no observable
toxicity was found. Pradhan et al. [115] found no significant changes in In vitro studies suggested that SPIONs are avidly taken up by fi-
haematological and biochemical parameters and suggested that the broblasts, macrophages and tumor cells. The surface property of the
high dose had raised the Serum glutamic pyruvic transaminase (SGPT) SPION has greater impression on the uptake inside the cell. For ex-
levels suggesting the hepatic toxicity while the detail histopathological ample, the system of carboxydextran-coated SPIONs of size ranging less
images suggested that there was no morphological changes was noted. than dextran-coated SPIONs had shown the higher percentage inter-
The study done by Lübbe et al. [116] developed a stable nanome- nalization inside the macrophage cell, but this uptake is not associated
dicine of magnetic nature and to which different molecules of drugs, with cell activation as no interleukine-1 release is observed [129].
cytokines and other molecules are chemically attached and directed Muller et al. [130] hypothesized that the cell toxicity was only con-
inside the cells through magnetic field. Various concentrations of the ferred after internalization into the cells [130]. Furthermore, Muller
magnetic fluid were tested in rats and immunosuppressed nude mice. et al. confirmed particle internalization into the granulocytes by la-
As a result, the Ferro-fluid did not cause major laboratory abnormal- beling the particles with luminal, a chemiluminescent dye, which nicely
ities. Hu et al. [117] coupled PEG-coated Fe3O4 nanocrystals with a correlate with intracellular iron uptake [85].
cancer-targeting antibody, rch 24 mAb as a MRI contrasting agent.
After completion of successful invitro cell line study the assembly was 5.2. Fate of SPIONs in vivo
used for in vivo experiments for identification of human colon carci-
noma After the experiment the nude mice recover anaesthesia and lived SPION once administered, the fate inside the body is dependent on
normally for weeks, which demonstrates that the bioconjugates have no various parameters which include size, shape, and most important
acute fatal toxicity. coating done on the surface of the particle. One study has reported that
initially the SPION once administered, enters into liver and spleen
4.2. Genotoxicity [131,132]. The system developed of oleic acid/pluronic-coated SPIONs
had shown that more than half of the drug were accumulated inside the
It has been seen that the any type of cellular stress has shown to liver of rats [133,134]. Similarly one study has reported that following
have expression of different signalling factor. Similarly, the SPIONs internalization of dextran coated SPIONs, the particles are accumulated
exposure uplifts the expression of genes which are involved in cell in lysosomes. The iron oxide is broken into iron ions via change in pH
signalling and shows the impact on signalling transduction pathways. and ultimately gets incorporated into haemoglobin. The dextranase
The, uplifted genes includes; tyrosine kinases, integrin subunits mem- further helps to break the dextran coating and facilitate the degradation
bers of the protein kinase C family, Ras-related protein, extracellular [129]. The important question here arise that this degree of degrada-
matrix proteins (ECM proteins) and matrix metalloproteinases [46]. It tion is highly dependent upon the protein corona present on the surface
is also reported that in vivo administration of dietary iron in rats had of SPION.
increased number of DNA breaks [118]. Polyaspartic acid-coated
magnetite NPs in vivo study demonstrated a time and dose-dependent 6. Conclusions
increase in micronucleus frequency [16].
Fig. 1 explain the possible mechanism of ROS after exposure of This review discusses the properties of SPIONs that may contribute
SPIONs following internalization via a number of possible mechanisms to their toxicity as well as some methods of assessing this toxicity in
is shown in Fig. 1, [119,120]. vitro. The importance of in vitro toxicity testing has increased in recent
times, mainly due to its desirable qualities over in vivo testing.
4.3. Immunotoxicity Specifically, in vitro tests are easier to manipulate, more cost effective
and easier to interpret.
Immunotoxicity is the study of toxicity effect of NPs on immune Toxicity of SPIONs is proved to be concentration dependent and it
cells [47]. Till date very limited data is available which can suggest the also depends on exposure time. No observable toxicity is seen at lower
interaction between immune system and SPION [121]. The study done levels of SPIONs as these particles can be cleared from body. While in
by Shen et al. [122] shown that administration of iron oxide nano- the case of high dose exposure, the particles may trigger cellular stress
particles, in a dose-dependent manner significantly weakened in- and altered response. Hence some more studies in this direction are
flammatory reactions and delayed the expression of interferon-γ, in- needed. In addition it is noted that the functionalization of SPION with
terleukin-6 and tumor necrosis factor-α at the inflammatory site [123]. biological moiety has shown least toxic effects, but it is critical to design
functionalized SPIONs which are able to meet sufficient internalization
4.4. Cellular stress property and are appropriately magnetizable, and also meet the de-
mands of a particular application without compromising on cellular
Cellular stress due to SPION is important factor for expression stress toxicity. The criteria to define toxicity of SPIONs needs to be redefined,
molecules. Gao et al. [124] reported that SPIONs lowers p53 expres- particularly as studies on SPIONs have begun to highlight aberrant
sion. He also studies the effects of SPIONs on cell cycle regulatory cellular responses including DNA damage, oxidative stress,

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