Genotoxicity and biocompatibility of superparamagnetic iron oxide nanoparticles: Influence of surface modification on biodistribution, retention, DNA damage and oxidative stress

Journal Pre-proof Genotoxicity and biocompatibility of superparamagnetic iron oxide nanoparticles: Influence of surface modification on biodistribution, retention, DNA damage and oxidative stress Swarupa Ghosh, Ilika Ghosh, Manoswini Chakrabarti, Anita Mukherjee PII:

S0278-6915(19)30779-3

DOI:

https://doi.org/10.1016/j.fct.2019.110989

Reference:

FCT 110989

To appear in:

Food and Chemical Toxicology

Received Date: 27 August 2019 Revised Date:

9 November 2019

Accepted Date: 19 November 2019

Please cite this article as: Ghosh, S., Ghosh, I., Chakrabarti, M., Mukherjee, A., Genotoxicity and biocompatibility of superparamagnetic iron oxide nanoparticles: Influence of surface modification on biodistribution, retention, DNA damage and oxidative stress, Food and Chemical Toxicology (2019), doi: https://doi.org/10.1016/j.fct.2019.110989. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Genotoxicity and biocompatibility of superparamagnetic iron oxide nanoparticles:

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Influence of surface modification on biodistribution, retention, DNA damage and

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oxidative stress

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SwarupaGhosha,b†, Ilika Ghosha,c,†, Manoswini Chakrabartia, Anita Mukherjeea*

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a

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Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata- 700 019. India.

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b

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c

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Sakura-ku, Saitama-shi, Saitama 338-8570.

Cell Biology and Genetic Toxicology Laboratory, Centre of Advanced Study, Department of

School of Life Science and Biotechnology, Adamas University, West Bengal, India.

Graduate School of Science and Engineering, Saitama University, 255, Shimo-okubo,

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* Corresponding author

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Anita Mukherjee

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Cell Biology and Genetic Toxicology Laboratory,

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Centre of Advanced Study, Department of Botany, University of Calcutta,

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35 Ballygunge Circular Road, Kolkata 700 019, India.

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Email: [email protected]

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Tel: 91-033-2461-5445; 9831061998

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†

These authors contributed equally to this work.

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Abstract

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Superparamagnetic iron oxide nanoparticles (SPION) require stable surface modifications to

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render safe nanocapsules for biomedical applications. Herein, two types of surface modified

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poly(lactic-co-glycolic acid)-encapsulated SPION were synthesized using either α-

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tocopheryl-polyetheleneglycol-succinate (TPGS) or didodecyl-dimethyl-ammonium-bromide

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(DMAB) as surfactants by emulsification. SPION-TPGS (180 nm) was larger than SPION-

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DMAB (25 nm) and uncoated SPION (10 nm). Both formulations were positively charged

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and induced lower cyto-genotoxicity and ROS generation than uncoated SPION in human

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lymphocytes. SPION-DMAB was least cyto-genotoxic among the three. Based on these

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results, mice were gavaged with the formulations for 5 consecutive days and biocompatibility

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studies were performed on the 7th and 21st days. ICP-AES and Prussian blue staining revealed

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the internalization of SPION-DMAB in brain and spleen, and SPION-TPGS in liver and

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kidney on day 7. This was correlated with high DNA damage and oxidative stress in the same

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organs. Substantial clearance of Fe was accompanied by reduced genotoxicity and oxidative

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stress on day 21. Therefore, SPION-DMAB can be further studied for oral drug delivery to

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the brain and imaging of cerebral tissue without any functional ligand or external magnetic

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field.

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Keywords: Comet assay, polymeric nanoparticles, biodistribution, oxidative stress, zeta

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potential

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1. Introduction

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Among the large number of metal oxide nanoparticles, only SPION are clinically approved.

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Under the action of an external magnetic field, SPION exhibit the unique ability of being

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guided to a target organ or tissue (Ye et al., 2014). This property makes them an excellent

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candidate for various theranostic applications such as MRI contrast agents, magnetic

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transfections, cellular labelling, chelation therapy, targeted drug and gene delivery. They also

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possess the unique ability of heat generation under an external magnetic field to enable their

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usage in magnetic hyperthermia for targeted destruction of tumour tissues and tissue

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engineering (Neuberger et al., 2005; Jain et al., 2008).

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However, bare or uncoated SPION have hydrophobic surfaces with large surface area to

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volume ratios. This facilitates their aggregation. Hence, to achieve a stable suspension

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feasible for clinical use, several types of coating materials such as dextran, chitosan,

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polyethylene glycol, etc have been used to modify the surface chemistry of SPION (Gupta

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and Gupta, 2005). To meet specific diagnostic and biomedical purposes like brain targeting,

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SPION are either directly delivered at the site or surface modified by functionalization using

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different ligands or subjected to an external magnetic field (Busquets et al., 2015).

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Hence, in order to produce a stable non-toxic aqueous dispersion of SPION a core-shell nano-

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capsule system was envisaged using the biodegradable polymer PLGA. In recent years,

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PLGA has been used for encapsulating a wide range of poorly water-soluble drugs (Ghosh et

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al., 2011; Reddy and Labhasetwar, 2009; Chakraborty et al., 2012; Ghosh et al., 2014).

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DMAB (didodecyl-dimethylammonium-bromide) and TPGS (α-tocopheryl-polyethelene-

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glycol-succinate) are two surfactants of varying chemical properties. DMAB is a cationic

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surfactant, reported to impede particle agglomeration and improve oral bioavailability

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(Hariharan et al., 2006). TPGS on the other is an efficient emulsifier, enhance cellular uptake 3

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and extend half-life of a drug (Win and Feng, 2006). They have been used earlier in

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experimental nanomedicine formulations supplemented with PLGA (Ghosh et al., 2011;

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Hariharan et al., 2006; Win and Feng, 2006). Addition of these surfactants in nanoparticle

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formulations did not impart particle toxicity but enhanced their ability of drug delivery. It

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was hypothesized that the use of DMAB and TPGS would make the SPION-polymer

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conjugate orally deliverable, the most convenient route for any biomedical application.

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The apparently interesting physico-chemical qualities of SPION allow them to cross diverse

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biological barriers and diffuse inside almost every cell type, thus giving rise to undesirable

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side effects. Cellular alterations manifested in the form of DNA damage, oxidative stress,

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chromosome condensation and formation of micronuclei are the common effects of SPION

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induced genotoxicity (Singh et al., 2010). Evaluation of safety and biocompatibility of novel

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nanoparticle formulations prior to biological use comprises genotoxicity study as an integral

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part (Moller, 2005). This necessitated the pressing need for assessment systems capable of

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identifying potential threats of nanoparticles application prior to their use in biomedicine

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(Oberdörster et al., 2005). Thus, the aim of this study was to assess the biocompatibility of

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DMAB and TPGS emulsified PLGA coated SPION in terms of genotoxicity, biodistribution

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and oxidative stress, in human lymphocytes in vitro and Swiss albino mice in vivo.

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2. Materials and methods

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2.1. Chemicals

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SPION, PLGA, DMAB, TPGS, RPMI 1640, normal melting point agarose (NMPA), low

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melting point agarose (LMPA), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium

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bromide (MTT), ethylenediaminetetraacetic acid (EDTA), dichloro-dihydro-fluorescein

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diacetate (DCFH-DA), tris buffer, ethidium bromide, Histopaque, trypan blue, hematoxylin,`

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eosin and potassium ferrocyanide were purchased from Sigma–Aldrich Co. (USA). All other

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chemicals were of analytical grade and were purchased from Himedia, Mumbai, India.

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2.2. Preparation and characterisation of PLGA encapsulated surface modified SPION

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Superparamagnetic iron oxide nanoparticles obtained commercially were modified in the

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laboratory to obtain nanocapsules of different sizes and surface properties.

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SPION-DMAB: SPION were polymer encapsulated at room temperature (25 ºC) by

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emulsion-diffusion - evaporation method. In brief 25 mg PLGA was dissolved in 2.5 ml ethyl

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acetate containing SPION suspension [(5mg/ml) in toluene] to prepare- suspension A. In a

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separate beaker DMAB taken in the molar ratio of 1:10 [PLGA: DMAB] was dissolved in 5

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ml of double distilled water at room temperature to prepare- solution B. Suspension A was

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added drop wise to solution B to form an emulsion. The resultant organic/water emulsion was

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left under stirring till the organic layer evaporated completely. Finally, the dark brown

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suspension was sonicated and stored at 4 ºC for future use.

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SPION-TPGS: Similarly, PLGA-SPION was prepared to get suspension A and for solution

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B, TPGS in the molar ratio of 1:10[PLGA: TPGS] was dissolved in 5 ml of double distilled

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water at room temperature. The process was similar to that of SPION-DMAB preparation.

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To remove the unentrapped SPION in the encapsulation, the SPION-DMAB or

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SPION-TPGS preparations were poured onto a Sephadex G50 mini gel column (pre-soaked

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in PBS for 12 h at 40C), and centrifuged at 1000 g for 10 min. The ‘free’ SPION was

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retained in the column while the filtrate containing the homogeneous suspension of SPION-

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DMAB /SPION-TPGS was collected. The concentration of encapsulated SPION was

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determined spectrophotometrically at 265 nm using a UV-visible spectrophotometer (model

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UV-1700 (E); Schimadzu, Kyoto, Japan) in triplicates. The encapsulation efficiency

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expressed as entrapment percentage was calculated through the following relationship:

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Encapsulation efficiency (%) =

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x 100

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Hydrodynamic size, zeta potential and polydispersity index of the prepared nanocapsules

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were analysed in a Malvern Zetasizer. Atomic force microscopy using a Veeco DICP2

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microscope with antimony doped silica cantilever (length 125µm, width 35 µm, thickness

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3.75 µm at a frequency of 300kHz in the tapping mode) was performed to visualize the 3D

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structure of the formulated particles and other physical properties including morphology,

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surface texture, roughness and size. Fourier transform infrared spectroscopy (FTIR) was done

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to study the surface chemistry of the formulated particles.

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2.3. In vitro toxicity evaluation

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Preliminary study on the toxic effects of the formulated SPION was carried out in vitro on

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human lymphocytes. Human peripheral blood was collected by venipuncture from 3 healthy

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volunteers with their consent (20–25 years old male donors, non-smokers, non-alcoholics and

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not consuming any medication), in vacutainers. The experiments were conducted in

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accordance with the Institutional guidelines (Institutional Ethical Committee, University of

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Calcutta, India, approval number (Registration #885/ac/04/IHEC). The lymphocytes were

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isolated using a Histopaque density gradient (Boyum, 1976) and were seeded onto 96 well

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plates. The initial cell viability was tested by Trypan Blue dye exclusion test (Tennant, 1967)

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and cells with viability > 98 % were used for subsequent experiments. Lymphocytes (1 x 106

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cells/mL) were treated for 3 h at 37 ºC with different concentrations of uncoated SPION or

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SPION-DMAB or SPION-TPGS. The concentrations of SPION (11.2-280 µg/mL) were

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selected based on the minimum iron concentration necessary for MRI imaging which is

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equivalent to 11.2 µg/ml (Astanina et al., 2014). Any dose below this critical dose might not

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be enough to produce clear MRI signals. The time point of 3 h was chosen according to the

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OECD guidelines for the detection of clastogens or aneugens (OECD, 2010).

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2.3.1. Cytotoxicity study

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Cell viability was studied by MTT assay following Mosmann, (1983) with modifications

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(Sinha et al., 2014). MTT is a yellow tetrazolium salt which is reduced into a purple

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formazan product by mitochondrial dehydrogenases present in metabolically active cells. The

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SPION treated lymphocytes (1 x 106 cells/mL) were incubated in MTT (Sigma–Aldrich, St.

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Louis, MO, USA) solution at a concentration of 0.5 mg/mL in RPMI-1640 (100 µl/well) for 4

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h at 37 ºC. At the end of the incubation period purple formazan crystals were observed.

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DMSO (100 µl/well) was used to dissolve the formazan crystals and the optical density was

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measured at 570 nm and using a reference wavelength of 630 nm using a microplate reader.

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2.3.2. Genotoxicity study

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Comet assay was performed using the method of Tice et al., (2000) with minor modifications

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(Ghosh et al., 2019). 100 µL of the treated lymphocyte suspension was added to 100 µL of 1

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% LMPA and mixed thoroughly. This suspension was placed on a base coated slide (1 %

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NMPA) and cover slips were mounted. The slides were kept on ice packs at 4 ºC till

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solidification of the second layer. Thereafter, a third layer containing 0.5 % LMPA was

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placed and slides were processed as above. After solidification of the third layer, coverslips

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were removed and the slides were placed in Coplin jars containing lysing solution (2.5 M

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NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl, 1%Triton-X-100, 10% (v/v) DMSO) for 4 h.

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The slides were rinsed in cold water, placed in an electrophoresis tank containing

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electrophoresis buffer (1 mM Na2EDTA, 300 mM NaOH, pH > 13) and incubated for 20

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minutes to allow unwinding of nuclear DNA. Electrophoresis was carried out at 26 V and 300

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mA for 30 min at 4 ºC. The slides were neutralized in 0.04 M Tris-HCl (pH 7.5) and stained

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with 5 µg/mL ethidium bromide. 300 nuclei (100 x 3) were scored per treatment using Komet

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5.5 (Andor Technology, Nottingham, UK) and % tail DNA was the chosen comet parameter.

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2.3.3. Study of ROS by flow cytometry

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The SPION treated lymphocytes were stained with DCFH-DA for the detection of ROS and

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analysed by a flow cytometer (Burow and Valet, 1987; Ghosh et al., 2019). The cells were

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washed with PBS and stained with DCFH-DA (25 µM in PBS) in dark for 30 min at 37 °C

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(Ghosh and Mukherjee, 2017). Approximately 10000 events were analysed per sample (BD

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FACS Verse™; Becton Dickinson, NJ, USA; λex = 485 nm and λem = 530-540 nm). Data

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were analysed using BD FACSuite software and results are expressed as % fluorescence

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intensity.

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Based on the above in vitro analyses the least toxic concentration and types of SPION were

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selected for biocompatibility studies in vivo.

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2.4. In vivo studies

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2.4.1. Animals and experimental design for in vivo administration

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Adult male Swiss albino mice (10-15 weeks), each weighing approximately 20-25 g, were

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acclimatized for 7 days under laboratory conditions (26–28°C; 60–80% relative humidity

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with 12-h light/dark cycle) prior to the commencement of treatment. The animals had access

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to food and drinking water ad libitum. All mice used in this study received proper care and

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handling in compliance with the Committee for the Purpose of Control and Supervision of

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Animals for Experiments [in India] (CPCSEA) and experimental procedures were approved

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by the ethical committee of the University Animal Care Unit, University of Calcutta

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(Registration #885/ac/05/CPCSEA). The mice were randomly divided into three groups

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(Groups A, B and C, n=12). A physiologically relevant concentration of iron (12.5 µg/mL)

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that is used clinically in MRI contrast agents was selected (Astanina et al., 2014). However, 8

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the uncoated SPION group was excluded from the in vivo experiment due to the high toxicity

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observed in in vitro experiments. The treatment groups have been divided as follows:

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Group A- Normal group, served as untreated controls were gavaged with 0.3 ml of normal

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saline.

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Group B – Animals were gavaged with SPION-TPGS (containing 12.5 µg of Fe/kg body

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weight)

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Group C - Animals were gavaged with SPION-DMAB (containing 12.5 µg of Fe/kg body

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weight)

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Six animals from each group were sacrificed on day 7 of the experiment. Rest of the six

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animals were sacrificed on day 21 post treatment cessation. These animals were provided

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with normal diet and drinking water without any SPION treatment till day 21. All animals

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were euthanatized for sacrifice. At the time of sacrifice final bodyweights were recorded and

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blood was collected from the heart of each animal. Major organs like brain, liver, kidney,

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spleen and testes were collected. A piece of each organ was used for comet assay and another

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was fixed overnight in 10% formalin for histology. The rest of the tissues were stored in -80

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ºC for further use.

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2.4.2. Hematology and clinical chemistry

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To study the systemic toxicity, intracardial blood samples were collected on day7 in separate

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vials containing 1.6 mg EDTA/mL. Blood glucose level, lipid profile, liver and kidney

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function of the animals were analysed on day 7 from serum.

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2.4.3. Histology and histochemistry

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Prefixed tissue samples collected from various organs of experimental animals (days 7 and

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21) were embedded in paraffin, and microtome 5-µm sections were prepared. The sections

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were stained with either HE (hematoxylin and eosin) and Perl’s Prussian blue stain (Zhuang

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et al., 2012). Histology was examined under a light microscope and accumulation of iron was

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visualized.

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2.4.4. ICP-AES Analyses

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Total iron accumulation in the collected organs (day7 and day21) was quantified by ICP-AES

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(ARCOS, SPECTRO Analytical Instruments GmbH, Germany) after digesting the tissue

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samples in HNO3 (Miller, 1998). 100 mg of tissue from brain, liver, spleen, kidney and testes

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was immersed in 3 mL HNO3 overnight and then digested in a fume hood. The remaining

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clear solution was diluted 1:10 using sterile deionized double distilled water. The metal

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concentrations were estimated by ICP-AES against appropriate standards and the Fe content

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was expressed as mg/kg tissue.

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2.4.5. Genotoxicity study

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Tissue samples from brain, liver, spleen, kidney and testes, collected on day 7 and 21 were

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assessed for SPION-induced DNA damage by comet assay according to the method of Tice et

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al., (2000) and Sasaki et al., (2002) with modifications (Ghosh et al., 2012). Brain, liver,

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spleen, kidney and testes tissues were collected in petri plates, teased and finely chopped in

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cold PBS using razor blades to release nuclei. The rest of the steps were the same as

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described for lymphocytes. Slides were prepared in triplicates and scored using Komet 5.5

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software (Kinetic imaging; Andor Technology, Nottingham, UK). % tail DNA was calculated

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from 300 nuclei (100 x 3) from each of the six animals of each treatment group.

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2.4.6. Biochemical analyses and antioxidant status

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To assess whether the formulated SPION caused oxidative stress in the animals oxidative

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stress markers like lipid peroxidation (Buege and Aust, 1978), GSH level (Sedlak and

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Lindsay, 1968), catalase (Aebi, 1984) and SOD (Marklund and Marklund, 1974) activities

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were measured in tissue samples collected on day 7 and 21. Protein was estimated by

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Bradford’s method (Kruger, 1994). 10

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2.4.7. Sperm Head Abnormality Assay

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To monitor whether formulated SPION administration induced any morphological

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abnormality in the germ cells, sperm head abnormality assay was performed on day 7. Sperm

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cells were isolated according to the method of Aduloju et al., (2008) with modifications. The

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morphological damage in the sperm head were categorized as close as described by Wyrobek

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and Bruce, (1975) which include hook less, amorphous, banana-shaped and pinhead. The

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frequency of abnormal sperm was expressed as percentage, calculated with the formula:

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2.5. Statistical Analyses

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All data are presented as mean ± SEM. One-way analysis of variance (ANOVA) was

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performed using SigmaPlot 12.1 software (Systat Software Inc., San Jose). The level of

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significance was established at P < 0.05. When significant differences were obtained by

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ANOVA, Holm-Sidak’s post-hoc test was performed at the same 5 % probability level.

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3. Results

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3.1. Surface modified SPION display higher primary size and superior colloidal stability

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than uncoated SPION

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The amount of SPION present in SPION-DMAB and SPION-TPGS was 61% and 67%,

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respectively, as estimated by UV-visible spectrophotometry. The physico-chemical characters

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of uncoated and surface modified SPION were assessed by AFM and DLS. The presence of

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PLGA coating stabilized with the surfactants DMAB and TPGS caused increased primary

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and hydrodynamic diameters. Figure 1A,B,C depicts the topography of the nanoparticles on

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the mica sheet surface. All three types of SPION were spherical and monodispersed. The

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PLGA encapsulated SPION were observed as dark particles coated within a polymer matrix

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with a halo-like appearance. Uncoated SPION showed a size range of 10.26 – 35.62 nm 11

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(Figure 1D). The size range of SPION-TPGS was 94.56 - 186.21 nm (Figure 1E) and SPION-

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DMAB was 28.26 - 57.1 nm (Figure 1F). Successful surface modification of SPION-TPGS

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(Figure 1G) and SPION-DMAB (Figure 1H) by PLGA was confirmed by the FTIR spectra.

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Appearance of a sharp peak at 1636 cm-1 in SPION-TPGS and SPION-DMAB indicates the

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presence of >C=O stretching of the lactide group in PLGA (Figure 1G,H). The mean

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hydrodynamic diameter (MHD) of uncoated SPION was 29.89 ± 13.31 nm with a zeta

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potential of -8.43 ± 6.22 mV. The MHD of SPION-DMAB (67.14±16.26 nm) was lesser than

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SPION-TPGS (178.6±106.0 nm). Addition of the positively charged surfactant DMAB

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provided superior colloidal stability by increasing the zeta potential to +53.3 ± 10.7 mV

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(Table 1).

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3.2. Surface modified SPION impart lower cyto-genotoxicity and ROS generation

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compared to their uncoated counterparts in human lymphocytes in vitro

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In this study, human lymphocyte cells were used to determine the in vitro toxicity of

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uncoated SPION, SPION-TPGS and SPION-DMAB. Cytotoxicity was evaluated by

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assessing lymphocyte cell viability by MTT assay. The results obtained demonstrate surface

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coating and Fe concentration dependent decline in cell viability (Figure 2A). Maximum

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cytotoxicity was induced by uncoated SPION while SPION-DMAB was least cytotoxic

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among the three. Cell viability was found to decrease in a dose dependent manner with

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increasing concentration.

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With regard to genotoxicity, the comet assay results showed that surface modification with

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PLGA using wither DMAB or TPGS as the surfactant could efficiently modulate the

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genotoxicity caused by uncoated SPION alone. However, SPION-DMAB and SPION-TPGS

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also caused significantly higher (p < 0.05) DNA damage than untreated cells, although the

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extent of DNA damage was significantly lower (p≤ 0.001) than that caused by uncoated 12

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SPION at all concentrations tested. DNA migration quantified as % tail DNA was observed

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to increase with increasing concentration of SPION. % tail DNA was less in SPION-DMAB

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than in SPION-TPGS treated cells at all concentrations (Figure 2B).

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The propensity of SPION to produce ROS such as •OH, ROO• and H2O2 was investigated by

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DCFH-DA staining followed by flow cytometry. Uncoated SPION caused a ~ 8 fold increase

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in ROS generation at the lowest concentration alone, followed by a linear increment in ROS

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accumulation at the subsequent higher concentrations (Figure 2C,D). On the other hand, ROS

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level was significantly lower in SPION-DMAB and SPION-TPGS when compared to

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uncoated SPION treated lymphocytes. Among the three groups, SPION-DMAB produced

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least amount of ROS at all the concentrations tested.

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3.3. SPION internalization is variable in different organs

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Based on the above in vitro results, only the two types of surface modified SPION were

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tested for in vivo biocompatibility. Administration of SPION-DMAB and SPION-TPGS had

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no effect on the body and organ weights of the animals. There were no clinical signs of

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reaction and no macroscopic changes. The internalization of SPION within different organs

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was qualitatively assessed by Pearl’s Prussian blue staining and quantitively by ICP-AES.

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Considerable Fe internalization was visualized as dark blue patches of Pearl’s Prussian blue

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deposition in the brain, liver, spleen, kidney and testes on the 7th day (Figure 3). Decreased

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occurrence of blue patches of Fe was evident in tissue sections from day 21 after treatment

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cessation. The control sections of both the time points also showed the normal occurrence of

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Fe, which was highest in spleen.

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Quantification of Fe internalization (7th day) and retention (21 days post treatment cessation)

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by ICP-AES from the same organs corroborated the qualitative analyses by Pearl’s Prussian

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blue staining (Table 2). SPION-TPGS uptake was highest in the liver followed by kidney and

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spleen. Highest uptake of SPION-DMAB occurred in the brain followed by spleen, liver and 13

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kidney. Administration of SPION-DMAB resulted in a ~ 4-fold higher concentration of Fe in

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brain and ~ 1.5-fold of Fe increment in spleen than that of untreated animals, on day 7. In

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case of SPION-TPGS treated animals, more than ~ 4-fold rise in the amount of Fe

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concentration was found in liver, followed by a ~ 3-fold increase in kidney compared to the

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untreated animals, on day 7. The elevated Fe content in the brain and spleen of SPION-

319

DMAB treated mice and in liver and kidney of SPION-TPGS treated animals was

320

significantly reduced after 21 days post treatment cessation. The Fe retention within other

321

organs of mice were found to be restored to the normal range on day 21.

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3.4. SPION internalization induces alterations in liver function

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Haematological parameters in animals gavaged with SPION-DMAB or SPION-TPGS

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(sacrificed on day 7) did not vary when compared to the respective controls (Table 3).

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SPION-DMAB and SPION-TPGS had no effect on the blood parameters, carbohydrate and

326

lipid metabolism. Mice gavaged with SPION-DMAB showed lesser alteration in liver

327

function than SPION-TPGS. The results of kidney function test were within the normal

328

range. Liver function tests in mice administered with SPION-TPGS showed significantly

329

higher ALT, AST and Alkaline Phosphatase activities indicating altered liver function.

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3.5. SPION induced genotoxicity is modulated after treatment cessation in various

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organs

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In vivo genotoxicity in brain, liver, spleen, kidney and testes of SPION-DMAB and SPION-

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TPGS treated mice was studied by alkaline comet assay on day 7 and 21, respectively (Figure

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4). On day 7, a significant (P< 0.05) increase in the amount of DNA damage was observed in

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all the organs of animals treated with both types of SPION. The magnitude of DNA damage

336

was highest in liver followed by kidney, brain, spleen and testes tissues of SPION-TPGS

337

administered animals. In mice treated with SPION-DMAB, highest % tail DNA was observed 14

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in brain, followed by spleen, kidney, liver and testes. The increase in DNA damage recorded

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on day 7 was significantly (P< 0.05) reduced on day 21in all the organs. This signifies the

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possible recovery of the initial DNA damage responses within 21 days.

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3.5. SPION exposure causes altered antioxidant defense responses in various organs

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Oxidative stress was evaluated in different organs collected from the animals on day 7 and on

343

day 21. The effect of SPION-DMAB and SPION-TPGS on lipid peroxidation, GSH level,

344

CAT and SOD activity in brain, liver, spleen, kidney and testes are presented in Figure 5. A

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significant increase in LPO and decrease in SOD and CAT activities, as well as GSH levels

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was observed on day 7. This was normalized in all organs after 21 days.

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3.6. Sperm head abnormality occurs as a result of SPION exposure

348

Sperm head abnormality was studied by light microscopy and recorded in animals gavaged

349

with SPION-TPGS and SPION-DMAB (Figure 6) on day 7. SPION-TPGS gavaged animals

350

showed a significant number of abnormal shaped sperm heads such as hook, pin-head,

351

banana and amorphous type. In mice treated with SPION-DMAB the frequency of abnormal

352

sperm heads was not significantly higher than the control values.

353

4. Discussion

354

The aim of the present investigation was to prepare a stable surface modified aqueous

355

dispersion of SPION suitable for biomedical applications. The orally deliverable nano-carrier

356

systems were tested for their biocompatibility in vitro and in vivo with respect to their

357

biodistribution, retention, genotoxicity and oxidative stress. To achieve a biocompatible

358

hydrophilic shell matrix, PLGA was the chosen polymer along with either DMAB or TPGS

359

as the surfactants. PLGA is a biodegradable amphiphilic co-polymer capable of forming a

360

core-shell nanoparticulate system for encapsulating a wide range of poorly water-soluble

361

drugs (Ghosh et al. 2011; Chakraborty et al., 2012; Ghosh et al., 2014). Polyesters like 15

362

PLGA, poly-lactic acid and poly-glycolic acid have attracted recent interest in stabilization of

363

iron oxide nanoparticles by optimizing electrostatic repulsion between similarly charged

364

particles (Jeong et al. 2004; Lee et al. 2005). The use of DMAB or TPGS as surfactants

365

during the emulsion-evaporation process produced high quality, monodisperse, orally

366

deliverable and water soluble PLGA-SPION-surfactant nano-carrier systems with two

367

different sizes. The presence of the surfactants DMAB and TPGS in the formulations

368

imparted positive charge to the negatively charged SPION core. The strong cationic nature of

369

DMAB rendered high positive ζ-potential (+53.3 ± 10.7), whereas SPION-TPGS suspensions

370

displayed a lower positive ζ-potential value (+35.6 ± 8.57). In terms of size, the presence of

371

polyethelene glycol in TPGS might have been responsible for its higher MHD than SPION-

372

DMAB (Ghosh et al., 2014). The poor solubility of uncoated SPION causes precipitation and

373

agglomeration under physiological conditions. Hence, they are not recommended for clinical

374

use (Singh et al., 2010). Therefore, a key objective of this study was to compare the in vivo

375

biocompatibility of SPION-DMAB and SPION-TPGS.

376

Results revealed that the formulated SPION-DMAB and SPION-TPGS aqueous dispersions

377

induced significantly less cytotoxicity, genotoxicity and ROS generation than uncoated

378

SPION, which displayed smallest size (29.89 ± 13.31) and negative ζ-potential (-8.43 ± 6.22).

379

Surface modification of nanoparticles changes size, surface charge and other vital physico-

380

chemical properties (Mahdavi et al., 2013; Ali et al., 2016) which govern their cellular entry

381

and fate in biological systems (Dowding et al., 2013; Salatin et al., 2015). The vesicular

382

entrapment of uncoated SPION by surface modification could effectively reduce toxicity in

383

lymphocytes. SPION-DMAB showed least toxicity in the tested parameters compared to

384

SPION-TPGS. The α-tochopherol component in TPGS is known to act as a prooxidant

385

(Martin-Rubio et al., 2018). This may explain the higher generation of ROS and subsequent

386

toxicity incurred upon SPION-TPGS exposure. Hence, SPION-DMAB and SPION-TPGS 16

387

were tested for biocompatibility in Swiss albino mice with normal saline as the control.

388

Uncoated SPION were not tested in mice as they induced high toxicity in vitro. In this regard,

389

PLGA coated SPION devoid of surfactants was not used as a control because that would

390

create a different nanoparticle with altered physico-chemical properties and in turn deviate

391

from the aim of this study.

392

On in vivo administration, SPION formulations did not cause any major visible impairment in

393

the overall health status of the mice as evident from the absence of mortality and no changes

394

in body/organ weights. Haematological parameters of RBC, WBC, platelet count and

395

haemoglobin content did not manifest haemolysis or aggregation of blood cells. Numerous

396

studies have demonstrated that biodistribution and retention of nanoparticles depend on their

397

size and zeta potential (Duan and Li, 2013; Janát-Amsbury et al., 2011). Accumulation of Fe

398

in different organs was measured by ICP-AES after 7 days of treatment and 21 days after

399

treatment was stopped. Both SPION-TPGS and SPION-DMAB were distributed in different

400

organs following intestinal absorption. The cationic nature of the formulated SPION might

401

have facilitated its absorption across the anionic mucin on intestinal epithelium layer

402

(Hariharan et al., 2006). A significant amount of Fe was detected in the brain of SPION-

403

DMAB treated mice by ICP-AES and histochemical staining. This indicated that these

404

vesicles of much smaller primary size range (~ 28 nm) were possibly able to reach the brain

405

by penetrating the blood brain barrier (BBB). Several reports confirm that nanoparticles <

406

100 nm can cross the BBB and extravasate the brain (Konduru et al., 2014). Kim et al.,

407

(2005) showed that magnetic nanoparticles of this size range can pass through BBB without

408

disturbing its structural integrity and function. Moreover, numerous studies reported that

409

surface modified SPION can internalize in the brain by crossing the BBB (Hoff et al., 2013;

410

Shi et al., 2016; Sillerud et al., 2013).

411

nanoparticles formed using PLGA and DMAB was reported to cross BBB and accumulate in

In a previous report by one of the authors,

17

412

brain cell mitochondria (Ghosh et al., 2017). The present study quantitatively (ICP-AES) and

413

qualitatively (Pearl’s Prussian blue staining) revealed the internalization of SPION-DMAB in

414

the brain. As an interesting future scope, transmission electron microscopy can be performed

415

to visualize the specific localization of SPION-DMAB in brain tissue. Moreover, energy-

416

dispersive X-ray analysis of the same samples can be performed to reaffirm the dissolution of

417

Fe in the brain.

418

Histochemical examination and ICP-AES data confirmed that SPION-TPGS having larger

419

primary size (~ 180 nm) remained concentrated in the liver on day 7 in concurrence with

420

earlier reports stating nanoparticles to first reach the liver when administered through the

421

gastro-intestinal route (Chen et al., 2006; Cui et al., 2011). Phagocytosed SPION were

422

observed within Kupffer cells due to oral administration. The Kupffer cells in liver with

423

phagocytic activity are known to readily take up colloidal particles (Mirshafiee et al., 2018).

424

Elevated levels of liver function enzymes like ALT and ALP as well as albumin, globulin and

425

total protein in the serum indicate a certain extent of liver damage arising due to Fe

426

internalization. Though Fe deposition was not very significant in liver of SPION-DMAB

427

treated mice, the AST enzyme level was found to be increased in this group reflecting a

428

burden on the reticuloendothelial system in the animals. Fe concentration was not

429

significantly high in brain of mice administered SPION-TPGS i.e., particles with more than

430

100 nm size. Substantial amount of Fe was detected in the kidney of SPION-TPGS and

431

SPION-DMAB treated animals indicating uptake of SPION by the resident renal

432

mononuclear phagocytes. But administration of these formulations did not change the levels

433

of creatinine, blood urea, bilirubin, potassium and salt balance indicating normal renal

434

function in the animals. A considerably high amount of Fe was detected in spleen, largely in

435

the red pulp region resided by macrophages over and above the natural iron build-up. Fe

436

concentration in the spleen of rodents fed on a standard diet is reported to be normally high 18

437

(Barrefelt et al., 2013). While iron overload was not detected in the testes, some amount of

438

sperm head abnormality was detected in SPION-TPGS treated group of animals. Our results

439

indicate the involvement of mononuclear phagocyte system in the uptake and accumulation

440

of formulated SPION particles.

441

Genetic errors increase from repeated DNA damage coupled with weakened DNA repair

442

enzymes and acquire the potential to initiate carcinogenesis (Ames et al., 1993). Comet assay

443

is capable of detecting most of the DNA oxidized products like oxidized bases, abasic sites,

444

DNA-DNA intra-strand adducts, DNA strand breaks and DNA-protein cross-links (Moller et

445

al., 2005). We employed this method to detect the genotoxic potential of the formulated

446

SPION in different tissues in mice. DNA damage (% tail DNA) was high in the tissues where

447

concentration of SPION was highest, for example, in liver (SPION-TPGS), in brain (SPION-

448

DMAB) and in kidney tissues of animals administered with both types of formulations of

449

SPION collected on day 7 of the experiment. Furthermore, the high surface charge of SPION

450

can plausibly be responsible in inducing DNA damage.

451

Oxidative stress has been considered as a probable mechanism of genotoxicity of

452

nanoparticles (Nel et al., 2006). Accumulation of iron oxide nanoparticles with an elevated

453

concentration of free iron ions generate oxidative burden in cells and tissues (Murray et al.,

454

2013; Novotna et al., 2012). Amount of lipid peroxidation in different tissues treated with

455

formulated SPION was considerably high on day 7. Lipid peroxidation is a characteristic

456

feature of ROS induced damage. The positive ζ-potential of SPION-DMAB (+53.3±10.7

457

mV) and SPION-TPGS (+35.6±8.57 mV) might have been responsible for inducing damage

458

to the membrane lipids and enzyme proteins thus contributing to oxidative stress. Besides Fe-

459

load, localization of SPION is also responsible for generating superoxide radicals and

460

oxidative stress (Singh et al., 2010). Significantly reduced catalase and superoxide dismutase

461

activities found in all the organs in both types of SPION-treated groups can be attributed to 19

462

the molecular oxidative damage of these enzyme proteins responsible for reducing oxidative

463

stress. GSH, the native antioxidant molecule of a cell, was significantly less in both the

464

treatment groups (SPION-TPGS and SPION-DMAB) that might have been oxidized to

465

GSSG in the oxidatively challenged milieu. An inverse correlation between lipid peroxidation

466

and GSH levels noted in our experiment was analogous to that of other reports (Mandal and

467

Das, 2005).

468

Sperm head abnormality assay can detect deleterious chemical agents (Wyrobek and Bruce,

469

1975). Oxidative stress detected in seminal fluids has been responsible in inducing sperm

470

head abnormalities (Ushijima et al., 2000; Meeker et al., 2008). We found significantly

471

reduced level of GSH and catalase activity on day 7 in testes of SPION-TPGS treated mice.

472

Asare et al., (2012) reported sub-micron sized metal nanoparticles (200 nm) to be more toxic

473

than nano sized ones in testicular cells. This is in agreement with our findings where SPION-

474

DMAB (~ 30 nm) induced less toxicity than SPION-TPGS (~ 180nm) in testes and sperm

475

cells.

476

SPION concentration was significantly reduced in liver and kidney of SPION-TPGS and in

477

brain, kidney and spleen of SPION-DMAB treated mice sacrificed on day21. These results

478

imply possible mobilization of iron load either in the formation of RBCs or excretion through

479

kidneys (Anzai et al., 2003). Samples processed on day 21 showed signs of recovery from

480

oxidative stress as evident from reduced MDA level, elevated catalase and superoxide

481

dismutase activity and increased GSH content. A significant moderation of DNA damage in

482

all the tissues was also observed in day 21 samples. These corroborate with the reduced

483

concentrations of Fe estimated in ICP-AES and in Perl’s Prussian blue stained sections.

484

When formulated SPION administration was stopped, no further accumulation occurred

485

along with Fe mobilization. The reduced DNA damage observed in day 21 samples might be

486

attributed to the efficient DNA repair enzymes that might have taken over when iron load 20

487

was low. However, day 21 ICP-AES data show higher Fe concentration in liver, spleen,

488

kidney and testes in SPION-TPGS treated group than in SPION-DMAB. The PEG

489

component of TPGS on one hand might be responsible in making the particles long

490

circulating but their sub-micron size collectively with positive zeta potential must have

491

governed their higher uptake in liver and retention therein (Jokerst et al., 2011). LPO level

492

continued to be higher in all organs on day 21 SPION-TPGS treated group than their control

493

counterparts. Reduced GSH level, lower SOD and CAT activity and higher % tail DNA also

494

observed in liver and kidney on day 21 in SPION-TPGS treated group reflects the retention

495

of iron load in the tissue due to continued uptake of particles from circulation.

496

The addition of surfactants is reported to stabilize SPION, affect clustering, modify

497

performance as contrasting agents and facilitate their biomedical applications (Luchini et al.,

498

2016; Liao et al., 2010; Filippousi et al., 2014). Surfactants are amphiphilic molecules that

499

align themselves such that the hydrophilic part is in solution. When SPION are encapsulated

500

within the hydrophobic core of the surfactant micelles, the exposed hydrophilic tails of the

501

surfactant molecules facilitate stabilization in suspension to render higher uptake within

502

target organs and increase contrast (Mok and Zhang, 2013). Therefore, results of the present

503

study indicate that SPION-DMAB can be a superior candidate over SPION-TPGS for further

504

investigation in nanomedicine.

505

Conclusions

506

In the present study, PLGA encapsulation along with the use of surfactants were cumulatively

507

responsible in decreasing cytotoxicity and genotoxicity caused by uncoated SPION. The

508

surfactants- TPGS and DMAB conferred the differences in size and zeta potential of SPION

509

nanocapsules. SPION-TPGS was a submicron sized (~ 180 nm) with ~ +35 mV surface

510

charge and SPION-DMAB were nano sized (~ 30 nm) having higher colloidal stability as

511

seen by a zeta potential of ~ +54 mV. Absorption from the intestinal tract was driven by 21

512

positive zeta potential upon both SPION-TPGS and SPION-DMAB exposure. Accumulation

513

in various organs was mainly governed by the size of the particles which determines their

514

ability to cross various physiological barriers. Larger sized SPION-TPGS were more easily

515

taken up by the cells of the mononuclear phagocyte system but could not penetrate the BBB

516

to enter brain cells. On the other hand, the small size of SPION-DMAB enabled possible

517

uptake in the brain (which is otherwise impermeable for xenobiotics) in considerable amount

518

and internalized in organs of the reticulo-endothelial system. Thus, SPION-DMAB is a

519

unique SPION system capable of accumulating in brain via oral route that did not require any

520

targeting ligand or application of external magnetic field or use of any BBB opening agent.

521

Initial signs of oxidative stress observed due to cellular internalization of SPION-TPGS and

522

SPION-DMAB can be attributed to the intracellular iron-load. However, oxidative stress and

523

DNA damage were reduced upon treatment cessation and the particles were possibly cleared

524

from the organs after 21 days. These toxicological data support the feasibility of SPION-

525

DMAB for further explorations as nano-vehicles for drug delivery and MRI.

526

Acknowledgement

527

SG acknowledges the University Grants’ Commission, Government of India. [Dr.

528

D.S.Kothari Post-Doctoral Fellowship Grant: (BSR)/BL/13-14/0478] for financial support.

529

The authors acknowledge Centre for Research in Nanoscience and Nanotechnology,

530

University of Calcutta, India, for instrumentation facilities.

531

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Table 1. Hydrodynamic characteristics of uncoated and surface modified SPION ζ-potential (mV)

Type of SPION

MHD (nm)

PDI

Uncoated SPION

29.89±13.31

0.325±0.012 -8.43±6.22

0.0136±0.0002

SPION-TPGS

178.6±106.0

0.274±0.030 +35.6±8.57

0.0201±0.0005

SPION-DMAB

67.14±16.26

0.366±0.041 +53.3±10.7

0.0286±0.001

Conductivity (mS/cm)

Values are expressed as mean ± SEM of three independent experiments. MHD- mean hydrodynamic diameter; PDI- polydispersity index; ζ-potential- zeta potential

Table 2. Quantitative estimation of Fe uptake and retention (mg/kg tissue weight) by ICPAES Organs

Control (mg/kg)

SPION-TPGS (mg/kg)

SPION-DMAB (mg/kg)

Day 7

Day 7

Day 21

Day 7

Day 21

2.90±0.22

9.73±1.54*

4.38±0.27#

Day 21

Brain

2.84±0.16 2.69±0.52

3.05±0.28

Liver

3.80±0.25 3.91±0.48

12.88±2.03* 6.11±0.57* 5.77±0.75#

Spleen

5.26±0.43 5.25±0.77

6.34±0.81

5.84±0.73

Kidney

2.34±0.19 2.37±0.62

7.23±0.65*

3.91±0.28* 5.69±0.22*

2.57±0.35

Testes

1.67±0.08 1.63±0.22

1.98±0.09

1.81±0.07

1.75±0.26

8.48±82#

2.18±0.13

3.92±0.56 5.83±0.71#

Results are expressed as mean mg/kg tissue weight. Values are expressed as mean ± SEM from six mice. SPION-TPGS (day7) and SPION-DMAB (day7) mice were compared with control mice and SPION-TPGS (day21) and SPION-DMAB (day21) mice were compared with SPION-TPGS (day7) and SPION-DMAB (day7) mice respectively. * indicates p < 0.001 and # indicates p < 0.05.

Table 3. Haematology and clinical chemistry parameters Parameters

Control

SPION-TPGS

SPION-DMAB

Haemoglobin (g/dL)

10.51 ± 0.62

8.83 ± 0.41

11.36 ± 0.58

RBC (1012/L)

8.68 ± 0.53

6.71 ± 0.34

8.59 ± 0.49

WBC (109/L)

6.92 ± 0.37

11.86 ± 0.64

14.74 ± 0.73

Neutrophils %

34.33 ± 0.49

15.33 ± 0.42

15.16 ± 0.48

Lymphocytes %

60.66 ± 0.42

80.16 ± 0.54

80.33 ± 0.72

Monocytes %

3.16 ± 0.40

2.33 ± 0.21

3.16 ± 0.31

Eosinophil %

1.83 ± 0.31

2.16 ± 0.31

1.33 ± 0.21

Bilirubin Total (mg/dL)

0.59 ± 0.03

0.42 ± 0.03

0.38 ± 0.02

Serum Protein (Total) (g/dL)

5.82 ± 0.26

8.07 ± 0.57

7.26 ± 0.38

Albumin (g/dL)

2.65 ± 0.16

2.44 ± 0.21

2.59 ± 0.13

Globulin (g/dL)

2.94 ± 0.25

5.68 ± 0.37

4.07 ± 0.31

AST (U/L)

68.52 ± 5.39

710.38 ± 18.57*

182.26 ± 8.94*

ALT (U/L)

52.85 ± 4.29

204.73 ± 9.46*

71.16 ± 5.44*

Alkaline Phosphatase (U/L)

65.03 ± 5.24

105.96 ± 7.61*

82.47 ± 6.83*

Glucose (F) (mg/dL)

87.68 ± 8.03

116.79 ± 8.94

92.42 ± 7.52

Cholesterol (mg/dL)

117.56 ± 9.32

110.28 ± 8.54

104.71 ± 6.63

Triglycerides (mg/dL)

74.32 ± 6.63

96.14 ± 4.58

94.29 ± 6.73

Urea (mg/dL)

47.51 ± 2.94

45.35 ± 2.29

54.82 ± 3.72

Creatinine (mg/dL)

0.83 ± 0.05

0.74 ± 0.06

0.87 ± 0.06

Potassium (mEq/L)

7.06 ± 0.32

6.65 ± 0.26

7.28 ± 0.41

Calcium (mg/dL)

9.14 ± 0.42

9.63 ± 0.58

8.44 ± 0.38

Phosphorous (mg/dL)

4.25 ± 0.27

10.11 ± 0.62

6.37 ± 0.47

Values are expressed as mean ±SD * P < 0.05 by One-way ANOVA followed by HolmSidak multiple comparison test.

Highlights Surface modified SPION show superior colloidal stability than uncoated SPION. SPION-DMAB and SPION-TPGS show lower cyto-genotoxicity and ROS generation than uncoated SPION in human lymphocytes. SPION induced genotoxicity and oxidative stress is modulated after treatment cessation in mice. SPION-DMAB shows high internalization in the brain of mice and can be used in brain imaging.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

The authors declare no conflict of interest.

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