Research Article Assessment of Immunotoxicity of Dextran ...downloads.hindawi.com/archive/2015/518527.pdf · thesis is used to coat iron nanoparticles. ... Antioxidant defense system

Research ArticleAssessment of Immunotoxicity of Dextran Coated FerriteNanoparticles in Albino Mice

Santhakumar Syama Viswanathan Gayathri and Parayanthala Valappil Mohanan

Toxicology Division Biomedical Technology Wing Sree Chitra Tirunal Institute for Medical Sciences and TechnologyThiruvananthapuram Kerala 695 012 India

Correspondence should be addressed to Parayanthala Valappil Mohanan mohanpv10gmailcom

Received 5 August 2015 Accepted 17 September 2015

Academic Editor Sharad S Singhal

Copyright copy 2015 Santhakumar Syama et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

In this study dextran coated ferrite nanoparticles (DFNPs) of size lt25 nm were synthesized characterized and evaluated forcytotoxicity immunotoxicity and oxidative stress by in vitro and in vivo methods Cytotoxicity was performed in vitro usingsplenocytes with different concentrations of DFNPs Gene expression of selected cytokines (IL-1 IL-10 and TNF 120573) secretion bysplenocytes was evaluated Also 100mg of DFNPs was injected intraperitoneally to 18 albino mice for immunological stimulationsSix animals eachwere sacrificed at the end of 7 14 and 21 days Spleenwas subjected to immunotoxic response and liverwas analyzedfor antioxidant parameters (lipid peroxidation reduced glutathione glutathione peroxidase superoxide dismutase and glutathionereductase) The results indicated that DFNPs failed to induce any immunological reactions and no significant alternation inantioxidant defense mechanism Also mRNA expression of the cytokines revealed an increase in IL-10 expression and subsequentdecreased expression of IL-1 and TNF 120573 Eventually DNA sequencing of liver actin gene revealed base alteration in nonconservedregions (10ndash20 bases) of all the treated groups when compared to control samples Hence it can be concluded that the DFNPs werenontoxic at the cellular level and nonimmunotoxic when exposed intraperitoneally to mice

1 Introduction

Nanoparticles are diverse class of small-scale (lt100 nm)substances with novel properties like small size large surfacearea specific shape and surface activity [1] Toxicity ofnanomaterials refers to the interaction of nanomaterials withthe biological systems and induction of toxic responses Onceinside the body they get distributed to various organs or mayremain in the same site and can be structurally modifiedor metabolized When inhaled they can translocate out ofthe respiratory tract via different pathways and mechanismsWhen ingested systemic uptake of nanomaterials via lymphcan occurWhen in blood circulation they can be distributedthroughout the organism andwill be taken up by liver spleenbone marrow heart and other major organs [2 3] Depend-ing on the duration of exposure these materials can translo-cate from circulation to internal organs Hence the potentialfor significant biological response at each of these sitesrequires investigation [4]

Magnetic nanoparticles are widely used in biomedicalapplications such as contrast agents in magnetic resonanceimaging (MRI) [1] and in tissue repairing [5] detoxificationof biological fluids hyperthermia [6] drug delivery [7] cellseparation [8] drug targeting [2] and so forth Ferrite parti-cles coated with biocompatible phases like hydroxyapatite areemployed for hyperthermia treatment of cancer [9] As baremetal and metal oxide nanoparticles are toxic to biologicalsystems coating with biocompatible phases is often doneto increase the biomimetic nature of the particles suitingfor various biomedical applications Dextran (C

6H10O5) a

branched polysaccharide obtained by microbiological syn-thesis is used to coat iron nanoparticles Dextran beingnontoxic biodegradable and hydrophilic may facilitate theintracellular uptake of dextran coatedmagnetic iron particles[3 4]

The major toxicological issue associated with the manu-factured nanomaterials is that some of them are redox activethey can transport across cell membranes and interact with

Hindawi Publishing CorporationMolecular Biology InternationalVolume 2015 Article ID 518527 10 pageshttpdxdoiorg1011552015518527

2 Molecular Biology International

subcellular organelles As a consequence of all these proper-ties nanoparticles can have direct interaction with individualtarget cells either with the external membrane or inside thecell at the site of action Advances in nanotechnology ledto the exposure of humans to engineered nanomaterials andhence it became necessary to evaluate the potential humanhealth effects before these materials are fully exploited [1 2]

Experimental evidences have shown that metal andmetaloxide nanoparticles induced DNA damage and apoptosisthrough ROS generation and oxidative stress [10 11] It hasbeen reported that magnetic nanoparticles administeredintraperitoneally can cross blood-brain barrier where theyhave the potential to affect cerebral functions [12] Due to thehigh reactivity of ROS most cellular components are likely tobe the targets of oxidative damage lipid peroxidation proteinoxidation GSH depletion and DNA single strand breaks Allof these events ultimately lead to cellular dysfunction andinjury [13] For this reason antioxidant enzymes are vitalmarkers for oxidative stress induced in the body Aerobicorganisms possess antioxidant defense systems that deal withthe removal of ROS As long as there exists a balance betweenoxidative stress and antioxidant defense system human bodyis maintained in an optimal health state

Antioxidant defense system includes both low-molecu-lar-weight free radical scavengers such as the tripeptide glu-tathione (GSH) as well as antioxidant enzymes such as super-oxide dismutase (SOD) glutathione reductase (GR) andglutathione peroxidase (GPx) [3]

The immunotoxicology of engineered nanoparticles is agrowing concern in the present era The potential immuno-toxicity and mechanisms of action of these particles have notreceived sufficient attention Nanoparticles can interact withimmune system in several ways and can enhance or suppressimmune function depending on their physicochemical char-acteristics [14] It was reported that the surface modificationof carbon nanotubes recognizes scavenger receptor andalleviates NF-120581B activation and reduces its immunotoxicity[15] Similarly shell-cross-linked knedel-like nanoparticlesinduced lower immunotoxicity than their non-cross-linkedanalogs as was demonstrated by Elsabahy et al [16]

Here DFNPs intended to be used for targeted drug deliv-ery applications were in-house synthesized and character-ized The primary focus of the present study was to examinethe immunotoxic potential and cellular response of DFNPs

2 Materials and Methods

21 Chemicals Thiobarbituric acid (TBA) reduced glu-tathione (GSH) oxidized glutathione (GSSG) and dithiobis-2-nitrobenzoic acid (DTNB) were purchased from SigmaChemical Co St Louis MO USA Pyrogallol (PG) diethy-lene triamine pentaacetic acid (DTPA) and trichloroaceticacid (TCA) were purchased from Merck Germany RNaseethanol bromophenol blue ethidium bromide 100 bp DNAladder and Taq polymerase were purchased from FermentasUSA mouse oligonucleotide primers for interleukin 1 (IL 1)interleukin 10 (IL 10) tumour necrosis factor beta (TNF 120573)and beta actin (120573 actin) were procured from Eurogentec Bel-gium RPMI-1640 from Himedia Mumbai India RT2 SYBR

green ROX q PCR master mix RNeasy lipid tissue minikit Qiazol lysis reagent and RT2 first strands kit were fromQiagen Hilden Germany GenElute mammalian genomicDNA Miniprep kit was from SigmaAldrich USA Mouse Tand B cell selection kit was from Stem Cell Technologies IncCanada 3H-thymidine was from BRIT India All the otherchemicals used were of analytical grade and were purchasedfrom qualified local vendors

22 Equipment The equipment used was as follows spec-trophotometer (Shimadzu Japan) laminar air flow (MarkAir particulars India) incubator shaker (New BrunswickScientific USA) biophotometer (Eppendorf Germany) andsteam sterilizer (Nat Steel India)

23 Animal Husbandry and Welfare All animals were han-dled humanely without making pain or distress and with duecare for their welfare The care and management of the ani-mals will comply with the regulations of the Committee forthe Purpose of Control and Supervision of Experimental Ani-mals (CPCSEA) Government of India All the animal experi-ments were carried out after prior approval from InstitutionalAnimal Ethics Committee and in accordance with approvedinstitutional protocol

Healthy albino mice were used for the study The bodyweight ranges between 17 and 23 g and wasmaintained in a 12h lightdark cycle at a constant temperature of 22 plusmn 3∘C withfree access to standard pellet diet and water Individual ani-mals were identified with picric acid marks on mice In addi-tion to this each animal cage was identified by labels havingdetails such as experiment number name animal number(s)and date of experiment All the animals were acclimatizedfor a period of 5 days before initiation of experiment

24 Synthesis of Dextran Coated Ferrite Nanoparticles(DFNPs) DFNPs were prepared using the coprecipitationmethod Briefly the stoichiometric mixtures of FeCl

3and

FeCl2sdot4H2O (Fe3+Fe2+ 2 1) were heated at 70∘C in N

2

atmosphere Ferrite nanoparticles were precipitated by theaddition of 3M NaOH dropwise for 1 h followed by heatingwith stirring for about another 1 hr The precipitate was thenwashed with deionized water three times to get uniformlydispersed spherical magnetite particles All reactions werecarried out in N

2atmosphere to prevent oxidation of

magnetite to magnemite Surface coating of ferrite nanopar-ticles with dextran was done by overnight stirring (at37∘C) of ferrite nanoparticles in a solution of dextran ofappropriate concentration The precipitate was then washedand lyophilized to get dextran coated ferrite nanoparticles(DFNPs) The size determination of synthesized DFNPswas studied by Transmission Electron Microscopy (TEM)analysis Further characterization of DFNPs was reported byus [17ndash19]

25 Cytotoxicity The cytotoxicity assay was performed byMTT assay using spleen cells [20] The spleen cells wereseeded at a density of 20000 cellswell in a 96-well plate at37∘C in 5 CO

2atmosphere After 24 h of culture 200 400

600 800 and 1000120583gmL of DFNPs (in triplicate cells alone

Molecular Biology International 3

as negative control) were added onto the suspension cultureof spleen cellsThe cells were incubated at 37plusmn1∘C for 24plusmn1 hand examined microscopically for morphological changesand quantitated by MTT assay 20 120583L of MTT dye solution(5mgmL in phosphate buffer pH 74) was added to each wellAfter 4 h of incubation theMTTwas removed and formazancrystals formed were solubilized with 200120583L of DMSO Theabsorbance of each well was read on amicroplate reader (ELx808iu ultramicroplate reader Bio-Tek instruments USA) at540 nmThe relative cell viability () was calculated

26 Reactive Oxygen Species (ROS) The generation of ROSwas monitored by employing 27dichlorodihydrofluoresceindiacetate (H

2DCFDA) [Invitrogen] which is nonfluorescent

unless oxidized by the intracellular ROS A dose dependentmeasurement of the ROS generation was done by prein-cubating 20000ndash40000 spleen cells with H

2DCFDA at a

concentration of 100120583MThen the cells were exposed to 200400 600 800 and 1000 120583gmL of DFNPs (in triplicate) for2 h at 37∘C Cells were then washed in serum-free mediumand resulting fluorescence intensitywas read in a fluorescencemicroplate reader using an excitation wavelength of 488 nmand emission wavelength of 535 nmThe values were normal-ized to the negative control (spleen cells alone)

27 Immunotoxicity Immunotoxicity is to assess the poten-tial adverse effects on the immune system which is an impor-tant component of the overall evaluation of drugchemicalnanomaterial toxicity In this study 100mg of DFNPs wasinjected intraperitoneally to 18 albino mice for immunolog-ical stimulations Six animals each were sacrificed at 0 days(animals not exposed to nanoparticles) and at the end of 7 14and 21 days [total 24 albino mice] after exposure At the endof each observation period gross necropsy was carried outand spleen and liver were collected for the following studies

28 T and B Lymphocyte Proliferation Assay Spleen wasexcised from experimental animals and splenocytes were iso-lated to study the cell proliferation by tritiated (3H) thymidineincorporation assay Viability of splenocytes was assessedusing trypan blue dye exclusion method Single cell spleno-cytes suspension was used to separate T and B lymphocytesusing kit (EasySep B cell enrichment kit T cell enrichmentkit) according to the protocol described in the kit using anautomated cell separator (ROBOSEP) Both the T and B lym-phocytes (200000 cellswell) were cultured in a 96-well platein RPMI-1640 medium supplemented with 10 FBS strep-tomycin (100 120583gmL) and penicillin (100 unitsmL) for 24 hat 37∘C in a CO

2incubator After 48 h of incubation 3H-

thymidine at a concentration of 1120583CimL was added toeach well and incubated further for 24 h at 37∘C Cells wereharvested after 72 h and radioactivity in terms of counts perminute (cpm) was measured by liquid scintillation counter[21 22]

29 Preparation of Liver Homogenate Liver was washedin normal saline and immediately placed in ice bath andhomogenized 10 liver tissue homogenate was prepared inphosphate buffer (01M pH 74) using an ice-chilled glass

homogenizing vessel in a rotor stator homogenizer at900 rpmThis was centrifuged at 3500 rpm for 10min at 4∘CThe resultant supernatants were maintained in an ice bathuntil being used for the estimation of total protein lipidperoxidation reduced glutathione and antioxidant enzymes(glutathione reductase glutathione peroxidase and superox-ide dismutase) using standard protocols with slightmodifica-tions

210 Total Protein Total proteins in the liver homogenate ofmice exposed to DFNPs were estimated by the method of[23] using bovine serum albumin as standard

211 Lipid Peroxidation The extent of lipid peroxidation(LPO) in the liver homogenate ofmice exposed toDFNPswasdetermined as the concentration of malondialdehyde (MDA)generated by the thiobarbituric acid reactive substances(TBARS) as described by [24] The amount of MDA formedwas measured spectrophotometrically at 532 nm

212 Reduced Glutathione Reduced glutathione (GSH) levelin the liver homogenate of mice exposed to DFNPs wasdetermined by the method of [25] with slight modifica-tions in which Ellmanrsquos reagent or DTNB (551015840-dithiobis-(2-nitrobenzoic acid)) reacts with GSH to form a spectropho-tometrically detectable product at 412 nm The change inabsorbance at 412 nm is a linear function of the GSH concen-tration in the reactionmixture and is based on the reaction ofGSHwithDTNB to give a compound 5-thionitrobenzoic acid(TNB) that is absorbed at 412 nm The amount of GSH wasexpressed as nmolmg protein

213 Antioxidant Enzymes The glutathione reductase (GR)activity in liver homogenate of mice exposed to DFNPs wasdetermined by measuring the reduction of GSSG in thepresence of NADPH as described by [26] Thus one GR unitis defined as the reduction of one 120583M of GSSG per minute at25∘C and pH 76

Activity of glutathione peroxidase (GPx) in the liverhomogenate of mice exposed to DFNPs was assayed by themethod described by [27] The remaining GSH after theenzyme catalyzed reactionwas complexedwithDTNBwhichabsorbs at maximum wavelength of 412 nm Enzyme activitywas expressed as 120583g of GSH consumedminmg protein

The superoxide dismutase (SOD) in liver homogenate ofmice exposed to DFNPs was done using modified pyrogallolautoxidation method spectrophotometrically measured at420 nm [28]

All measurements were carried out using UV Spec-trophotometer-1601 Shimadzu Japan

214 Real Time PCR Analysis for Determining m-RNA ofSpecific Cytokines Total m-RNA was isolated from spleno-cytes exposed to DFNPs (200 600 and 800120583gmL) and bareferrite nanoparticles (600 800 120583gmL) following the manu-facturerrsquos protocol using Trizol reagent (Sigma USA) 150 ngof m-RNA was used for c-DNA synthesis of IL-10 IL-1 TNF120573 and 120573 actin in a reaction volume of 20 120583L using RT2 firststrand Kit (Qiagen Germany) and the synthesis was carried


Table 1 The mouse oligonucleotide forward and reverse primer sequence used to determine specific m-RNA gene expressions

Number Primer Primer sequence Accession number

1 Interleukin 10 (IL-10) f-CCAGTCGGCCAGAGCCACAT NM 0105482r-GGCCATGCTTCTCTGCCTGGG

2 Interleukin 1 (IL-1) f-CTCTCCCCAGCTTTTCCAGG NM 0011779751r-TCTCTGGGCTTGACTGCTTG

3 Tumour necrosis factor beta (TNF 120573) f-TGCCAGCTCCAGGATTTCAG NM 0116103r-CTCAGCCCTCACTTGACCTG

5 Beta actin (120573 actin) f-GCGTGGGGACAGCCGCATCTT BC 0231961r-ATCGGCAGAAGGGGCGGAGA

out on the Eppendorf master cycler Germany The mouseoligonucleotide forward and reverse primer sequences usedto determine specific m-RNA gene expressions are depictedin Table 1 The real time PCR reaction was carried out withRT2 SYBR green ROX q PCR master mix of total reactionvolume of 25 120583L and real time PCR amplifications were doneusing a Chromo 4 System Bio-Rad (MJ Research CA) for40 cycles as per manufacturerrsquos protocol The level of geneexpression is reported as the ratio between the mRNA levelof the target gene and the 120573 actin a reference gene using thecomparative 2ΔΔCt method [29]

215 Nuclear DNA (nDNA) Isolation and Amplification of120573 Actin nDNA was isolated from liver tissues of miceused for immunotoxicity studies as per the manufacturerrsquosprotocol (Sigma USA) The quantity and quality of theisolated nDNA were estimated (nanodrop Eppendorf Ger-many) nDNA isolated from in vivo experimental groupswas amplified using mouse 120573 actin specific forward primer(f-GCGTGGGGACAGCCGCATCTT) and reverse primer(r-ATCGGCAGAAGGGGCGGAGA) (Eurogentec BelgiumAccession number HQ6750311) at a concentration of 100 ng120583L per reaction mixture PCR of nDNA was carried out asper standard conditions [30] in Eppendorf master cyclerGermany Purity and integrity of the amplified productswere checked by purity factor (260280 nm) and agarose gelelectrophoresis

216 Sequence Analysis of 120573 Actin Nuclear DNA (nDNA)The amplified PCR product (120573 actin) from the aboveimmunotoxicity studies was purified with a QIAquick PCRpurification kit (Qiagen Hilden Germany) according to themanufacturerrsquos instructions DNA sequencing was done bySangerrsquos dideoxy chain termination method [31] 120573 actinnDNAproductswere sequenced usingmouse120573 actin forwardand reverse primers in AB1 prism 3730 Genetic analyzerDNA sequencer with a Big Dye terminator cycle sequencingready reaction kit (Applied Biosystems JapanCo Ltd TokyoJapan) ABI Sequence Scanner was used for sequencing andalignment of sequence was done using Bioedit software Aftersequence analysis and alignment using sequence navigatorprogram version 21 all sequences were submitted to NCBIwebsite (httpblastncbinlmnihgovBlastcgi) and BLASTsequence similarity search was conducted

Figure 1 TEM image of DFNPs (X30K)

217 Statistical Analysis All values are expressed as Mean plusmnSD unless otherwise stated Statistical significance betweenthe control and experimental values was compared by Stu-dentrsquos 119905-test For all comparisons 119875 lt 005 was consideredsignificant

3 Results

31 Synthesis and Characterization of DFNPs Ferrite nano-particles were prepared by the standard coprecipitationmethod and coatedwith dextran to yieldDFNPsThedetailedcharacterization of thismaterial was published by [17 18]TheTransmission Electron Microscopic (TEM) images indicatethat a very uniform size distribution of DFNPs particles andwas found to be less than 25 nm (Figure 1)

32 Cytotoxicity Cytotoxicity of DFNPs was evaluated incultured splenocytes the results are depicted in Figure 2(a)Nomarked decrease in cell viability was observed after 24 hrsof exposure for all the concentration tested

33 Reactive Oxygen Species It is well known thatmost of themanufactured nanoparticles cause cell injury by increasingreactive oxygen species formation Thus the measurementof ROS upon exposure to DFNPs is far more important Theamount of ROS generated after 24 hrs of exposure toDFNPs isillustrated in Figure 2(b)The results of the study suggest thatthe ROS generation in all the samples was well comparablewith control values and was not statistically significant


020406080

100120

Control 200 400 600 800 1000

Cel

l via

bilit

y (

)MTT assay

Concentration of DFNM (120583gmL)

(a)

020000400006000080000

100000120000140000160000180000

Control 200 400 600 800 1000

Relat

ive fl

uore

scen

ce u

nits

ROS production


(b)

Figure 2 (a) Viability of splenocytes incubated with DFNPs (values are Mean plusmn SD 119899 = 3) (b) ROS production in splenocytes exposed toDFNPs (values are Mean plusmn SD 119899 = 3)

0

5000

10000

15000

20000

25000

Control 7 days 14 days 21 days

(cp

m)

T cells

T cells

3H

thym

idin

e inc

orpo

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n

lowast

(a)

0

10000

20000

30000

40000


(cp

m)

B cells

B cells

3H

thym

idin

e inc

orpo

ratio

nlowast

lowast

(b)

Figure 3 Proliferative activity in spleen cells of exposed to DFNPs (a) T lymphocyte cells (b) B lymphocyte cells (values are Mean plusmn SD119899 = 5) lowaststatistically significant 119875 lt 005

34 Immunotoxicity The animals exposed to DFNPs weremonitored throughout the experimental period for anyunusual changes The general physical conditions of theexperimental animals were normal The increase in bodyweight and feed intake was normal and none of the animalsshowed any abnormality or behavioral changes during theexperimental period Gross examination of carcasses of con-trol and DFNPs exposedmice did not reveal any abnormalityin the organs examined

35 T and B Lymphocyte Proliferation Assay Lymphocyteproliferation is one of the markers to predict the immuneresponse induced upon nanoparticle exposureThe prolifera-tion assaywas evaluated in bothT andB lymphocytes isolatedfrom spleen of animals exposed to DFNPs for different timeperiods The result of the proliferation assay is shown inFigure 3 It was observed that at 7 days after exposure a slightincrease in thymidine incorporation was observed whencompared to control although the effect was statisticallyinsignificant Decrease in lymphocyte proliferation wasobserved for T cells after 21 days of exposure and after 14 and21 days for B cellsThe differences were statistically significanton comparison to control

36 Lipid Peroxidation Figure 4(a) shows the malondialde-hyde (MDA) production in the liver homogenate ofmice afterexposure to DFNPsThe results indicate that the level of LPOproduction in the control and 21 days postexposure groupwas451 plusmn 17 and 583 plusmn 05 nmolesmg proteins respectivelyIt was observed that the level of LPO was not significantlyincreased at any time periods (7 14 and 21 days afterexposure) when compared to control

37 Reduced Glutathione The level of reduced glutathione(GSH) in the liver homogenate of mice exposed to DFNPsis illustrated in Figure 4(b) The result suggests that a timedependent decrease in the level of GSH was observed in theliver homogenates of mice with the significant decrease in21 days postexposure group (112 plusmn 02) when compared tocontrol (149 plusmn 01)

38 Antioxidant Enzymes The level of glutathione reductase(GR) glutathione peroxidase (GPx) and superoxide dismu-tase (SOD) was illustrated in Figure 5 The result of thestudy indicates that the production of GR was significantlyincreased at the end of 7 days and decreased at the endof 21 days after exposure of DFNPs (034 plusmn 002) in micewhen compared to control (029 plusmn 002) Similarly there was


012345678


Con

cent

ratio

n of

MD

A

(nm

olm

g pr

otei

n)Lipid peroxidation

(a)

002040608

112141618


Am

ount

of r

educ

ed G

SH

(nm

olm

g pr

otei

n)

lowast

Reduced glutathione

(b)

Figure 4 (a) Level of malondialdehyde in liver cells of mice exposed to DFNPs (values are Mean plusmn SD 119899 = 3) (b) Level of GSH in liver cellsof mice exposed to DFNPs (values are Mean plusmn SD 119899 = 3) lowaststatistically significant 119875 lt 005

000

010

020

030

040

050


Enzy

me a

ctiv

ity (u

nits

mg

prot

ein)

Antioxidant enzymes

GRGPxSOD

lowast

lowast

lowast

Figure 5 Level of GR GPx and SOD in liver cells of mice exposedto DFNPs (values are Mean plusmn SD 119899 = 3) lowaststatistically significant119875 lt 005

significant increase in the production of SOD in the liverhomogenate of mice 21 days after exposure of DFNPs (015 plusmn000) when compared to control (012 plusmn 001) In additionthere was no increase in the level of GPx observed at any timepoints which was well comparable to control values

39 Real Time PCR Analysis for Determining m-RNA of Spe-cific Cytokines As evident from Figure 6(a) IL-10 mRNAexpression was increased by 3-fold in 600120583gmLDFNPs cellsand 2-fold in 800 120583g DFNPs exposed cells on comparisonof cells treated with similar concentration of bare ferriteparticles No marked difference was noticed in IL-1 mRNAexpression for both DFNPs and bare ferrite nanoparticlestreated cells (Figure 6(b)) Eventually there was a dramaticdecrease in m-RNA expression of TNF 120573 in DFNPs particlesin a concentration dependent manner when compared withbare ferrite particles Differences were observed in both con-centrations (600120583g and 800120583gmL) of DFNPs when com-pared to bare ferrite nanoparticles (Figure 6(c)) The valuesare expressed in Mean plusmn SE

310 Sequence Analysis of 120573 Actin Nuclear DNA (nDNA)From Figure 7(a) ((B) (C) and (D)) (treated groups forward

120573 actin primer) when compared to Figure 7(a) (A) (Controlgroup forward 120573 actin primer) it was evident that bases 10to 20 of the sequence were altered in 7 14 and 21 daysrsquotreated mice liver respectively Likewise it is apparent fromFigure 7(b) ((B) (C) and (D)) (treated groups reverse120573 actinprimer)when comparedwith Figure 7(b) (A) (Control groupreverse 120573 actin primer) bases 10 to 20 showed irregular basemismatches Figure 7(b) (A) (Control group reverse 120573 actinprimer) had a poly A tail whereas in Figure 7(b) (B) (7 daysrsquotreated group) such tail was tainted in the sequence

4 Discussion

Bioengineered nanoparticles are being considered for a widerange of biomedical applications from magnetic resonanceimaging to drug delivery systems The development of novelnanomaterials for biomedical applications must be accompa-nied by careful scrutiny of their biocompatibility Attentionshould be paid on the possible interactions between nanopar-ticles and cells of the immune system In the present studyDFNPs intended to be used for targeted drug delivery appli-cations were in-house synthesized characterized and exam-ined for the immunotoxic potential and cellular responseDFNPs were synthesized using the coprecipitation methodThe detailed characterization of DFNPs was carried out usingDynamic Light Scattering for hydrodynamic size profilingtransmission electron microscope for particle size analysisX-ray diffraction technique for phase purity analysis thermogravimetric analysis for quantifying the dextran in DFNPsand Fourier transform infrared spectral analysis for coatingefficiency The result of the study indicated that the synthe-sized DFNPs were an authentic particle and have a size lessthan 25 nm [17 18]

On entry into the body nanoparticles can move aheadand get absorbed in liver spleen and bone marrow throughblood and lymph It was also known that nanoparticles expo-suremay result in ROSproduction and splenocytes apoptosischange cytokine production and decrease immune responseImmune cells are the ones that act as defense against anypathogen access Thus it is extremely crucial to evaluate thealtered immune responses induced upon nanoparticle entryIn this study systemic immune response induced by DFNPswas evaluated under both in vitro and in vivo conditions


005

115

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200 600 800 600 800

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ive m

RNA

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essio

nIL 10

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Concentration of nanoparticles (120583gmL)

(a)

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001

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1

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200 600 800 600 800

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ive m

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expr

essio

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IL 1


(b)

DFNPFerrite NP

005

115

225

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200 600 800 600 800

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ive m

RNA

expr

essio

n


TNF 120573

(c)

Figure 6 (a) Relative mRNA expression (IL 10) when splenocytes were exposed to different concentrations of DFNPs (values are Mean plusmnSE 119899 = 3) (b) Relative mRNA expression (IL 1) when splenocytes were exposed to different concentrations of DFNPs (values are Mean plusmn SE119899 = 3) (c) Relative mRNA expression (TNF 120573) when splenocytes were exposed to different concentrations of DFNPs (values are Mean plusmn SE119899 = 3)

Spleen is the largest lymphoid organwhich plays a critical rolein both innate and adaptive immune responses splenocyteswere opted for initial cytotoxicity screening It was found thatno obvious cytotoxicity was noted in spleen cells after DFNPsexposure even though a marginal increase in splenocytesactivity was observedThe oxidative stress experienced by thecell was assessed by the measurement of intracellular reactiveoxygen species (ROS) level Increases in intracellular ROS(oxidative stress) are potentially toxic to the cells which ifnot neutralized by antioxidant defenses (eg glutathione andantioxidant enzymes) could lead to membrane dysfunctionprotein degradation and DNA damage and finally cell deathFor measuring the ROS the cell permeable probe (ie 2101584071015840-dichlorodihydro-fluorescein diacetate H

2DCFDA) was used

for the present study From the results it was very clear thatthe ROS generation was well comparable with control valuesthat predict a nontoxic response at the cellular level

The systemic administration of DFNPs (7 14 and 21 days)for immunotoxicity evaluation in albinomice showed that thegeneral physical conditions of the experimental animals werenormal during the experimental period All the animals were

sacrificed and there were no abnormalities observed duringthe gross necropsies of animal at the end of each observa-tion period The results of the proliferation assay indicatedthat there was a significant reduction in the 3H-Thymidineincorporation in both the T and B cells after 14 and 21 daysafter exposure of DFNPs which needs further investigation

The concentration of MDA in biological materials hasbeen widely utilized as an indicator of oxidative damageto unsaturated lipid Measurement of MDA the byproductof LPO provides an exact and well-established index ofoxidative damage since it is very reactive and takes part incross-linking with biomolecules [32] It is well known thatlipid peroxidation occurs naturally in small amounts in thebody mainly by the effect of several ROS or by the action ofseveral phagocytes [33 34] In the present study no changesin the level of lipid peroxidation in liver were observed Thiscorrelates with the results obtained in in vitro study using flu-orescent probe But there was a significant reduction of GSHobserved after 21 days of exposure and this may be the resultof direct reaction ofDFNPswithGSH [35] reported that afteradministration of nanoparticles GSH can act as a conjugating


(A) Control nuclear DNA 120573 actin sequence (forward primer)

(B) 7 daysrsquo treated nuclear DNA 120573 actin sequence (forward primer)

(C) 14 daysrsquo treated nuclear DNA 120573 actin sequence (forward primer)

(D) 21 daysrsquo treated nuclear DNA 120573 actin sequence (forward primer)

(a)

(A) Control nuclear DNA 120573 actin sequence (reverse primer)

(C) 14 daysrsquo treated nuclear DNA 120573 actin sequence (reverse primer)

(D) 21 daysrsquo treated nuclear DNA 120573 actin sequence (reverse primer)

(B) 7 daysrsquo treated nuclear DNA 120573 actin sequence (reverse primer)

(b)

Figure 7 Sequence analysis of cytoskeletal 120573 actin from mouse liver

agent in their metabolism When these nanoparticles induceoxidative stress by generating H

2O2or hydroperoxides GSH

can also be oxidized in a reaction catalyzed by GSH-Px Thedepletion ofGSH in tissues leads to impairment of the cellulardefense against ROS and may result in peroxidative injury[35 36]

Regarding the antioxidant enzymes it was observed thatthe level of GR and SODwas significantly altered 21 days afterexposure period Intracellular GSH scavenge free radicalsformed inside the cells The cells replenish the lost GSH byconverting the peroxides formed from free radicals intoGSHThis reaction was catalyzed by GRThus in the present studythe increase in GR activity observed after 7 days of exposuremay result in the conversion of peroxides formed by ROS intoGSH After 21 days of exposure although a slight decreasein GR activity was observed it was significantly highercompared to control It was reported that GPx GR andSOD protect cells against ROS [37] GR and GPx are the twomost important enzymes in the GSH-GSSG cycle andmay be

activated by increased hydrogen andor lipid peroxide pro-duction Antioxidant activity of GSH-Px involves neutral-ization of H

2O2 reduction of lipid hydroperoxidases and

maintenance of normal membrane permeability [38] It wasalso observed that there was no increase in the GPx pro-duction when DFNPs were exposed to mice The SOD levelwas also comparable to control values in mice exposed toDFNPs

The finding of the cytokines analysis of the presentstudy are in line with the literature suggesting that tumornecrosis factor-beta (TNF 120573) is a cytokine that is inhibitedby interleukin 10 It was observed that the increase in theconcentration of DFNPs increases the expression of IL-10an anti-inflammatory cytokine and decreases the expressionof TNF 120573 which mediates a large variety of inflammatoryimmunostimulatory and antiviral responses [39] Earlierstudies revealed no macrophage or dendritic cell secretion ofproinflammatory cytokines when silicone-coated iron oxidenanoparticles were administered [40] Similarly the present


study revealed that no increase in proinflammatory cytokinesoccurs upon administration of DFNPs

Sequence analysis of cytoskeletal 120573 actin revealed regionsfrom 20 to 50 bases were conserved in treated mice Earlierstudies of cytoskeletal 120573 actin mRNA sequencing of mouseprovided information about the conserved sequences as wellas the posttranscriptional regions in the sequence [41] Theconserved sequence of 120573 actin in mice for miR-644a targetsite was provided in 2012 [42]The 51015840 end of themouse120573 actingene contains sequence elements which mediate the stim-ulatory effects of serum growth factors and are responsiveto both positive and negative regulators of gene expressionof several genes [43] Base mismatches in the nonconservedregion observed may not significantly lead to mutations andaltered protein synthesis but may result in the short frameshift mutations

5 Conclusion

Animals appeared to be normal during the course of theexperimental period after exposure to DFNPs The DFNPs(lt25 nm) are found to be noncytotoxic in spleen cells It wasalso evident that the DFNPs do not influence cellular prolif-eration LPO GSH and antioxidant enzymes Hence it canbe concluded that the DFNPs were nontoxic at cellular leveland nonimmunotoxic when exposed to albino mice underlaboratory conditions simulation Base alterations in the non-conserved regionmay not significantly lead to mutations anddistorted protein synthesis of the cytoskeletal 120573 actin genebut might result in the diminutive frame shift mutations

Abbreviations

DFNPs dextran coated ferrite nanoparticlesROS reactive oxygen speciesLPO lipid peroxidationGSH reduced glutathioneGR glutathione reductaseGPx glutathione peroxidaseSOD superoxide dismutaseIL1 interleukin 1IL10 interleukin 10TNF 120573 tumor necrosis factor 120573

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

Theauthors are thankful to theDirector andHead of Biomed-ical Technology Wing Sree Chitra Tirunal Institute forMedical Sciences and Technology for providing the facilitiesto carry out the work The authors also acknowledge thetechnical assistance of Mr Shaji S and Mr Harikumar GThework was financially supported by the Nanomission Depart-ment of Science andTechnology (Government of India) NewDelhi (Grant no SRNMNS-902008)

References

[1] C Corot P Robert J-M Idee and M Port ldquoRecent advancesin iron oxide nanocrystal technology for medical imagingrdquoAdvanced Drug Delivery Reviews vol 58 no 14 pp 1471ndash15042006

[2] A Kumar B Sahoo AMontpetit S Behera R F Lockey and SS Mohapatra ldquoDevelopment of hyaluronic acid-Fe

2O3hybrid

magnetic nanoparticles for targeted delivery of peptidesrdquoNano-medicine Nanotechnology Biology and Medicine vol 3 no 2pp 132ndash137 2007

[3] L Hovgaard and H Broslashndsted ldquoCurrent applications ofpolysaccharides in colon targetingrdquo Critical Reviews in Thera-peutic Drug Carrier Systems vol 13 no 3-4 pp 185ndash223 1996

[4] A S Arbab L A Bashaw B RMiller et al ldquoCharacterization ofbiophysical andmetabolic properties of cells labeledwith super-paramagnetic iron oxide nanoparticles and transfection agentfor cellularMR imagingrdquoRadiology vol 229 no 3 pp 838ndash8462003

[5] D Bahadur and J Giri ldquoBiomaterials andmagnetismrdquo Sadhanavol 28 no 3 pp 639ndash656 2003

[6] D-H Kim D E Nikles D T Johnson and C S Brazel ldquoHeatgeneration of aqueously dispersed CoFe

2O4nanoparticles as

heating agents for magnetically activated drug delivery andhyperthermiardquo Journal of Magnetism and Magnetic Materialsvol 320 no 19 pp 2390ndash2396 2008

[7] B Gleich and J Weizenecker ldquoTomographic imaging using thenonlinear response of magnetic particlesrdquo Nature vol 435 no7046 pp 1214ndash1217 2005

[8] Y X Wang S M Hussain and G P Krestin ldquoSuperparamag-netic iron oxide contrast agents physicochemical characteris-tics and applications in MR imagingrdquo European Radiology vol11 no 11 pp 2319ndash2331 2001

[9] S Deb J Giri S Dasgupta D Datta andD Bahadur ldquoSynthesisand characterization of biocompatible hydroxyapatite coatedferriterdquo Bulletin of Materials Science vol 26 no 7 pp 655ndash6602003

[10] M-H Tan C A Commens L Burnett and P J Snitch ldquoA pilotstudy on the percutaneous absorption of microfine titaniumdioxide from sunscreensrdquo Australasian Journal of Dermatologyvol 37 no 4 pp 185ndash187 1996

[11] O Lunov T Syrovets B Buchele et al ldquoThe effect of carboxy-dextran-coated superparamagnetic iron oxide nanoparticles onc-Jun N-terminal kinase-mediated apoptosis in human macro-phagesrdquo Biomaterials vol 31 no 19 pp 5063ndash5071 2010

[12] K G Desai and H J Park ldquoPreparation characterization andprotein loading of hexanoyl-modified chitosan nanoparticlesrdquoDrug Delivery vol 13 no 5 pp 375ndash381 2006

[13] A Sutton G E Harrison T E F Carr and D BarltropldquoReduction in the absorption of dietary strontium in children byan alginate derivativerdquo International Journal of Radiation Biol-ogy vol 19 no 1 pp 79ndash85 1971

[14] M Elsabahy and K L Wooley ldquoCytokines as biomarkers ofnanoparticle immunotoxicityrdquo Chemical Society Reviews vol42 no 12 pp 5552ndash5576 2013

[15] N Gao Q Zhang Q Mu et al ldquoSteering carbon nanotubes toscavenger receptor recognition by nanotube surface chemistrymodification partially alleviatesNF120581B activation and reduces itsimmunotoxicityrdquo ACS Nano vol 5 no 6 pp 4581ndash4591 2011

[16] M Elsabahy S Samarajeewa J E Raymond C Clark and KL Wooley ldquoShell-crosslinked knedel-like nanoparticles induce


lower immunotoxicity than their non-crosslinked analogsrdquoJournal of Materials Chemistry B vol 1 no 39 pp 5241ndash52552013

[17] P V Mohanan C S Geetha S Syama and H K Varma ldquoInter-facing of dextran coated ferrite nanomaterials with cellularsystem and delayed hypersensitivity on Guinea pigsrdquo Colloidsand Surfaces B Biointerfaces vol 116 pp 633ndash642 2014

[18] P V Mohanan S Syama A Sabareeswaran P J Sreekanthand H K Varma ldquoMolecular toxicity of dextran coated ferritenanoparticles after dermal exposure to Wistar ratsrdquo Journal ofToxicology and Health vol 104 pp 406ndash422 2014

[19] S Syama S C Reshma B Leji et al ldquoToxicity evaluation ofdextran coated ferrite nanomaterials after acute oral exposuretoWistar ratsrdquo Journal of Allergy ampTherapy vol 5 no 2 article166 2014

[20] A A van de Loosdrecht R H J Beelen G J Ossenkoppele MG Broekhoven andMMAC Langenhuijsen ldquoA tetrazolium-based colorimetric MTT assay to quantitate human monocytemediated cytotoxicity against leukemic cells from cell lines andpatientswith acutemyeloid leukemiardquo Journal of ImmunologicalMethods vol 174 no 1-2 pp 311ndash320 1994

[21] V Gayathri C S Geetha and P V Mohanan ldquoProtectivemechanism of melatonin on kainic acid induced immunemodulatory effect on lymphocytes derived frommouse spleenrdquoJournal of Clinical amp Cellular Immunology vol 4 article 172 9pages 2013

[22] V Gayathri C S Geetha and P V Mohanan ldquoAttenuation bymelatonin of kainic acid-induced alterations in the expressionof mRNAs of some immune-modulatory cytokines and mito-chondrial DNA damage in micerdquoThe Journal of Toxicology andHealth vol 103 pp 150ndash161 2013

[23] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[24] A Okado-Matsumoto and I Fridovich ldquoSubcellular distribu-tion of superoxide dismutases (SOD) in rat liver Cu Zn-SODin mitochondriardquo Journal of Biological Chemistry vol 276 no42 pp 38388ndash38393 2001

[25] M S Moron J W Depierre and B Mannervik ldquoLevels of glu-tathione glutathione reductase and glutathione S-transferaseactivities in rat lung and liverrdquo Biochimica et Biophysica Actavol 582 no 1 pp 67ndash78 1979

[26] C E Mize and R G Langdon ldquoHepatic glutathione reductaseI Purification and general kinetic propertiesrdquo The Journal ofBiological Chemistry vol 237 pp 1589ndash1595 1962

[27] J T Rotruck A L Pope H E Ganther A B Swanson D GHafeman and W G Hoekstra ldquoSelenium biochemical role asa component of glutathione peroxidaserdquo Science vol 179 no4073 pp 588ndash590 1973

[28] S Marklund and G Marklund ldquoInvolvement of the superoxideanion radical in the autoxidation of pyrogallol and a convenientassay for superoxide dismutaserdquo European Journal of Biochem-istry vol 47 no 3 pp 469ndash474 1974

[29] A-A Dussault and M Pouliot ldquoRapid and simple comparisonof messenger RNA levels using real-time PCRrdquo BiologicalProcedures Online vol 8 no 1 pp 1ndash10 2006

[30] T C Koekemoer T G Downing and W Oelofsen ldquoAn alter-native PCR assay for quantifying mitochondrial DNA in crudepreparationsrdquo Nucleic Acids Research vol 26 no 11 pp 2829ndash2830 1998

[31] F Sanger and A R Coulson ldquoA rapid method for determiningsequences in DNA by primed synthesis with DNA polymeraserdquoJournal of Molecular Biology vol 94 no 3 pp 441ndash448 1975

[32] S Kubow ldquoToxicity of dietary lipid peroxidation productsrdquoTrends in Food Science and Technology vol 1 pp 67ndash71 1990

[33] CHoskins LWangW P Cheng andA Cuschieri ldquoDilemmasin the reliable estimation of the in-vitro cell viability inmagneticnanoparticle engineering which tests and what protocolsrdquoNanoscale Research Letters vol 7 article 77 2012

[34] Y Cho R Shi and R Ben Borgens ldquoChitosan nanoparticle-based neuronal membrane sealing and neuroprotection follow-ing acrolein-induced cell injuryrdquo Journal of Biological Engineer-ing vol 4 article 2 2010

[35] H Sies and P M T Akerboom ldquoGlutathione disulfide effluxfrom cells and tissuesrdquo Methods in Enzymology vol 105 pp445ndash451 1984

[36] T Berivan and N U Nuray ldquoKinetic mechanism andmolecularproperties of glutathione reductaserdquoFABAD Journal of Pharma-ceutical Sciences vol 31 pp 230ndash237 2006

[37] L Flohe ldquoGlutathione peroxidase brought to focusrdquo in FreeRadicals in Biology and Medicine W Pryor Ed vol 5 pp 223ndash253 Academic Press New York NY USA 1982

[38] B A Zachara U Trafikowska H Labedzka A Sosnowskiand R Kanarkowski ldquoEffect of selenium supplementation onglutathione peroxidase synthesis and element accumulation insheep erythrocytesrdquo Biomedica Biochimica Acta vol 49 no 2-3pp S186ndashS191 1990

[39] C Waltenbaugh T Doan R Melvold and S Viselli Immunol-ogy Wolters Kluwer Health Lippincott Williams amp WilkinsPhiladelphia Pa USA 2008

[40] A Kunzmann B Andersson C Vogt et al ldquoEfficient inter-nalization of silica-coated iron oxide nanoparticles of differentsizes by primary human macrophages and dendritic cellsrdquoToxicology and Applied Pharmacology vol 253 no 2 pp 81ndash932011

[41] K Toicunaga H Taniguchi K Yoda M Shimizu and S Sal-ciyama ldquoNucleotide sequence of a full-length cdna for mousecytosketetal 120573-actin raRNArdquoNucleic Acids Research vol 14 no6 article 2829 1986

[42] K Sikand J Singh J S Ebron andG C Shukla ldquoHousekeepinggene selection advisory glyceraldehyde-3-phosphate dehydro-genase (GAPDH) and 120573-actin are targets of miR-644ardquo PLoSONE vol 7 no 10 Article ID e47510 2012

[43] P K Elder C L French M Subramaniam L J Schmidt andM J Getz ldquoEvidence that the functional 120573-actin gene is singlecopy inmostmice and is associatedwith 51015840 sequences capable ofconferring serum- and cycloheximide-dependent regulationrdquoMolecular and Cellular Biology vol 8 no 1 pp 480ndash485 1988

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


subcellular organelles As a consequence of all these proper-ties nanoparticles can have direct interaction with individualtarget cells either with the external membrane or inside thecell at the site of action Advances in nanotechnology ledto the exposure of humans to engineered nanomaterials andhence it became necessary to evaluate the potential humanhealth effects before these materials are fully exploited [1 2]

Experimental evidences have shown that metal andmetaloxide nanoparticles induced DNA damage and apoptosisthrough ROS generation and oxidative stress [10 11] It hasbeen reported that magnetic nanoparticles administeredintraperitoneally can cross blood-brain barrier where theyhave the potential to affect cerebral functions [12] Due to thehigh reactivity of ROS most cellular components are likely tobe the targets of oxidative damage lipid peroxidation proteinoxidation GSH depletion and DNA single strand breaks Allof these events ultimately lead to cellular dysfunction andinjury [13] For this reason antioxidant enzymes are vitalmarkers for oxidative stress induced in the body Aerobicorganisms possess antioxidant defense systems that deal withthe removal of ROS As long as there exists a balance betweenoxidative stress and antioxidant defense system human bodyis maintained in an optimal health state

Antioxidant defense system includes both low-molecu-lar-weight free radical scavengers such as the tripeptide glu-tathione (GSH) as well as antioxidant enzymes such as super-oxide dismutase (SOD) glutathione reductase (GR) andglutathione peroxidase (GPx) [3]

The immunotoxicology of engineered nanoparticles is agrowing concern in the present era The potential immuno-toxicity and mechanisms of action of these particles have notreceived sufficient attention Nanoparticles can interact withimmune system in several ways and can enhance or suppressimmune function depending on their physicochemical char-acteristics [14] It was reported that the surface modificationof carbon nanotubes recognizes scavenger receptor andalleviates NF-120581B activation and reduces its immunotoxicity[15] Similarly shell-cross-linked knedel-like nanoparticlesinduced lower immunotoxicity than their non-cross-linkedanalogs as was demonstrated by Elsabahy et al [16]

Here DFNPs intended to be used for targeted drug deliv-ery applications were in-house synthesized and character-ized The primary focus of the present study was to examinethe immunotoxic potential and cellular response of DFNPs

2 Materials and Methods

21 Chemicals Thiobarbituric acid (TBA) reduced glu-tathione (GSH) oxidized glutathione (GSSG) and dithiobis-2-nitrobenzoic acid (DTNB) were purchased from SigmaChemical Co St Louis MO USA Pyrogallol (PG) diethy-lene triamine pentaacetic acid (DTPA) and trichloroaceticacid (TCA) were purchased from Merck Germany RNaseethanol bromophenol blue ethidium bromide 100 bp DNAladder and Taq polymerase were purchased from FermentasUSA mouse oligonucleotide primers for interleukin 1 (IL 1)interleukin 10 (IL 10) tumour necrosis factor beta (TNF 120573)and beta actin (120573 actin) were procured from Eurogentec Bel-gium RPMI-1640 from Himedia Mumbai India RT2 SYBR

green ROX q PCR master mix RNeasy lipid tissue minikit Qiazol lysis reagent and RT2 first strands kit were fromQiagen Hilden Germany GenElute mammalian genomicDNA Miniprep kit was from SigmaAldrich USA Mouse Tand B cell selection kit was from Stem Cell Technologies IncCanada 3H-thymidine was from BRIT India All the otherchemicals used were of analytical grade and were purchasedfrom qualified local vendors

22 Equipment The equipment used was as follows spec-trophotometer (Shimadzu Japan) laminar air flow (MarkAir particulars India) incubator shaker (New BrunswickScientific USA) biophotometer (Eppendorf Germany) andsteam sterilizer (Nat Steel India)

23 Animal Husbandry and Welfare All animals were han-dled humanely without making pain or distress and with duecare for their welfare The care and management of the ani-mals will comply with the regulations of the Committee forthe Purpose of Control and Supervision of Experimental Ani-mals (CPCSEA) Government of India All the animal experi-ments were carried out after prior approval from InstitutionalAnimal Ethics Committee and in accordance with approvedinstitutional protocol

Healthy albino mice were used for the study The bodyweight ranges between 17 and 23 g and wasmaintained in a 12h lightdark cycle at a constant temperature of 22 plusmn 3∘C withfree access to standard pellet diet and water Individual ani-mals were identified with picric acid marks on mice In addi-tion to this each animal cage was identified by labels havingdetails such as experiment number name animal number(s)and date of experiment All the animals were acclimatizedfor a period of 5 days before initiation of experiment

24 Synthesis of Dextran Coated Ferrite Nanoparticles(DFNPs) DFNPs were prepared using the coprecipitationmethod Briefly the stoichiometric mixtures of FeCl

3and

FeCl2sdot4H2O (Fe3+Fe2+ 2 1) were heated at 70∘C in N

2

atmosphere Ferrite nanoparticles were precipitated by theaddition of 3M NaOH dropwise for 1 h followed by heatingwith stirring for about another 1 hr The precipitate was thenwashed with deionized water three times to get uniformlydispersed spherical magnetite particles All reactions werecarried out in N

2atmosphere to prevent oxidation of

magnetite to magnemite Surface coating of ferrite nanopar-ticles with dextran was done by overnight stirring (at37∘C) of ferrite nanoparticles in a solution of dextran ofappropriate concentration The precipitate was then washedand lyophilized to get dextran coated ferrite nanoparticles(DFNPs) The size determination of synthesized DFNPswas studied by Transmission Electron Microscopy (TEM)analysis Further characterization of DFNPs was reported byus [17ndash19]

25 Cytotoxicity The cytotoxicity assay was performed byMTT assay using spleen cells [20] The spleen cells wereseeded at a density of 20000 cellswell in a 96-well plate at37∘C in 5 CO

2atmosphere After 24 h of culture 200 400

600 800 and 1000120583gmL of DFNPs (in triplicate cells alone






2DCFDA at a




























3 Results





020406080

100120

Control 200 400 600 800 1000

Cel

l via

bilit

y (

)MTT assay


(a)

020000400006000080000

100000120000140000160000180000

Control 200 400 600 800 1000

Relat

ive fl

uore

scen

ce u

nits

ROS production


(b)


0

5000

10000

15000

20000

25000


(cp

m)

T cells

T cells

3H

thym

idin

e inc

orpo

ratio

n

lowast

(a)

0

10000

20000

30000

40000


(cp

m)

B cells

B cells

3H

thym

idin

e inc

orpo

ratio

nlowast

lowast

(b)








012345678


Con

cent

ratio

n of

MD

A

(nm

olm

g pr

otei


(a)

002040608

112141618


Am

ount

of r

educ

ed G

SH

(nm

olm

g pr

otei

n)

lowast

Reduced glutathione

(b)


000

010

020

030

040

050


Enzy

me a

ctiv

ity (u

nits

mg

prot

ein)

Antioxidant enzymes

GRGPxSOD

lowast

lowast

lowast






4 Discussion




005

115

225

335

445

200 600 800 600 800

Relat

ive m

RNA

expr

essio

nIL 10

DFNPFerrite NP


(a)

DFNPFerrite NP

001

01

1

10

200 600 800 600 800

Relat

ive m

RNA

expr

essio

n

IL 1


(b)

DFNPFerrite NP

005

115

225

335

200 600 800 600 800

Relat

ive m

RNA

expr

essio

n


TNF 120573

(c)



2DCFDA) was used










(a)





(b)













5 Conclusion


Abbreviations




Acknowledgments


References



2O3hybrid






2O4nanoparticles as
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






2DCFDA at a




























3 Results





020406080

100120

Control 200 400 600 800 1000

Cel

l via

bilit

y (

)MTT assay


(a)

020000400006000080000

100000120000140000160000180000

Control 200 400 600 800 1000

Relat

ive fl

uore

scen

ce u

nits

ROS production


(b)


0

5000

10000

15000

20000

25000


(cp

m)

T cells

T cells

3H

thym

idin

e inc

orpo

ratio

n

lowast

(a)

0

10000

20000

30000

40000


(cp

m)

B cells

B cells

3H

thym

idin

e inc

orpo

ratio

nlowast

lowast

(b)








012345678


Con

cent

ratio

n of

MD

A

(nm

olm

g pr

otei


(a)

002040608

112141618


Am

ount

of r

educ

ed G

SH

(nm

olm

g pr

otei

n)

lowast

Reduced glutathione

(b)


000

010

020

030

040

050


Enzy

me a

ctiv

ity (u

nits

mg

prot

ein)

Antioxidant enzymes

GRGPxSOD

lowast

lowast

lowast






4 Discussion




005

115

225

335

445

200 600 800 600 800

Relat

ive m

RNA

expr

essio

nIL 10

DFNPFerrite NP


(a)

DFNPFerrite NP

001

01

1

10

200 600 800 600 800

Relat

ive m

RNA

expr

essio

n

IL 1


(b)

DFNPFerrite NP

005

115

225

335

200 600 800 600 800

Relat

ive m

RNA

expr

essio

n


TNF 120573

(c)



2DCFDA) was used










(a)





(b)













5 Conclusion


Abbreviations




Acknowledgments


References



2O3hybrid






2O4nanoparticles as
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













3 Results





020406080

100120

Control 200 400 600 800 1000

Cel

l via

bilit

y (

)MTT assay


(a)

020000400006000080000

100000120000140000160000180000

Control 200 400 600 800 1000

Relat

ive fl

uore

scen

ce u

nits

ROS production


(b)


0

5000

10000

15000

20000

25000


(cp

m)

T cells

T cells

3H

thym

idin

e inc

orpo

ratio

n

lowast

(a)

0

10000

20000

30000

40000


(cp

m)

B cells

B cells

3H

thym

idin

e inc

orpo

ratio

nlowast

lowast

(b)








012345678


Con

cent

ratio

n of

MD

A

(nm

olm

g pr

otei


(a)

002040608

112141618


Am

ount

of r

educ

ed G

SH

(nm

olm

g pr

otei

n)

lowast

Reduced glutathione

(b)


000

010

020

030

040

050


Enzy

me a

ctiv

ity (u

nits

mg

prot

ein)

Antioxidant enzymes

GRGPxSOD

lowast

lowast

lowast






4 Discussion




005

115

225

335

445

200 600 800 600 800

Relat

ive m

RNA

expr

essio

nIL 10

DFNPFerrite NP


(a)

DFNPFerrite NP

001

01

1

10

200 600 800 600 800

Relat

ive m

RNA

expr

essio

n

IL 1


(b)

DFNPFerrite NP

005

115

225

335

200 600 800 600 800

Relat

ive m

RNA

expr

essio

n


TNF 120573

(c)



2DCFDA) was used










(a)





(b)













5 Conclusion


Abbreviations




Acknowledgments


References



2O3hybrid






2O4nanoparticles as
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


020406080

100120

Control 200 400 600 800 1000

Cel

l via

bilit

y (

)MTT assay


(a)

020000400006000080000

100000120000140000160000180000

Control 200 400 600 800 1000

Relat

ive fl

uore

scen

ce u

nits

ROS production


(b)


0

5000

10000

15000

20000

25000


(cp

m)

T cells

T cells

3H

thym

idin

e inc

orpo

ratio

n

lowast

(a)

0

10000

20000

30000

40000


(cp

m)

B cells

B cells

3H

thym

idin

e inc

orpo

ratio

nlowast

lowast

(b)








012345678


Con

cent

ratio

n of

MD

A

(nm

olm

g pr

otei


(a)

002040608

112141618


Am

ount

of r

educ

ed G

SH

(nm

olm

g pr

otei

n)

lowast

Reduced glutathione

(b)


000

010

020

030

040

050


Enzy

me a

ctiv

ity (u

nits

mg

prot

ein)

Antioxidant enzymes

GRGPxSOD

lowast

lowast

lowast






4 Discussion




005

115

225

335

445

200 600 800 600 800

Relat

ive m

RNA

expr

essio

nIL 10

DFNPFerrite NP


(a)

DFNPFerrite NP

001

01

1

10

200 600 800 600 800

Relat

ive m

RNA

expr

essio

n

IL 1


(b)

DFNPFerrite NP

005

115

225

335

200 600 800 600 800

Relat

ive m

RNA

expr

essio

n


TNF 120573

(c)



2DCFDA) was used










(a)





(b)













5 Conclusion


Abbreviations




Acknowledgments


References



2O3hybrid






2O4nanoparticles as
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


012345678


Con

cent

ratio

n of

MD

A

(nm

olm

g pr

otei


(a)

002040608

112141618


Am

ount

of r

educ

ed G

SH

(nm

olm

g pr

otei

n)

lowast

Reduced glutathione

(b)


000

010

020

030

040

050


Enzy

me a

ctiv

ity (u

nits

mg

prot

ein)

Antioxidant enzymes

GRGPxSOD

lowast

lowast

lowast






4 Discussion




005

115

225

335

445

200 600 800 600 800

Relat

ive m

RNA

expr

essio

nIL 10

DFNPFerrite NP


(a)

DFNPFerrite NP

001

01

1

10

200 600 800 600 800

Relat

ive m

RNA

expr

essio

n

IL 1


(b)

DFNPFerrite NP

005

115

225

335

200 600 800 600 800

Relat

ive m

RNA

expr

essio

n


TNF 120573

(c)



2DCFDA) was used










(a)





(b)













5 Conclusion


Abbreviations




Acknowledgments


References



2O3hybrid






2O4nanoparticles as
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


005

115

225

335

445

200 600 800 600 800

Relat

ive m

RNA

expr

essio

nIL 10

DFNPFerrite NP


(a)

DFNPFerrite NP

001

01

1

10

200 600 800 600 800

Relat

ive m

RNA

expr

essio

n

IL 1


(b)

DFNPFerrite NP

005

115

225

335

200 600 800 600 800

Relat

ive m

RNA

expr

essio

n


TNF 120573

(c)



2DCFDA) was used










(a)





(b)













5 Conclusion


Abbreviations




Acknowledgments


References



2O3hybrid






2O4nanoparticles as
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






(a)





(b)













5 Conclusion


Abbreviations




Acknowledgments


References



2O3hybrid






2O4nanoparticles as
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




5 Conclusion


Abbreviations




Acknowledgments


References



2O3hybrid






2O4nanoparticles as
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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