research article iterative cellular screening system for...

Research ArticleIterative Cellular Screening System for NanoparticleSafety Testing

Franziska Sambale1 Frank Stahl1 Ferdinand Ruumldinger1 Dror Seliktar2 Cornelia Kasper3

Detlef Bahnemann14 and Thomas Scheper1

1 Institute for Technical Chemistry Gottfried Wilhelm Leibniz University Hanover Callinstraszlige 5 30167 Hanover Germany2Faculty of Biomedical Engineering Technion Israel Institute of Technology 32000 Haifa Israel3Institute of Applied Microbiology University of Natural Resources and Life Science (Boku) Muthgasse 18 1190 Vienna Austria4Laboratory ldquoPhotoactive Nanocomposite Materialsrdquo Saint Petersburg State University Ulyanovskaya Street 1 PeterhofSaint Petersburg 198504 Russia

Correspondence should be addressed to Frank Stahl stahliftcuni-hannoverde

Received 14 April 2015 Revised 1 July 2015 Accepted 8 July 2015

Academic Editor Jin-Ho Choy

Copyright copy 2015 Franziska Sambale 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

Nanoparticles have the potential to exhibit risks to human beings and to the environment due to the wide applications ofnanoproducts extensive risk management must not be neglected Therefore we have constructed a cell-based iterative screeningsystem to examine a variety of nanoproducts concerning their toxicity during development The sensitivity and application ofvarious cell-based methods were discussed and proven by applying the screening to two different nanoparticles zinc oxide andtitanium dioxide nanoparticles They were used as benchmarks to set up our methods and to examine their effects on mammaliancell lines Different biological processes such as cell viability gene expression of interleukin-8 and heat shock protein 70 as well asmorphology changes were investigated Within our screening system both nanoparticle suspensions and coatings can be testedElectric cell impedancemeasurements revealed to be a goodmethod for onlinemonitoring of cellular behaviorThe implementationof three-dimensional cell culture is essential to better mimic in vivo conditions In conclusion our screening system is highlyefficient cost minimizing and reduces the need for animal studies

1 Introduction

Nanoparticles have been used in many applications overthe last decades such as cosmetics medicine and paints In2012 more than 100 products containing nanoparticles wereavailable [1] However the constantly increasing amount ofindustrial as well as consumer nanoproducts requires reliableand extensive risk management as interactions with humanbeings and the environment are becoming more frequentVarious public exposure paths are possible [2] but up to nowno standardized guideline for nanoparticle and nanomateri-als testing exists [1 3] Currently for nanoparticle testing invitro only the commonly used assays validated for drug test-ing are available However nanoparticles differ significantlyfrom normal chemicals [2] and can interfere with the assays[1ndash4]

In particular oxidized nanoparticles are able to reduceassay dyes causing an underestimation of the cytotoxicity byoverestimating cell viability [5] Furthermore a key challengeof in vitro nanoparticle safety testing is determining thestability of nanoparticles in biological media For a pluralityof nanoparticles stable nanoparticle suspensions exhibit anacidic pH value andor contain different stabilizing agentsThus adding nanoparticles for in vitro cytotoxicity assaysdirectly to the physiological aqueous culture media oftenresults in nanoparticle aggregation and agglomeration [2 67]

While these limitations in nanoparticle cytotoxicity mea-surements are known as long as alternative assays havenot been developed careful examinations of the resultsare needed Furthermore when choosing a method fornanoparticle testing it must be excluded that false-positive

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2015 Article ID 691069 16 pageshttpdxdoiorg1011552015691069

2 Journal of Nanomaterials

results can be misinterpreted Nevertheless for nanoparticlecytotoxicity studies in vitro cell-exposure studies can yieldthe first evidence of their potential risk These tests offer theadvantages to be simple and to clarify the basic interaction[1] Additionally these methods are rapid cheaper and morereproducible than in vivo systems [1] Thus in vitro studiesare often used before animal studies are applied Howeverthere are concerns whether the results of these studies canreally predict in vivo cell behavior A comparative study ofthe effects of TiO

2nanoparticles demonstrated a good corre-

lation between acute toxicity measurements of in vitro and invivo tests based on the steepest slopes of dose response curves[8] Moreover Kim et al could identify higher cytotoxicityof nanosized SiO

2as compared with microsized particles in

vitro as well as in vivo [9]To improve the prediction of in vivo effects a set of in

vitro assays investigating different mechanism is needed [9]As in vitro assays cannot mimic real tissue conditions alsomore complex in vitro models like three-dimensional (3D)cell culture models could bridge the gap between classicaltwo-dimensional (2D) in vitromodels and in vivo studies

The development of consumer nanomaterials implieslong optimization procedures resulting in the formation ofplenty of precursors and intermediates All these materialsmay pose risks to human beings and the environmentAll these materials must be tested for toxicity especiallywith regards to occupational safety provision and consumerprotection However the number of in vitro cytotoxicityassays currently available is huge consequently all of theseassays cannot be applied for all precursors intermediates andproductsMoreover animal studies are ethically controversialand therefore limited Thus a broad and complex study canoften not be performed because the bottlenecks are in vitrocell-based toxicity tests and in vivo animal studies which arevery time consuming and costly

Therefore the aim of our study was the developmentof an in vitro hierarchical nanoparticle screening system toexamine any kind of nanoparticles concerning their toxicityFirst the structure of the screening system is presentedin general Next is a separate section of the prescreeningfine-screening and complex-screening where the sensitivityand difficulties of the cytotoxicity assays are discussed Theapplication of the methods in each screening level wasproven exemplary with two different kinds of nanoparticlesThis screening system includes different cytotoxic assays andcomplex cell culture systems such as a three-dimensionalcell culture or dynamic cultivation to increase the relevanceof in vitro assays As nanoparticle suspension testing haslimitations regarding the cell culture we studied whethersimilar results can be obtained with nanoparticle suspensionsand with coating experiments respectively According to thepossible nanoparticle incorporation into the human organ-ism we used human lung carcinoma A549 cells (respiratorytract) and murine fibroblasts NIH-3T3 cells (skin) As abenchmark-system for the development of the screeningsystem we investigated the effects of zinc oxide nanoparticles(ZnO-NP) and titanium dioxide nanoparticles (TiO

2-NP)

on mammalian cell lines These two nanoparticle types havebeen used in different applications and their influence on 2D

monolayer cultures has been analyzed previously For ZnO-NP cytotoxic effects have been published [3 10ndash12] while thecytotoxicity of TiO

2-NP is still a point of discussion Some

studies have reported nontoxic effects for TiO2-NP [13 14]

whereas other studies suggested cytotoxic effects of TiO2-NP

[14ndash16] In the present study both types of nanoparticles werescreened with the developed screening system

2 Materials and Methods

21 Nanoparticles In this study ZnO-NP (with 01 Ru)were used which were synthesized and characterized by Blohet al (2012 2014) [17 18] The ZnO-NP have a BrunauerndashEmmettndashTeller (BET) surface of 654m2g and a particlesize of 50 plusmn 10 nm (X-ray) [17] ZnO-NP exhibited anaverage hydrodynamic diameter (dynamic light scattering)of 41 plusmn 5 nm in water 190 plusmn 3 nm in Dulbeccorsquos ModifiedEagle Medium (DMEM) and 106 plusmn 11 nm in the standardculturemedium (meanplusmn standard derivation) [19]TheTiO

2-

NP (Hombikat XXS 700) were obtained from SachtlebenDuisburg Germany exhibiting a primary particle size of7 nm (REM) in the anatase form according to the data sheetThe hydrodynamic diameter of TiO

2-NP has been reported

previously by Sambale et al [19] In water TiO2-NP have a

diameter of 79 plusmn 25 nm in DMEM 142 plusmn 29 nm and in theculture medium 118 plusmn 28 nm [19]

22 Cell Culture A549 human lung carcinoma cells (DSMZnumber ACC 107) and NIH-3T3 mouse fibroblasts cells(DMSZ number ACC 59) were purchased from the GermanCollection of Microorganisms and Cell Cultures (DSMZ)Both cell lines used here were cultivated in DulbeccorsquosModified Eaglersquos Medium (DMEM) (D7777 Sigma-AldrichSteinheimGermany) supplementedwith 10 fetal calf serum(FCS) and 100 120583gmL antibiotics (penicillinstreptomycin) ina humidified environment at 37∘C5 CO

2 Every 3 or 4 days

cells were subcultivated when the cultures reached 70ndash80confluenceThe passage number of all used cells was less than20

23 3D Cell Culture For 3D cell cultures two different 3Dcell culture models were performed 20000 cells were seededon each side of a round scaffold of 1mm thickness and7mm diameter via pipetting (Matriderm Medskin SolutionDr Suwelack AG Germany) The scaffolds were placed in a24-well plate and were incubated at 37∘C5 CO

2for 72 h

to allow the cells to adhere and to build a 3D structureAfterwards the cells were treated in triplicate with differentconcentrations of ZnO-NP or TiO

2-NP in the cell culture

medium for 24 h As a second model cell encapsulationin a semisynthetic PEG-fibrinogen-based hydrogel (Facultyof Biomedical Engineering Technion Haifa Israel) wasinvestigated Here 1mL hydrogel (fibrinogen concentrationof 8mgmL) contains 1 times 106 cells and 10 120583L photoinitiator(10 (wv) in 70 ethanol) For generating defined hydrogelconstructs 50 120583L of the hydrogel-cell-mixture was added toa 6mm silicon gasket (Grace Bio-Labs silicone gasket forProPlate microarray system Sigma-Aldrich USA) Covalent

Journal of Nanomaterials 3

cross-linking of the hydrogel was performed with UV light(6W 365 nm VL-6L Vilber Lourmat France) for 2minThen each hydrogel construct was placed in a 24-wellplate containing 500120583L cell culture medium Additionallyhydrogel constructs without cells were used as controls Toallow the cells to adhere in the hydrogel to proliferate and tobe connected to 3D network the cells were incubated for 48 hbefore nanoparticle exposure for 24 h was performed

24 Nanoparticle Testing According to their doubling timea defined number of cells were seeded for each cell lineThe A549 cells exhibit a doubling time of 40 hours whereasNIH-3T3 cells exhibit a doubling time of 20 hours For 2Dcell culture experiments 8000 A549 cellswell and 6000NIH-3T3 cellswell were seeded in 96-well plates (SarstedtAG) and different exposure methods of ZnO-NP and TiO

2-

NP were investigated For nanoparticle testing in suspensionan aqueous nanoparticle suspension was diluted with cellculture media The cells were cultured for 24 h in 100 120583Lstandard culture medium before nanoparticle treatment andwere exposed to different concentrations of nanoparticlesfor 24 h (triplicate) For coating experiments the cells wereseeded directly on nanoparticle coatings (triplicate) and cellviability was determined after 48 h In order to analyzewhether nanoparticles have entered the solution from thenanoparticle coatings extracts were prepared according tothe ISO standard 10993-122012 (Biological Evaluation ofMedical DevicesndashPart 12 Sample Preparation and ReferenceMaterials) [20]Therefore 3 cm2mL cell culturemediumwasadded to the nanoparticles coatings (different concentrationsin triplicate) and were placed in an incubator at 37∘C and 5CO2for 24 hThen standard culture medium was replaced to

the extract medium and cells were cultured for further 24 hin the incubator

25 Cell Morphology The effect of ZnO-NP and TiO2-NP

suspensions on cell morphology in 2D and in 3D cell cul-tures (hydrogel) was examined by phase contrast microcopy(Olympus IX 50 Olympus Corporation Tokio Japan) afterthe cells were exposed to nanoparticles for 24 h

26 Cell Viability After nanoparticle exposure the cell viabil-ity was determined using either the CTB analysis (CellTiter-Blue Cell Viability Assay Promega Madison USA) or theMTT assay (Sigma-Aldrich Munich Germany) The MTTassay is based on the reduction of the yellow tetrazoliumdye MTT to the insoluble blue formazan by metabolicactive cells [21] To perform the MTT assay the cell cul-ture medium was removed from each well and 100120583L (2Dcell culture)300120583L (3D scaffolds) of 10 MTT solution(90 120583L DMEM) and 10 120583L MTT stock solution (5mgmLphosphate buffered saline) were added to each well andincubated for 4 h at 37∘C5 CO

2 Afterwards 100 120583L (2D

cell culture)200120583L (3D scaffolds) sodium dodecyl sulfate(SDS Solution) (1 g SDS in 10mL 001M HCl) was added toeach well and samples were incubated for further 18 h Toensure that the formazan was released completely from thescaffold the samples were shaken at a speed of 700 rpm The

absorption signal at 570 nm630 nm was determined using amicroplate reader (Bio-Rad Munich Germany) to quantifythe results Scaffolds without cells were used as backgroundcontrols

Comparable to the MTT assay the CTB assay is basedon the reduction of the blue dye resazurin to the purpledye resorufin by metabolically active cells monitored viafluorescence For the 3D hydrogel cell cultures 100 120583L CTBstock solution was added to achieve a final concentra-tion of 10 CTB solution and the dye was incubated for18 h at 37∘C5 CO

2 Samples with hydrogel and different

nanoparticle concentrations but without cells were used asbackground control The fluorescence signals at an extinc-tion wavelength of 544 nm and an emission wavelength of590 nm were determined using a microplate reader (Fluo-roskan AcentThermo Fisher Scientific Inc Waltham USA)For both assays the absorptionfluorescence signals of cell-free controls with different nanoparticle concentration weredetermined to prevent misinterpretations of the assays

27 Electric Cell-Substrate Impedance Sensing Electric Cell-Substrate Impedance Sensing (ECIS) measurements wereperformed tomonitor the cellular behavior onlineThereforecells were grown on 8W1E (8 well 1 electrode) ECIS slides(Applied BioPhysics USA) Each of the eight wells containsan electrode covered with a gold film used to apply an alter-nating current (AC) signal Cell attachment cell spreadingand cell morphological changes affect themeasured electrodeimpedance which can be detected by ECIS Model 1600R(Applied BioPhysics USA) Initially each slide was equili-brated overnight with 400 120583L of standard culture mediumThen the mediumwas removed and 125000 cells (A549 cellsor NIH-3T3 cells) in 400120583L standard culture medium wereseeded per well One well remained cell-free as referenceAfter approximately 48 h the cells had grown to confluenceand the impedance signal was stable ZnO-NP or TiO

2-NP

suspension in culture medium was added to the cells at leastin duplicate at a concentration of the calculated half maximalinhibitory concentration (IC

50) value In addition at least two

wells were filled with standard culture medium as controlwhile nanoparticles to the cell-free well were added at thesame concentration The impedance signal was monitoredduring the entire time of the measurement

28 Quantification of the Expression Levels of il-8 and hsp70mRNA The expression of il-8 and hsp70 genes was analyzedwith quantitative real-time PCR (qPCR) In a T75 culture-flask 25000 cellscm2 of A549 cells or NIH-3T3 cells wereincubated at 37∘C for 24 h before the cells were exposed toZnO-NP or TiO

2-NP at a concentration of the IC

50value

for 24 h NIH-3T3 cells were exposed to 20120583gmL ZnO-NPTiO

2-NP and A549 cells to 40 120583gmL Afterwards the

total RNA was isolated by using the RNeasy Plus Mini Kit(QIAGEN Hilden Germany) following the manufacturerrsquosinstructions and the RNA concentration was measuredat 260 nm by Nanodrop ND-1000 (Peqlab BiotechnologieGmbH Germany) For cDNA synthesis 2 120583g of RNA and3 120583L oligo (dt) primers (poly d(T) 12ndash18 Primer Roth


Table 1 Sequences of the primers used in qPCR

Gene name Gene bank accessionnumber (NCBI GenBank) Location (bp) Expected size of PCR

amplicon (bp) Sequence (51015840-31015840)

GAPDH NM 0020463880ndash900

126F AAGGTGGTGAAGCAGGCGTCG

1005ndash984 R AATGCCAGCCCCAGCGTCAAAG

HPRT NM 0001942650ndash669

267F AAGCTTGCTGGTGAAAAGGA

897ndash916 R AAGCAGATGCCCACACAACTFor A549 cells

IL-8 NM 0005843221ndash240

131F CAGTTTTGCCAAGGAGTGCT

351ndash332 R AATTTCTGTGTTGGCGCAGT

Hsp70 (HSPA1A) NM 00534552049ndash2068

138F GTGTAACCCCATCATCAGCG

2186ndash2166 R CAATCTTGGAAAGGCCCCTAAFor NIH-3T3 cells

IL-8 NM 0113392338ndash357

110F GAATTTCCACCGGCAATGAA

447ndash428 R TCCCGAATTGGAAAGGGAAA


104F CATCGAGGAGGTGGATTAGAG

2244ndash2225 R ACCTTGACAGTAATCGGTGC

Germany) in a total volume of 27120583L (add with ddH2O)

were incubated at 65∘C for 5min and then held at 4∘C for1min Subsequently 8120583L M-MLV RT 5x Buffer (PromegaUSA) 4 120583L dNTPs (dNTP set Thermo Scientific USA)and 1 120583L M-MLV Reverse Transcriptase (Promega USA)were added and the mixture was incubated at 37∘C for 1 hThe qPCR was performed with a reaction volume of 25120583Lcontaining 05 120583L (02 120583M) of each of the forward and reverseprimers (see Table 1) 25 ng cDNA template and 125 120583L IQSYBR Green Supermix (Bio-Rad USA) The measurementswere carried out on an iQ5 Multicolor Real-Time PCRDetection System (Bio-Rad USA) and all samples were runin triplicate In addition the efficiency of each primer pairwas determined using serial dilutions of the cDNA templatePCR reactions were performed at 95∘C for 3min followedby 40 cycles of 95∘C for 10 sec and of 57∘C for 20 secThe data (comparative threshold (ct) values) were analyzedusing the Gene Expression Analysis for iCycler iQ Real-time PCRDetection System (Bio-Rad USA) Glyceraldehyde3-phosphate dehydrogenase (GAPDH) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) were used asendogenous control genes to normalize the expression of thetarget genes Therefore the expression levels of hsp70 and ofil-8 are expressed as 119899-fold differences relative to the referencegenes

29 Data and Statistical Analysis For the determination ofthe IC

50values dose response curves were fitted to the data

obtained by theMTT or the CTB assay using OriginPro 850SR1 (nonlinear curve fitting growthsigmoidal DoseResp)The data shown are from at least three independent exper-iments triplicate (119899 ge 3) The ANOVA one-way analysis(OriginPro 850 SR1) was performed for statistical analysisand a significant effect was reported at lowast119901 lt 005

3 Results and Discussion

31 Development of a Cellular Screening System In ourrecent study we have constructed and validated a cell-basedhierarchical screening system in order to realize the analysisof a variety of nanoparticles and nanomaterials under definedand reproducible conditions (Figure 1) For this purpose thescheme for biomaterials in tissue engineering published byBruns et al (2007) served as a template for our screeningsystem for nanoparticles [22] The idea of the screeningsystem is that toxicity measurements are done during thecourse of nanomaterial developmentThe screening system isdivided into a prescreening a fine-screening and a complex-screening (Figure 1) In the prescreening simple methods areused for all materials whereas more complicated techniquesare used for fine- and complex-screening which are appliedonly to selected materials Especially in fine screening theassays cover various impact areas on the cells

After each screening step cells exhibiting a significantvariation as compared to nontreated cells are identified(119901 lt 005 ANOVA one way) Materials causing such sig-nificant variations are classified as cytotoxic and thus notfurther investigated and rejected whereas the remainingnanomaterials are subjected to further analyses Continu-ous communication (feedback) between the process chainand the screening system is essential in order to opti-mize nanoparticle development and associated safety testingFeedback should be given with regard to the toxicity and theparticle properties in order to select nanomaterials which areprofitable and which should be further analyzed Thus thehierarchical structure facilitatesmaking decisions as to whichnanomaterials need complex costly investigations or in vivostudies thus avoiding unnecessary testing of the remain-ing nanomaterials Finally only nanomaterials which pass


Significant variation

All nanomaterials

Selected nanomaterials

+

Human cell linesCell viability

Cell morphology

Prescreening

Human cell linesEnergy metabolism

ProliferationDNA damage

Cell deathROS detection

Gene expressionCell adhesion

Nanoparticle uptake

Fine-screening

Human cell lines and primary cellsCell systems 3D cell culture and

dynamic conditions(1) Methods from prescreening(2) Methods from fine-screening(3) Combination of cell systems

Complex-screening

In vivo studies

Screening system for nanoparticles

TypeSuspension coating and extract

+

+

Feedback

Feedback

Feedback

minus

minus

minus

Significant variationlowast


minus toxic + nontoxiclowastp lt 005

Data sheetParticle size pH value and stabilization

Figure 1 Hierarchical cell-based screening system for nanoparticle safety testing The screening system is divided in prescreening fine-screening and complex-screening and allows examining of a variety of nanoparticles during nanoproduct development Prescreening isperformed with all nanomaterials whereas complex cell systems are only applied to selected nanomaterials

the whole screening system without significant side-effectscan reach the application level Thus our screening systemis highly efficient and cost minimizing and reduces animalstudies

Before nanoparticles or nanomaterials can be analyzedin the screening system a thorough characterization isneeded Therefore a data-sheet is required which providesinformation about the size the surface modification the zetapotential the aqueous stability and the sterilizationmethods

According to the data-sheet information and the desiredapplication of the nanomaterials one specific type ofnanoparticle testing is applicable such as suspension coatingor extraction (detailed description is given below) For theevaluation of the nanoparticle screening system two differ-ent commonly used kinds of nanoparticles were selectedZnO-NP andTiO

2-NPWhile the focus in this study has been

on short-term nanoparticle exposure investigations long-term conditions can also be tested in this complex-screeninglayout

Concentrating on the desired application of the nanopar-ticles and on their potential incorporation into the human

organism different cell lines were selected for the prescreen-ing and for the fine-screeningThemain absorption routes arevia skin via respiratory tract and via gastrointestinal tract [1]The skin being the largest organ [1] provides a large targetsurface for nanoparticles Recent studies have reported thepenetration of nanoparticles [1] through the skin tissue whichmeans investigations of in vitro skin test systems are neededIn addition nanoparticles can be inhaled and can be absorbedby pulmonary alveoli [1 8] Moreover via the gastrointestinaltract nanoparticles can reach the liver and accumulate therein [1] With regard to the described absorption routes amatrix of relevant cell lines is required to identify cytotoxiceffects of nanoparticles in vitro because various cell linesdisplay different sensitivities and cell responses [1 23]

To avoid cell-type specific behavior it is important touse different cell lines Therefore commonly used cell linessuch as A549 cells (lung model) and NIH-3T3 cells (skinmodel) have been applied here The use of mammaliancell lines in the screening system has the advantage thatthey provide reproducible results as cells are uniform stan-dardized and well characterized Thus cell lines provide


the first evidence concerning the toxicity of the nanomateri-als

However using standardized cell lines bears the disad-vantage that their cell behavior cannot be easily transferredto in vivo conditions because they are often immortalizedor isolated from tumors [6] In addition the proliferationrate of immortalized cell lines is often higher than that of invivo cells [6]Therefore in the screening system primary cellsand complex cell systems are examined to better mimic realtissue conditions in a human organismThese cell systems arethree-dimensional (3D) cell cultures where cells can developmore in vivo-like cell-cell interactions and an extracellularmatrix [24 25] Secondly dynamic conditions are performedto mimic for instance the blood stream Both cell systemscan finally be combined for a higher complexity and toperform long-term exposure analysis Since these modelsrequire much more complex settings they are used only withselected nanomaterials in complex-screening

32 Prescreening Morphology Studies and Comparison ofNanoparticle Suspensions Coatings and Extract Medium

321 Morphology Studies of A549 Cells and NIH-3T3 CellsExposed to ZnO-NP and TiO2-NP in 2D and 3D Cell CulturesFor in vitro studies 2D cell cultures are needed in theprescreening because they allow high-throughput screeningThus different nanoparticles nanoparticle concentrationsand various cell types can be tested in parallelThe prescreen-ing reduces the huge amount of nanomaterials obtained fromthe process chain to a more manageable number Decisionshave to be made concerning the question which nanoma-terial requires further investigation in the fine-screeningor complex-screening The impact of the cell viability afternanoparticle exposure indicating cytotoxic effects is analyzedwith commonly used assays such as the MTT the CTBor the WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-13-benzene disulfonate) assay With all threeassays comparable results can be produced which only differin their sensitivity Besides these viability measurementsmorphology studies are applied in the prescreening Cellmorphology studies are important to support the resultsobtained with cytotoxicity assays especially with regard topotential interference by the nanoparticles on the assaycompounds Some harmful effects can be identified via cellmorphology changes so in these cases false-positive resultsmay be detected

To validate the significance of cell morphology studies forscreening cytotoxic effects of nanoparticles A549 and NIH-3T3 cells were treatedwith ZnO-NP or TiO

2-NP respectively

In addition the cells were grown in 2D as well as in 3D For3D cell cultures the cells were encapsulated in a hydrogelThe cell morphology of nontreated and treated cells wasanalyzedThe specific IC

50value calculated from the viability

results for both cell lines and with both cell systems wasdisplayed In Figure 2 the cell morphology of NIH-3T3 (c)and A549 cells (g) grown in 2D and treated with ZnO-NPis shown In the standard culture media the NIH-3t3 cellsexhibit their characteristic contact arms to their neighboring

cells (Figures 2(a) and 2(b)) ZnO-NP treated NIH-3T3cells at the IC

50value changed their morphology turning

smaller and rounder characteristic of dead cells At highernanoparticle concentrations they became detached from thesurface For A549 cells grown in 2D the control cells hada triangular shape and became round when ZnO-NP wereadded (IC

50value) (Figure 2(e)) In 3D the NIH-3T3 cells

displayed the same changes as observed in the 2D cell culture(Figure 2(b)) In the hydrogel NIH-3T3 cells are highlynetworked (Figure 2(b)) At the IC

50value the cells turned

round (Figure 2(d)) On the contrary A549 cells grown inthe hydrogel were round in the standard culture media aswell as in the ZnO-NP treated media (Figures 2(f) and 2(h))Thus no significant differences in their morphology couldbe revealed For TiO

2-NP no reduction in the viability was

determined and both cells lines did not show anymorphologychanges (data not shown)

In summary the morphology studies should be regardedas an important tool in the nanoparticle safety screeningsystem In some cases however the viability assay is notsufficient to prescreen the nanoparticles accurately In thiscontext for in vitro drug testing Quent et al have reported anoverestimation of the viability of daunorubicin-treated cellsdetermined by the MTT assay [4] The morphology studyclearly displays the release of detached cells indicating celldeath whereas the MTT was still reduced in the cells [4] Inour study ZnO-NP morphology changes of NIH-3T3 cellscould be observed in 2D as well as in the 3D hydrogelsAlso for A549 cells grown in 2D the toxic effect of ZnO-NPwas determined by the cell morphology studies However forA549 cells growing in hydrogel this method failed to identifytoxic effects Here no changes in the morphology of theA549 cells after adding ZnO-NP to the culture were observedwhile the cell viability assay showed a significant reduction atthis concentration (Table 1) Furthermore different 3D cellculture models based on cell aggregation (spheroids) cellencapsulation (hydrogel) and cell adhesion on scaffolds arecurrently used to mimic a 3D environment In particularthe scaffold model hinders morphology studies as scaffoldsare not transparent In conclusion morphology studies cansupport other cytotoxic assays while they can only detectspecific impacts of nanoparticle exposure

322 Cell Viability Measurements of A549 Cells and NIH-3T3 Cells Exposed to ZnO-NP and TiO2-NP in Suspensionin Extract Medium or on Coatings For in vitro nanoparticlesafety testing the methodological challenge is the prepara-tion of stable aqueous physiological nanoparticle suspen-sions because nanoparticles often agglomerate in biologicalmedia As reported by Jones and Grainger (2009) thesenanoparticle aggregations can affect several nanoparticleproperties such as size and cellular uptake [6] Currentlydifferent dispersion protocols and various biocompatible sta-bilizers are being examined for generating stable nanoparticlesuspensions [7 26] Protocols have thus been developedfor the dispersion of nanoparticles in FCS using variouscentrifugation steps [7] in culture media [7] or in anaqueous suspension [26] Only with gum arabic as a stabilizer


2D cell culture

(h)(g)

(f)(e)

(d)(c)

(b)(a)

3D cell culture

Con

trol

ZnO

-NP

ZnO

-NP

Con

trol

NIH-3T3 cells

A549 cells

Figure 2 Morphology studies of NIH-3T3 cells (andashd) and A549 cells (endashh) grown on 2Dmonolayers (a c e g) or encapsulated in a hydrogel(3D cell culture) (b d f h) NIH-3T3 cells were cultivated in standard culture medium (a b) or were treated with 10120583gmL ZnO-NP in 2D (c)and in 3D (d) For A549 cells 40 120583gmL ZnO-NP were added in 2D (g) and 300 120583gmL ZnO-NP in 3D (h) The A549 control cells cultivatedin standard culture medium were displayed in (e) and (f) Morphology changes after ZnO-NP exposure were observed in 2D for both celllines whereas in 3D effects were only determined for NIH-3T3 cells


0 5 10 15 200

20

40

60

80

100

120Vi

abili

ty (

)

SuspensionCoatingExtraction

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

Concentration (120583gcm2)

(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100

120

Viab

ility

()

lowast lowast


lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast lowast

lowast


(b) A549 cells

Figure 3 Viability of NIH-3T3 cells (a) and A549 cells (b) after ZnO-NP exposure determined with the MTT assay The cells were treatedwith nanoparticle suspension coating or extract medium The signals of untreated cells were set as 100 Data points are means plusmn SD for119899 ge 3 lowast119901 lt 005

the ZnO-NP suspension was found to be stable for morethan five days [26] In this study alternative in vitro tech-niques were developed and validated to avoid the describedproblemswith nanoparticle suspensionsWe studiedwhethersimilar cell viability results can be obtained with nanoparticlesuspensions extract medium and coatings For this purposeboth cell lines were seeded on nanoparticle coatings and via-bility measurements were carried out Additionally extractmedia from TiO

2-NP and ZnO-NP coatings respectively

were prepared according to the ISO 10993-122012 standardfor medical devices and subsequently used for the treatmentof the cells [20] Thereby we wanted to monitor whethernanoparticles are dissolved from the coatings thus influenc-ing the viability of the cells

For TiO2-NP no significant reduction in the viability of

either cell line tested was observed for all three methods (seesupporting information Figure 8 see Supplementary Mate-rial available online at httpdxdoiorg1011552015691069)On the other hand the viability of the cells was reducedby ZnO-NP in a dose-dependent manner In Figure 3the viability of ZnO-NP suspensions coatings and extractmedium upon exposure to NIH-3T3 and to A549 cells isdisplayed In addition in Table 2 the IC

50value of the viability

measurements are summarizedFor ZnO-NP toxic effects were detected with all three

methods and with both cell lines performing the MTTassay NIH-3T3 cells were found to be more sensitive toZnO-NP than A549 cells thus different cell lines apparentlyshow different sensitivity to nanoparticles The NIH-3T3cells exhibited the same IC

50value for ZnO-NP suspensions

coatings and extract medium On the contrary the IC50

value determined for ZnO-NP suspensions in A549 cellswas found to be half of the IC

50value determined with

Table 2 Calculated IC50 value of NIH-3T3 cells and A549 cellstreated with ZnO-NP suspensions coatings or extract medium(MTT assay) For A549 cells the IC50 value for nanoparticlesuspensions is twice lower as compared with the respective coatingsData points are mean plusmn SD for 119899 ge 3

IC50 value [120583gcm2]

NIH-3T3 cells A549 cellsSuspension 3 plusmn 1 18 plusmn 5Coating 2 plusmn 1 31 plusmn 8Extract medium 2 plusmn 2 30 plusmn 8

the coating experiments Therefore instead of suspensiontesting for the identification of toxic nanoparticles coatinganalysis can be a good alternative especially for nanoparticleswhich agglomerate in standard culturemedia For the coatingand extract medium results no differences in the IC

50values

were observed for either cell line Thus the determinedreduction in the cell viability is apparently caused by therelease of ZnO-NP from the coating Our results clearlydemonstrate that ZnO-NP exposure induces a decrease of thecell viability indicating toxic effects

Obviously nanoparticle effects to human cell lines canbe investigated either with stable suspensions with coatingsor with extract medium The choice of strategy dependson the nanoparticle properties as well as on the desiredapplication of the nanoproductWhile for somenanoparticlesa stable aqueous suspension in the cell culture medium canbe produced other nanoparticles agglomerate under theseconditions When evaluating the potential risk of nanopar-ticles to human beings it should be considered that nanopar-ticles often may not reach the human organism as individ-ual nanoparticles but rather as nanoparticle agglomerates


In in vitro studies proteins present in the media often adsorbto the nanoparticles [2] thus affecting the cell response of thenanoparticle treatmentThe same effect may take place in thehuman organism Consequently nanoparticle agglomerateanalysis can make sense for example to predict the in vivocell behavior On the contrary for the in vitro investigation ofnanoparticle effects in the respiratory tract stable nanoparti-cle suspensions are required Inhaled particles with diametersbelow 25 120583m can reach the pulmonary alveoli where theycan be absorbed [8] For any nanoparticle test system it istherefore important to consider the relevance for humanhealth effects [27]

As an alternative technique for nanoparticle in vitro test-ingwe performed the so-called coatingmethodHowever theproliferation rate of adherent cells can be affected by nanos-tructured surfaces Several studies have demonstrated thatthe surface roughness of the cell culture plates influences thecellular behavior [28ndash30] Therefore the stability of certainnanoparticle coatings (nanomaterials and nanocomposites)and the effect of potentially solved nanoparticles to thehumanorganismmay bemore relevantThus the preparationof extract media is the chosen strategy for certain nanomate-rial applications such as for medical devices window glassesor sanitation Comparable with our extract experimentsPaddle-Ledinek et al (2006) have used this method beforeto test wound dressings coated with nanoparticles [31] In thepresent study it could be shown that nonstable nanoparticlecoatings require particular care during the testing processHence measurements of the toxic effect of ZnO-NP withsuspensions with coatings and with the extraction methoddemonstrated that extract testing is an alternative indirectmethod to investigate the cytotoxicity of nanomaterials

In summary the nanoparticle screening system intro-duced here is capable of analyzing suspensions coatings aswell as coating extractions regarding the respective appli-cation of the nanomaterials Moreover the analysis of theextract also monitors the stability of the coating

33 Fine-Screening Gene Expression Analysis and ECIS Mea-surements of A549 Cells and NIH-3T3 Cells Exposed to ZnO-NP and TiO2-NP Following the prescreening assays fine-screening tests were performed to provide higher sensitiv-ity and to yield detailed information concerning the cellresponses induced by the nanoparticle exposure To avoidpotentialmisinterpretations resulting from for example pos-sible interference of nanoparticleswith the assay componentsdifferent methods based on fluorescence or luminescence areused here in the fine-screening Additionally several positiveand negative controls are needed Moreover for an accuratein vitro nanoparticle fine-screening various biological effectsof the nanoparticles were analyzed The energy metabolismin the cells can for example be investigated with adenosinetriphosphate (ATP) For cell death analysis the release oflactate dehydrogenase (LDH) indicates necrosis and thecaspase activity or the annexin V-FITCPI assay indicatingapoptosis can be performed Changes in the proliferationrate of the cells can be detected with the bromodeoxyuridine(BrdU) assay which is based on the integration of BrdU achemical analogue of the nucleoside thymidine BrdU can be

detected by labeled antibodies and indicates the proliferationrate of the cell Moreover DNA damage in cells caused bythe exposure to nanoparticles can be observed by the alkalinemicroelectrophoresis (comet assay) Also the generation ofreactive oxygen species (ROS) induced by nanoparticles canbe determined thus demonstrating stress for the cells Acommonly used assay for the ROS detection is the DCF assayIn addition nanoparticlesmay also affect the gene regulationso the expression of selected marker genes can be quanti-fied using real-time PCR or microarray analysis Molecularmarkers can for instance be mitosis markers heat-shockproteins interleukins or apoptosis markers While all thedescribed methods are endpoint assays the Electric Cell-Substrate Impedance Sensing (ECIS) measurement providesonline data Cell morphology changes such as cell roundingor surface detachment can be detected immediately afterthe addition of nanoparticles [2] Moreover no additionalcompounds are required for this alternative technique mini-mizing false-positive results [32]

In our study we investigated the effects of ZnO-NP andTiO2-NP on gene expression using real-time PCR analy-

sis We selected two established biomarkers il-8 indicatinginflammation [33 34] and hsp70 indicating oxidative stressresponses [35 36] In NIH-3t3 cells (Figure 4(a)) as well as inA549 cells (Figure 4(b)) the il-8 mRNA level was increasedwhen cells were exposed to ZnO-NP Furthermore ZnO-NP induced hsp70 expression in A549 cells (Figure 4(b))On the contrary exposing NIH-3T3 cells and A549 cells toTiO2-NP did not lead to higher levels of the investigated

biomarkers The real-time PCR results clearly demonstratedthat ZnO-NP induce inflammation in both investigated celllines and additionally oxidative stress in A549 cells Severalstudies confirmed our investigation In human bronchialepithelial cells for example ZnO-NP exposure increased theexpression of il-8mRNA [33] as well as the protein level [3337] Also il-8mRNA secretion has been used for identifyingcytotoxic effects of other nanoparticles such as carbonnanotubes and polystyrene nanoparticles [38] Exposure ofTiO2-NP to keratinocytes [7] human monocytes and lung

epithelial cells [36] did not cause inflammation Furthermorefor TiO

2-NP an increase of the il-8 gene expression in A549

cells was only achieved at unrealistically high concentration(400 120583gcm2) with regard to in vivo conditions [34]Howeverin vivo studies with mice demonstrated that TiO

2-NP induce

increased levels of several interleukins such as il-8 mRNA[39 40]

The second chosen biomarker hsp70 was already vali-dated for investigation of the effect of several nanoparticlesHsp70rsquos virtues include that it is a very sensitive biomarker forcellular stress responses it is well characterized [41 42] and itprecludes false protein-folding [43] The toxic effect of silvernanoparticles was determined in vivo by an increased level ofhsp70 mRNA [44 45] and of Hsp70 protein [41] indicatingoxidative stress Furthermore Cu-NP [42] carbon-blacknanoparticles [46] andCdSeZnSnanoparticles [47] inducedhsp70 expressionWhile in our study ZnO-NP exposure leadsto a higher hsp70 expression in A549 cells this effect was notobserved for NIH-3T3 cells According to Chen et al (2012)NIH-3T3 cells exhibited a lower heat-shock response than


Control ZnO0

2

4

6

8

10

12Re

lativ

e mRN

A ex

pres

sion

hsp70


il-8

TiO2

lowast

lowast

(a)

Relat

ive m

RNA

expr

essio

n

0

2

4

6

8

10

12

hsp70il-8

Control ZnOTiO2

lowast

lowast

(b)

Figure 4 Relative mRNA expression of il-8 and hsp70 from NIH-3T3 cells (a) and A549 cells (b) upon treatment with ZnO-NP or TiO2-NP

for 24 h NIH-3T3 cells were exposed to 20120583gmL and A549 cells to 40 120583gmL ZnO-NP or TiO2-NP Data points are means plusmn SD for 119899 ge 4

Significant differences versus the control group are indicated in the figure (ANOVA one way) lowast119901 lt 005

other cell lines thus the sensitivity to nanoparticle responsedetection was reduced [43] In addition different cell linesrevealed a variance in their exposure time-course response[43] For TiO

2-NP no significant changes of hsp70 expression

relative to the control cells were observed for Thp-1 cells(human monocytes) or for NCI-H292 cells (human lungepithelial cells) [36] but not for other cell lines such as NIH-3T3 cells or HepG2 cells [43] On the contrary the hsp70expression in mice (in vivo) was downregulated after TiO

2-

NP exposure [39 40] Presumably hsp70 reduction couldindicate a detoxification process of the cells [40]

ECIS measurements were used for the online monitoringof nanoparticle effects in the cells Instead of endpointmeasurements cellular response to nanoparticle additioncan be observed directly by this method and no additionaldye is needed Thus interference of assay compounds withnanoparticles can beminimizedThe results of the impedancemeasurements of NIH-3T3 cells (Figure 5(a)) and of A549cells (Figure 5(c)) cultivated with ZnO-NP were comparedwith the control cells

After addition of ZnO-NP to the cells the impedancesignal dropped immediately for the NIH-3T3 cells Interest-ingly for A549 cells the impedance signal increased afterZnO-NP addition during the first 10 h before it decreasedagain The cells treated with ZnO-NP lost their cell-cellinteraction became smaller and detached from the surfaceThe decrease of the impedance signal indicates the cell deaththus ZnO-NP exhibited a toxic effect to the cells The slightincrease of the impedance signal for A549 cells after ZnO-NPaddition had been already reported by Seiffert et al (2012)

[32] Such an effect cannot be explained with proliferation asA549 cells have doubling times of 24 h thus another cellularresponse must have occurred [32] For TiO

2-NP no changes

in the impedance signal were revealed with both cell lines(Figures 5(b) and 5(d)) indicating no cellular changes Alsothe impedance of a cell-free control did not changeThus thenanoparticles themselves did not affect the impedance signal

ECIS measurements have previously been used for cyto-toxicity analysis of nanoparticles A significant decrease inthe impedance signal indicating cytotoxicity was determinedfor ZnO-NP as well as for CuO-NP but not for TiO

2-NP

[32] Furthermore the calculated IC50value determined with

the viability assay was found to be comparable with the onedetermined by the cell-impedance measurements after 24 h[32] For anatase TiO

2-NP no significant cytotoxic effect was

observed [48 49] and for rutile TiO2-NP it was observed only

at high concentrations (IC50

value gt 200120583gmL) [48 50]Therefore the effect of TiO

2-NP on the cellular behavior

appears to be dependent on the particle size as well as on theshape

In our study during the fine-screening stage TiO2-NP

were not found to cause a significant variation either in thegene expression analysis or in the ECIS measurements Thusthese particles should be further analyzed in the complex-screening mode

34 Complex-Screening Comparison of Cell Viability of A549Cells and of NIH-3T3 Cells Exposed to ZnO-NP and TiO2-NPin 2D and in Different 3D Cell Culture Models The complex-screening is performed for the remaining nanomaterials not


0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35ZnO-NP

Nor

m i

mpe

danc

e (O

hm)

Time (h)MediumControl cells Cells with NP

(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)

MediumControl cells Cells with NP

Time (h)

TiO2-NP

(b) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(c) A549 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(d) A549 cells

Figure 5 Electric Cell-Substrate Impedance Sensing of NIH-3T3 cells (a b) and A549 cells (c d) to monitor nanoparticle toxicity The cellswere grown to confluence before nanoparticle exposure Dynamic changes in the impedance signal of NIH-3T3 cells to 0 and 20120583gmLsuspensions of ZnO-NP (a) or TiO

2-NP (b) are displayed For A549 cells the effect of 40120583gmL of ZnO-NP (c) and TiO

2-NP (d) was

investigated Cell-free medium with nanoparticles was used as reference The measurements were performed with a 16 kHz ac source Thetime point for nanoparticle addition is indicated with the black arrow Data points are means plusmn SD for 119899 ge 3

exhibiting any clear toxic effects during the fine-screeningto apply more extensive in vitro methods As currently used2D cell culture models have several limitations [25 51] 3Dcell culture systems are employed here The cells were grownin a more physiological cell environment to better predictthe cellular behavior in the organism The 3D cell cultureexhibits a higher complexity compared to the conventional2D cell culture and is more expensive Currently different

models have been developed to enable in vitro 3D cell growthsuch as cell spheroids cell encapsulation in hydrogel [52]or cell adhesion on scaffolds Secondly the bloodstreamcan be mimicked by cells growing under continuous flowconditions (2D cell culture) Here a shear stress is applied tothe cells during nanoparticle exposure Therefore to realizeboth aspects in the complex-screening it is divided into threesections (Figure 1) In the first section the methods used in


the prescreening are performed either on a 3D cell culturemodel or under continuous flow conditions Afterwards inSection 2 the methods of the fine-screening are performedFinally after the nanoparticles have been tested separately inthe cell culture systems a combination of 3D cell culture andcontinuous flow conditions should be performed (Section 3)In contrast to the previously described methods long-termexposure analysis can also be carried out hereby Theseexperiments provide more relevant results so in vivo studiesshould follow when no significant variation is detectedThusthe complex-screening can bridge the gap between the cell-based in vitro testing and the in vivo studies thereby reducingthe amount of required animal studies

In our studywe compared different 3D cell culturemodelsand their applicability for the investigation of nanoparticletoxicity NIH-3T3 cells and A549 cells were seeded on colla-gen scaffolds or were encapsulated in a hydrogelThe scaffoldmodel as well as the encapsulation of cells in the hydrogelwas developed within the present study For both of these 3Dcell culture models we have optimized parts of the procedurelike the cell density and incubation for cell growth and theassay performance The composition of the semisyntheticPEG-fibrinogen-based hydrogel mix was tuned to create adefined structure and to enable cell growth The cells weretreated with different concentrations of ZnO-NP or TiO

2-NP

and the viability of the cells was determined In addition thetwo 3D cell culture models were compared with the already-published data for cell spheroids [19] As described abovenot all assays can be combined with all cell culture systemsThe viability assays differ in the solubility of the detection-dye Whereas the MTT assay generates an insoluble productthe reagent used in the CTB assay is soluble In our study wepreferred the CTB assay to measure the viability of the cellsgrown in the hydrogel because no additional solution stepwasneeded for detection On the other hand for the cells grownon the collagen scaffold diffusion limitations of the reactivedye may affect the results To avoid only cells on the outerscaffold surfacemetabolizing the dye we used theMTT assayinstead Here the formed blue formazan was trapped in thecells once MTT was metabolized Thus the MTT was ableto reach the cells inside the scaffolds as well and the viabilityof all living cells can be revealed For a better comparisonof the 3D cell culture results both assays were performedwith the 2D cell culture as well Figure 6 displays the viabilityof both cell lines after ZnO-NP exposure determined withthe CTB assay or the MTT assay The cells were encapsu-lated in hydrogel seeded on scaffolds or cultured on 2Dmonolayers

In Table 3 the calculated IC50

values for ZnO-NP in the2D cell culture as well as in the 3D cell culture models aresummarized for A549 and for NIH-3T3 cells Additionallythe spheroid results from our previous study are listed toallow a comparison of all three 3D cell culture models [19]Again ZnO-NP reduced the viability of the cells in a dose-dependent manner in the 2D as well as in the 3D cellcultures The results of the two different cell viability assaysdid not show a significant difference for the 2D cell cultureThe choice of the viability assay for cells growing in 3Dis dependent on the used 3D model For the 3D scaffold

Table 3 Comparison of calculated IC50 values for ZnO-NP of2D cell culture and different 3D cell culture models NIH-3T3 cellsand A549 cells were attached on collagen scaffolds encapsulated inhydrogel or aggregated in spheroids to grow in 3D After exposureto ZnO-NP the viability of the cells was determined using either theMTT assay or the CTB assay Values are means plusmn SD for 119899 ge 3 lowastDatawere already published by Sambale et al 2015 [19]

Viability assay IC50 value [120583gmL]NIH-3T3 cells A549 cells

2D MTT 10 plusmn 3 58 plusmn 162Dlowast CTB 15 plusmn 6 54 plusmn 123D scaffold MTT 11 plusmn 2 75 plusmn 183D hydrogel CTB 7 plusmn 3 328 plusmn 133D spheroidslowast CTB 9 plusmn 1 42 plusmn 13

model the MTT assay and for 3D hydrogel the CTB assaygave reliable results According to the IC

50value the NIH-

3T3 cells were more sensitive to the ZnO-NP than the A549cells For NIH-3T3 cells only minor differences in the IC

50

values were observed but for A549 cells in the hydrogela more than fivefold higher IC

50value was determined

Also the value for the scaffold model was found to behigher However the spheroid model displayed a slightlylower IC

50value in comparison to the 2D monolayer culture

[19]For TiO

2-NP no significant reductionwas observed in 2D

as well as in 3D (hydrogel and scaffold model) with eithercell line (Figure 7) Interestingly in our previous study weobserved that TiO

2-NP induced the formation of several

smaller spheroids [19]Differences of 2D and 3D cell cultures for toxicity testing

have already been reported in the literature Controversialresults showed increased decreased or equal cell sensitivityin 3D cultures when compared to 2D monolayers [2453] Lee and colleagues showed a reduced toxic effect inHepG2 spheroids for cadmium telluride (CdTe) and for goldnanoparticles in comparison to the 2D cell culture [54]Drug screening analysis of aflatoxin B1 amiodarone valproicacid and chlorpromazine with HepaRG spheroids [24] andof staurosporine and chlorambucil with HCT116 spheroids[53] demonstrated differences in their half maximal effectiveconcentration (EC

50) value for 2D and 3D cell cultures In

our study we discerned A549 cells in the hydrogel to beless sensitive to ZnO-NP than the cells in 2D In recentstudies it was shown that gold nanoparticles can bind tohyaluronic acid hydrogel thus limiting the cell-nanoparticleinteraction [55] However for NIH-3T3 cells no significantdifference in the sensitivity of 2D cells or 3D hydrogel modelcells was observed Thus limitations of nanoparticle pene-tration through the hydrogel can be excluded In additionXu et al also demonstrated that cells grown in hyaluronicacid hydrogel were less sensitive to doxorubicin-loadedpolymer nanoparticles than cells grown on 2D monolayers[56]

In summary the three 3D cell culture models pro-vide different critical concentrations in vitro for ZnO-NP


0 5 10 15 20 25 30 350

20

40

60

80

100

1203D hydrogel

Viab

ility

()

Concentration (120583gmL)

lowastlowast

lowast

lowast

lowast

lowast

lowast

2D cell culture 3D cell culture (hydrogel)

(a) NIH-3T3 cells

3D scaffold

0 5 10 15 20 25 30 350

20

40

60

80

100

120

Viab

ility

()


lowast

lowast lowast

lowast

lowast

2D cell culture 3D cell culture (scaffold)

(b) NIH-3T3 cells


0 100 200 300 400 5000

20

40

60

80

100

120

Viab

ility

()


lowastlowast

lowast

lowastlowast

lowast

lowastlowastlowastlowast

lowast

lowast

lowast

(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


lowastlowast

lowast

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

(d) A549 cells

Figure 6 Viability of NIH-3T3 cells (a b) and A549 cells (c d) after ZnO-NP exposure determined with the CTB assay (a c) or the MTTassay (b d) The cells were encapsulated in hydrogel (a c) seeded on scaffolds (b d) or cultured on 2D monolayers The signals of untreatedcells were set as 100 Data points are means plusmn SD for 119899 ge 3 lowast119901 lt 005

Therefore for a solid prediction for the subsequent in vivostudies the suitability of the 3D model and the later appli-cation of the tested nanomaterial has to be in the focusWith spheroids limitations of nutrients oxygen and othermetabolites present in tumor tissues can be investigated[51] Thus spheroids are interesting for tumor modelingThis 3D model can be used for nanoparticle developmentfor tumor therapies and nanomedicine In contrast thehydrogel and the scaffold 3D model are more representativeto mimic real tissue conditions in the human organismFormation of model tissues and organs could be realized[51] These models can find applications to clarify nanopar-ticle risks and to support industrial nanoparticle develop-ment

4 Conclusions

We have developed a hierarchical cell-based screening sys-tem for nanomaterial toxicity testing which is divided ina pre- a fine- and a complex-screening Therefore a set ofhigh-throughput cytotoxicity assays as well as complex cellculture models such as 3D cell culture or dynamic cultivationwere integrated In future work the screening system couldalso be extended to long-term studies using a bioreactorInitially in the current study we focused on ZnO-NP andTiO2-NP because of their frequent use in many applications

In our future studies we will extend our investigationsto a large set of different nanoparticles Therefore othernanoparticles will be screened as well Regarding the later


0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D hydrogel



(a) NIH-3T3 cells

0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D scaffold



(b) NIH-3T3 cells

Viab

ility

()


0 100 200 300 400 5000

20

40

60

80

100

120


(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


(d) A549 cells

Figure 7 Viability of NIH-3T3 cells (a b) and A549 cells (c d) after TiO2-NP exposure determined with the CTB assay (a c) or the MTT

assay (b d) The cells were encapsulated in hydrogel (a c) seeded on scaffolds (b d) or cultured on 2D monolayers The signals of untreatedcells were set as 100 Data points are means plusmn SD for 119899 ge 3

application the effects of the nanoparticles can be examinedin suspension coatings or extract media A high amount ofnanomaterials can be analyzed extensively so that only forselected nanoparticles the application of in vivo studies willbe needed Indeed in vivo studies cannot be replaced entirelybut can be significantly reduced applying our screeningsystem As case studies the effects of zinc oxide (ZnO-NP) and titanium dioxide nanoparticles (TiO

2-NP) were

screened ZnO-NP revealed cytotoxic effects to mammaliancells in 2D cell culture as well as in 3D cell culture thusreducing cell viability and inducing inflammation and oxida-tive stress On the contrary for TiO

2-NP no significant

variation was observed with the used methods We clearlydemonstrated that assays have limitations and that the choice

of the cell line may affect the results In comparison to NIH-3T3 cells A549 cells were less sensitive to the investigatednanoparticles and did not adhere to the 3D hydrogelsWhile microscopic detection of morphology changes waspossible in 2D cell cultures this method was critical in 3Dcell cultures to identify toxic nanoparticles Electric Cell-Substrate Impedance Sensing (ECIS) measurements providean excellent method for the noninvasive online monitoringof cellular responses to nanoparticles and were hence placedin the fine-screening part of the overall assay Interference ofdyes with the nanoparticles can be excluded employing thismethod

In conclusion the developed screening system can bridgethe gap between a constantly increasing nanotechnology and


comprehensive risk assessment to define safety provisions forworkers and customers

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the European RegionalDevelopment Fund (EFRE Project ldquoNanokomprdquo Grant no60421066) The authors acknowledge support by DeutscheForschungsgemeinschaft and Open Access Publishing Fundof Leibniz Universitat Hannover

References

[1] S Arora J M Rajwade and K M Paknikar ldquoNanotoxicologyand in vitro studies the need of the hourrdquo Toxicology andApplied Pharmacology vol 258 no 2 pp 151ndash165 2012

[2] A Kroll M H Pillukat D Hahn and J SchnekenburgerldquoCurrent in vitro methods in nanoparticle risk assessmentlimitations and challengesrdquo European Journal of Pharmaceuticsand Biopharmaceutics vol 72 no 2 pp 370ndash377 2009

[3] S J Soenen W J Parak J Rejman and B Manshianldquo(Intra)Cellular stability of inorganic nanoparticles effects oncytotoxicity particle functionality and biomedical applica-tionsrdquo Chemical Reviews vol 115 no 5 pp 2109ndash2135 2015

[4] V M C Quent D Loessner T Friis J C Reichert andD W Hutmacher ldquoDiscrepancies between metabolic activityand DNA content as tool to assess cell proliferation in cancerresearchrdquo Journal of Cellular and Molecular Medicine vol 14no 4 pp 1003ndash1013 2010

[5] S GWangH T Yu and J KWickliffe ldquoLimitation of theMTTand XTT assays for measuring cell viability due to superoxideformation induced by nano-scale TiO

2rdquoToxicology in Vitro vol

25 no 8 pp 2147ndash2151 2011[6] C F Jones and D W Grainger ldquoIn vitro assessments of

nanomaterial toxicityrdquoAdvanced Drug Delivery Reviews vol 61no 6 pp 438ndash456 2009

[7] K Fujita M Horie H Kato et al ldquoEffects of ultrafine TiO2

particles on gene expression profile in human keratinocyteswithout illumination involvement of extracellular matrix andcell adhesionrdquo Toxicology Letters vol 191 no 2-3 pp 109ndash1172009

[8] X Han N Corson P Wade-Mercer et al ldquoAssessing therelevance of in vitro studies in nanotoxicology by examiningcorrelations between in vitro and in vivo datardquo Toxicology vol297 no 1ndash3 pp 1ndash9 2012

[9] H W Kim E-K Ahn B K Jee H-K Yoon K H Lee and YLim ldquoNanoparticulate-induced toxicity and relatedmechanismin vitro and in vivordquo Journal of Nanoparticle Research vol 11 no1 pp 55ndash65 2009

[10] I-L Hsiao and Y-J Huang ldquoTitanium oxide shell coatingsdecrease the cytotoxicity of ZnO nanoparticlesrdquo ChemicalResearch in Toxicology vol 24 no 3 pp 303ndash313 2011

[11] H Fukui M Horie S Endoh et al ldquoAssociation of zinc ionrelease and oxidative stress induced by intratracheal instillationof ZnO nanoparticles to rat lungrdquo Chemico-Biological Interac-tions vol 198 no 1ndash3 pp 29ndash37 2012

[12] S Li W Song and M Gao ldquoSingle and combined cytotoxicityresearch of propiconazole and nano-zinc oxide on the NIH3T3cellrdquo Procedia Environmental Sciences vol 18 pp 100ndash105 2013Proceedings of the International Symposium on EnvironmentalScience and Technology (ISEST rsquo13)

[13] S Wagner S Munzer P Behrens T Scheper D Bahnemannand C Kasper ldquoCytotoxicity of titanium and silicon dioxidenanoparticlesrdquo Journal of Physics Conference Series vol 170Article ID 012022 2009

[14] E Demir H Akca F Turna et al ldquoGenotoxic and cell-transforming effects of titanium dioxide nanoparticlesrdquo Envi-ronmental Research vol 136 pp 300ndash308 2015

[15] H Shi R Magaye V Castranova and J Zhao ldquoTitaniumdioxide nanoparticles a review of current toxicological datardquoParticle and Fibre Toxicology vol 10 no 1 article 15 2013

[16] S Aueviriyavit D Phummiratch K Kulthong and R Mani-ratanachote ldquoTitanium dioxide nanoparticles-mediated invitro cytotoxicity does not induce Hsp70 and Grp78 expressionin human bronchial epithelial A549 cellsrdquo Biological TraceElement Research vol 149 no 1 pp 123ndash132 2012

[17] J Z Bloh R Dillert and D W Bahnemann ldquoRuthenium-modified zinc oxide a highly active vis-photocatalyst thenature and reactivity of photoactive centresrdquo Physical ChemistryChemical Physics vol 16 no 12 pp 5833ndash5845 2014

[18] J Z Bloh R Dillert and D W Bahnemann ldquoTransition metal-modified zinc oxides for UV and visible light photocatalysisrdquoEnvironmental Science and Pollution Research vol 19 no 9 pp3688ndash3695 2012

[19] F Sambale A Lavrentieva F Stahl et al ldquoThree dimensionalspheroid cell culture for nanoparticle safety testingrdquo Journal ofBiotechnology vol 205 pp 120ndash129 2015

[20] National Standards Authority of Ireland (NSAI) BiologicalEvaluation of Medical DevicesmdashPart 12 Sample Preparation andReference Materials (ISO 10993-122012) National StandardsAuthority of Ireland (NSAI) 2012

[21] F Denizot and R Lang ldquoRapid colorimetric assay for cellgrowth and survival Modifications to the tetrazolium dyeprocedure giving improved sensitivity and reliabilityrdquo Journalof Immunological Methods vol 89 no 2 pp 271ndash277 1986

[22] S Bruns Y Stark M Wieland F Stahl C Kasper and TScheper ldquoFast and efficient screening system for new bio-materials in tissue engineering a model for peripheral nerveregenerationrdquo Journal of Biomedical Materials Research A vol81 no 3 pp 736ndash747 2007

[23] A Kroll C Dierker C Rommel et al ldquoCytotoxicity screeningof 23 engineered nanomaterials using a test matrix of ten celllines and three different assaysrdquo Particle and Fibre Toxicologyvol 8 article 9 2011

[24] D Mueller L Kramer E Hoffmann S Klein and F Noor ldquo3Dorganotypic HepaRG cultures as in vitro model for acute andrepeated dose toxicity studiesrdquo Toxicology in Vitro vol 28 no1 pp 104ndash112 2014

[25] E L da Rocha L M Porto and C R Rambo ldquoNanotechnologymeets 3D in vitro models tissue engineered tumors and cancertherapiesrdquo Materials Science and Engineering C vol 34 no 1pp 270ndash279 2014

[26] L Taccola V Raffa C Riggio et al ldquoZinc oxide nanoparticlesas selective killers of proliferating cellsrdquo International Journal ofNanomedicine vol 6 pp 1129ndash1140 2011

[27] U G Sauer S Vogel A Hess et al ldquoIn vivo-in vitro comparisonof acute respiratory tract toxicity using human 3D airway


epithelial models and human A549 and murine 3T3 monolayercell systemsrdquo Toxicology in Vitro vol 27 no 1 pp 174ndash190 2013

[28] C Zink H Hall D M Brunette and N D Spencer ldquoOrthog-onal nanometer-micrometer roughness gradients probe mor-phological influences on cell behaviorrdquo Biomaterials vol 33 no32 pp 8055ndash8061 2012

[29] D D Deligianni N D Katsala P G Koutsoukos and Y F Mis-sirlis ldquoEffect of surface roughness of hydroxyapatite on humanbone marrow cell adhesion proliferation differentiation anddetachment strengthrdquo Biomaterials vol 22 no 1 pp 87ndash962001

[30] L Ventrelli T Fujie S D Turco G Basta B Mazzolai andV Mattoli ldquoInfluence of nanoparticle-embedded polymericsurfaces on cellular adhesion proliferation and differentiationrdquoJournal of Biomedical Materials Research A vol 102 no 8 pp2652ndash2661 2014

[31] J E Paddle-Ledinek Z Nasa and H J Cleland ldquoEffect ofdifferent wound dressings on cell viability and proliferationrdquoPlastic amp Reconstructive Surgery vol 117 no 7 pp 110Sndash118S2006

[32] J M Seiffert M-O Baradez V Nischwitz T Lekishvili HGoenaga-Infante and D Marshall ldquoDynamic monitoring ofmetal oxide nanoparticle toxicity by label free impedancesensingrdquoChemical Research in Toxicology vol 25 no 1 pp 140ndash152 2012

[33] Z Yan L Xu J Han et al ldquoTranscriptional and posttranscrip-tional regulation and endocytosis were involved in zinc oxidenanoparticle-induced interleukin-8 overexpression in humanbronchial epithelial cellsrdquo Cell Biology and Toxicology vol 30no 2 pp 79ndash88 2014

[34] S Singh T Shi R Duffin et al ldquoEndocytosis oxidative stressand IL-8 expression in human lung epithelial cells upon treat-ment with fine and ultrafine TiO

2 role of the specific surface

area and of surface methylation of the particlesrdquo Toxicology andApplied Pharmacology vol 222 no 2 pp 141ndash151 2007

[35] J T Silver and E G Noble ldquoRegulation of survival gene hsp70rdquoCell Stress and Chaperones vol 17 no 1 pp 1ndash9 2012

[36] J Okuda-Shimazaki S Takaku K Kanehira S Sonezaki andATaniguchi ldquoEffects of titanium dioxide nanoparticle aggregatesize on gene expressionrdquo International Journal of MolecularSciences vol 11 no 6 pp 2383ndash2392 2010

[37] J K Chen C Ho H Chang et al ldquoParticulate nature of inhaledzinc oxide nanoparticles determines systemic effects andmech-anisms of pulmonary inflammation in micerdquo Nanotoxicologyvol 9 no 1 pp 43ndash53 2015

[38] E Frohlich C Meindl KWagner G Leitinger and E RobleggldquoUse ofwhole genome expression analysis in the toxicity screen-ing of nanoparticlesrdquoToxicology andApplied Pharmacology vol280 no 2 pp 272ndash284 2014

[39] Q Sun D Tan Y Ze et al ldquoPulmotoxicological effects causedby long-term titanium dioxide nanoparticles exposure inmicerdquoJournal of Hazardous Materials vol 235-236 pp 47ndash53 2012

[40] S Gui Z Zhang L Zheng et al ldquoMolecular mechanism ofkidney injury of mice caused by exposure to titanium dioxidenanoparticlesrdquo Journal of Hazardous Materials vol 195 pp365ndash370 2011

[41] M Ahamed R Posgai T J Gorey M Nielsen S M Hussainand J J Rowe ldquoSilver nanoparticles induced heat shock protein70 oxidative stress and apoptosis in Drosophila melanogasterrdquoToxicology and Applied Pharmacology vol 242 no 3 pp 263ndash269 2010

[42] M Ahamed M A Siddiqui M J Akhtar I Ahmad A BPant and H A Alhadlaq ldquoGenotoxic potential of copper oxidenanoparticles in human lung epithelial cellsrdquo Biochemical andBiophysical Research Communications vol 396 no 2 pp 578ndash583 2010

[43] P Chen K Kanehira S Sonezaki and A Taniguchi ldquoDetectionof cellular response to titanium dioxide nanoparticle agglom-erates by sensor cells using heat shock protein promoterrdquoBiotechnology and Bioengineering vol 109 no 12 pp 3112ndash31182012

[44] Y J Chae C H Pham J Lee E Bae J Yi and M B GuldquoEvaluation of the toxic impact of silver nanoparticles onJapanese medaka (Oryzias latipes)rdquo Aquatic Toxicology vol 94no 4 pp 320ndash327 2009

[45] C H Pham J Yi and M B Gu ldquoBiomarker gene responsein male Medaka (Oryzias latipes) chronically exposed to silvernanoparticlerdquo Ecotoxicology and Environmental Safety vol 78pp 239ndash245 2012

[46] L Foucaud S Goulaouic A Bennasroune et al ldquoOxidativestress induction by nanoparticles in THP-1 cells with 4-HNEproduction stress biomarker or oxidative stress signallingmoleculerdquo Toxicology in Vitro vol 24 no 6 pp 1512ndash15202010

[47] R Posgai M Ahamed S M Hussain J J Rowe and M GNielsen ldquoInhalationmethod for delivery of nanoparticles to theDrosophila respiratory system for toxicity testingrdquo Science of theTotal Environment vol 408 no 2 pp 439ndash443 2009

[48] K B Male M Hamzeh J Montes A C W Leung and J H TLuong ldquoMonitoring of potential cytotoxic and inhibitory effectsof titanium dioxide using on-line and non-invasive cell-basedimpedance spectroscopyrdquo Analytica Chimica Acta vol 777 pp78ndash85 2013

[49] X Zhu E Hondroulis W Liu and C-Z Li ldquoBiosensingapproaches for rapid genotoxicity and cytotoxicity assays uponnanomaterial exposurerdquo Small vol 9 no 9-10 pp 1821ndash18302013

[50] B Moe S Gabos and X-F Li ldquoReal-time cell-microelectronicsensing of nanoparticle-induced cytotoxic effectsrdquo AnalyticaChimica Acta vol 789 pp 83ndash90 2013

[51] Z Li and Z Cui ldquoThree-dimensional perfused cell culturerdquoBiotechnology Advances vol 32 no 2 pp 243ndash254 2014

[52] F Ruedinger A Lavrentieva C Blume I Pepelanova andT Scheper ldquoHydrogels for 3D mammalian cell culture astarting guide for laboratory practicerdquoAppliedMicrobiology andBiotechnology vol 99 no 2 pp 623ndash636 2015

[53] M Drewitz M Helbling N Fried et al ldquoTowards automatedproduction and drug sensitivity testing using scaffold-freespherical tumor microtissuesrdquo Biotechnology Journal vol 6 no12 pp 1488ndash1496 2011

[54] J Lee D Lilly C Doty P Podsiadlo and N Kotov ldquoIn vitrotoxicity testing of nanoparticles in 3D cell culturerdquo Small vol 5no 10 pp 1213ndash1221 2009

[55] A I Astashkina C F Jones GThiagarajan et al ldquoNanoparticletoxicity assessment using an in vitro 3-D kidney organoidculturemodelrdquoBiomaterials vol 35 no 24 pp 6323ndash6331 2014

[56] X Xu C R Sabanayagam D A Harrington M C Farach-Carson and X Jia ldquoA hydrogel-based tumormodel for the eval-uation of nanoparticle-based cancer therapeuticsrdquoBiomaterialsvol 35 no 10 pp 3319ndash3330 2014

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


results can be misinterpreted Nevertheless for nanoparticlecytotoxicity studies in vitro cell-exposure studies can yieldthe first evidence of their potential risk These tests offer theadvantages to be simple and to clarify the basic interaction[1] Additionally these methods are rapid cheaper and morereproducible than in vivo systems [1] Thus in vitro studiesare often used before animal studies are applied Howeverthere are concerns whether the results of these studies canreally predict in vivo cell behavior A comparative study ofthe effects of TiO

2nanoparticles demonstrated a good corre-

lation between acute toxicity measurements of in vitro and invivo tests based on the steepest slopes of dose response curves[8] Moreover Kim et al could identify higher cytotoxicityof nanosized SiO

2as compared with microsized particles in

vitro as well as in vivo [9]To improve the prediction of in vivo effects a set of in

vitro assays investigating different mechanism is needed [9]As in vitro assays cannot mimic real tissue conditions alsomore complex in vitro models like three-dimensional (3D)cell culture models could bridge the gap between classicaltwo-dimensional (2D) in vitromodels and in vivo studies

The development of consumer nanomaterials implieslong optimization procedures resulting in the formation ofplenty of precursors and intermediates All these materialsmay pose risks to human beings and the environmentAll these materials must be tested for toxicity especiallywith regards to occupational safety provision and consumerprotection However the number of in vitro cytotoxicityassays currently available is huge consequently all of theseassays cannot be applied for all precursors intermediates andproductsMoreover animal studies are ethically controversialand therefore limited Thus a broad and complex study canoften not be performed because the bottlenecks are in vitrocell-based toxicity tests and in vivo animal studies which arevery time consuming and costly

Therefore the aim of our study was the developmentof an in vitro hierarchical nanoparticle screening system toexamine any kind of nanoparticles concerning their toxicityFirst the structure of the screening system is presentedin general Next is a separate section of the prescreeningfine-screening and complex-screening where the sensitivityand difficulties of the cytotoxicity assays are discussed Theapplication of the methods in each screening level wasproven exemplary with two different kinds of nanoparticlesThis screening system includes different cytotoxic assays andcomplex cell culture systems such as a three-dimensionalcell culture or dynamic cultivation to increase the relevanceof in vitro assays As nanoparticle suspension testing haslimitations regarding the cell culture we studied whethersimilar results can be obtained with nanoparticle suspensionsand with coating experiments respectively According to thepossible nanoparticle incorporation into the human organ-ism we used human lung carcinoma A549 cells (respiratorytract) and murine fibroblasts NIH-3T3 cells (skin) As abenchmark-system for the development of the screeningsystem we investigated the effects of zinc oxide nanoparticles(ZnO-NP) and titanium dioxide nanoparticles (TiO

2-NP)

on mammalian cell lines These two nanoparticle types havebeen used in different applications and their influence on 2D

monolayer cultures has been analyzed previously For ZnO-NP cytotoxic effects have been published [3 10ndash12] while thecytotoxicity of TiO

2-NP is still a point of discussion Some

studies have reported nontoxic effects for TiO2-NP [13 14]

whereas other studies suggested cytotoxic effects of TiO2-NP

[14ndash16] In the present study both types of nanoparticles werescreened with the developed screening system

2 Materials and Methods

21 Nanoparticles In this study ZnO-NP (with 01 Ru)were used which were synthesized and characterized by Blohet al (2012 2014) [17 18] The ZnO-NP have a BrunauerndashEmmettndashTeller (BET) surface of 654m2g and a particlesize of 50 plusmn 10 nm (X-ray) [17] ZnO-NP exhibited anaverage hydrodynamic diameter (dynamic light scattering)of 41 plusmn 5 nm in water 190 plusmn 3 nm in Dulbeccorsquos ModifiedEagle Medium (DMEM) and 106 plusmn 11 nm in the standardculturemedium (meanplusmn standard derivation) [19]TheTiO

2-

NP (Hombikat XXS 700) were obtained from SachtlebenDuisburg Germany exhibiting a primary particle size of7 nm (REM) in the anatase form according to the data sheetThe hydrodynamic diameter of TiO

2-NP has been reported

previously by Sambale et al [19] In water TiO2-NP have a

diameter of 79 plusmn 25 nm in DMEM 142 plusmn 29 nm and in theculture medium 118 plusmn 28 nm [19]

22 Cell Culture A549 human lung carcinoma cells (DSMZnumber ACC 107) and NIH-3T3 mouse fibroblasts cells(DMSZ number ACC 59) were purchased from the GermanCollection of Microorganisms and Cell Cultures (DSMZ)Both cell lines used here were cultivated in DulbeccorsquosModified Eaglersquos Medium (DMEM) (D7777 Sigma-AldrichSteinheimGermany) supplementedwith 10 fetal calf serum(FCS) and 100 120583gmL antibiotics (penicillinstreptomycin) ina humidified environment at 37∘C5 CO

2 Every 3 or 4 days

cells were subcultivated when the cultures reached 70ndash80confluenceThe passage number of all used cells was less than20

23 3D Cell Culture For 3D cell cultures two different 3Dcell culture models were performed 20000 cells were seededon each side of a round scaffold of 1mm thickness and7mm diameter via pipetting (Matriderm Medskin SolutionDr Suwelack AG Germany) The scaffolds were placed in a24-well plate and were incubated at 37∘C5 CO

2for 72 h

to allow the cells to adhere and to build a 3D structureAfterwards the cells were treated in triplicate with differentconcentrations of ZnO-NP or TiO

2-NP in the cell culture

medium for 24 h As a second model cell encapsulationin a semisynthetic PEG-fibrinogen-based hydrogel (Facultyof Biomedical Engineering Technion Haifa Israel) wasinvestigated Here 1mL hydrogel (fibrinogen concentrationof 8mgmL) contains 1 times 106 cells and 10 120583L photoinitiator(10 (wv) in 70 ethanol) For generating defined hydrogelconstructs 50 120583L of the hydrogel-cell-mixture was added toa 6mm silicon gasket (Grace Bio-Labs silicone gasket forProPlate microarray system Sigma-Aldrich USA) Covalent




2-













2-NP






50value














IL-8 NM 0005843221ndash240






IL-8 NM 0113392338ndash357
















All nanomaterials


+


Cell morphology

Prescreening





Nanoparticle uptake

Fine-screening



Complex-screening

In vivo studies



+

+

Feedback

Feedback

Feedback

minus

minus

minus




















2-NP respectively
















2D cell culture

(h)(g)

(f)(e)

(d)(c)

(b)(a)

3D cell culture

Con

trol

ZnO

-NP

ZnO

-NP

Con

trol

NIH-3T3 cells

A549 cells



0 5 10 15 200

20

40

60

80

100

120Vi

abili

ty (

)


lowast

lowast

lowast

lowastlowast

lowastlowast


lowastlowast


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100

120

Viab

ility

()

lowast lowast


lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast lowast

lowast


(b) A549 cells


















50values















2-NP induce




Control ZnO0

2

4

6

8

10

12Re

lativ

e mRN

A ex

pres

sion

hsp70


il-8

TiO2

lowast

lowast

(a)

Relat

ive m

RNA

expr

essio

n

0

2

4

6

8

10

12

hsp70il-8

Control ZnOTiO2

lowast

lowast

(b)







2-





2-NP no changes



2-NP













0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35ZnO-NP

Nor

m i

mpe

danc

e (O

hm)


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

TiO2-NP

(b) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(c) A549 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(d) A549 cells



2-NP (d) was







2-NP








50value the NIH-


50





[19]For TiO










0 5 10 15 20 25 30 350

20

40

60

80

100

1203D hydrogel

Viab

ility

()


lowastlowast

lowast

lowast

lowast

lowast

lowast


(a) NIH-3T3 cells

3D scaffold

0 5 10 15 20 25 30 350

20

40

60

80

100

120

Viab

ility

()


lowast

lowast lowast

lowast

lowast


(b) NIH-3T3 cells


0 100 200 300 400 5000

20

40

60

80

100

120

Viab

ility

()


lowastlowast

lowast

lowastlowast

lowast


lowast

lowast

lowast

(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


lowastlowast

lowast

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

(d) A549 cells



4 Conclusions




0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D hydrogel



(a) NIH-3T3 cells

0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D scaffold



(b) NIH-3T3 cells

Viab

ility

()


0 100 200 300 400 5000

20

40

60

80

100

120


(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


(d) A549 cells




2-NP) were


2-NP no significant








Acknowledgments


References







































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






2-













2-NP






50value














IL-8 NM 0005843221ndash240






IL-8 NM 0113392338ndash357
















All nanomaterials


+


Cell morphology

Prescreening





Nanoparticle uptake

Fine-screening



Complex-screening

In vivo studies



+

+

Feedback

Feedback

Feedback

minus

minus

minus




















2-NP respectively
















2D cell culture

(h)(g)

(f)(e)

(d)(c)

(b)(a)

3D cell culture

Con

trol

ZnO

-NP

ZnO

-NP

Con

trol

NIH-3T3 cells

A549 cells



0 5 10 15 200

20

40

60

80

100

120Vi

abili

ty (

)


lowast

lowast

lowast

lowastlowast

lowastlowast


lowastlowast


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100

120

Viab

ility

()

lowast lowast


lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast lowast

lowast


(b) A549 cells


















50values















2-NP induce




Control ZnO0

2

4

6

8

10

12Re

lativ

e mRN

A ex

pres

sion

hsp70


il-8

TiO2

lowast

lowast

(a)

Relat

ive m

RNA

expr

essio

n

0

2

4

6

8

10

12

hsp70il-8

Control ZnOTiO2

lowast

lowast

(b)







2-





2-NP no changes



2-NP













0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35ZnO-NP

Nor

m i

mpe

danc

e (O

hm)


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

TiO2-NP

(b) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(c) A549 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(d) A549 cells



2-NP (d) was







2-NP








50value the NIH-


50





[19]For TiO










0 5 10 15 20 25 30 350

20

40

60

80

100

1203D hydrogel

Viab

ility

()


lowastlowast

lowast

lowast

lowast

lowast

lowast


(a) NIH-3T3 cells

3D scaffold

0 5 10 15 20 25 30 350

20

40

60

80

100

120

Viab

ility

()


lowast

lowast lowast

lowast

lowast


(b) NIH-3T3 cells


0 100 200 300 400 5000

20

40

60

80

100

120

Viab

ility

()


lowastlowast

lowast

lowastlowast

lowast


lowast

lowast

lowast

(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


lowastlowast

lowast

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

(d) A549 cells



4 Conclusions




0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D hydrogel



(a) NIH-3T3 cells

0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D scaffold



(b) NIH-3T3 cells

Viab

ility

()


0 100 200 300 400 5000

20

40

60

80

100

120


(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


(d) A549 cells




2-NP) were


2-NP no significant








Acknowledgments


References







































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials













IL-8 NM 0005843221ndash240






IL-8 NM 0113392338ndash357
















All nanomaterials


+


Cell morphology

Prescreening





Nanoparticle uptake

Fine-screening



Complex-screening

In vivo studies



+

+

Feedback

Feedback

Feedback

minus

minus

minus




















2-NP respectively
















2D cell culture

(h)(g)

(f)(e)

(d)(c)

(b)(a)

3D cell culture

Con

trol

ZnO

-NP

ZnO

-NP

Con

trol

NIH-3T3 cells

A549 cells



0 5 10 15 200

20

40

60

80

100

120Vi

abili

ty (

)


lowast

lowast

lowast

lowastlowast

lowastlowast


lowastlowast


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100

120

Viab

ility

()

lowast lowast


lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast lowast

lowast


(b) A549 cells


















50values















2-NP induce




Control ZnO0

2

4

6

8

10

12Re

lativ

e mRN

A ex

pres

sion

hsp70


il-8

TiO2

lowast

lowast

(a)

Relat

ive m

RNA

expr

essio

n

0

2

4

6

8

10

12

hsp70il-8

Control ZnOTiO2

lowast

lowast

(b)







2-





2-NP no changes



2-NP













0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35ZnO-NP

Nor

m i

mpe

danc

e (O

hm)


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

TiO2-NP

(b) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(c) A549 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(d) A549 cells



2-NP (d) was







2-NP








50value the NIH-


50





[19]For TiO










0 5 10 15 20 25 30 350

20

40

60

80

100

1203D hydrogel

Viab

ility

()


lowastlowast

lowast

lowast

lowast

lowast

lowast


(a) NIH-3T3 cells

3D scaffold

0 5 10 15 20 25 30 350

20

40

60

80

100

120

Viab

ility

()


lowast

lowast lowast

lowast

lowast


(b) NIH-3T3 cells


0 100 200 300 400 5000

20

40

60

80

100

120

Viab

ility

()


lowastlowast

lowast

lowastlowast

lowast


lowast

lowast

lowast

(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


lowastlowast

lowast

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

(d) A549 cells



4 Conclusions




0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D hydrogel



(a) NIH-3T3 cells

0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D scaffold



(b) NIH-3T3 cells

Viab

ility

()


0 100 200 300 400 5000

20

40

60

80

100

120


(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


(d) A549 cells




2-NP) were


2-NP no significant








Acknowledgments


References







































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





All nanomaterials


+


Cell morphology

Prescreening





Nanoparticle uptake

Fine-screening



Complex-screening

In vivo studies



+

+

Feedback

Feedback

Feedback

minus

minus

minus




















2-NP respectively
















2D cell culture

(h)(g)

(f)(e)

(d)(c)

(b)(a)

3D cell culture

Con

trol

ZnO

-NP

ZnO

-NP

Con

trol

NIH-3T3 cells

A549 cells



0 5 10 15 200

20

40

60

80

100

120Vi

abili

ty (

)


lowast

lowast

lowast

lowastlowast

lowastlowast


lowastlowast


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100

120

Viab

ility

()

lowast lowast


lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast lowast

lowast


(b) A549 cells


















50values















2-NP induce




Control ZnO0

2

4

6

8

10

12Re

lativ

e mRN

A ex

pres

sion

hsp70


il-8

TiO2

lowast

lowast

(a)

Relat

ive m

RNA

expr

essio

n

0

2

4

6

8

10

12

hsp70il-8

Control ZnOTiO2

lowast

lowast

(b)







2-





2-NP no changes



2-NP













0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35ZnO-NP

Nor

m i

mpe

danc

e (O

hm)


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

TiO2-NP

(b) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(c) A549 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(d) A549 cells



2-NP (d) was







2-NP








50value the NIH-


50





[19]For TiO










0 5 10 15 20 25 30 350

20

40

60

80

100

1203D hydrogel

Viab

ility

()


lowastlowast

lowast

lowast

lowast

lowast

lowast


(a) NIH-3T3 cells

3D scaffold

0 5 10 15 20 25 30 350

20

40

60

80

100

120

Viab

ility

()


lowast

lowast lowast

lowast

lowast


(b) NIH-3T3 cells


0 100 200 300 400 5000

20

40

60

80

100

120

Viab

ility

()


lowastlowast

lowast

lowastlowast

lowast


lowast

lowast

lowast

(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


lowastlowast

lowast

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

(d) A549 cells



4 Conclusions




0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D hydrogel



(a) NIH-3T3 cells

0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D scaffold



(b) NIH-3T3 cells

Viab

ility

()


0 100 200 300 400 5000

20

40

60

80

100

120


(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


(d) A549 cells




2-NP) were


2-NP no significant








Acknowledgments


References







































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials









2-NP respectively
















2D cell culture

(h)(g)

(f)(e)

(d)(c)

(b)(a)

3D cell culture

Con

trol

ZnO

-NP

ZnO

-NP

Con

trol

NIH-3T3 cells

A549 cells



0 5 10 15 200

20

40

60

80

100

120Vi

abili

ty (

)


lowast

lowast

lowast

lowastlowast

lowastlowast


lowastlowast


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100

120

Viab

ility

()

lowast lowast


lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast lowast

lowast


(b) A549 cells


















50values















2-NP induce




Control ZnO0

2

4

6

8

10

12Re

lativ

e mRN

A ex

pres

sion

hsp70


il-8

TiO2

lowast

lowast

(a)

Relat

ive m

RNA

expr

essio

n

0

2

4

6

8

10

12

hsp70il-8

Control ZnOTiO2

lowast

lowast

(b)







2-





2-NP no changes



2-NP













0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35ZnO-NP

Nor

m i

mpe

danc

e (O

hm)


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

TiO2-NP

(b) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(c) A549 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(d) A549 cells



2-NP (d) was







2-NP








50value the NIH-


50





[19]For TiO










0 5 10 15 20 25 30 350

20

40

60

80

100

1203D hydrogel

Viab

ility

()


lowastlowast

lowast

lowast

lowast

lowast

lowast


(a) NIH-3T3 cells

3D scaffold

0 5 10 15 20 25 30 350

20

40

60

80

100

120

Viab

ility

()


lowast

lowast lowast

lowast

lowast


(b) NIH-3T3 cells


0 100 200 300 400 5000

20

40

60

80

100

120

Viab

ility

()


lowastlowast

lowast

lowastlowast

lowast


lowast

lowast

lowast

(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


lowastlowast

lowast

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

(d) A549 cells



4 Conclusions




0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D hydrogel



(a) NIH-3T3 cells

0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D scaffold



(b) NIH-3T3 cells

Viab

ility

()


0 100 200 300 400 5000

20

40

60

80

100

120


(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


(d) A549 cells




2-NP) were


2-NP no significant








Acknowledgments


References







































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




2D cell culture

(h)(g)

(f)(e)

(d)(c)

(b)(a)

3D cell culture

Con

trol

ZnO

-NP

ZnO

-NP

Con

trol

NIH-3T3 cells

A549 cells



0 5 10 15 200

20

40

60

80

100

120Vi

abili

ty (

)


lowast

lowast

lowast

lowastlowast

lowastlowast


lowastlowast


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100

120

Viab

ility

()

lowast lowast


lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast lowast

lowast


(b) A549 cells


















50values















2-NP induce




Control ZnO0

2

4

6

8

10

12Re

lativ

e mRN

A ex

pres

sion

hsp70


il-8

TiO2

lowast

lowast

(a)

Relat

ive m

RNA

expr

essio

n

0

2

4

6

8

10

12

hsp70il-8

Control ZnOTiO2

lowast

lowast

(b)







2-





2-NP no changes



2-NP













0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35ZnO-NP

Nor

m i

mpe

danc

e (O

hm)


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

TiO2-NP

(b) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(c) A549 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(d) A549 cells



2-NP (d) was







2-NP








50value the NIH-


50





[19]For TiO










0 5 10 15 20 25 30 350

20

40

60

80

100

1203D hydrogel

Viab

ility

()


lowastlowast

lowast

lowast

lowast

lowast

lowast


(a) NIH-3T3 cells

3D scaffold

0 5 10 15 20 25 30 350

20

40

60

80

100

120

Viab

ility

()


lowast

lowast lowast

lowast

lowast


(b) NIH-3T3 cells


0 100 200 300 400 5000

20

40

60

80

100

120

Viab

ility

()


lowastlowast

lowast

lowastlowast

lowast


lowast

lowast

lowast

(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


lowastlowast

lowast

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

(d) A549 cells



4 Conclusions




0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D hydrogel



(a) NIH-3T3 cells

0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D scaffold



(b) NIH-3T3 cells

Viab

ility

()


0 100 200 300 400 5000

20

40

60

80

100

120


(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


(d) A549 cells




2-NP) were


2-NP no significant








Acknowledgments


References







































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0 5 10 15 200

20

40

60

80

100

120Vi

abili

ty (

)


lowast

lowast

lowast

lowastlowast

lowastlowast


lowastlowast


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100

120

Viab

ility

()

lowast lowast


lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast lowast

lowast


(b) A549 cells


















50values















2-NP induce




Control ZnO0

2

4

6

8

10

12Re

lativ

e mRN

A ex

pres

sion

hsp70


il-8

TiO2

lowast

lowast

(a)

Relat

ive m

RNA

expr

essio

n

0

2

4

6

8

10

12

hsp70il-8

Control ZnOTiO2

lowast

lowast

(b)







2-





2-NP no changes



2-NP













0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35ZnO-NP

Nor

m i

mpe

danc

e (O

hm)


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

TiO2-NP

(b) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(c) A549 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(d) A549 cells



2-NP (d) was







2-NP








50value the NIH-


50





[19]For TiO










0 5 10 15 20 25 30 350

20

40

60

80

100

1203D hydrogel

Viab

ility

()


lowastlowast

lowast

lowast

lowast

lowast

lowast


(a) NIH-3T3 cells

3D scaffold

0 5 10 15 20 25 30 350

20

40

60

80

100

120

Viab

ility

()


lowast

lowast lowast

lowast

lowast


(b) NIH-3T3 cells


0 100 200 300 400 5000

20

40

60

80

100

120

Viab

ility

()


lowastlowast

lowast

lowastlowast

lowast


lowast

lowast

lowast

(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


lowastlowast

lowast

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

(d) A549 cells



4 Conclusions




0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D hydrogel



(a) NIH-3T3 cells

0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D scaffold



(b) NIH-3T3 cells

Viab

ility

()


0 100 200 300 400 5000

20

40

60

80

100

120


(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


(d) A549 cells




2-NP) were


2-NP no significant








Acknowledgments


References







































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials















2-NP induce




Control ZnO0

2

4

6

8

10

12Re

lativ

e mRN

A ex

pres

sion

hsp70


il-8

TiO2

lowast

lowast

(a)

Relat

ive m

RNA

expr

essio

n

0

2

4

6

8

10

12

hsp70il-8

Control ZnOTiO2

lowast

lowast

(b)







2-





2-NP no changes



2-NP













0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35ZnO-NP

Nor

m i

mpe

danc

e (O

hm)


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

TiO2-NP

(b) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(c) A549 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(d) A549 cells



2-NP (d) was







2-NP








50value the NIH-


50





[19]For TiO










0 5 10 15 20 25 30 350

20

40

60

80

100

1203D hydrogel

Viab

ility

()


lowastlowast

lowast

lowast

lowast

lowast

lowast


(a) NIH-3T3 cells

3D scaffold

0 5 10 15 20 25 30 350

20

40

60

80

100

120

Viab

ility

()


lowast

lowast lowast

lowast

lowast


(b) NIH-3T3 cells


0 100 200 300 400 5000

20

40

60

80

100

120

Viab

ility

()


lowastlowast

lowast

lowastlowast

lowast


lowast

lowast

lowast

(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


lowastlowast

lowast

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

(d) A549 cells



4 Conclusions




0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D hydrogel



(a) NIH-3T3 cells

0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D scaffold



(b) NIH-3T3 cells

Viab

ility

()


0 100 200 300 400 5000

20

40

60

80

100

120


(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


(d) A549 cells




2-NP) were


2-NP no significant








Acknowledgments


References







































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Control ZnO0

2

4

6

8

10

12Re

lativ

e mRN

A ex

pres

sion

hsp70


il-8

TiO2

lowast

lowast

(a)

Relat

ive m

RNA

expr

essio

n

0

2

4

6

8

10

12

hsp70il-8

Control ZnOTiO2

lowast

lowast

(b)







2-





2-NP no changes



2-NP













0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35ZnO-NP

Nor

m i

mpe

danc

e (O

hm)


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

TiO2-NP

(b) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(c) A549 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(d) A549 cells



2-NP (d) was







2-NP








50value the NIH-


50





[19]For TiO










0 5 10 15 20 25 30 350

20

40

60

80

100

1203D hydrogel

Viab

ility

()


lowastlowast

lowast

lowast

lowast

lowast

lowast


(a) NIH-3T3 cells

3D scaffold

0 5 10 15 20 25 30 350

20

40

60

80

100

120

Viab

ility

()


lowast

lowast lowast

lowast

lowast


(b) NIH-3T3 cells


0 100 200 300 400 5000

20

40

60

80

100

120

Viab

ility

()


lowastlowast

lowast

lowastlowast

lowast


lowast

lowast

lowast

(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


lowastlowast

lowast

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

(d) A549 cells



4 Conclusions




0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D hydrogel



(a) NIH-3T3 cells

0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D scaffold



(b) NIH-3T3 cells

Viab

ility

()


0 100 200 300 400 5000

20

40

60

80

100

120


(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


(d) A549 cells




2-NP) were


2-NP no significant








Acknowledgments


References







































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35ZnO-NP

Nor

m i

mpe

danc

e (O

hm)


(a) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

TiO2-NP

(b) NIH-3T3 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(c) A549 cells

0 10 20 30 40 50 60 70 80 90 10000

05

10

15

20

25

30

35

Nor

m i

mpe

danc

e (O

hm)


Time (h)

(d) A549 cells



2-NP (d) was







2-NP








50value the NIH-


50





[19]For TiO










0 5 10 15 20 25 30 350

20

40

60

80

100

1203D hydrogel

Viab

ility

()


lowastlowast

lowast

lowast

lowast

lowast

lowast


(a) NIH-3T3 cells

3D scaffold

0 5 10 15 20 25 30 350

20

40

60

80

100

120

Viab

ility

()


lowast

lowast lowast

lowast

lowast


(b) NIH-3T3 cells


0 100 200 300 400 5000

20

40

60

80

100

120

Viab

ility

()


lowastlowast

lowast

lowastlowast

lowast


lowast

lowast

lowast

(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


lowastlowast

lowast

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

(d) A549 cells



4 Conclusions




0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D hydrogel



(a) NIH-3T3 cells

0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D scaffold



(b) NIH-3T3 cells

Viab

ility

()


0 100 200 300 400 5000

20

40

60

80

100

120


(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


(d) A549 cells




2-NP) were


2-NP no significant








Acknowledgments


References







































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






2-NP








50value the NIH-


50





[19]For TiO










0 5 10 15 20 25 30 350

20

40

60

80

100

1203D hydrogel

Viab

ility

()


lowastlowast

lowast

lowast

lowast

lowast

lowast


(a) NIH-3T3 cells

3D scaffold

0 5 10 15 20 25 30 350

20

40

60

80

100

120

Viab

ility

()


lowast

lowast lowast

lowast

lowast


(b) NIH-3T3 cells


0 100 200 300 400 5000

20

40

60

80

100

120

Viab

ility

()


lowastlowast

lowast

lowastlowast

lowast


lowast

lowast

lowast

(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


lowastlowast

lowast

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

(d) A549 cells



4 Conclusions




0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D hydrogel



(a) NIH-3T3 cells

0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D scaffold



(b) NIH-3T3 cells

Viab

ility

()


0 100 200 300 400 5000

20

40

60

80

100

120


(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


(d) A549 cells




2-NP) were


2-NP no significant








Acknowledgments


References







































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0 5 10 15 20 25 30 350

20

40

60

80

100

1203D hydrogel

Viab

ility

()


lowastlowast

lowast

lowast

lowast

lowast

lowast


(a) NIH-3T3 cells

3D scaffold

0 5 10 15 20 25 30 350

20

40

60

80

100

120

Viab

ility

()


lowast

lowast lowast

lowast

lowast


(b) NIH-3T3 cells


0 100 200 300 400 5000

20

40

60

80

100

120

Viab

ility

()


lowastlowast

lowast

lowastlowast

lowast


lowast

lowast

lowast

(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


lowastlowast

lowast

lowast

lowast

lowast lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

(d) A549 cells



4 Conclusions




0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D hydrogel



(a) NIH-3T3 cells

0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D scaffold



(b) NIH-3T3 cells

Viab

ility

()


0 100 200 300 400 5000

20

40

60

80

100

120


(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


(d) A549 cells




2-NP) were


2-NP no significant








Acknowledgments


References







































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D hydrogel



(a) NIH-3T3 cells

0 50 100 150 2000

20

40

60

80

100

120

Viab

ility

()

3D scaffold



(b) NIH-3T3 cells

Viab

ility

()


0 100 200 300 400 5000

20

40

60

80

100

120


(c) A549 cells


0 100 200 300 400 5000

20

40

60

80

100

120Vi

abili

ty (

)


(d) A549 cells




2-NP) were


2-NP no significant








Acknowledgments


References







































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







Acknowledgments


References







































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



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