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Toxicology Letters 212 (2012) 75–82Contents lists available at SciVerse ScienceDirectToxicologyLettersj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /t o x l etBiological evaluation of Fe 3O 4–poly(l -lactide)–poly(ethyleneglycol)–poly(l -lactide)magnetic microspheres prepared in supercritical CO 2Ai-Zheng Chen a ,b ,∗,Xiao-Fen Lin a ,Shi-Bin Wang a ,b ,∗∗,Li Li a ,Yuan-Gang Liu a ,b ,Li Ye a ,Guang-Ya Wang aa College of Chemical Engineering,Huaqiao University,Xiamen 361021,ChinabInstitute of Biomaterials and Tissue Engineering,Huaqiao University,Xiamen 361021,Chinah i g h l i g h t sWe used the SpEDS process to pre-pare Fe 3O 4–PLLA–PEG–PLLA MMPs. A low cytotoxicity was evaluated of Fe 3O 4–PLLA–PEG–PLLA MMPs at the cellular level.Fe 3O 4–PLLA–PEG–PLLA MMPs create low genotoxic and immuntoxic at the molecular level.Acute toxicity tests of MMPs show quite a low toxicity.Fe 3O 4–PLLA–PEG–PLLA MMPs have great potential for use in biomedical applications.g r a p h i c a la b s t r a cta r t i c l ei n f oArticle history:Received 12March 2012Received in revised form 6May 2012Accepted 8May 2012Available online 15 May 2012Keywords:Biocompatibility Fe 3O 4PLLA–PEG–PLLAMagnetic microspheres Supercritical CO 2a b s t r a c tThe biocompatibility of Fe 3O 4–poly(l -lactide)–poly(ethylene glycol)–poly(l -lactide)magnetic micro-spheres (Fe 3O 4–PLLA–PEG–PLLA MMPs)prepared in a process of suspension-enhanced dispersion by supercritical CO 2(SpEDS)was evaluated at various levels:cellular,molecular,and integrated.At the cellular level,the investigations of cytotoxicity and intracellular reactive oxygen species (ROS)gener-ation indicate that the polymer-coated MMPs (2.0mg/mL)had a higher toxicity than uncoated Fe 3O 4nanoparticles,which led to about 20%loss of cell viability and an increase (0.2fold)in ROS gener-ation;the differences were not statistically significant (p >0.05).However,an opposite phenomenon was observed in tests of hemolysis,which showed that the MMPs displayed the weakest hemolytic activity,namely only about 6%at the highest concentration (20mg/mL).This phenomenon reveals that polymer-coated MMPs created less toxicity in red blood cells than uncoated Fe 3O 4nanoparticles.At the molecular level,the MMPs were shown to be less genotoxic than Fe 3O 4nanoparticles by measuring the micronucleus (MN)frequency in CHO-K1cells.Furthermore,the mRNA expression of pro-inflammatory cytokines demonstrates that polymer-coated MMPs elicited a less intense secretion of pro-inflammatory cytokines than uncoated Fe 3O 4nanoparticles.Acute toxicity tests of MMPs show quite a low toxic-ity,with an LD 50>1575.00mg/kg.The evidence of low toxicity presented in the results indicates that the Fe 3O 4–PLLA–PEG–PLLA MMPs from the SpEDS process have great potential for use in biomedical applications.© 2012 Elsevier Ireland Ltd. All rights reserved.∗Corresponding author at:College of Chemical Engineering,Huaqiao University,Xiamen 361021,China.Tel.:+865926162326;fax:+865926162326.∗∗Corresponding author at:College of Chemical Engineering,Huaqiao University,Xiamen 361021,China.Tel.:+865926162288;fax:+865926162288.E-mail addresses:biomat@ ,azchen@ (A.-Z.Chen),sbwang@ (S.-B.Wang).1.IntroductionApplications for the use of magnetic nanoparticles are mostly in biomedicine and bioengineering,including the areas of hyper-thermia,targeted drug delivery,magnetic resonance imaging (MRI),biological separation,protein immobilization,and biosen-sors (Evanochko et al.,1984;Widder et al.,1980;Yuk et al.,2011;0378-4274/$–see front matter © 2012 Elsevier Ireland Ltd. All rights reserved./10.1016/j.toxlet.2012.05.00976 A.-Z.Chen et al./Toxicology Letters212 (2012) 75–82Baselt et al.,1998;Gu et al.,2006;Lewin et al.,2000).Among dif-ferent kinds of magnetic nanoparticles,Fe3O4and␥-Fe2O3are two promising candidates due to their distinguished biocompatibility and the relative ease with which they can be combined with organic materials such as chitosan(Liang and Zhang,2007),polyethylene glycol(PEG)(Sun et al.,2006),and polyvinyl alcohol(PVA)(Petri-Fink et al.,2005)to enhance their stability and enlarge the range of their application.The core–shell structured MMPs generally consist of iron oxide as the core and polymer coating as the shell(Zhang and Misra,2007;Kim et al.,2008;Yang et al.,2009).Fe3O4–PLLA–PEG–PLLA MMPs have been successfully prepared by the SpEDS process in our previous work.These surface-coated MMPs possess unique physicochemical properties,such as high surface-volume ratios and magnetic features.They may exhibit unknown nanoscale phenomena in biological manifesta-tions(Karlsson et al.,2009)and it is therefore necessary to perform biological evaluations before their clinical trials.As they are candidates for biomedical applications,in particular for drug delivery,the evaluation of MMPs has traditionally been focused on cell toxicity,viability,proliferation,and differentiation (Fischer and Chan,2007;Hohnholt et al.,2011).Because testing of biomaterials at the cellular level is not comprehensive or specific, interactions between MMPs and animal cells have not been inves-tigated systematically and completely.For the sake of harvesting more reliable results from various aspects,methods for evaluat-ing the biocompatibility of the MMPs will be implemented at three levels:cellular,molecular,and integrated.Analysis of the biocom-patibility of MMPs at various levels focuses on their interaction with different cell lines and innate biological systems.In particular, assessments of cytotoxicity,immunotoxicity,genotoxicity,acute toxicity,hemolysis,and the potential induction of cell stress are considered highly relevant.Some previous studies have evaluated the biocompatibility of various types of MMPs;however,studies at a variety of levels are still limited(Hafeli and Pauer,1999;Brayner,2008).In1995,the concept of the molecular level was addressed by Chou et al.(1995), who reported that the biological effects of biomaterials on an innate system would be more precisely observed from the molecular level. However,the aim of this study is to evaluate the biocompatibility of Fe3O4–PLLA–PEG–PLLA MMPs using a systematic approach at var-ious levels,including cell viability,induction of intracellular ROS, secretion of pro-inflammatory cytokines,and DNA damage.2.Materials and methods2.1.Cell culture and materialsL929cells(a mousefibroblast cell line),RAW264.7cells(a murine macrophage-like cell line),and CHO-K1cells(a Chinese hamster ovarian cell line)(all from CTCCCAS,Shanghai,China)were cultured in RPMI Medium1640(Gibco,USA),Dul-becco’s Minimum Essential Medium(DMEM,Gibco,USA),and Nutrient mixture F12Ham,Kaighn’s Modification(F12K,Sigma Aldrich Ltd.,Germany),respectively, supplemented with10%heat-inactivated fetal bovine serum(Biochrom,Germany) and2mM l-glutamine,100U/mL penicillin,and100mg/mL streptomycin(Hyclone, USA).The cells were cultured in a humidified incubator at37◦C(95%humidity,5% CO2).Alamar Blue was purchased from Invitrogen(USA).The2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT)Cell Proliferation Kit was purchased from Roche(Germany).DCFH-DA was purchased from the Beyotime Institute of Biotechnology(Jiangsu,China).Kunming mice were purchased from the Slac Laboratory Animal Co.Ltd.(Shanghai,China).Giemsa stain and Cytochalasin B (Cyt B)were purchased from Sigma Aldrich Ltd.(Germany).Mitomycine C(MMC) was purchased from Roche(Switzerland).The Prime Script TM RT reagent Kit and SYBR Premix Ex Taq TM were purchased from Takara Bio Ltd.(Dalian,China).The Fe3O4–PLLA–PEG–PLLA MMPs were prepared in a SpEDS process by coating Fe3O4nanoparticles(mean size30nm)with PLLA-PEG-PLLA.The schematic diagram of the SpEDS apparatus and the coating process is shown in Fig.S1of the Supplemen-tary data;the surface morphology of the resulting Fe3O4–PLLA–PEG–PLLA MMPs is shown in Fig.S2in the same section.The physicochemical parameters of the MMPs are as follows:mean size629nm,SPAN1.17,Fe3O4content13.2%(w/w),saturation magnetization16.3emu/g.2.2.Cellular level evaluation2.2.1.Cytotoxicity assaysFor the cytotoxicity test,the L929cells were seeded at a density of 1×105cells/well in a96-well plate(Corning,USA)and kept in the incubator for 24h for better cell attachment.After incubation for24h,the culture medium was replaced by fresh medium containing MMPs at different concentrations or control media of cell culture medium,and analyzed after24h or72h incubation for cell viability.All incubations were performed at37◦C in a5%CO2humidified incubator. Assays were implemented according to the manufacturer’s instructions.Briefly,the cells were rinsed with PBS,then100␮L of10%(v/v)Alamar Blue solution,prepared in fresh media(without FBS or supplements),was added to each well when the control media or test samples were removed.After incubation for5h,Alamar Blue fluorescence was determined at the respective excitation and emission wavelengths of570nm and600nm using a microplate reader(Molecular Devices,SpectraMax M5,USA).The spectrophotometer was calibrated to zero absorbance,using culture medium without cells.In addition,an XTT assay was performed in order to deter-mine the cell viability.By the same process,after L929cells had been incubated with MMPs for24h or72h,the treated cells were washed with100␮L of PBS and incubated for4h with50␮L of XTT solution(final concentration0.3mg/mL)at37◦C in a5%CO2humidified incubator.After incubation,the absorbance was measured using a microplate reader at a wavelength of500nm(reference wavelength600nm). The relative cell viability(%)related to wells containing cell culture medium with MMPs was calculated by[A]test/[A]control×100.Here[A]test is the absorbance of the test sample and[A]control is the absorbance of the control sample(0mg/mL).Data analysis was carried out using SPSS for one-way ANOVA.2.2.2.Intracellular ROSIntracellular ROS levels were investigated by measuring the oxidative conver-sion of2 ,7 -dichlorofluorescin diacetate(DCFH-DA)to thefluorescent compound dichlorofluorescin(DCF).In brief,RAW264.7cells were seeded into96-well and6-well plates with a density of1×105cells/well and1×106cells/well,respectively; 24h later,the cells were treated with increasing concentrations of MMPs or con-trol medium for24h.Thereafter,the medium was removed and replaced with serum-free medium,and100␮L of10␮M DCFH-DA(the Beyotime Institute of Biotechnology,Jiangsu,China)was added to each well.The cells were incubated for30min and then the results of intracellular oxidation of DCFH-DA to DCF were obtained quantitatively by using the multifunctional microplate reader(Molecular Devices,SpectraMax M5,USA)and qualitatively by using confocal laser scanning microscopy(Zeiss,LSM710,Germany).High DMEM culture medium was used as a negative control.Rousp(hydrogen peroxide)was used as a positive control to validate the protocol.Data analysis was carried out using SPSS for one-way ANOVA.2.2.3.Hemolysis testHuman whole blood samples were collected from a healthy23-year-old vol-unteer.Red blood cells(RBCs)were obtained by centrifugation at1000rpm for5min and then washed three times with physiological saline buffer.The MMP solutions(9800␮L)with different concentrations were added to the RBC solutions(200␮L)and incubated for60min at37◦C with shaking at200rpm. After incubation,intact RBCs were removed by centrifugation(1000rpm for 5min)and100␮L of supernatant was harvested.The amount of hemoglobin released was investigated by reading the absorbance of the resulting super-natant at540nm using a microplate reader.Physiological saline buffer(0% lysis)and double-distilled water(100%lysis)were employed as control sam-ples in all experiments.Less than10%hemolysis was regarded as a non-toxic level in our experiments.Data analysis was carried out using SPSS for one-way ANOVA.2.3.Integrated level evaluation2.3.1.Acute toxicity testThe median lethal dose(LD50)values of the MMPs were investigated in Kun-ming mice(male/female,4–6weeks,18–22g).Ten mice were used per group and a single intraperitoneal(i.p.)dose of525.00mg/kg,420.00mg/kg,336.00mg/kg, 268.80mg/kg,or215.04mg/kg of MMPs was used for each mouse.The same volume of either physiological saline or MMP solution was used in corresponding groups. Signs of toxicity and/or death,behavioral changes,and the latency of death were observed over seven days.The maximum tolerated dose(MTD)was then deter-mined based on the results obtained from the LD50investigation.Briefly,an i.p. dose of525.00mg/kg was determined to be the MTD in mice by a triple-treated test(the dose given once every5h).The definition of the MTD is the highest pos-sible dose resulting in no animal deaths,less than20%weight loss of the control animals,and no particular changes in general signs.The experiments were carried out in accordance with the guidelines issued by the Ethical Committee of Huaqiao University.A.-Z.Chen et al./Toxicology Letters212 (2012) 75–82772.4.Molecular level evaluation2.4.1.Cytokinesis block micronucleus(CBMN)assayThe CBMN assay is one of the most widespread assays used for DNA-damage screening,using Cyt B according to the Fenech protocol(Fenech,2007).The CBMN assay was performed according to the following procedure.Briefly,the CHO-K1cells(1×106cells),firstly cultured for24h in seeding medium,were exposed to different concentrations of MMP suspensions and incubated for another 24h.Untreated cells were used as a negative control,and cells incubated with 200ng/mL of MMC were used as a positive control.The Cyt B(5␮g/mL)was added to the cultures before they were re-incubated at37◦C for24h.Cells were then treated according to the same procedure described for the chromosomal aberration assay.Slides were coded and the assay was carried out using an opti-cal microscope.Following the criteria for characterizing binucleated(BN)cells, the numbers of MN and1000BN cells per slide were scored.The assay was carried out in triplicate.Data analysis was carried out using SPSS for one-way ANOVA.2.4.2.Real-time reverse transcriptase polymerase chain reaction(RT-PCR)The cytokine secretion was further investigated by real-time RT-PCR deter-mined at the mRNA level.RAW264.7cells(1×106cells/mL)were collected after being treated with different concentrations of MMPs for24h.The intracellular total RNA was extracted with Trizol reagent.The Prime Script TM RT reagent Kit was then used to construct the cDNA library,and the constructed cDNA was further accessed as the template of the PCR.Specific primers for murine IL-16,TNF-␣,and iNOS(Takara Bio Ltd.,Dalian,China)were used for the PCR,as shown in Table1;␤-actin was used as an internal control and LPS,an inducer of RAW264.7cell acti-vation,was used as a positive control.Real-time PCR reactions were performed using a SYBR Premix Ex Taq TM Kit on the ABI Prism®7500system.Each reaction was run in a20␮L mixture containing10␮L of SYBR Premix Ex Taq(2×),0.4␮L of each primer(10␮M),2␮L of cDNA,0.4␮L of ROX Reference Dye II(50×),and 6.8␮L of ddH2O.The primer and␤-actin sequences are listed in Table1.A two-step real-time PCR cycle was carried out;the steps included an initial predenaturation by heating to95◦C for30s,followed by40cycles involving denaturation at95◦C for15s,and annealing and elongation at60◦C for34s.Thefluorescence signal was harvested at the end of each cycle.Melting curve analysis was used to con-firm the specificity of the products.Results were analyzed by the2− C T method (Livak and Schmittgen,2001).Data analysis was carried out using SPSS for one-way ANOVA.3.Results and discussion3.1.Alamar Blue assayThe cytotoxicity of different concentrations of MMPs was deter-mined using the Alamar Blue assay for an exposure time of24h or 72h,and the results are shown in Fig.1a and b.They demonstrate a dose-dependent reduction in Alamar Blue absorbance in L929cells treated with MMPs after24h or72h.After24h of treatment,the Fe3O4–PLLA–PEG–PLLA MMPs and Fe3O4nanoparticles were rela-tively non-toxic at the low concentration of0.5mg/mL,with a cell viability of greater than90%.Approximately20%loss of cell viabil-ity occurred at the higher concentration of Fe3O4–PLLA–PEG–PLLA MMPs(2.0mg/mL),as shown in Fig.1a.Even after72h of exposure, the Fe3O4nanoparticles and MMPs were relatively non-toxic;as is shown in Fig.1b,the cell viability at the highest concentration was still greater than80%.Furthermore,no statistically significant dif-ferences(p>0.05)in cell viability were demonstrated by the Alamar Blue assay.Our results indicate that the iron-oxide nanoparticles would induce less cytotoxicity,afinding which has been demon-strated by previous studies(Gupta and Gupta,2005;Soenen et al., 2011).This can be explained by the fact that some of the nanoparti-cles are taken up by the cells as a result of endocytosis and increased apoptosis due to weak cell adhesion interactions with the nanopar-ticles(Berry et al.,2003;Gupta et al.,2003).The cell toxicity of Fe3O4–PLLA–PEG–PLLA MMPs may also be attributed to the fact that the polymer-coated surface would improve the uptake of nano and micromolecules(Zhang et al.,2002).However,after microen-capsulation with polymers,the biocompatibility of the resulting composite microspheres is not significantly different at the cellular level from that of the Fe3O4nanoparticles.A similar phenomenon was also observed in the results of the XTT assay.(The dataare Fig. 1.Relative cell viability detected at different concentrations of Fe3O4–PLLA–PEG–PLLA MMPs and Fe3O4nanoparticles by Alamar Blue assay following exposure for(a)24h and(b)72h.Values are represented as the mean±the standard error of the mean(n=6).shown in Figs.S3a and S3b of the Supplementary data;no signif-icant differences were found between the Fe3O4–PLLA–PEG–PLLA MMPs and the Fe3O4nanoparticles.)3.2.Intracellular ROS productionDue to the catalytic function of iron oxide nanoparticles in the production of ROS in the Fenton reaction,free iron oxide released from the magnetite core of MMPs could be toxic.The potential for iron oxide nanoparticles to induce oxidative stress was tested by evaluating intracellular ROS using the DCFH-DA assay.ROS generation in RAW264.7cells following24h of expo-sure to test samples at different concentrations is shown in Fig.2. There was obviously a dose-dependent increase in the ROS level. Briefly,a rise of the ROS level in cells was not detected during treatment with Fe3O4nanoparticles,but an increase of ROS gen-eration over the control level was found after treatment with Fe3O4–PLLA–PEG–PLLA MMPs(400␮g/mL);however,there were still no significant statistical differences(p>0.05)between the ROS levels they induced.The increase with Fe3O4–PLLA–PEG–PLLA MMPs was probably because the polymer-coated surfaces allow the Fe3O4nanoparticles to be better dispersed and enhance the chance of their interaction with cells.Similar studies have also investi-gated such surface-modified nanoparticles(Ahamed et al.,2008; Mu et al.,2010).The ROS results above also correspond with those78A.-Z.Chen et al./Toxicology Letters 212 (2012) 75–82Table 1Primer sequences for the cytokines.NamePrimer sequences (5 –3 )Amplification length (bp)IL-6IL6-FW 5 -CCACTTCACAAGTCGGAGGCTTA-3 169IL6-RV 5 -CCAGTTTGGTAGCATCCATCATTTC-3 TNF-␣TNF-FW 5 -TATGGCCCAGACCCTCACA-3199TNF-RV5 -GGAGTAGACAAGGTACAACCCATC-3iNOS iNOS-FW 5 -CAAGCTGAACTTGAGCGAGGA-3 185iNOS-RV5 -TTTACTCAGTGCCAGAAGCTGGA-3␤-actin␤-actin-FW 5 -CATCCGTAAAGACCTCTATGCCAAC-3 171␤-actin-RV5 -GGAGGAAGAGGATGCGGCAGT-3from the Alamar Blue and XTT assays.In terms of cytotoxicity,a slightly higher toxicity was found with the Fe 3O 4–PLLA–PEG–PLLA MMPs,but again,no significant differences were found between the Fe 3O 4–PLLA–PEG–PLLA MMPs and the Fe 3O 4nanoparticles.The intracellular ROS production in the macrophage cells after 24h of exposure to Fe 3O 4–PLLA–PEG–PLLA MMPs at different concentra-tions was also visualized by confocal fluorescence microscopy and is shown in Fig.3a and e.3.3.HemolysisErythrocyte-induced hemolysis in vitro can be considered as a simple and reliable measure for estimating membrane damage caused in vivo (Pape et al.,1987;Reer et al.,1994).The behavior of Fe 3O 4–PLLA–PEG–PLLA MMPs in vivo therefore was predicted by evaluating the degree of hemolysis in vitro.Hemolysis results shown in Fig.4reveal that a negligible hemolysis (lower than 2%)was produced at the lower concentration (5mg/mL)of all sam-ples.At the same time,hemolysis in all samples was observed to be concentration-dependent,rising along with the increasing concentration,but still remaining below 10%.Among them,the hemolytic activity of the Fe 3O 4–PLLA–PEG–PLLA MMPs was the weakest,namely about 6%at the highest concentration (20mg/mL).Meanwhile,a significant difference (p <0.01)was found when this was compared with the hemolytic activity of Fe 3O 4nanoparticles (nearly 8%)at 20mg/mL.In previous reports,superparamagnetic iron oxide particles have been studied by intravenous injection in vivo for biomedical application due to their negligible hemol-ysis (Wagner et al.,2002;Lawaczeck et al.,1997).Our results on polymer-coated iron oxide particles reveal that therewasFig.2.Intracellular ROS generation of iron oxide;RAW264.7cells were treated with different concentrations of Fe 3O 4–PLLA–PEG–PLLA MMPs or Fe 3O 4nanoparticles for 24h.The ROS level of the positive control was set at 100%.The negative control was high DMEM culture medium,and the positive control was Rousp (hydrogen peroxide).Values are represented as the mean ±the standard error of the mean (n =6).even less hemolysis after the particles had been surface-coated.It has also been reported that some special designs for surfactant polymers could improve the blood compatibility of biomaterials (Sagnella and Mai-Ngam,2005).Even though no significant differ-ences were found previously in cell toxicity,we are still gratified to have predicted that Fe 3O 4–PLLA–PEG–PLLA MMPs would not cause membrane damage in vivo and then be able to demonstrate negligible hemolysis.3.4.Acute toxicityNo mortality or toxicity was observed after intraperitoneal injection of Fe 3O 4–PLLA–PEG–PLLA MMPs or the control groups.This result reveals that the MMPs did not create acute toxi-city.Considering the dispersion of the Fe 3O 4–PLLA–PEG–PLLA MMPs,sediment would be an aggregation due to the increase of solute.The LD 50of the MMPs was difficult to elucidate,so the MTD was determined by a triple-treated test within 24h.The MTD of the Fe 3O 4–PLLA–PEG–PLLA MMPs was equal to 1575.00mg/kg.Detailed results are shown in Table 2and Fig.5.(The corresponding histopathological observation is shown in Fig.S4of the Supplementary data .)For the purpose of studying the biological effects of magnetic nanoparticles,intraperitoneal injection is highly recommended (Lacava et al.,1999).Through observing the effects of poisoning and the probable resultant deaths in animals,researchers can study the toxicity of iron oxide nanopar-ticles more comprehensively,and in particular,the polymer-coated Fe 3O 4nanoparticles.Prior to those results,we demonstrated that the Fe 3O 4–PLLA–PEG–PLLA MMPs showed quite a low acute toxic-ity,with an LD 50>1575.00mg/kg.3.5.MN frequencyThe formation of an MN in BN cells was observed after treatment with different concentrations of MMPs and the control groups.The MN formation in CHO-K1BN cells treated with 2.0mg/mL of Fe 3O 4–PLLA–PEG–PLLA MMPs is shown in Fig.6.As shown in Fig.7,a very significant increase in MN fre-quency (p <0.01)was observed in the cells treated with different concentrations of MMPs compared with the negative control (F12-K medium,0mg/mL)and the positive control (MMC).The Fe 3O 4–PLLA–PEG–PLLA MMPs induced a concentration-dependent response of MN frequency in the whole study,while the Fe 3O 4nanoparticles produced a greater increase in response to the same concentration.Furthermore,there was a significant dif-ference (p <0.01)between the Fe 3O 4–PLLA–PEG–PLLA MMPs and the Fe 3O 4nanoparticles at 2.0mg/mL.The results above demonstrate genotoxic effects on CHO-K1cells exposed to the Fe 3O 4–PLLA–PEG–PLLA MMPs.These findings are consistent with previous studies which demonstrated that inorganic nanoparticles increase MN frequencies in cultured cells (Lindberg et al.,2009;Pfaller et al.,2010).Furthermore,the Fe 3O 4–PLLA–PEG–PLLA MMPs caused less DNA damage than the Fe 3O 4nanoparticles.The issueA.-Z.Chen et al./Toxicology Letters212 (2012) 75–8279Fig.3.Confocal laser scanning micrographs of intracellular ROS generation in RAW264.7cells following exposure to(a)negative control,(b)positive control,(c)100␮g/mL of Fe3O4–PLLA–PEG–PLLA MMPs,(d)200␮g/mL of Fe3O4–PLLA–PEG–PLLA MMPs,and(e)400␮g/mL of Fe3O4–PLLA–PEG–PLLA MMPs.The data show the cells after exposure to Fe3O4–PLLA–PEG–PLLA MMPs for24h.of whether or not the polymer coating of iron oxide nanoparticles influences their genetoxicity in terms of DNA damage is still lim-ited to this study.Our study has demonstrated that the polymer coating may reduce the DNA damage caused by the iron oxide.It has been suggested that MN formation may be due to physical dis-turbance of the particles around the mitotic apparatus(Hesterberg et al.,1986).Hence,the polymer coating might have affected the physical presence of iron oxide nanoparticles.However,the preciseTable2Results of Fe3O4–PLLA–PEG–PLLA MMPs intraperitoneal injection of mice(n=10,days=7,“↓”means decrease,“—”means no change or normal,“↑”means increase). Dose(mg/kg)Observation indexAppetite Diarrhea Sleepiness Activities Weight Death 525.00↓——↓↓None 420.00———↓↓None 336.00————↑None 268.80————↑None 215.04————↑None Physiological saline————↑None80 A.-Z.Chen et al./Toxicology Letters 212 (2012) 75–82Fig.4.Erythrocyte membrane damage induced by Fe 3O 4–PLLA–PEG–PLLA MMPs and Fe 3O 4nanoparticles at various concentrations.Values are represented as the mean ±the standard error of the mean (n =3).When appropriate,statistical signifi-cance is indicated:“*”:p <0.05;“**”:p <0.01.mechanism of MN formation caused by inorganic nanoparticles remains unclear.Additional work needs to be undertaken to under-stand the mechanisms of damage.3.6.Cytokine releaseThe immune modulatory end-point in MMPs was studied by investigating cytokine secretion.Expression of the mRNA level was measured by real-time RT-PCR;the effects of MMPs on cytokine production in RAW264.7cells are shown in Fig.8a and c.There is a dose-response increase in cytokine secretion in the super-natant after incubation with Fe 3O 4–PLLA–PEG–PLLA MMPs and the other samples.The presence of different concentrations of Fe 3O 4–PLLA–PEG–PLLA MMPs and Fe 3O 4nanoparticles elicited the production of pro-inflammatory cytokines such as IL-6,TNF-␣,and inducible nitric oxide synthase (iNOS),but their levels were much lower than those with LPS (200ng/mL)stimulation,and this difference was statistically significant (p <0.01).Most reports about the effects of MMPs on macrophage cytokine secretion are in agreement with our finding.Ferucarbotran above 100␮g Fe/mL showed a significant (p <0.01)increase in cytokine secretionandFig.5.Weights of mice before and seven days after i.p.Fe 3O 4–PLLA–PEG–PLLA MMPs.Values are represented as the mean ±the standard error of the mean (n =10).Fig. 6.MN formation in CHO-K1BN cells treated with2mg/mLofFe 3O 4–PLLA–PEG–PLLA MMPs.The bar indicates 50.0␮m.mRNA expression,followed by nitric oxide (NO)secretion and iNOS mRNA expression (Yeh et al.,2010).Incubation of mouse peritoneal macrophages with 500␮g/mL of ferumoxides triggered a slight increase in IL-1production (177pg/mL)which remained far below that obtained with the pro-inflammatory stimulant LPS (1012pg/mL)(Muller et al.,2007).In addition,our results indicate that the IL-6,TNF-␣,and iNOS expressions after treatment with Fe 3O 4–PLLA–PEG–PLLA MMPs were lower than those following treatment with Fe 3O 4nanoparticles.This is probably because the polymer-coated surface decreased the pro-inflammatory cytokine and iNOS production stimulated by the Fe 3O 4–PLLA–PEG–PLLA MMPs.In previous reports and our own studies,surface-coated magnetic nanoparticles did not induce a significant release of the pro-inflammatory cytokines (Kunzmann et al.,2011)and did pre-vent some of the response of the complement system (Aqil et al.,2008),easing the immunological impact of the magnetic nanopar-ticles.Prior to the results of cell toxicity,although no significant differences were found in cell toxicity and ROS levels,the results of MN frequency and the expression of pro-inflammatory cytokines at the molecular level suggest that such polymer-coated MMPs do have a better biocompatibility than uncoated Fe 3O 4nanoparticles.Fig.7.MN induction in CHO-K1cells exposed to Fe 3O 4–PLLA–PEG–PLLA MMPs or Fe 3O 4nanoparticles for 24h.F12-K medium (0mg/mL)and MMC were used as the negative control and positive control,respectively.A very significant statistical difference (p <0.01)between Fe 3O 4–PLLA–PEG–PLLA MMPs and Fe 3O 4nanoparti-cles compared with the control groups was indicated.“**”indicates a significant difference at p <0.01for Fe 3O 4–PLLA–PEG–PLLA MMPs compared with Fe 3O 4nanoparticles.Values are represented as the mean ±the standard error of the mean (n =3).When appropriate,statistical significance is indicated:“**”:p <0.01.。