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Copyright copy 2011 American Scientific PublishersAll rights reservedPrinted in the United States of America

Journal ofBiomedical Nanotechnology

Vol 7 677ndash684 2011

Physiological Investigation of Magnetic Iron OxideNanoparticles Towards Chinese Mung Bean

Hong-Xuan Ren12 dagger Ling Liu3 dagger Chong Liu4 Shi-Ying He1 Jin Huang5Jun-Li Li5 Yu Zhang1 Xing-Jiu Huang6lowast and Ning Gu1lowast

1School of Biological Science and Medical Engineering Southeast University Nanjing 210098 PR China2National Center for Nanoscience and Technology of China Beijing 100080 PR China

3College of Resources and Environmental Science Nanjing Agricultural University Nanjing 210095 PR China4Institute of Agricultural Sciences of Jiangsu Coastal District Yancheng 224002 PR China5School of Mechanical and Electronic Engineering and College of Chemical Engineering

Wuhan University of Technology Wuhan 430070 China6Research Center for Biomimetic Functional Materials and Sensing Devices Institute of Intelligent Machines

Chinese Academy of Sciences Hefei 230031 PR China

While few publications have documented systematic physiological effects of nanoparticles on plantcells and tissues this is the first study describing a detailed evidence of impact of -Fe2O3 magnetitenanoparticles (MNPs) on Chinese mung bean plants by measuring the physiological parameterssuch as germination root activity activity of catalase (CAT) peroxidase (POD) and superoxidedismutase (SOD) content of chlorophyll soluble protein and content of malondialdehyde (MDA)Our results will help answer the question on how both positive and negative or inconsequentialeffects take place in plants after treatment by the nutrient solution containing nanoparticles

Keywords -Fe2O3 Magnetite Nanoparticles (MNPs) Physiological Investigation Plants Cellsand Tissues

1 INTRODUCTION

Nanomaterials such as carbon nanotubes and nanoparti-cles show signs of toxicity1ndash2 Until now most of studieson the adverse outcomes of nanomaterials were limited toanimal cells or whole organisms The impact of nanoma-terials on plants has scantly been examined in the currentliterature In the past two years the impact of nanomateri-als on plants is attracting increasing attention For exampleXing et al3ndash5 studied the effects of multi-walled carbonnanotube aluminum alumina zinc and zinc oxide on seedgermination and root growth of radish rape ryegrass let-tuce corn and cucumber and found that seed germina-tion was not affected except for the inhibition of nanoscalezinc (nano-Zn) on ryegrass and zinc oxide (nano-ZnO) oncorn at 2000 mg Lminus1 and the inhibition occurred dur-ing the seed incubation process rather than seed soakingstage An et al6 found that Cu nanoparticles can cross thecell membrane and might agglomerate in the cells of plantspecies were Phaseolus radiatus (mung bean) and Triticum

lowastAuthors to whom correspondence should be addresseddaggerTwo authors have the equivalent contribution to this work

aestivum (wheat) Shah et al7 studied the short term influ-ence of Si Pd Au and Cu nanoparticles on the germinationof lettuce seeds at two different concentrations of nanopar-ticles Trapp et al8 found that manufactured TiO2 nanopar-ticles have low toxic effects on willow trees Font et al9

studied the risk of the release to the environment of Fe3O4Ag and Au nanoparticles by seed germination test (cucum-ber and lettuce) anaerobic toxicity test and biolumines-cent test (Photobacterium phosphoreum) In all cases lowor zero toxicity was observed at the assayed concentrationsWhite et al10 investigated the effects of five nanomateri-als (multiwalled carbon nanotubes Ag Cu ZnO Si) andtheir corresponding bulk counterparts on seed germinationroot elongation and biomass of Cucurbita pepo (zucchini)Zhang et al11 reported the phytotoxicity of four rare earthoxide nanoparticles nano-CeO2 nano-La2O3 nano-Gd2O3

and nano-Yb2O3 on seven higher plant species (radishrape tomato lettuce wheat cabbage and cucumber) bymeans of root elongation experiments Gardea-Torresdeyet al12 studied the fate transport and possible toxicityof cerium oxide nanoparticles (nanoceria CeO2 on seedsof alfalfa (Medicago sativa) corn (Zea mays) cucumber(Cucumis sativus) and tomato (Lycopersicon esculentum)

J Biomed Nanotechnol 2011 Vol 7 No 5 1550-703320117677008 doi101166jbn20111338 677

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

Smalle et al13 described uptake and distribution of theultrasmall anatase TiO2 in the plant model system Ara-bidopsis Ke et al14ndash15 characterized the dynamic uptakecompartment distribution and transformation of fullereneC70 in rice plants and have detected the transmission ofC70 to the progeny through seeds Alvarez et al16 investi-gated the effects of aluminum oxide (nAl2O3 silicon diox-ide (nSiO2 magnetite (nFe3O4 and zinc oxide (nZnO) onthe development of Arabidopsis thaliana (Mouse-ear cress)from three toxicity indicators (seed germination root elon-gation and number of leaves)Very recently Ma et al17 reviewed the current knowl-

edge on the phytotoxicity and interactions of engineerednanoparticles (ENPs) with plants at seedling and cellularlevels and discussed the information gap and some imme-diate research needs to further our knowledge on this topicKumar et al18 reviewed the delivery of nanoparticulatematerials to plants and their ultimate effects which couldprovide some insights for the safe use of this novel tech-nology for the improvement of crops Obviously Most ofthese studies were focused on the study of potential toxi-city (seed germination and root growth) of ENPs to plantsvia several standard methods and both positive and nega-tive or inconsequential effects have been reported How-ever little is known about systematic physiological effectsof nanoparticle on plantsIron oxide magnetite nanoparticles (MNPs) eg Fe3O4

possess an intrinsic enzyme mimetic activity similar to thatfound in natural peroxidases which are widely used to oxi-dize organic substrates in the treatment of wastewater oras detection tools19ndash27 A few studies have been conductedto assess the effects of iron oxide MNPs on ecologicalterrestrial species such as plants The first study was byJin and co-workers28 who demonstrated significant uptakeof Fe3O4 MNPs by pumpkin plants and their subsequenttranslocation and accumulation in various tissues How-ever fundamental questions remain regarding the system-atic physiological effects on plant cells and tissues and theimpact of these processes on plant reproduction Here weprovide a detailed evidence of impact of -Fe2O3 MNPson Chinese mung bean plants by measuring the physiolog-ical parameters such as germination root activity activ-ity of catalase (CAT) peroxidase (POD) and superoxidedismutase (SOD) and content of chlorophyll soluble pro-tein and content of malondialdehyde (MDA) The datapresented in this article suggest the potential impact ofnanomaterial exposure on plant development and the foodchain and prompt further investigation into the geneticconsequences through plant nanomaterial interactions

2 MATERIALS AND METHODS

21 Synthesis of -Fe2O3 MNPs

Fe3O4 MNPs with diameters of approximately 9 nm and18 nm were first prepared according to the chemical

co-precipitation method29 The synthesized Fe3O4 parti-cles were diluted to 50 L and the pH of the solution wasadjusted to 3 with 2 M HCl under nitrogen gas protectionand vigorous stirring using nonmagnetic stirrer Then theFe3O4 nanoparticles were transferred into a double-layerglass reaction vessel to react with oxygen through bub-bling air under continuous stirring at 90 C After stirringfor 5 h the obtained -Fe2O3 nanoparticles precipitate wasseparated from the reaction medium by magnetic field andwashed with 200 mL deionized water four times With thissynthesis once the synthetic conditions are fixed the qual-ity of the magnetite nanoparticles is fully reproducibleThe prepared -Fe2O3 nanoparticles were dispersed indeionized water and the pH of the solution was adjustedto 27 1365 g DMSA dissolved in 50 mL DMSO wasadded to the dispersion with continuous stirring After thereaction for 5 h at room temperature the products werecollected with a magnet and were dispersed in (CH34NOHsolution and the pH of the solution was adjusted to 10The stable magnetic fluids were obtained after the pH ofthe solution was adjusted to neutral and reverse osmosis

22 Superoxide Dismutase (SOD) Activity Assay

A 05 g of fresh samples were ground in a mortar with001 g PVP 5 mL of 01 mol middotLminus1 phosphate buffer con-taining 02 mM EDTA and 04 mM -mercaptoethanol(pH 78) After extraction in an ice bath the homogenatewas centrifuged at 1200 rpm for 10 min The supernatantwas collected for further measurementsTake 50 L supernatant and add to 15 mL of 005 M

phosphate buffer (pH 78) 03 mL of 130 mM methio-nine 03 mL of 750 M Nitrotetrazolium blue chloride03 mL of 100 M EDTA-Na 03 mL 20 M lactochromeand 025 mL of distilled water Another two control sam-ples were prepared using buffer instead of enzyme solu-tion After uniform mixing one control test was put inthe dark and others were illuminated under fluorescencelamp for 20 min Activity was measured by the changesin absorbance at 560 nm

23 Peroxidase (POD) Activity Assay

Peroxidase activity was determined by guaiacol oxidationPOD reaction mixture was first prepared by the addition of28 L guaiacol into 50 mL of 100 mM phosphate buffer(pH 70) when completely dissolving and cooling at roomtemperature 19 l of 30 H2O2 was introduced (Notesuch mixture was required to freshly prepared before useand stored in fridge) The assay mixture contained in100 L of the diluted supernatant 2 mL POD reactionmixture and 1 mL of 02 M potassium dihydrophospatebuffer The hydrogen peroxide can oxidize guaiacol andproduce dark brown substance in the presence of perox-idase The conversion of the dye was monitored by themeasurement of the changes in absorbance at 470 nm per

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Ren et al Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

30 sec The POD activity was expressed as the change ofabsorbance per minute (A470mingFW)

24 Catalase (CAT) Activity Assay

Catalase (CAT) activity was determined by the addi-tion of 100 L supernatant to 3 ml mixture contain-ing 01 H2O2 and 100 mM phosphate buffer (pH 70)The results were monitored by the measurements of thechanges in absorbance at 240 nm per 30 sec The activ-ity was expressed as the change of absorbance per minute(A240ming FW (fresh weigh))

25 Soluble Protein Content Assay

Soluble protein content was analyzed using dying methodwith Coomasie Brilliant Fluka G-250 Protein standardsolution was firstly prepared and stored in the refrigeratorCoomassie Brilliant Blue G-250 solution was prepared byweighing coomassie brilliant blue G-250 100 mg adding50 mL of 95 ethanol and 100 mL of 85 (wv) H3PO4

to the final volume with distilled water 1000 mL at roomtemperature The test solution was prepared by weighinga dried sample 05 g adding a small amount of quartzsand with distilled water the mixture was ground into ahomogenate After standing 10ndash30 min (3ndash5 min shakingtime) via a filter (such as active carbon containing pigmentavailable) the volume of the solution was set to 100 mLfor further use

26 Malondialdehyde Content Assay

Take 1 ml supernatant and add to 5 mL of 20trichloroacetic acid (TCA) containing 05 M thiobarbi-turic acid (TBA) such a mixture was then manually agi-tated The container was placed in a boiling water bath for30 min After cooling down to room temperature it wascentrifuged at 4000 rpm for 10 min Using 20 TCA as areference the supernatant was collected for measurementsof the changes in absorbance at 450 532 and 600 nm

27 Chlorophyl Content Assay

01 g of fresh leaves were ground and transferred to atest-tube Chlorophyl was extracted with 10 ml mixture ofethanol and acetone (11 vv) under a controlled environ-ment of room temperature and 24 h dark photoperiod Datawas collected by the changes in absorbance at 645 and663 nm

28 Determination of Root Activity

The root activity was estimated by triphenyl tetrazoliumchloride (TTC) reduction30 Control roots were boiled for10 min in distilled water to insure that enzymes were dena-tured All roots were cut into 1 cm pieces submerged in

3 mL of 06 (wv) 235-triphenyl tetrazolium chloridein 005 M Na2HPO4-KH2PO4 (pH 74)+ 005 wettingagent (Triton X-100) and vacuum-infiltrated for 5 minto insure infiltration of TTC Samples were incubated at30 C for 24 h rinsed twice with distilled water andextracted four times in 4 mL of 95 (vv) ethanol for5 min in a waterbath at 85 C The total solution extractedwas brought up to a volume of 25 mL and measured witha spectrophotometer (Shimadzu UV160U Kyoto Japan)at 490 nm

3 RESULTS AND DISCUSSION

Chinese mung bean was selected as a model plant becauseitrsquos a very popular plant and widely cultivated in China Asin the case of the potential biological effects of nanopar-ticles in the green agriculture the physiological investi-gation of magnetic iron oxide nanoparticles could be ofenormous benefit In this study a complex ecosystem ina greenhouse was constructed to model the growth envi-ronment of Chinese mung bean for further measuring thebehaviour of -Fe2O3 magnetic nanoparticles (Fig 1(A))Considering that the properties of nanoscale materialsare often dependent on size and nanoparticles have thepotential to pass across physiological barriers or targetspecific cells and organs and administer small quanti-ties of drugs and most importantly individual -Fe2O3

particles of 20 nm can be existed in the suspension-Fe2O3 MNPs with size of 9 and 18 nm were cho-sen and prepared for further experiments The -Fe2O3

MNPs appeared spherical and homogeneous and were ofthe expected size (Figs 1(BndashC)) After immersing into-Fe2O3 MNPs nutrient solution Chinese mung beanseeds were transferred into separate temporary contain-ers filled with natural silica sediment to allow the growthtill the germination These seedlings were then transferredinto plastic pots (filled with silica sediment) and supple-mented with 12 Hoagland solution containing -Fe2O3

MNPs everyday till the appearance of leaf Two differentconcentrations of 10 and 20 mg middotLminus1 were prepared for theinvestigation Together with a control test each treatmentwas conducted with three replicates and the results werestatistical analysis and presented as mean plusmnSD (standarddeviation) The details can be found in MethodsFigure 2(A) shows the results of germination of Chi-

nese mung bean seeds after treatment for 10 days by thenutrient solution containing 9 and 18 nm -Fe2O3 MNPs(10 and 20 mg middotLminus1 respectively In this test 100 seedswere selected for the experiments Seed germinations wereaffected by the concentration of nanoparticles in nutrientsolution As seen the germination of seeds was inhibitedby the lower concentration of nanoparticles 10 mg middotLminus1The percentage is 56 and 52 for size 9 and 18 nm ofnanoparticles respectively It is quite lower than the nor-mal case 72 in the absence of nanoparticles However

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

Fig 1 Experimental setup (A) Growth of Chinese mung bean modelling the natural environment was maintained in a greenhouse Chinese mungbean is shown growing in the sediment pots Each pot contained natural silica sediment and received a certain dose of -Fe2O3 MNPs nutritive medium(BndashC) TEM images of -Fe2O3 magnetic iron oxide nanopartiles from deionized water used in this study (B) 18 nm (C) 9 nm

the germination was promoted by the higher concentrationof nanoparticles 20 mg middotLminus1 The percentage is 78 forboth 9 nm and 18 nm of nanoparticles The results arequite different to the situation which ryegrass and cornwere inhibited by nano-Zn and nano-ZnO This might bedue to that Magnetic -Fe2O3 nanoparticles are generallyconsidered to be biologically and chemically inert Mag-netic -Fe2O3nanoparticles have been coated with metalcatalysts or conjugated with enzymes to combine the sep-arating power of the magnetic properties with the catalytic

(A) (B) (C)

Fig 2 Effect of -Fe2O3 magnetic nanoparticles on the germination of Chinese mung bean seeds (A) Percentage of germination of Chinese mungbean seeds inclubated in size 918 nm of 10 and 20 mg middotLminus1-Fe2O3 magnetic nanoparticles nutritive medium (B) Length of sprout as a functionof growth time under different sizes of nanoparticle Concentration 20 mg middotLminus1 (C) Length of sprout as a function of growth time under differentconcentrations -Fe2O3 MNPs 18 nm

activity of the metal surface or enzyme conjugate It isconcluded that the higher concentration has a promotionimpact on the seed germinationWe therefore chose 20 mg middot Lminus1 of nutrient solution

containing 9 and 18 nm -Fe2O3 MNPs to investigatethe length of sprout during the growth as presented inFigure 2(B) In contrast to the control test (that is in theabsence of nanoparticles) -Fe2O3 MNPs nutrient solu-tion can accelerated the growth of sprout This indicatesthat the existing of -Fe2O3 MNPs helps root cell open

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Ren et al Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

(A) (B)

Fig 3 (A) Root activity in the presence of 9 nm and 18 nm -Fe2O3

magnetic nanoparticles of various concentrations (B) Root activity versusconcentrations (0 10 20 40 60 80 and 100 mg middotLminus1 in the presenceof 9 nm -Fe2O3 magnetic nanoparticles

water channels and effectively promotes cell better adsorp-tion to water inorganic ions and other nutritional compo-nents and that the influence of smaller nanoparticle (sizeof 9 nm) is more obvious than that of bigger one (sizeof 18 nm) The nanoparticles with small size readily per-meate through the plant cell wall driven by a concentra-tion gradient This contribution is excellent consistent withthe effect of -Fe2O3 MNPs on the root activity as willbe discussed in the following section Figure 2(C) is theresults of length of sprout as a function of growth timeunder different concentrations It is seen that the growingof sprout is well at lower concentration (10 mg middotLminus1 Thisphenomenon might be attributed to that the higher con-centration can form clusters and tends to block the porousplant cell wallThe absorption of plant roots is an active organ and

synthetic organs root growth and vigor of shoot directly

(A) (B) (C)

Fig 4 Activity of a group of enzymes in the presence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles of various concentrations (A) Activityof catalase (CAT) (B) Activity of peroxidase (POD) (C) Activity of superoxide dismutase (SOD)

affect the level of the nutritional status and produc-tion levels Figure 3 shows the root activity of Chinesemung bean seedlings in the presence of 9-nm and 18-nm-Fe2O3 MNPs of various concentrations As seen fromFigure 3(A) no changes were observed for 18 nm MNPsat 20 mg middotLminus1 compared to the control At the same con-centration 9 nm -Fe2O3 MNPs have a positive effectit proposed an increase of 503 percent for root activityThe best root activity can be seen in the case of 9 nmMNPs of 10 mg middot Lminus1 an increase of 1172 percent isobtained For both of 9 nm and 18 nm nanoparicles rootactivity influenced by lower concentration is more obviousthan that influenced by higher concentration This experi-ment gives a supportive evidence for the growth of sproutshown in Figures 2(B)ndash(C) Figure 3(B) shows the rootactivity under different concentrations in the presence of9 nm -Fe2O3 MNPs It is seen that as increasing the con-centration to 40 mg middotLminus1 the root activity sharply decreasesand consequently again increases to normal case (see dot-ted line) This might be explained either by the significantaggregation of those MNPs at high concentration or by theaccumulation of many -Fe2O3 nanoparticles on the rootsurface (ie the accumulation inhibits the transmission ofwater and other nutritional components)Peroxide accumulation may cause changes in plant

metabolism in several ways They may oxidize sulflhydrylgroups and in combination with superoxides they canform hydroxyl radicals which may be involved in theaging process H2O2 may be involved also in the oxidativebreakdown of indoleacetic acid It has been shown thatincreased H2O2 levels inactivate indoleacetic acid Thisinactivation was reversed upon the introduction of catalaseFigure 4 shows the activity of a group of enzymes such astatalase peroxidase and superoxide dismutase in the pres-ence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

of various concentrations Figure 4(A) shows the effect of-Fe2O3 MNPs on the function of the enzyme catalasewhich is found in all plant tissues As seen a nanoparticleaddition in nutrient solution results in a decrease of activ-ity of catalase while the effect of nanoparticles in theseplants showed no significant concentration or size depen-dence A decrease in catalase activity could lead to theobserved accumulation of H2O2 in tissue since there is anincrease in peroxidase activity (Fig 4(B)) to compensatefor the H2O2 removal by catalase even though the activ-ity is still lower than normal levels Taking 20 mg middotLminus1

of -Fe2O3 MNPs solution as an example the catalaseactivity treated by 18 nm MNPs is a litter higher than thattreated by 9 nm (Fig 4(A)) Peroxidase activity is oppo-site to this result (Fig 4(B)) Similar result can also befound in the case of 10 mg Lminus1-Fe2O3 MNPs solutionAs seen from Figure 4(C) the activity of superoxide dis-mutase was inhibited by the suspension of -Fe2O3 MNPsand shows a size and concentration dependence A signifi-cant effect was found at higher concentration (20 mg middotLminus1and larger size (18 nm) Superoxide dismutase (SOD) inplant catalyzes the destruction of the O2minus free radical thedecrease of activity of SOD result in the accumulation ofO2minus free radical in plant leaf Consequently these freeradicals react with H2O2 produced by chloroplast to formOHbull free radicals which might result in the degradation ofchlorophyll However we observed a quite different phe-nomenon from the test of chlorophyl content as shown inFigure 5(A)As can be seen from Figure 5(A) a slight increasing

on the content of chlorophyll was observed after treatedby -Fe2O3 MNPs It is obvious that the introduction ofnanoparticles is contributed to a synthesis of chlorophyllHowever it is not clear whether this is due to a decreasein peroxidase activity intrinsic peroxidase-like activity of-Fe2O3 nanoparticles possess or both Content of soluble

(A) (B) (C)

Fig 5 Physiological parameters in the presence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles of various concentrations (A) Content ofchlorophyl (B) Content of soluble protein (SP) (C) Content of malondialdehyde (MDA)

protein was highly correlated with the age of plants iecontent of soluble protein decreases with the increasingof the age The results shown in Figure 5(B) reveal that-Fe2O3 MNPs can stimulate the growth of plants espe-cially for those samples treated using 18 nm of 10 mg middotLminus1

nanoparticle suspension Soluble protein was also strongrelative to the Content of chlorophyl of plant leaf With theincrease of content of chlorophyl photosynthesis of plantsincreases resulting in the accumulation of soluble proteinMalondialdehyde (MDA) is an important lipid peroxida-tion product when plant is in stress conditions of agingor injured Its content is closely related to plant senes-cence and stress injury The extent of the damage of plantmembrane system and plant resistance can be known bymeasuring the MDA level of lipid peroxidation The lowcontent of MDA is useful for protecting the structure andfunction of cell membrane As depicted in Figure 5(C)the content of MDA decreases in the present conditionsexcept the treatment using 9 nm of 20 mg middotLminus1 nanopar-ticles indicating that the proxidation of unsaturated fattyacid in cell membrane is weaker than that of the controlThis situation should be due to the increase of the con-tent of SOD CAT and POD in theory It is quite differ-ent from the above mentioned experimental results FromTEM image we are sure that -Fe2O3 MNPs were intro-duced to plant (as will be discussed later) suggesting thatthere may have another mechanism Maybe one of thesethree enzymes or boththree of them waswere replaced by-Fe2O3 MNPs The unusual result obtained under 9 nmof 20 mg middotLminus1 nanoparticles is likely owing to the glomer-ation of small particles Larger particles cannot easily pen-etrate the cell wall and membrane resulting in the lowerutilization efficient and consequently affect the synthesisof chlorophyll and increasing of MDA contentFigure 6 shows TEM (Transmission electron

microscopy JEM-2010 microscope equipped with Oxford

682 J Biomed Nanotechnol 7 677ndash684 2011

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Ren et al Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

Fig 6 TEM images of mitochondria in the absence (A) and presence (B) of 9 nm of 20 mg middotLminus1-Fe2O3 MNPs suspension (CndashD) Detection ofnanoparticles in cytoplasm of Chinese mung bean treated with 9 nm of 20 mg middotLminus1-Fe2O3 MNPs suspension

INCA EDS operated at 200 kV accelerating voltage)images of the plant tissues after treating in 9 nm of20 mg middot Lminus1-Fe2O3 MNPs suspension Figure 6(A) isunder treatments of control of epidermal cell of Chinesemung bean root However the cristae of mitochondriabecomes rough and the color of the cytoplasm gets lighter(Fig 6(B)) The tumefaction of mitochondria cannot beobserved indicating that the membrane of mitochondriawas not damaged by the nanoparticles Taking account ofabove mentioned results CAT POD and SOD content ofplant tissue decrease after treating by -Fe2O3 nanoparti-cles this observation demonstrates that the peroxidationof the membrane does not occur Black aggregates werefrequently found in the cytoplasm (Figs 6(C and D))indicating that the sequence of nanoparticle uptakewas from the plant seeds and roots to the stems andleaves

4 CONCLUSIONS

This manuscript reports the first study describing a detailedevidence of impact of -Fe2O3 magnetite nanoparticles

(MNPs) on Chinese mung bean plants by measuring thephysiological parameters such as germination root activ-ity activity of catalase (CAT) peroxidase (POD) andsuperoxide dismutase (SOD) content of chlorophyll sol-uble protein and content of malondialdehyde (MDA) Itshould be noted that although the nanoparticles used inthis study have a diameter size within the nanometrerange aggregates of different sizes were formed in theplant cells Thus it can be concluded that the -Fe2O3

nanoparticles could enter into the tissues or cells fromthe roots Even though more works need further attentionin order to obtain aspects of mechanism including uptakeand translocation and the interactions between the particleswith plant tissue at the cellular level this research never-theless provides convincing evidence that plant uptake isa potential transport pathway of nanoparticles in the envi-ronment Another main significance of this study couldprovide a guideline for the production of so-called sele-nium (Se)-riched rice in China After our inspection sele-nium (Se)-riched rice were produced in two ways One ismanual spraying of Se solution during rice growing andthen through bio-transformation the inorganic selenium

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

into organic selenium and stored in the rice so absorbedby the body Another is the local soil rich in selenium con-tent the rice produced natural selenium Our results willhelp answer the question on how significant physiologicalchanges of plant occur

Acknowledgments X-J Huang thanks ldquoOne HundredPerson Projectrdquo of the Chinese Academy of SciencesChina and N Gu thanks State Key Development Programfor Basic Research of China (Grant No 2006CB933200)and National Natural Science Funds for DistinguishedYoung Scholar (NSFC-60725101) for their financialsupports

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2 H Ren and X J Huang Polyacrylate nanoparticles Toxicity or newnanomedicine Eur Respir J 36 218 (2010)

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4 D H Lin and B S Xing Root uptake and phytotoxicity of ZnOnanoparticles Environ Sci Technol 42 5580 (2008)

5 D H Lin and B S Xing Phytotoxicity of nanoparticles Inhibi-tion of seed germination and root growth Environ Pollut 150 243(2007)

6 W M Lee Y J An H Yoon and H S Kweon Toxicity andbioavailability of copper nanoparticles to the terrestrial plants mungbean (Phaseolus radiatus) and wheat (Triticum aestivum) Plantagar test for water-insoluble nanoparticles Environ Toxicol Chem27 1915 (2008)

7 V Shah and I Belozerova Influence of metal nanoparticles on thesoil microbial community and germination of Lettuce Seeds WaterAir Soil Poll 197 143 (2009)

8 E Seeger A Baun M Kastner and S Trapp Insignificant acutetoxicity of TiO2 nanoparticles to willow trees J Soil Sediment 9 46(2009)

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10 D Stampoulis S K Sinha and J C White Assay-dependent phy-totoxicity of nanoparticles to plants Environ Sci Technol 43 9473(2009)

11 Y H Ma L L Kuang X He W Bai Y Y Ding Z Y ZhangY L Zhao and Z F Chai Effects of rare earth oxide nanoparticleson root elongation of plants Chemosphere 78 273 (2010)

12 M L Lopez-Moreno G de la Rosa J A Hernandez-Viezcas J RPeralta-Videa and J L Gardea-Torresdey X-ray absorption spec-troscopy (XAS) corroboration of the uptake and storage of CeO2

nanoparticles and assessment of their differential toxicity in fouredible plant species J Agr Food Chem 58 3689 (2010)

13 J Kurepa T Paunesku S Vogt H Arora B M Rabatic J JLu M B Wanzer G E Woloschak and J A Smalle Uptake anddistribution of ultrasmall anatase TiO2 alizarin red S nanoconjugatesin arabidopsis thaliana Nano Lett 10 2296 (2010)

14 S J Lin J Reppert Q Hu J S Hudson M L Reid T ARatnikova A M Rao H Luo and P C Ke Uptake Transloca-tion and transmission of carbon nanomaterials in rice plants Small5 1128 (2009)

15 R Chen T A Ratnikova M B Stone S Lin M Lard G HuangJ S Hudson and P C Ke Differential uptake of carbon nanoparti-cles by plant and mammalian Cells Small 6 612 (2010)

16 C W Lee S Mahendra K Zodrow D Li Y C Tsai JBraam and P J J Alvarez Developmental phytotoxicity of metaloxide nanoparticles to arabidopsis thaliana Environ Toxicol Chem29 1399 (2010)

17 X M Ma J Geiser-Lee Y Deng and A Kolmakov Interactionsbetween engineered nanoparticles (ENPs) and plants Phytotoxicityuptake and accumulation Sci Total Environ 408 3053 (2010)

18 R Nair S H Varghese B G Nair T Maekawa Y Yoshida andD S Kumar Nanoparticulate material delivery to plants Plant Sci179 154 (2010)

19 L Z Gao J Zhuang L Nie J B Zhang Y Zhang N GuT H Wang J Feng D L Yang S Perrett and X Yan Intrinsicperoxidase-like activity of ferromagnetic nanoparticles Nat Nano-technol 2 577 (2007)

20 S P Singh Multifuncational magnetic quantum dots for cancer ther-anostics J Biomed Nanotechnol 7 95 (2011)

21 J Ding J H Zhao K LCheng G F Liu and D H Xiu In Vivophotodynamic therapy and magnetic resonance imaging of cancer byTSPP-coated Fe3O4 nanoconjugates J Biomed Nanotechnol 6 683(2010)

22 K Men S Zeng M L Gou G Guo Y C Gu F LuoX Zhao Y Q Wei and Z Y Qian Preparation of magnetic micro-spheres based on poly(e-caprolactone)-poly(ethylene glycol)-poly(e-caprolactone) copolymers by modified solvent diffusion methodJ Biomed Nanotechnol 6 287 (2010)

23 A Arkhis A Elaissari T Delair B Verrier and B Mandrand Cap-ture of enveloped viruses using polymer tentacles containing mag-netic latex particles J Biomed Nanotechnol 6 28 (2010)

24 L Tian S Li H N Liu Z F Wang and N Y He An automatedmagstation for high-throughput single nucleotide polymorphismgenotyping and the dual-color hybridization J Biomed Nanotech-nol 5 511 (2009)

25 Z Y Li L He Z Y Shi H Wang S Li H N Liu Y B Dai Z FWang and N Y He Preparation of SiO2(PMMAFe3O4 nanopar-ticles and its application in detecting Ecoli O157H7 using chemi-luminescent immunological method J Biomed Nanotechnol 5 505(2009)

26 Z Y Li L He N Y He Z Y Shi H Wang S Li H N LiuX L Li Y B Dai and Z F Wang Chemiluminescent detect ofEcoli O157H7 using immunological method based on magneticnanoparticles J Nanosci Nanotechnol 10 696 (2010)

27 L He Z Y Li J Fu F Wang C Ma Y Deng Z Y Shi H Wangand N Y He Preparation of SiO2(PMMAFe3O4 nanoparticlesusing linolenic acid as crosslink agent for nucleic acid detectionusing chemiluminescent method J Nanosci Nanotechnol 11 2256(2011)

28 H Zhu J Han J Q Xiao and Y Jin Uptake translocation andaccumulation of manufactured iron oxide nanoparticles by pumpkinplants J Environ Monitor 10 713 (2008)

29 A H Lu E L Salabas and F Schuth Magnetic nanoparticles Syn-thesis protection functionalization and application Angew ChemInt Ed 46 1222 (2007)

30 L H Comas D M Eissenstat and A N Lakso Assessing rootdeath and root system dynamics in a study of grape canopy pruningNew Phytol 147 171 (2000)

Received 10 June 2011 Accepted 20 June 2011

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

Smalle et al13 described uptake and distribution of theultrasmall anatase TiO2 in the plant model system Ara-bidopsis Ke et al14ndash15 characterized the dynamic uptakecompartment distribution and transformation of fullereneC70 in rice plants and have detected the transmission ofC70 to the progeny through seeds Alvarez et al16 investi-gated the effects of aluminum oxide (nAl2O3 silicon diox-ide (nSiO2 magnetite (nFe3O4 and zinc oxide (nZnO) onthe development of Arabidopsis thaliana (Mouse-ear cress)from three toxicity indicators (seed germination root elon-gation and number of leaves)Very recently Ma et al17 reviewed the current knowl-

edge on the phytotoxicity and interactions of engineerednanoparticles (ENPs) with plants at seedling and cellularlevels and discussed the information gap and some imme-diate research needs to further our knowledge on this topicKumar et al18 reviewed the delivery of nanoparticulatematerials to plants and their ultimate effects which couldprovide some insights for the safe use of this novel tech-nology for the improvement of crops Obviously Most ofthese studies were focused on the study of potential toxi-city (seed germination and root growth) of ENPs to plantsvia several standard methods and both positive and nega-tive or inconsequential effects have been reported How-ever little is known about systematic physiological effectsof nanoparticle on plantsIron oxide magnetite nanoparticles (MNPs) eg Fe3O4

possess an intrinsic enzyme mimetic activity similar to thatfound in natural peroxidases which are widely used to oxi-dize organic substrates in the treatment of wastewater oras detection tools19ndash27 A few studies have been conductedto assess the effects of iron oxide MNPs on ecologicalterrestrial species such as plants The first study was byJin and co-workers28 who demonstrated significant uptakeof Fe3O4 MNPs by pumpkin plants and their subsequenttranslocation and accumulation in various tissues How-ever fundamental questions remain regarding the system-atic physiological effects on plant cells and tissues and theimpact of these processes on plant reproduction Here weprovide a detailed evidence of impact of -Fe2O3 MNPson Chinese mung bean plants by measuring the physiolog-ical parameters such as germination root activity activ-ity of catalase (CAT) peroxidase (POD) and superoxidedismutase (SOD) and content of chlorophyll soluble pro-tein and content of malondialdehyde (MDA) The datapresented in this article suggest the potential impact ofnanomaterial exposure on plant development and the foodchain and prompt further investigation into the geneticconsequences through plant nanomaterial interactions

2 MATERIALS AND METHODS

21 Synthesis of -Fe2O3 MNPs

Fe3O4 MNPs with diameters of approximately 9 nm and18 nm were first prepared according to the chemical

co-precipitation method29 The synthesized Fe3O4 parti-cles were diluted to 50 L and the pH of the solution wasadjusted to 3 with 2 M HCl under nitrogen gas protectionand vigorous stirring using nonmagnetic stirrer Then theFe3O4 nanoparticles were transferred into a double-layerglass reaction vessel to react with oxygen through bub-bling air under continuous stirring at 90 C After stirringfor 5 h the obtained -Fe2O3 nanoparticles precipitate wasseparated from the reaction medium by magnetic field andwashed with 200 mL deionized water four times With thissynthesis once the synthetic conditions are fixed the qual-ity of the magnetite nanoparticles is fully reproducibleThe prepared -Fe2O3 nanoparticles were dispersed indeionized water and the pH of the solution was adjustedto 27 1365 g DMSA dissolved in 50 mL DMSO wasadded to the dispersion with continuous stirring After thereaction for 5 h at room temperature the products werecollected with a magnet and were dispersed in (CH34NOHsolution and the pH of the solution was adjusted to 10The stable magnetic fluids were obtained after the pH ofthe solution was adjusted to neutral and reverse osmosis

22 Superoxide Dismutase (SOD) Activity Assay

A 05 g of fresh samples were ground in a mortar with001 g PVP 5 mL of 01 mol middotLminus1 phosphate buffer con-taining 02 mM EDTA and 04 mM -mercaptoethanol(pH 78) After extraction in an ice bath the homogenatewas centrifuged at 1200 rpm for 10 min The supernatantwas collected for further measurementsTake 50 L supernatant and add to 15 mL of 005 M

phosphate buffer (pH 78) 03 mL of 130 mM methio-nine 03 mL of 750 M Nitrotetrazolium blue chloride03 mL of 100 M EDTA-Na 03 mL 20 M lactochromeand 025 mL of distilled water Another two control sam-ples were prepared using buffer instead of enzyme solu-tion After uniform mixing one control test was put inthe dark and others were illuminated under fluorescencelamp for 20 min Activity was measured by the changesin absorbance at 560 nm

23 Peroxidase (POD) Activity Assay

Peroxidase activity was determined by guaiacol oxidationPOD reaction mixture was first prepared by the addition of28 L guaiacol into 50 mL of 100 mM phosphate buffer(pH 70) when completely dissolving and cooling at roomtemperature 19 l of 30 H2O2 was introduced (Notesuch mixture was required to freshly prepared before useand stored in fridge) The assay mixture contained in100 L of the diluted supernatant 2 mL POD reactionmixture and 1 mL of 02 M potassium dihydrophospatebuffer The hydrogen peroxide can oxidize guaiacol andproduce dark brown substance in the presence of perox-idase The conversion of the dye was monitored by themeasurement of the changes in absorbance at 470 nm per

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Ren et al Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

30 sec The POD activity was expressed as the change ofabsorbance per minute (A470mingFW)

24 Catalase (CAT) Activity Assay

Catalase (CAT) activity was determined by the addi-tion of 100 L supernatant to 3 ml mixture contain-ing 01 H2O2 and 100 mM phosphate buffer (pH 70)The results were monitored by the measurements of thechanges in absorbance at 240 nm per 30 sec The activ-ity was expressed as the change of absorbance per minute(A240ming FW (fresh weigh))

25 Soluble Protein Content Assay

Soluble protein content was analyzed using dying methodwith Coomasie Brilliant Fluka G-250 Protein standardsolution was firstly prepared and stored in the refrigeratorCoomassie Brilliant Blue G-250 solution was prepared byweighing coomassie brilliant blue G-250 100 mg adding50 mL of 95 ethanol and 100 mL of 85 (wv) H3PO4

to the final volume with distilled water 1000 mL at roomtemperature The test solution was prepared by weighinga dried sample 05 g adding a small amount of quartzsand with distilled water the mixture was ground into ahomogenate After standing 10ndash30 min (3ndash5 min shakingtime) via a filter (such as active carbon containing pigmentavailable) the volume of the solution was set to 100 mLfor further use

26 Malondialdehyde Content Assay

Take 1 ml supernatant and add to 5 mL of 20trichloroacetic acid (TCA) containing 05 M thiobarbi-turic acid (TBA) such a mixture was then manually agi-tated The container was placed in a boiling water bath for30 min After cooling down to room temperature it wascentrifuged at 4000 rpm for 10 min Using 20 TCA as areference the supernatant was collected for measurementsof the changes in absorbance at 450 532 and 600 nm

27 Chlorophyl Content Assay

01 g of fresh leaves were ground and transferred to atest-tube Chlorophyl was extracted with 10 ml mixture ofethanol and acetone (11 vv) under a controlled environ-ment of room temperature and 24 h dark photoperiod Datawas collected by the changes in absorbance at 645 and663 nm

28 Determination of Root Activity

The root activity was estimated by triphenyl tetrazoliumchloride (TTC) reduction30 Control roots were boiled for10 min in distilled water to insure that enzymes were dena-tured All roots were cut into 1 cm pieces submerged in

3 mL of 06 (wv) 235-triphenyl tetrazolium chloridein 005 M Na2HPO4-KH2PO4 (pH 74)+ 005 wettingagent (Triton X-100) and vacuum-infiltrated for 5 minto insure infiltration of TTC Samples were incubated at30 C for 24 h rinsed twice with distilled water andextracted four times in 4 mL of 95 (vv) ethanol for5 min in a waterbath at 85 C The total solution extractedwas brought up to a volume of 25 mL and measured witha spectrophotometer (Shimadzu UV160U Kyoto Japan)at 490 nm

3 RESULTS AND DISCUSSION

Chinese mung bean was selected as a model plant becauseitrsquos a very popular plant and widely cultivated in China Asin the case of the potential biological effects of nanopar-ticles in the green agriculture the physiological investi-gation of magnetic iron oxide nanoparticles could be ofenormous benefit In this study a complex ecosystem ina greenhouse was constructed to model the growth envi-ronment of Chinese mung bean for further measuring thebehaviour of -Fe2O3 magnetic nanoparticles (Fig 1(A))Considering that the properties of nanoscale materialsare often dependent on size and nanoparticles have thepotential to pass across physiological barriers or targetspecific cells and organs and administer small quanti-ties of drugs and most importantly individual -Fe2O3

particles of 20 nm can be existed in the suspension-Fe2O3 MNPs with size of 9 and 18 nm were cho-sen and prepared for further experiments The -Fe2O3

MNPs appeared spherical and homogeneous and were ofthe expected size (Figs 1(BndashC)) After immersing into-Fe2O3 MNPs nutrient solution Chinese mung beanseeds were transferred into separate temporary contain-ers filled with natural silica sediment to allow the growthtill the germination These seedlings were then transferredinto plastic pots (filled with silica sediment) and supple-mented with 12 Hoagland solution containing -Fe2O3

MNPs everyday till the appearance of leaf Two differentconcentrations of 10 and 20 mg middotLminus1 were prepared for theinvestigation Together with a control test each treatmentwas conducted with three replicates and the results werestatistical analysis and presented as mean plusmnSD (standarddeviation) The details can be found in MethodsFigure 2(A) shows the results of germination of Chi-

nese mung bean seeds after treatment for 10 days by thenutrient solution containing 9 and 18 nm -Fe2O3 MNPs(10 and 20 mg middotLminus1 respectively In this test 100 seedswere selected for the experiments Seed germinations wereaffected by the concentration of nanoparticles in nutrientsolution As seen the germination of seeds was inhibitedby the lower concentration of nanoparticles 10 mg middotLminus1The percentage is 56 and 52 for size 9 and 18 nm ofnanoparticles respectively It is quite lower than the nor-mal case 72 in the absence of nanoparticles However

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

Fig 1 Experimental setup (A) Growth of Chinese mung bean modelling the natural environment was maintained in a greenhouse Chinese mungbean is shown growing in the sediment pots Each pot contained natural silica sediment and received a certain dose of -Fe2O3 MNPs nutritive medium(BndashC) TEM images of -Fe2O3 magnetic iron oxide nanopartiles from deionized water used in this study (B) 18 nm (C) 9 nm

the germination was promoted by the higher concentrationof nanoparticles 20 mg middotLminus1 The percentage is 78 forboth 9 nm and 18 nm of nanoparticles The results arequite different to the situation which ryegrass and cornwere inhibited by nano-Zn and nano-ZnO This might bedue to that Magnetic -Fe2O3 nanoparticles are generallyconsidered to be biologically and chemically inert Mag-netic -Fe2O3nanoparticles have been coated with metalcatalysts or conjugated with enzymes to combine the sep-arating power of the magnetic properties with the catalytic

(A) (B) (C)

Fig 2 Effect of -Fe2O3 magnetic nanoparticles on the germination of Chinese mung bean seeds (A) Percentage of germination of Chinese mungbean seeds inclubated in size 918 nm of 10 and 20 mg middotLminus1-Fe2O3 magnetic nanoparticles nutritive medium (B) Length of sprout as a functionof growth time under different sizes of nanoparticle Concentration 20 mg middotLminus1 (C) Length of sprout as a function of growth time under differentconcentrations -Fe2O3 MNPs 18 nm

activity of the metal surface or enzyme conjugate It isconcluded that the higher concentration has a promotionimpact on the seed germinationWe therefore chose 20 mg middot Lminus1 of nutrient solution

containing 9 and 18 nm -Fe2O3 MNPs to investigatethe length of sprout during the growth as presented inFigure 2(B) In contrast to the control test (that is in theabsence of nanoparticles) -Fe2O3 MNPs nutrient solu-tion can accelerated the growth of sprout This indicatesthat the existing of -Fe2O3 MNPs helps root cell open

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Ren et al Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

(A) (B)

Fig 3 (A) Root activity in the presence of 9 nm and 18 nm -Fe2O3

magnetic nanoparticles of various concentrations (B) Root activity versusconcentrations (0 10 20 40 60 80 and 100 mg middotLminus1 in the presenceof 9 nm -Fe2O3 magnetic nanoparticles

water channels and effectively promotes cell better adsorp-tion to water inorganic ions and other nutritional compo-nents and that the influence of smaller nanoparticle (sizeof 9 nm) is more obvious than that of bigger one (sizeof 18 nm) The nanoparticles with small size readily per-meate through the plant cell wall driven by a concentra-tion gradient This contribution is excellent consistent withthe effect of -Fe2O3 MNPs on the root activity as willbe discussed in the following section Figure 2(C) is theresults of length of sprout as a function of growth timeunder different concentrations It is seen that the growingof sprout is well at lower concentration (10 mg middotLminus1 Thisphenomenon might be attributed to that the higher con-centration can form clusters and tends to block the porousplant cell wallThe absorption of plant roots is an active organ and

synthetic organs root growth and vigor of shoot directly

(A) (B) (C)

Fig 4 Activity of a group of enzymes in the presence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles of various concentrations (A) Activityof catalase (CAT) (B) Activity of peroxidase (POD) (C) Activity of superoxide dismutase (SOD)

affect the level of the nutritional status and produc-tion levels Figure 3 shows the root activity of Chinesemung bean seedlings in the presence of 9-nm and 18-nm-Fe2O3 MNPs of various concentrations As seen fromFigure 3(A) no changes were observed for 18 nm MNPsat 20 mg middotLminus1 compared to the control At the same con-centration 9 nm -Fe2O3 MNPs have a positive effectit proposed an increase of 503 percent for root activityThe best root activity can be seen in the case of 9 nmMNPs of 10 mg middot Lminus1 an increase of 1172 percent isobtained For both of 9 nm and 18 nm nanoparicles rootactivity influenced by lower concentration is more obviousthan that influenced by higher concentration This experi-ment gives a supportive evidence for the growth of sproutshown in Figures 2(B)ndash(C) Figure 3(B) shows the rootactivity under different concentrations in the presence of9 nm -Fe2O3 MNPs It is seen that as increasing the con-centration to 40 mg middotLminus1 the root activity sharply decreasesand consequently again increases to normal case (see dot-ted line) This might be explained either by the significantaggregation of those MNPs at high concentration or by theaccumulation of many -Fe2O3 nanoparticles on the rootsurface (ie the accumulation inhibits the transmission ofwater and other nutritional components)Peroxide accumulation may cause changes in plant

metabolism in several ways They may oxidize sulflhydrylgroups and in combination with superoxides they canform hydroxyl radicals which may be involved in theaging process H2O2 may be involved also in the oxidativebreakdown of indoleacetic acid It has been shown thatincreased H2O2 levels inactivate indoleacetic acid Thisinactivation was reversed upon the introduction of catalaseFigure 4 shows the activity of a group of enzymes such astatalase peroxidase and superoxide dismutase in the pres-ence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

of various concentrations Figure 4(A) shows the effect of-Fe2O3 MNPs on the function of the enzyme catalasewhich is found in all plant tissues As seen a nanoparticleaddition in nutrient solution results in a decrease of activ-ity of catalase while the effect of nanoparticles in theseplants showed no significant concentration or size depen-dence A decrease in catalase activity could lead to theobserved accumulation of H2O2 in tissue since there is anincrease in peroxidase activity (Fig 4(B)) to compensatefor the H2O2 removal by catalase even though the activ-ity is still lower than normal levels Taking 20 mg middotLminus1

of -Fe2O3 MNPs solution as an example the catalaseactivity treated by 18 nm MNPs is a litter higher than thattreated by 9 nm (Fig 4(A)) Peroxidase activity is oppo-site to this result (Fig 4(B)) Similar result can also befound in the case of 10 mg Lminus1-Fe2O3 MNPs solutionAs seen from Figure 4(C) the activity of superoxide dis-mutase was inhibited by the suspension of -Fe2O3 MNPsand shows a size and concentration dependence A signifi-cant effect was found at higher concentration (20 mg middotLminus1and larger size (18 nm) Superoxide dismutase (SOD) inplant catalyzes the destruction of the O2minus free radical thedecrease of activity of SOD result in the accumulation ofO2minus free radical in plant leaf Consequently these freeradicals react with H2O2 produced by chloroplast to formOHbull free radicals which might result in the degradation ofchlorophyll However we observed a quite different phe-nomenon from the test of chlorophyl content as shown inFigure 5(A)As can be seen from Figure 5(A) a slight increasing

on the content of chlorophyll was observed after treatedby -Fe2O3 MNPs It is obvious that the introduction ofnanoparticles is contributed to a synthesis of chlorophyllHowever it is not clear whether this is due to a decreasein peroxidase activity intrinsic peroxidase-like activity of-Fe2O3 nanoparticles possess or both Content of soluble

(A) (B) (C)

Fig 5 Physiological parameters in the presence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles of various concentrations (A) Content ofchlorophyl (B) Content of soluble protein (SP) (C) Content of malondialdehyde (MDA)

protein was highly correlated with the age of plants iecontent of soluble protein decreases with the increasingof the age The results shown in Figure 5(B) reveal that-Fe2O3 MNPs can stimulate the growth of plants espe-cially for those samples treated using 18 nm of 10 mg middotLminus1

nanoparticle suspension Soluble protein was also strongrelative to the Content of chlorophyl of plant leaf With theincrease of content of chlorophyl photosynthesis of plantsincreases resulting in the accumulation of soluble proteinMalondialdehyde (MDA) is an important lipid peroxida-tion product when plant is in stress conditions of agingor injured Its content is closely related to plant senes-cence and stress injury The extent of the damage of plantmembrane system and plant resistance can be known bymeasuring the MDA level of lipid peroxidation The lowcontent of MDA is useful for protecting the structure andfunction of cell membrane As depicted in Figure 5(C)the content of MDA decreases in the present conditionsexcept the treatment using 9 nm of 20 mg middotLminus1 nanopar-ticles indicating that the proxidation of unsaturated fattyacid in cell membrane is weaker than that of the controlThis situation should be due to the increase of the con-tent of SOD CAT and POD in theory It is quite differ-ent from the above mentioned experimental results FromTEM image we are sure that -Fe2O3 MNPs were intro-duced to plant (as will be discussed later) suggesting thatthere may have another mechanism Maybe one of thesethree enzymes or boththree of them waswere replaced by-Fe2O3 MNPs The unusual result obtained under 9 nmof 20 mg middotLminus1 nanoparticles is likely owing to the glomer-ation of small particles Larger particles cannot easily pen-etrate the cell wall and membrane resulting in the lowerutilization efficient and consequently affect the synthesisof chlorophyll and increasing of MDA contentFigure 6 shows TEM (Transmission electron

microscopy JEM-2010 microscope equipped with Oxford

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Ren et al Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

Fig 6 TEM images of mitochondria in the absence (A) and presence (B) of 9 nm of 20 mg middotLminus1-Fe2O3 MNPs suspension (CndashD) Detection ofnanoparticles in cytoplasm of Chinese mung bean treated with 9 nm of 20 mg middotLminus1-Fe2O3 MNPs suspension

INCA EDS operated at 200 kV accelerating voltage)images of the plant tissues after treating in 9 nm of20 mg middot Lminus1-Fe2O3 MNPs suspension Figure 6(A) isunder treatments of control of epidermal cell of Chinesemung bean root However the cristae of mitochondriabecomes rough and the color of the cytoplasm gets lighter(Fig 6(B)) The tumefaction of mitochondria cannot beobserved indicating that the membrane of mitochondriawas not damaged by the nanoparticles Taking account ofabove mentioned results CAT POD and SOD content ofplant tissue decrease after treating by -Fe2O3 nanoparti-cles this observation demonstrates that the peroxidationof the membrane does not occur Black aggregates werefrequently found in the cytoplasm (Figs 6(C and D))indicating that the sequence of nanoparticle uptakewas from the plant seeds and roots to the stems andleaves

4 CONCLUSIONS

This manuscript reports the first study describing a detailedevidence of impact of -Fe2O3 magnetite nanoparticles

(MNPs) on Chinese mung bean plants by measuring thephysiological parameters such as germination root activ-ity activity of catalase (CAT) peroxidase (POD) andsuperoxide dismutase (SOD) content of chlorophyll sol-uble protein and content of malondialdehyde (MDA) Itshould be noted that although the nanoparticles used inthis study have a diameter size within the nanometrerange aggregates of different sizes were formed in theplant cells Thus it can be concluded that the -Fe2O3

nanoparticles could enter into the tissues or cells fromthe roots Even though more works need further attentionin order to obtain aspects of mechanism including uptakeand translocation and the interactions between the particleswith plant tissue at the cellular level this research never-theless provides convincing evidence that plant uptake isa potential transport pathway of nanoparticles in the envi-ronment Another main significance of this study couldprovide a guideline for the production of so-called sele-nium (Se)-riched rice in China After our inspection sele-nium (Se)-riched rice were produced in two ways One ismanual spraying of Se solution during rice growing andthen through bio-transformation the inorganic selenium

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

into organic selenium and stored in the rice so absorbedby the body Another is the local soil rich in selenium con-tent the rice produced natural selenium Our results willhelp answer the question on how significant physiologicalchanges of plant occur

Acknowledgments X-J Huang thanks ldquoOne HundredPerson Projectrdquo of the Chinese Academy of SciencesChina and N Gu thanks State Key Development Programfor Basic Research of China (Grant No 2006CB933200)and National Natural Science Funds for DistinguishedYoung Scholar (NSFC-60725101) for their financialsupports

References and Notes

1 H X Ren X Chen J H Liu N Gu and X J Huang Toxicity ofsingle-walled carbon nanotube How we were wrong Mater Today13 6 (2010)

2 H Ren and X J Huang Polyacrylate nanoparticles Toxicity or newnanomedicine Eur Respir J 36 218 (2010)

3 S G Xing Y B Jun Z W Hau and L Y Liang Higher accumu-lation of gamma-aminobutyric acid induced by salt stress throughstimulating the activity of diarnine oxidases in Glycine max (L)Merr roots Plant Physiol Biochem 45 560 (2007)

4 D H Lin and B S Xing Root uptake and phytotoxicity of ZnOnanoparticles Environ Sci Technol 42 5580 (2008)

5 D H Lin and B S Xing Phytotoxicity of nanoparticles Inhibi-tion of seed germination and root growth Environ Pollut 150 243(2007)

6 W M Lee Y J An H Yoon and H S Kweon Toxicity andbioavailability of copper nanoparticles to the terrestrial plants mungbean (Phaseolus radiatus) and wheat (Triticum aestivum) Plantagar test for water-insoluble nanoparticles Environ Toxicol Chem27 1915 (2008)

7 V Shah and I Belozerova Influence of metal nanoparticles on thesoil microbial community and germination of Lettuce Seeds WaterAir Soil Poll 197 143 (2009)

8 E Seeger A Baun M Kastner and S Trapp Insignificant acutetoxicity of TiO2 nanoparticles to willow trees J Soil Sediment 9 46(2009)

9 R Barrena E Casals J Colon X Font A Sanchez and V PuntesEvaluation of the ecotoxicity of model nanoparticles Chemosphere75 850 (2009)

10 D Stampoulis S K Sinha and J C White Assay-dependent phy-totoxicity of nanoparticles to plants Environ Sci Technol 43 9473(2009)

11 Y H Ma L L Kuang X He W Bai Y Y Ding Z Y ZhangY L Zhao and Z F Chai Effects of rare earth oxide nanoparticleson root elongation of plants Chemosphere 78 273 (2010)

12 M L Lopez-Moreno G de la Rosa J A Hernandez-Viezcas J RPeralta-Videa and J L Gardea-Torresdey X-ray absorption spec-troscopy (XAS) corroboration of the uptake and storage of CeO2

nanoparticles and assessment of their differential toxicity in fouredible plant species J Agr Food Chem 58 3689 (2010)

13 J Kurepa T Paunesku S Vogt H Arora B M Rabatic J JLu M B Wanzer G E Woloschak and J A Smalle Uptake anddistribution of ultrasmall anatase TiO2 alizarin red S nanoconjugatesin arabidopsis thaliana Nano Lett 10 2296 (2010)

14 S J Lin J Reppert Q Hu J S Hudson M L Reid T ARatnikova A M Rao H Luo and P C Ke Uptake Transloca-tion and transmission of carbon nanomaterials in rice plants Small5 1128 (2009)

15 R Chen T A Ratnikova M B Stone S Lin M Lard G HuangJ S Hudson and P C Ke Differential uptake of carbon nanoparti-cles by plant and mammalian Cells Small 6 612 (2010)

16 C W Lee S Mahendra K Zodrow D Li Y C Tsai JBraam and P J J Alvarez Developmental phytotoxicity of metaloxide nanoparticles to arabidopsis thaliana Environ Toxicol Chem29 1399 (2010)

17 X M Ma J Geiser-Lee Y Deng and A Kolmakov Interactionsbetween engineered nanoparticles (ENPs) and plants Phytotoxicityuptake and accumulation Sci Total Environ 408 3053 (2010)

18 R Nair S H Varghese B G Nair T Maekawa Y Yoshida andD S Kumar Nanoparticulate material delivery to plants Plant Sci179 154 (2010)

19 L Z Gao J Zhuang L Nie J B Zhang Y Zhang N GuT H Wang J Feng D L Yang S Perrett and X Yan Intrinsicperoxidase-like activity of ferromagnetic nanoparticles Nat Nano-technol 2 577 (2007)

20 S P Singh Multifuncational magnetic quantum dots for cancer ther-anostics J Biomed Nanotechnol 7 95 (2011)

21 J Ding J H Zhao K LCheng G F Liu and D H Xiu In Vivophotodynamic therapy and magnetic resonance imaging of cancer byTSPP-coated Fe3O4 nanoconjugates J Biomed Nanotechnol 6 683(2010)

22 K Men S Zeng M L Gou G Guo Y C Gu F LuoX Zhao Y Q Wei and Z Y Qian Preparation of magnetic micro-spheres based on poly(e-caprolactone)-poly(ethylene glycol)-poly(e-caprolactone) copolymers by modified solvent diffusion methodJ Biomed Nanotechnol 6 287 (2010)

23 A Arkhis A Elaissari T Delair B Verrier and B Mandrand Cap-ture of enveloped viruses using polymer tentacles containing mag-netic latex particles J Biomed Nanotechnol 6 28 (2010)

24 L Tian S Li H N Liu Z F Wang and N Y He An automatedmagstation for high-throughput single nucleotide polymorphismgenotyping and the dual-color hybridization J Biomed Nanotech-nol 5 511 (2009)

25 Z Y Li L He Z Y Shi H Wang S Li H N Liu Y B Dai Z FWang and N Y He Preparation of SiO2(PMMAFe3O4 nanopar-ticles and its application in detecting Ecoli O157H7 using chemi-luminescent immunological method J Biomed Nanotechnol 5 505(2009)

26 Z Y Li L He N Y He Z Y Shi H Wang S Li H N LiuX L Li Y B Dai and Z F Wang Chemiluminescent detect ofEcoli O157H7 using immunological method based on magneticnanoparticles J Nanosci Nanotechnol 10 696 (2010)

27 L He Z Y Li J Fu F Wang C Ma Y Deng Z Y Shi H Wangand N Y He Preparation of SiO2(PMMAFe3O4 nanoparticlesusing linolenic acid as crosslink agent for nucleic acid detectionusing chemiluminescent method J Nanosci Nanotechnol 11 2256(2011)

28 H Zhu J Han J Q Xiao and Y Jin Uptake translocation andaccumulation of manufactured iron oxide nanoparticles by pumpkinplants J Environ Monitor 10 713 (2008)

29 A H Lu E L Salabas and F Schuth Magnetic nanoparticles Syn-thesis protection functionalization and application Angew ChemInt Ed 46 1222 (2007)

30 L H Comas D M Eissenstat and A N Lakso Assessing rootdeath and root system dynamics in a study of grape canopy pruningNew Phytol 147 171 (2000)

Received 10 June 2011 Accepted 20 June 2011

684 J Biomed Nanotechnol 7 677ndash684 2011

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Ren et al Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

30 sec The POD activity was expressed as the change ofabsorbance per minute (A470mingFW)

24 Catalase (CAT) Activity Assay

Catalase (CAT) activity was determined by the addi-tion of 100 L supernatant to 3 ml mixture contain-ing 01 H2O2 and 100 mM phosphate buffer (pH 70)The results were monitored by the measurements of thechanges in absorbance at 240 nm per 30 sec The activ-ity was expressed as the change of absorbance per minute(A240ming FW (fresh weigh))

25 Soluble Protein Content Assay

Soluble protein content was analyzed using dying methodwith Coomasie Brilliant Fluka G-250 Protein standardsolution was firstly prepared and stored in the refrigeratorCoomassie Brilliant Blue G-250 solution was prepared byweighing coomassie brilliant blue G-250 100 mg adding50 mL of 95 ethanol and 100 mL of 85 (wv) H3PO4

to the final volume with distilled water 1000 mL at roomtemperature The test solution was prepared by weighinga dried sample 05 g adding a small amount of quartzsand with distilled water the mixture was ground into ahomogenate After standing 10ndash30 min (3ndash5 min shakingtime) via a filter (such as active carbon containing pigmentavailable) the volume of the solution was set to 100 mLfor further use

26 Malondialdehyde Content Assay

Take 1 ml supernatant and add to 5 mL of 20trichloroacetic acid (TCA) containing 05 M thiobarbi-turic acid (TBA) such a mixture was then manually agi-tated The container was placed in a boiling water bath for30 min After cooling down to room temperature it wascentrifuged at 4000 rpm for 10 min Using 20 TCA as areference the supernatant was collected for measurementsof the changes in absorbance at 450 532 and 600 nm

27 Chlorophyl Content Assay

01 g of fresh leaves were ground and transferred to atest-tube Chlorophyl was extracted with 10 ml mixture ofethanol and acetone (11 vv) under a controlled environ-ment of room temperature and 24 h dark photoperiod Datawas collected by the changes in absorbance at 645 and663 nm

28 Determination of Root Activity

The root activity was estimated by triphenyl tetrazoliumchloride (TTC) reduction30 Control roots were boiled for10 min in distilled water to insure that enzymes were dena-tured All roots were cut into 1 cm pieces submerged in

3 mL of 06 (wv) 235-triphenyl tetrazolium chloridein 005 M Na2HPO4-KH2PO4 (pH 74)+ 005 wettingagent (Triton X-100) and vacuum-infiltrated for 5 minto insure infiltration of TTC Samples were incubated at30 C for 24 h rinsed twice with distilled water andextracted four times in 4 mL of 95 (vv) ethanol for5 min in a waterbath at 85 C The total solution extractedwas brought up to a volume of 25 mL and measured witha spectrophotometer (Shimadzu UV160U Kyoto Japan)at 490 nm

3 RESULTS AND DISCUSSION

Chinese mung bean was selected as a model plant becauseitrsquos a very popular plant and widely cultivated in China Asin the case of the potential biological effects of nanopar-ticles in the green agriculture the physiological investi-gation of magnetic iron oxide nanoparticles could be ofenormous benefit In this study a complex ecosystem ina greenhouse was constructed to model the growth envi-ronment of Chinese mung bean for further measuring thebehaviour of -Fe2O3 magnetic nanoparticles (Fig 1(A))Considering that the properties of nanoscale materialsare often dependent on size and nanoparticles have thepotential to pass across physiological barriers or targetspecific cells and organs and administer small quanti-ties of drugs and most importantly individual -Fe2O3

particles of 20 nm can be existed in the suspension-Fe2O3 MNPs with size of 9 and 18 nm were cho-sen and prepared for further experiments The -Fe2O3

MNPs appeared spherical and homogeneous and were ofthe expected size (Figs 1(BndashC)) After immersing into-Fe2O3 MNPs nutrient solution Chinese mung beanseeds were transferred into separate temporary contain-ers filled with natural silica sediment to allow the growthtill the germination These seedlings were then transferredinto plastic pots (filled with silica sediment) and supple-mented with 12 Hoagland solution containing -Fe2O3

MNPs everyday till the appearance of leaf Two differentconcentrations of 10 and 20 mg middotLminus1 were prepared for theinvestigation Together with a control test each treatmentwas conducted with three replicates and the results werestatistical analysis and presented as mean plusmnSD (standarddeviation) The details can be found in MethodsFigure 2(A) shows the results of germination of Chi-

nese mung bean seeds after treatment for 10 days by thenutrient solution containing 9 and 18 nm -Fe2O3 MNPs(10 and 20 mg middotLminus1 respectively In this test 100 seedswere selected for the experiments Seed germinations wereaffected by the concentration of nanoparticles in nutrientsolution As seen the germination of seeds was inhibitedby the lower concentration of nanoparticles 10 mg middotLminus1The percentage is 56 and 52 for size 9 and 18 nm ofnanoparticles respectively It is quite lower than the nor-mal case 72 in the absence of nanoparticles However

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ARTIC

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

Fig 1 Experimental setup (A) Growth of Chinese mung bean modelling the natural environment was maintained in a greenhouse Chinese mungbean is shown growing in the sediment pots Each pot contained natural silica sediment and received a certain dose of -Fe2O3 MNPs nutritive medium(BndashC) TEM images of -Fe2O3 magnetic iron oxide nanopartiles from deionized water used in this study (B) 18 nm (C) 9 nm

the germination was promoted by the higher concentrationof nanoparticles 20 mg middotLminus1 The percentage is 78 forboth 9 nm and 18 nm of nanoparticles The results arequite different to the situation which ryegrass and cornwere inhibited by nano-Zn and nano-ZnO This might bedue to that Magnetic -Fe2O3 nanoparticles are generallyconsidered to be biologically and chemically inert Mag-netic -Fe2O3nanoparticles have been coated with metalcatalysts or conjugated with enzymes to combine the sep-arating power of the magnetic properties with the catalytic

(A) (B) (C)

Fig 2 Effect of -Fe2O3 magnetic nanoparticles on the germination of Chinese mung bean seeds (A) Percentage of germination of Chinese mungbean seeds inclubated in size 918 nm of 10 and 20 mg middotLminus1-Fe2O3 magnetic nanoparticles nutritive medium (B) Length of sprout as a functionof growth time under different sizes of nanoparticle Concentration 20 mg middotLminus1 (C) Length of sprout as a function of growth time under differentconcentrations -Fe2O3 MNPs 18 nm

activity of the metal surface or enzyme conjugate It isconcluded that the higher concentration has a promotionimpact on the seed germinationWe therefore chose 20 mg middot Lminus1 of nutrient solution

containing 9 and 18 nm -Fe2O3 MNPs to investigatethe length of sprout during the growth as presented inFigure 2(B) In contrast to the control test (that is in theabsence of nanoparticles) -Fe2O3 MNPs nutrient solu-tion can accelerated the growth of sprout This indicatesthat the existing of -Fe2O3 MNPs helps root cell open

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RESEARCH

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Ren et al Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

(A) (B)

Fig 3 (A) Root activity in the presence of 9 nm and 18 nm -Fe2O3

magnetic nanoparticles of various concentrations (B) Root activity versusconcentrations (0 10 20 40 60 80 and 100 mg middotLminus1 in the presenceof 9 nm -Fe2O3 magnetic nanoparticles

water channels and effectively promotes cell better adsorp-tion to water inorganic ions and other nutritional compo-nents and that the influence of smaller nanoparticle (sizeof 9 nm) is more obvious than that of bigger one (sizeof 18 nm) The nanoparticles with small size readily per-meate through the plant cell wall driven by a concentra-tion gradient This contribution is excellent consistent withthe effect of -Fe2O3 MNPs on the root activity as willbe discussed in the following section Figure 2(C) is theresults of length of sprout as a function of growth timeunder different concentrations It is seen that the growingof sprout is well at lower concentration (10 mg middotLminus1 Thisphenomenon might be attributed to that the higher con-centration can form clusters and tends to block the porousplant cell wallThe absorption of plant roots is an active organ and

synthetic organs root growth and vigor of shoot directly

(A) (B) (C)

Fig 4 Activity of a group of enzymes in the presence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles of various concentrations (A) Activityof catalase (CAT) (B) Activity of peroxidase (POD) (C) Activity of superoxide dismutase (SOD)

affect the level of the nutritional status and produc-tion levels Figure 3 shows the root activity of Chinesemung bean seedlings in the presence of 9-nm and 18-nm-Fe2O3 MNPs of various concentrations As seen fromFigure 3(A) no changes were observed for 18 nm MNPsat 20 mg middotLminus1 compared to the control At the same con-centration 9 nm -Fe2O3 MNPs have a positive effectit proposed an increase of 503 percent for root activityThe best root activity can be seen in the case of 9 nmMNPs of 10 mg middot Lminus1 an increase of 1172 percent isobtained For both of 9 nm and 18 nm nanoparicles rootactivity influenced by lower concentration is more obviousthan that influenced by higher concentration This experi-ment gives a supportive evidence for the growth of sproutshown in Figures 2(B)ndash(C) Figure 3(B) shows the rootactivity under different concentrations in the presence of9 nm -Fe2O3 MNPs It is seen that as increasing the con-centration to 40 mg middotLminus1 the root activity sharply decreasesand consequently again increases to normal case (see dot-ted line) This might be explained either by the significantaggregation of those MNPs at high concentration or by theaccumulation of many -Fe2O3 nanoparticles on the rootsurface (ie the accumulation inhibits the transmission ofwater and other nutritional components)Peroxide accumulation may cause changes in plant

metabolism in several ways They may oxidize sulflhydrylgroups and in combination with superoxides they canform hydroxyl radicals which may be involved in theaging process H2O2 may be involved also in the oxidativebreakdown of indoleacetic acid It has been shown thatincreased H2O2 levels inactivate indoleacetic acid Thisinactivation was reversed upon the introduction of catalaseFigure 4 shows the activity of a group of enzymes such astatalase peroxidase and superoxide dismutase in the pres-ence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

of various concentrations Figure 4(A) shows the effect of-Fe2O3 MNPs on the function of the enzyme catalasewhich is found in all plant tissues As seen a nanoparticleaddition in nutrient solution results in a decrease of activ-ity of catalase while the effect of nanoparticles in theseplants showed no significant concentration or size depen-dence A decrease in catalase activity could lead to theobserved accumulation of H2O2 in tissue since there is anincrease in peroxidase activity (Fig 4(B)) to compensatefor the H2O2 removal by catalase even though the activ-ity is still lower than normal levels Taking 20 mg middotLminus1

of -Fe2O3 MNPs solution as an example the catalaseactivity treated by 18 nm MNPs is a litter higher than thattreated by 9 nm (Fig 4(A)) Peroxidase activity is oppo-site to this result (Fig 4(B)) Similar result can also befound in the case of 10 mg Lminus1-Fe2O3 MNPs solutionAs seen from Figure 4(C) the activity of superoxide dis-mutase was inhibited by the suspension of -Fe2O3 MNPsand shows a size and concentration dependence A signifi-cant effect was found at higher concentration (20 mg middotLminus1and larger size (18 nm) Superoxide dismutase (SOD) inplant catalyzes the destruction of the O2minus free radical thedecrease of activity of SOD result in the accumulation ofO2minus free radical in plant leaf Consequently these freeradicals react with H2O2 produced by chloroplast to formOHbull free radicals which might result in the degradation ofchlorophyll However we observed a quite different phe-nomenon from the test of chlorophyl content as shown inFigure 5(A)As can be seen from Figure 5(A) a slight increasing

on the content of chlorophyll was observed after treatedby -Fe2O3 MNPs It is obvious that the introduction ofnanoparticles is contributed to a synthesis of chlorophyllHowever it is not clear whether this is due to a decreasein peroxidase activity intrinsic peroxidase-like activity of-Fe2O3 nanoparticles possess or both Content of soluble

(A) (B) (C)

Fig 5 Physiological parameters in the presence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles of various concentrations (A) Content ofchlorophyl (B) Content of soluble protein (SP) (C) Content of malondialdehyde (MDA)

protein was highly correlated with the age of plants iecontent of soluble protein decreases with the increasingof the age The results shown in Figure 5(B) reveal that-Fe2O3 MNPs can stimulate the growth of plants espe-cially for those samples treated using 18 nm of 10 mg middotLminus1

nanoparticle suspension Soluble protein was also strongrelative to the Content of chlorophyl of plant leaf With theincrease of content of chlorophyl photosynthesis of plantsincreases resulting in the accumulation of soluble proteinMalondialdehyde (MDA) is an important lipid peroxida-tion product when plant is in stress conditions of agingor injured Its content is closely related to plant senes-cence and stress injury The extent of the damage of plantmembrane system and plant resistance can be known bymeasuring the MDA level of lipid peroxidation The lowcontent of MDA is useful for protecting the structure andfunction of cell membrane As depicted in Figure 5(C)the content of MDA decreases in the present conditionsexcept the treatment using 9 nm of 20 mg middotLminus1 nanopar-ticles indicating that the proxidation of unsaturated fattyacid in cell membrane is weaker than that of the controlThis situation should be due to the increase of the con-tent of SOD CAT and POD in theory It is quite differ-ent from the above mentioned experimental results FromTEM image we are sure that -Fe2O3 MNPs were intro-duced to plant (as will be discussed later) suggesting thatthere may have another mechanism Maybe one of thesethree enzymes or boththree of them waswere replaced by-Fe2O3 MNPs The unusual result obtained under 9 nmof 20 mg middotLminus1 nanoparticles is likely owing to the glomer-ation of small particles Larger particles cannot easily pen-etrate the cell wall and membrane resulting in the lowerutilization efficient and consequently affect the synthesisof chlorophyll and increasing of MDA contentFigure 6 shows TEM (Transmission electron

microscopy JEM-2010 microscope equipped with Oxford

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Ren et al Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

Fig 6 TEM images of mitochondria in the absence (A) and presence (B) of 9 nm of 20 mg middotLminus1-Fe2O3 MNPs suspension (CndashD) Detection ofnanoparticles in cytoplasm of Chinese mung bean treated with 9 nm of 20 mg middotLminus1-Fe2O3 MNPs suspension

INCA EDS operated at 200 kV accelerating voltage)images of the plant tissues after treating in 9 nm of20 mg middot Lminus1-Fe2O3 MNPs suspension Figure 6(A) isunder treatments of control of epidermal cell of Chinesemung bean root However the cristae of mitochondriabecomes rough and the color of the cytoplasm gets lighter(Fig 6(B)) The tumefaction of mitochondria cannot beobserved indicating that the membrane of mitochondriawas not damaged by the nanoparticles Taking account ofabove mentioned results CAT POD and SOD content ofplant tissue decrease after treating by -Fe2O3 nanoparti-cles this observation demonstrates that the peroxidationof the membrane does not occur Black aggregates werefrequently found in the cytoplasm (Figs 6(C and D))indicating that the sequence of nanoparticle uptakewas from the plant seeds and roots to the stems andleaves

4 CONCLUSIONS

This manuscript reports the first study describing a detailedevidence of impact of -Fe2O3 magnetite nanoparticles

(MNPs) on Chinese mung bean plants by measuring thephysiological parameters such as germination root activ-ity activity of catalase (CAT) peroxidase (POD) andsuperoxide dismutase (SOD) content of chlorophyll sol-uble protein and content of malondialdehyde (MDA) Itshould be noted that although the nanoparticles used inthis study have a diameter size within the nanometrerange aggregates of different sizes were formed in theplant cells Thus it can be concluded that the -Fe2O3

nanoparticles could enter into the tissues or cells fromthe roots Even though more works need further attentionin order to obtain aspects of mechanism including uptakeand translocation and the interactions between the particleswith plant tissue at the cellular level this research never-theless provides convincing evidence that plant uptake isa potential transport pathway of nanoparticles in the envi-ronment Another main significance of this study couldprovide a guideline for the production of so-called sele-nium (Se)-riched rice in China After our inspection sele-nium (Se)-riched rice were produced in two ways One ismanual spraying of Se solution during rice growing andthen through bio-transformation the inorganic selenium

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

into organic selenium and stored in the rice so absorbedby the body Another is the local soil rich in selenium con-tent the rice produced natural selenium Our results willhelp answer the question on how significant physiologicalchanges of plant occur

Acknowledgments X-J Huang thanks ldquoOne HundredPerson Projectrdquo of the Chinese Academy of SciencesChina and N Gu thanks State Key Development Programfor Basic Research of China (Grant No 2006CB933200)and National Natural Science Funds for DistinguishedYoung Scholar (NSFC-60725101) for their financialsupports

References and Notes

1 H X Ren X Chen J H Liu N Gu and X J Huang Toxicity ofsingle-walled carbon nanotube How we were wrong Mater Today13 6 (2010)

2 H Ren and X J Huang Polyacrylate nanoparticles Toxicity or newnanomedicine Eur Respir J 36 218 (2010)

3 S G Xing Y B Jun Z W Hau and L Y Liang Higher accumu-lation of gamma-aminobutyric acid induced by salt stress throughstimulating the activity of diarnine oxidases in Glycine max (L)Merr roots Plant Physiol Biochem 45 560 (2007)

4 D H Lin and B S Xing Root uptake and phytotoxicity of ZnOnanoparticles Environ Sci Technol 42 5580 (2008)

5 D H Lin and B S Xing Phytotoxicity of nanoparticles Inhibi-tion of seed germination and root growth Environ Pollut 150 243(2007)

6 W M Lee Y J An H Yoon and H S Kweon Toxicity andbioavailability of copper nanoparticles to the terrestrial plants mungbean (Phaseolus radiatus) and wheat (Triticum aestivum) Plantagar test for water-insoluble nanoparticles Environ Toxicol Chem27 1915 (2008)

7 V Shah and I Belozerova Influence of metal nanoparticles on thesoil microbial community and germination of Lettuce Seeds WaterAir Soil Poll 197 143 (2009)

8 E Seeger A Baun M Kastner and S Trapp Insignificant acutetoxicity of TiO2 nanoparticles to willow trees J Soil Sediment 9 46(2009)

9 R Barrena E Casals J Colon X Font A Sanchez and V PuntesEvaluation of the ecotoxicity of model nanoparticles Chemosphere75 850 (2009)

10 D Stampoulis S K Sinha and J C White Assay-dependent phy-totoxicity of nanoparticles to plants Environ Sci Technol 43 9473(2009)

11 Y H Ma L L Kuang X He W Bai Y Y Ding Z Y ZhangY L Zhao and Z F Chai Effects of rare earth oxide nanoparticleson root elongation of plants Chemosphere 78 273 (2010)

12 M L Lopez-Moreno G de la Rosa J A Hernandez-Viezcas J RPeralta-Videa and J L Gardea-Torresdey X-ray absorption spec-troscopy (XAS) corroboration of the uptake and storage of CeO2

nanoparticles and assessment of their differential toxicity in fouredible plant species J Agr Food Chem 58 3689 (2010)

13 J Kurepa T Paunesku S Vogt H Arora B M Rabatic J JLu M B Wanzer G E Woloschak and J A Smalle Uptake anddistribution of ultrasmall anatase TiO2 alizarin red S nanoconjugatesin arabidopsis thaliana Nano Lett 10 2296 (2010)

14 S J Lin J Reppert Q Hu J S Hudson M L Reid T ARatnikova A M Rao H Luo and P C Ke Uptake Transloca-tion and transmission of carbon nanomaterials in rice plants Small5 1128 (2009)

15 R Chen T A Ratnikova M B Stone S Lin M Lard G HuangJ S Hudson and P C Ke Differential uptake of carbon nanoparti-cles by plant and mammalian Cells Small 6 612 (2010)

16 C W Lee S Mahendra K Zodrow D Li Y C Tsai JBraam and P J J Alvarez Developmental phytotoxicity of metaloxide nanoparticles to arabidopsis thaliana Environ Toxicol Chem29 1399 (2010)

17 X M Ma J Geiser-Lee Y Deng and A Kolmakov Interactionsbetween engineered nanoparticles (ENPs) and plants Phytotoxicityuptake and accumulation Sci Total Environ 408 3053 (2010)

18 R Nair S H Varghese B G Nair T Maekawa Y Yoshida andD S Kumar Nanoparticulate material delivery to plants Plant Sci179 154 (2010)

19 L Z Gao J Zhuang L Nie J B Zhang Y Zhang N GuT H Wang J Feng D L Yang S Perrett and X Yan Intrinsicperoxidase-like activity of ferromagnetic nanoparticles Nat Nano-technol 2 577 (2007)

20 S P Singh Multifuncational magnetic quantum dots for cancer ther-anostics J Biomed Nanotechnol 7 95 (2011)

21 J Ding J H Zhao K LCheng G F Liu and D H Xiu In Vivophotodynamic therapy and magnetic resonance imaging of cancer byTSPP-coated Fe3O4 nanoconjugates J Biomed Nanotechnol 6 683(2010)

22 K Men S Zeng M L Gou G Guo Y C Gu F LuoX Zhao Y Q Wei and Z Y Qian Preparation of magnetic micro-spheres based on poly(e-caprolactone)-poly(ethylene glycol)-poly(e-caprolactone) copolymers by modified solvent diffusion methodJ Biomed Nanotechnol 6 287 (2010)

23 A Arkhis A Elaissari T Delair B Verrier and B Mandrand Cap-ture of enveloped viruses using polymer tentacles containing mag-netic latex particles J Biomed Nanotechnol 6 28 (2010)

24 L Tian S Li H N Liu Z F Wang and N Y He An automatedmagstation for high-throughput single nucleotide polymorphismgenotyping and the dual-color hybridization J Biomed Nanotech-nol 5 511 (2009)

25 Z Y Li L He Z Y Shi H Wang S Li H N Liu Y B Dai Z FWang and N Y He Preparation of SiO2(PMMAFe3O4 nanopar-ticles and its application in detecting Ecoli O157H7 using chemi-luminescent immunological method J Biomed Nanotechnol 5 505(2009)

26 Z Y Li L He N Y He Z Y Shi H Wang S Li H N LiuX L Li Y B Dai and Z F Wang Chemiluminescent detect ofEcoli O157H7 using immunological method based on magneticnanoparticles J Nanosci Nanotechnol 10 696 (2010)

27 L He Z Y Li J Fu F Wang C Ma Y Deng Z Y Shi H Wangand N Y He Preparation of SiO2(PMMAFe3O4 nanoparticlesusing linolenic acid as crosslink agent for nucleic acid detectionusing chemiluminescent method J Nanosci Nanotechnol 11 2256(2011)

28 H Zhu J Han J Q Xiao and Y Jin Uptake translocation andaccumulation of manufactured iron oxide nanoparticles by pumpkinplants J Environ Monitor 10 713 (2008)

29 A H Lu E L Salabas and F Schuth Magnetic nanoparticles Syn-thesis protection functionalization and application Angew ChemInt Ed 46 1222 (2007)

30 L H Comas D M Eissenstat and A N Lakso Assessing rootdeath and root system dynamics in a study of grape canopy pruningNew Phytol 147 171 (2000)

Received 10 June 2011 Accepted 20 June 2011

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RESEARCH

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

Fig 1 Experimental setup (A) Growth of Chinese mung bean modelling the natural environment was maintained in a greenhouse Chinese mungbean is shown growing in the sediment pots Each pot contained natural silica sediment and received a certain dose of -Fe2O3 MNPs nutritive medium(BndashC) TEM images of -Fe2O3 magnetic iron oxide nanopartiles from deionized water used in this study (B) 18 nm (C) 9 nm

the germination was promoted by the higher concentrationof nanoparticles 20 mg middotLminus1 The percentage is 78 forboth 9 nm and 18 nm of nanoparticles The results arequite different to the situation which ryegrass and cornwere inhibited by nano-Zn and nano-ZnO This might bedue to that Magnetic -Fe2O3 nanoparticles are generallyconsidered to be biologically and chemically inert Mag-netic -Fe2O3nanoparticles have been coated with metalcatalysts or conjugated with enzymes to combine the sep-arating power of the magnetic properties with the catalytic

(A) (B) (C)

Fig 2 Effect of -Fe2O3 magnetic nanoparticles on the germination of Chinese mung bean seeds (A) Percentage of germination of Chinese mungbean seeds inclubated in size 918 nm of 10 and 20 mg middotLminus1-Fe2O3 magnetic nanoparticles nutritive medium (B) Length of sprout as a functionof growth time under different sizes of nanoparticle Concentration 20 mg middotLminus1 (C) Length of sprout as a function of growth time under differentconcentrations -Fe2O3 MNPs 18 nm

activity of the metal surface or enzyme conjugate It isconcluded that the higher concentration has a promotionimpact on the seed germinationWe therefore chose 20 mg middot Lminus1 of nutrient solution

containing 9 and 18 nm -Fe2O3 MNPs to investigatethe length of sprout during the growth as presented inFigure 2(B) In contrast to the control test (that is in theabsence of nanoparticles) -Fe2O3 MNPs nutrient solu-tion can accelerated the growth of sprout This indicatesthat the existing of -Fe2O3 MNPs helps root cell open

680 J Biomed Nanotechnol 7 677ndash684 2011

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IP 12842202150Mon 23 Jul 2012 190458

RESEARCH

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LE

Ren et al Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

(A) (B)

Fig 3 (A) Root activity in the presence of 9 nm and 18 nm -Fe2O3

magnetic nanoparticles of various concentrations (B) Root activity versusconcentrations (0 10 20 40 60 80 and 100 mg middotLminus1 in the presenceof 9 nm -Fe2O3 magnetic nanoparticles

water channels and effectively promotes cell better adsorp-tion to water inorganic ions and other nutritional compo-nents and that the influence of smaller nanoparticle (sizeof 9 nm) is more obvious than that of bigger one (sizeof 18 nm) The nanoparticles with small size readily per-meate through the plant cell wall driven by a concentra-tion gradient This contribution is excellent consistent withthe effect of -Fe2O3 MNPs on the root activity as willbe discussed in the following section Figure 2(C) is theresults of length of sprout as a function of growth timeunder different concentrations It is seen that the growingof sprout is well at lower concentration (10 mg middotLminus1 Thisphenomenon might be attributed to that the higher con-centration can form clusters and tends to block the porousplant cell wallThe absorption of plant roots is an active organ and

synthetic organs root growth and vigor of shoot directly

(A) (B) (C)

Fig 4 Activity of a group of enzymes in the presence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles of various concentrations (A) Activityof catalase (CAT) (B) Activity of peroxidase (POD) (C) Activity of superoxide dismutase (SOD)

affect the level of the nutritional status and produc-tion levels Figure 3 shows the root activity of Chinesemung bean seedlings in the presence of 9-nm and 18-nm-Fe2O3 MNPs of various concentrations As seen fromFigure 3(A) no changes were observed for 18 nm MNPsat 20 mg middotLminus1 compared to the control At the same con-centration 9 nm -Fe2O3 MNPs have a positive effectit proposed an increase of 503 percent for root activityThe best root activity can be seen in the case of 9 nmMNPs of 10 mg middot Lminus1 an increase of 1172 percent isobtained For both of 9 nm and 18 nm nanoparicles rootactivity influenced by lower concentration is more obviousthan that influenced by higher concentration This experi-ment gives a supportive evidence for the growth of sproutshown in Figures 2(B)ndash(C) Figure 3(B) shows the rootactivity under different concentrations in the presence of9 nm -Fe2O3 MNPs It is seen that as increasing the con-centration to 40 mg middotLminus1 the root activity sharply decreasesand consequently again increases to normal case (see dot-ted line) This might be explained either by the significantaggregation of those MNPs at high concentration or by theaccumulation of many -Fe2O3 nanoparticles on the rootsurface (ie the accumulation inhibits the transmission ofwater and other nutritional components)Peroxide accumulation may cause changes in plant

metabolism in several ways They may oxidize sulflhydrylgroups and in combination with superoxides they canform hydroxyl radicals which may be involved in theaging process H2O2 may be involved also in the oxidativebreakdown of indoleacetic acid It has been shown thatincreased H2O2 levels inactivate indoleacetic acid Thisinactivation was reversed upon the introduction of catalaseFigure 4 shows the activity of a group of enzymes such astatalase peroxidase and superoxide dismutase in the pres-ence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles

J Biomed Nanotechnol 7 677ndash684 2011 681

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IP 12842202150Mon 23 Jul 2012 190458

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Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

of various concentrations Figure 4(A) shows the effect of-Fe2O3 MNPs on the function of the enzyme catalasewhich is found in all plant tissues As seen a nanoparticleaddition in nutrient solution results in a decrease of activ-ity of catalase while the effect of nanoparticles in theseplants showed no significant concentration or size depen-dence A decrease in catalase activity could lead to theobserved accumulation of H2O2 in tissue since there is anincrease in peroxidase activity (Fig 4(B)) to compensatefor the H2O2 removal by catalase even though the activ-ity is still lower than normal levels Taking 20 mg middotLminus1

of -Fe2O3 MNPs solution as an example the catalaseactivity treated by 18 nm MNPs is a litter higher than thattreated by 9 nm (Fig 4(A)) Peroxidase activity is oppo-site to this result (Fig 4(B)) Similar result can also befound in the case of 10 mg Lminus1-Fe2O3 MNPs solutionAs seen from Figure 4(C) the activity of superoxide dis-mutase was inhibited by the suspension of -Fe2O3 MNPsand shows a size and concentration dependence A signifi-cant effect was found at higher concentration (20 mg middotLminus1and larger size (18 nm) Superoxide dismutase (SOD) inplant catalyzes the destruction of the O2minus free radical thedecrease of activity of SOD result in the accumulation ofO2minus free radical in plant leaf Consequently these freeradicals react with H2O2 produced by chloroplast to formOHbull free radicals which might result in the degradation ofchlorophyll However we observed a quite different phe-nomenon from the test of chlorophyl content as shown inFigure 5(A)As can be seen from Figure 5(A) a slight increasing

on the content of chlorophyll was observed after treatedby -Fe2O3 MNPs It is obvious that the introduction ofnanoparticles is contributed to a synthesis of chlorophyllHowever it is not clear whether this is due to a decreasein peroxidase activity intrinsic peroxidase-like activity of-Fe2O3 nanoparticles possess or both Content of soluble

(A) (B) (C)

Fig 5 Physiological parameters in the presence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles of various concentrations (A) Content ofchlorophyl (B) Content of soluble protein (SP) (C) Content of malondialdehyde (MDA)

protein was highly correlated with the age of plants iecontent of soluble protein decreases with the increasingof the age The results shown in Figure 5(B) reveal that-Fe2O3 MNPs can stimulate the growth of plants espe-cially for those samples treated using 18 nm of 10 mg middotLminus1

nanoparticle suspension Soluble protein was also strongrelative to the Content of chlorophyl of plant leaf With theincrease of content of chlorophyl photosynthesis of plantsincreases resulting in the accumulation of soluble proteinMalondialdehyde (MDA) is an important lipid peroxida-tion product when plant is in stress conditions of agingor injured Its content is closely related to plant senes-cence and stress injury The extent of the damage of plantmembrane system and plant resistance can be known bymeasuring the MDA level of lipid peroxidation The lowcontent of MDA is useful for protecting the structure andfunction of cell membrane As depicted in Figure 5(C)the content of MDA decreases in the present conditionsexcept the treatment using 9 nm of 20 mg middotLminus1 nanopar-ticles indicating that the proxidation of unsaturated fattyacid in cell membrane is weaker than that of the controlThis situation should be due to the increase of the con-tent of SOD CAT and POD in theory It is quite differ-ent from the above mentioned experimental results FromTEM image we are sure that -Fe2O3 MNPs were intro-duced to plant (as will be discussed later) suggesting thatthere may have another mechanism Maybe one of thesethree enzymes or boththree of them waswere replaced by-Fe2O3 MNPs The unusual result obtained under 9 nmof 20 mg middotLminus1 nanoparticles is likely owing to the glomer-ation of small particles Larger particles cannot easily pen-etrate the cell wall and membrane resulting in the lowerutilization efficient and consequently affect the synthesisof chlorophyll and increasing of MDA contentFigure 6 shows TEM (Transmission electron

microscopy JEM-2010 microscope equipped with Oxford

682 J Biomed Nanotechnol 7 677ndash684 2011

Delivered by Ingenta toRice University Fondren Library

IP 12842202150Mon 23 Jul 2012 190458

RESEARCH

ARTIC

LE

Ren et al Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

Fig 6 TEM images of mitochondria in the absence (A) and presence (B) of 9 nm of 20 mg middotLminus1-Fe2O3 MNPs suspension (CndashD) Detection ofnanoparticles in cytoplasm of Chinese mung bean treated with 9 nm of 20 mg middotLminus1-Fe2O3 MNPs suspension

INCA EDS operated at 200 kV accelerating voltage)images of the plant tissues after treating in 9 nm of20 mg middot Lminus1-Fe2O3 MNPs suspension Figure 6(A) isunder treatments of control of epidermal cell of Chinesemung bean root However the cristae of mitochondriabecomes rough and the color of the cytoplasm gets lighter(Fig 6(B)) The tumefaction of mitochondria cannot beobserved indicating that the membrane of mitochondriawas not damaged by the nanoparticles Taking account ofabove mentioned results CAT POD and SOD content ofplant tissue decrease after treating by -Fe2O3 nanoparti-cles this observation demonstrates that the peroxidationof the membrane does not occur Black aggregates werefrequently found in the cytoplasm (Figs 6(C and D))indicating that the sequence of nanoparticle uptakewas from the plant seeds and roots to the stems andleaves

4 CONCLUSIONS

This manuscript reports the first study describing a detailedevidence of impact of -Fe2O3 magnetite nanoparticles

(MNPs) on Chinese mung bean plants by measuring thephysiological parameters such as germination root activ-ity activity of catalase (CAT) peroxidase (POD) andsuperoxide dismutase (SOD) content of chlorophyll sol-uble protein and content of malondialdehyde (MDA) Itshould be noted that although the nanoparticles used inthis study have a diameter size within the nanometrerange aggregates of different sizes were formed in theplant cells Thus it can be concluded that the -Fe2O3

nanoparticles could enter into the tissues or cells fromthe roots Even though more works need further attentionin order to obtain aspects of mechanism including uptakeand translocation and the interactions between the particleswith plant tissue at the cellular level this research never-theless provides convincing evidence that plant uptake isa potential transport pathway of nanoparticles in the envi-ronment Another main significance of this study couldprovide a guideline for the production of so-called sele-nium (Se)-riched rice in China After our inspection sele-nium (Se)-riched rice were produced in two ways One ismanual spraying of Se solution during rice growing andthen through bio-transformation the inorganic selenium

J Biomed Nanotechnol 7 677ndash684 2011 683

Delivered by Ingenta toRice University Fondren Library

IP 12842202150Mon 23 Jul 2012 190458

RESEARCH

ARTIC

LE

Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

into organic selenium and stored in the rice so absorbedby the body Another is the local soil rich in selenium con-tent the rice produced natural selenium Our results willhelp answer the question on how significant physiologicalchanges of plant occur

Acknowledgments X-J Huang thanks ldquoOne HundredPerson Projectrdquo of the Chinese Academy of SciencesChina and N Gu thanks State Key Development Programfor Basic Research of China (Grant No 2006CB933200)and National Natural Science Funds for DistinguishedYoung Scholar (NSFC-60725101) for their financialsupports

References and Notes

1 H X Ren X Chen J H Liu N Gu and X J Huang Toxicity ofsingle-walled carbon nanotube How we were wrong Mater Today13 6 (2010)

2 H Ren and X J Huang Polyacrylate nanoparticles Toxicity or newnanomedicine Eur Respir J 36 218 (2010)

3 S G Xing Y B Jun Z W Hau and L Y Liang Higher accumu-lation of gamma-aminobutyric acid induced by salt stress throughstimulating the activity of diarnine oxidases in Glycine max (L)Merr roots Plant Physiol Biochem 45 560 (2007)

4 D H Lin and B S Xing Root uptake and phytotoxicity of ZnOnanoparticles Environ Sci Technol 42 5580 (2008)

5 D H Lin and B S Xing Phytotoxicity of nanoparticles Inhibi-tion of seed germination and root growth Environ Pollut 150 243(2007)

6 W M Lee Y J An H Yoon and H S Kweon Toxicity andbioavailability of copper nanoparticles to the terrestrial plants mungbean (Phaseolus radiatus) and wheat (Triticum aestivum) Plantagar test for water-insoluble nanoparticles Environ Toxicol Chem27 1915 (2008)

7 V Shah and I Belozerova Influence of metal nanoparticles on thesoil microbial community and germination of Lettuce Seeds WaterAir Soil Poll 197 143 (2009)

8 E Seeger A Baun M Kastner and S Trapp Insignificant acutetoxicity of TiO2 nanoparticles to willow trees J Soil Sediment 9 46(2009)

9 R Barrena E Casals J Colon X Font A Sanchez and V PuntesEvaluation of the ecotoxicity of model nanoparticles Chemosphere75 850 (2009)

10 D Stampoulis S K Sinha and J C White Assay-dependent phy-totoxicity of nanoparticles to plants Environ Sci Technol 43 9473(2009)

11 Y H Ma L L Kuang X He W Bai Y Y Ding Z Y ZhangY L Zhao and Z F Chai Effects of rare earth oxide nanoparticleson root elongation of plants Chemosphere 78 273 (2010)

12 M L Lopez-Moreno G de la Rosa J A Hernandez-Viezcas J RPeralta-Videa and J L Gardea-Torresdey X-ray absorption spec-troscopy (XAS) corroboration of the uptake and storage of CeO2

nanoparticles and assessment of their differential toxicity in fouredible plant species J Agr Food Chem 58 3689 (2010)

13 J Kurepa T Paunesku S Vogt H Arora B M Rabatic J JLu M B Wanzer G E Woloschak and J A Smalle Uptake anddistribution of ultrasmall anatase TiO2 alizarin red S nanoconjugatesin arabidopsis thaliana Nano Lett 10 2296 (2010)

14 S J Lin J Reppert Q Hu J S Hudson M L Reid T ARatnikova A M Rao H Luo and P C Ke Uptake Transloca-tion and transmission of carbon nanomaterials in rice plants Small5 1128 (2009)

15 R Chen T A Ratnikova M B Stone S Lin M Lard G HuangJ S Hudson and P C Ke Differential uptake of carbon nanoparti-cles by plant and mammalian Cells Small 6 612 (2010)

16 C W Lee S Mahendra K Zodrow D Li Y C Tsai JBraam and P J J Alvarez Developmental phytotoxicity of metaloxide nanoparticles to arabidopsis thaliana Environ Toxicol Chem29 1399 (2010)

17 X M Ma J Geiser-Lee Y Deng and A Kolmakov Interactionsbetween engineered nanoparticles (ENPs) and plants Phytotoxicityuptake and accumulation Sci Total Environ 408 3053 (2010)

18 R Nair S H Varghese B G Nair T Maekawa Y Yoshida andD S Kumar Nanoparticulate material delivery to plants Plant Sci179 154 (2010)

19 L Z Gao J Zhuang L Nie J B Zhang Y Zhang N GuT H Wang J Feng D L Yang S Perrett and X Yan Intrinsicperoxidase-like activity of ferromagnetic nanoparticles Nat Nano-technol 2 577 (2007)

20 S P Singh Multifuncational magnetic quantum dots for cancer ther-anostics J Biomed Nanotechnol 7 95 (2011)

21 J Ding J H Zhao K LCheng G F Liu and D H Xiu In Vivophotodynamic therapy and magnetic resonance imaging of cancer byTSPP-coated Fe3O4 nanoconjugates J Biomed Nanotechnol 6 683(2010)

22 K Men S Zeng M L Gou G Guo Y C Gu F LuoX Zhao Y Q Wei and Z Y Qian Preparation of magnetic micro-spheres based on poly(e-caprolactone)-poly(ethylene glycol)-poly(e-caprolactone) copolymers by modified solvent diffusion methodJ Biomed Nanotechnol 6 287 (2010)

23 A Arkhis A Elaissari T Delair B Verrier and B Mandrand Cap-ture of enveloped viruses using polymer tentacles containing mag-netic latex particles J Biomed Nanotechnol 6 28 (2010)

24 L Tian S Li H N Liu Z F Wang and N Y He An automatedmagstation for high-throughput single nucleotide polymorphismgenotyping and the dual-color hybridization J Biomed Nanotech-nol 5 511 (2009)

25 Z Y Li L He Z Y Shi H Wang S Li H N Liu Y B Dai Z FWang and N Y He Preparation of SiO2(PMMAFe3O4 nanopar-ticles and its application in detecting Ecoli O157H7 using chemi-luminescent immunological method J Biomed Nanotechnol 5 505(2009)

26 Z Y Li L He N Y He Z Y Shi H Wang S Li H N LiuX L Li Y B Dai and Z F Wang Chemiluminescent detect ofEcoli O157H7 using immunological method based on magneticnanoparticles J Nanosci Nanotechnol 10 696 (2010)

27 L He Z Y Li J Fu F Wang C Ma Y Deng Z Y Shi H Wangand N Y He Preparation of SiO2(PMMAFe3O4 nanoparticlesusing linolenic acid as crosslink agent for nucleic acid detectionusing chemiluminescent method J Nanosci Nanotechnol 11 2256(2011)

28 H Zhu J Han J Q Xiao and Y Jin Uptake translocation andaccumulation of manufactured iron oxide nanoparticles by pumpkinplants J Environ Monitor 10 713 (2008)

29 A H Lu E L Salabas and F Schuth Magnetic nanoparticles Syn-thesis protection functionalization and application Angew ChemInt Ed 46 1222 (2007)

30 L H Comas D M Eissenstat and A N Lakso Assessing rootdeath and root system dynamics in a study of grape canopy pruningNew Phytol 147 171 (2000)

Received 10 June 2011 Accepted 20 June 2011

684 J Biomed Nanotechnol 7 677ndash684 2011

Delivered by Ingenta toRice University Fondren Library

IP 12842202150Mon 23 Jul 2012 190458

RESEARCH

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LE

Ren et al Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

(A) (B)

Fig 3 (A) Root activity in the presence of 9 nm and 18 nm -Fe2O3

magnetic nanoparticles of various concentrations (B) Root activity versusconcentrations (0 10 20 40 60 80 and 100 mg middotLminus1 in the presenceof 9 nm -Fe2O3 magnetic nanoparticles

water channels and effectively promotes cell better adsorp-tion to water inorganic ions and other nutritional compo-nents and that the influence of smaller nanoparticle (sizeof 9 nm) is more obvious than that of bigger one (sizeof 18 nm) The nanoparticles with small size readily per-meate through the plant cell wall driven by a concentra-tion gradient This contribution is excellent consistent withthe effect of -Fe2O3 MNPs on the root activity as willbe discussed in the following section Figure 2(C) is theresults of length of sprout as a function of growth timeunder different concentrations It is seen that the growingof sprout is well at lower concentration (10 mg middotLminus1 Thisphenomenon might be attributed to that the higher con-centration can form clusters and tends to block the porousplant cell wallThe absorption of plant roots is an active organ and

synthetic organs root growth and vigor of shoot directly

(A) (B) (C)

Fig 4 Activity of a group of enzymes in the presence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles of various concentrations (A) Activityof catalase (CAT) (B) Activity of peroxidase (POD) (C) Activity of superoxide dismutase (SOD)

affect the level of the nutritional status and produc-tion levels Figure 3 shows the root activity of Chinesemung bean seedlings in the presence of 9-nm and 18-nm-Fe2O3 MNPs of various concentrations As seen fromFigure 3(A) no changes were observed for 18 nm MNPsat 20 mg middotLminus1 compared to the control At the same con-centration 9 nm -Fe2O3 MNPs have a positive effectit proposed an increase of 503 percent for root activityThe best root activity can be seen in the case of 9 nmMNPs of 10 mg middot Lminus1 an increase of 1172 percent isobtained For both of 9 nm and 18 nm nanoparicles rootactivity influenced by lower concentration is more obviousthan that influenced by higher concentration This experi-ment gives a supportive evidence for the growth of sproutshown in Figures 2(B)ndash(C) Figure 3(B) shows the rootactivity under different concentrations in the presence of9 nm -Fe2O3 MNPs It is seen that as increasing the con-centration to 40 mg middotLminus1 the root activity sharply decreasesand consequently again increases to normal case (see dot-ted line) This might be explained either by the significantaggregation of those MNPs at high concentration or by theaccumulation of many -Fe2O3 nanoparticles on the rootsurface (ie the accumulation inhibits the transmission ofwater and other nutritional components)Peroxide accumulation may cause changes in plant

metabolism in several ways They may oxidize sulflhydrylgroups and in combination with superoxides they canform hydroxyl radicals which may be involved in theaging process H2O2 may be involved also in the oxidativebreakdown of indoleacetic acid It has been shown thatincreased H2O2 levels inactivate indoleacetic acid Thisinactivation was reversed upon the introduction of catalaseFigure 4 shows the activity of a group of enzymes such astatalase peroxidase and superoxide dismutase in the pres-ence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles

J Biomed Nanotechnol 7 677ndash684 2011 681

Delivered by Ingenta toRice University Fondren Library

IP 12842202150Mon 23 Jul 2012 190458

RESEARCH

ARTIC

LE

Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

of various concentrations Figure 4(A) shows the effect of-Fe2O3 MNPs on the function of the enzyme catalasewhich is found in all plant tissues As seen a nanoparticleaddition in nutrient solution results in a decrease of activ-ity of catalase while the effect of nanoparticles in theseplants showed no significant concentration or size depen-dence A decrease in catalase activity could lead to theobserved accumulation of H2O2 in tissue since there is anincrease in peroxidase activity (Fig 4(B)) to compensatefor the H2O2 removal by catalase even though the activ-ity is still lower than normal levels Taking 20 mg middotLminus1

of -Fe2O3 MNPs solution as an example the catalaseactivity treated by 18 nm MNPs is a litter higher than thattreated by 9 nm (Fig 4(A)) Peroxidase activity is oppo-site to this result (Fig 4(B)) Similar result can also befound in the case of 10 mg Lminus1-Fe2O3 MNPs solutionAs seen from Figure 4(C) the activity of superoxide dis-mutase was inhibited by the suspension of -Fe2O3 MNPsand shows a size and concentration dependence A signifi-cant effect was found at higher concentration (20 mg middotLminus1and larger size (18 nm) Superoxide dismutase (SOD) inplant catalyzes the destruction of the O2minus free radical thedecrease of activity of SOD result in the accumulation ofO2minus free radical in plant leaf Consequently these freeradicals react with H2O2 produced by chloroplast to formOHbull free radicals which might result in the degradation ofchlorophyll However we observed a quite different phe-nomenon from the test of chlorophyl content as shown inFigure 5(A)As can be seen from Figure 5(A) a slight increasing

on the content of chlorophyll was observed after treatedby -Fe2O3 MNPs It is obvious that the introduction ofnanoparticles is contributed to a synthesis of chlorophyllHowever it is not clear whether this is due to a decreasein peroxidase activity intrinsic peroxidase-like activity of-Fe2O3 nanoparticles possess or both Content of soluble

(A) (B) (C)

Fig 5 Physiological parameters in the presence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles of various concentrations (A) Content ofchlorophyl (B) Content of soluble protein (SP) (C) Content of malondialdehyde (MDA)

protein was highly correlated with the age of plants iecontent of soluble protein decreases with the increasingof the age The results shown in Figure 5(B) reveal that-Fe2O3 MNPs can stimulate the growth of plants espe-cially for those samples treated using 18 nm of 10 mg middotLminus1

nanoparticle suspension Soluble protein was also strongrelative to the Content of chlorophyl of plant leaf With theincrease of content of chlorophyl photosynthesis of plantsincreases resulting in the accumulation of soluble proteinMalondialdehyde (MDA) is an important lipid peroxida-tion product when plant is in stress conditions of agingor injured Its content is closely related to plant senes-cence and stress injury The extent of the damage of plantmembrane system and plant resistance can be known bymeasuring the MDA level of lipid peroxidation The lowcontent of MDA is useful for protecting the structure andfunction of cell membrane As depicted in Figure 5(C)the content of MDA decreases in the present conditionsexcept the treatment using 9 nm of 20 mg middotLminus1 nanopar-ticles indicating that the proxidation of unsaturated fattyacid in cell membrane is weaker than that of the controlThis situation should be due to the increase of the con-tent of SOD CAT and POD in theory It is quite differ-ent from the above mentioned experimental results FromTEM image we are sure that -Fe2O3 MNPs were intro-duced to plant (as will be discussed later) suggesting thatthere may have another mechanism Maybe one of thesethree enzymes or boththree of them waswere replaced by-Fe2O3 MNPs The unusual result obtained under 9 nmof 20 mg middotLminus1 nanoparticles is likely owing to the glomer-ation of small particles Larger particles cannot easily pen-etrate the cell wall and membrane resulting in the lowerutilization efficient and consequently affect the synthesisof chlorophyll and increasing of MDA contentFigure 6 shows TEM (Transmission electron

microscopy JEM-2010 microscope equipped with Oxford

682 J Biomed Nanotechnol 7 677ndash684 2011

Delivered by Ingenta toRice University Fondren Library

IP 12842202150Mon 23 Jul 2012 190458

RESEARCH

ARTIC

LE

Ren et al Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

Fig 6 TEM images of mitochondria in the absence (A) and presence (B) of 9 nm of 20 mg middotLminus1-Fe2O3 MNPs suspension (CndashD) Detection ofnanoparticles in cytoplasm of Chinese mung bean treated with 9 nm of 20 mg middotLminus1-Fe2O3 MNPs suspension

INCA EDS operated at 200 kV accelerating voltage)images of the plant tissues after treating in 9 nm of20 mg middot Lminus1-Fe2O3 MNPs suspension Figure 6(A) isunder treatments of control of epidermal cell of Chinesemung bean root However the cristae of mitochondriabecomes rough and the color of the cytoplasm gets lighter(Fig 6(B)) The tumefaction of mitochondria cannot beobserved indicating that the membrane of mitochondriawas not damaged by the nanoparticles Taking account ofabove mentioned results CAT POD and SOD content ofplant tissue decrease after treating by -Fe2O3 nanoparti-cles this observation demonstrates that the peroxidationof the membrane does not occur Black aggregates werefrequently found in the cytoplasm (Figs 6(C and D))indicating that the sequence of nanoparticle uptakewas from the plant seeds and roots to the stems andleaves

4 CONCLUSIONS

This manuscript reports the first study describing a detailedevidence of impact of -Fe2O3 magnetite nanoparticles

(MNPs) on Chinese mung bean plants by measuring thephysiological parameters such as germination root activ-ity activity of catalase (CAT) peroxidase (POD) andsuperoxide dismutase (SOD) content of chlorophyll sol-uble protein and content of malondialdehyde (MDA) Itshould be noted that although the nanoparticles used inthis study have a diameter size within the nanometrerange aggregates of different sizes were formed in theplant cells Thus it can be concluded that the -Fe2O3

nanoparticles could enter into the tissues or cells fromthe roots Even though more works need further attentionin order to obtain aspects of mechanism including uptakeand translocation and the interactions between the particleswith plant tissue at the cellular level this research never-theless provides convincing evidence that plant uptake isa potential transport pathway of nanoparticles in the envi-ronment Another main significance of this study couldprovide a guideline for the production of so-called sele-nium (Se)-riched rice in China After our inspection sele-nium (Se)-riched rice were produced in two ways One ismanual spraying of Se solution during rice growing andthen through bio-transformation the inorganic selenium

J Biomed Nanotechnol 7 677ndash684 2011 683

Delivered by Ingenta toRice University Fondren Library

IP 12842202150Mon 23 Jul 2012 190458

RESEARCH

ARTIC

LE

Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

into organic selenium and stored in the rice so absorbedby the body Another is the local soil rich in selenium con-tent the rice produced natural selenium Our results willhelp answer the question on how significant physiologicalchanges of plant occur

Acknowledgments X-J Huang thanks ldquoOne HundredPerson Projectrdquo of the Chinese Academy of SciencesChina and N Gu thanks State Key Development Programfor Basic Research of China (Grant No 2006CB933200)and National Natural Science Funds for DistinguishedYoung Scholar (NSFC-60725101) for their financialsupports

References and Notes

1 H X Ren X Chen J H Liu N Gu and X J Huang Toxicity ofsingle-walled carbon nanotube How we were wrong Mater Today13 6 (2010)

2 H Ren and X J Huang Polyacrylate nanoparticles Toxicity or newnanomedicine Eur Respir J 36 218 (2010)

3 S G Xing Y B Jun Z W Hau and L Y Liang Higher accumu-lation of gamma-aminobutyric acid induced by salt stress throughstimulating the activity of diarnine oxidases in Glycine max (L)Merr roots Plant Physiol Biochem 45 560 (2007)

4 D H Lin and B S Xing Root uptake and phytotoxicity of ZnOnanoparticles Environ Sci Technol 42 5580 (2008)

5 D H Lin and B S Xing Phytotoxicity of nanoparticles Inhibi-tion of seed germination and root growth Environ Pollut 150 243(2007)

6 W M Lee Y J An H Yoon and H S Kweon Toxicity andbioavailability of copper nanoparticles to the terrestrial plants mungbean (Phaseolus radiatus) and wheat (Triticum aestivum) Plantagar test for water-insoluble nanoparticles Environ Toxicol Chem27 1915 (2008)

7 V Shah and I Belozerova Influence of metal nanoparticles on thesoil microbial community and germination of Lettuce Seeds WaterAir Soil Poll 197 143 (2009)

8 E Seeger A Baun M Kastner and S Trapp Insignificant acutetoxicity of TiO2 nanoparticles to willow trees J Soil Sediment 9 46(2009)

9 R Barrena E Casals J Colon X Font A Sanchez and V PuntesEvaluation of the ecotoxicity of model nanoparticles Chemosphere75 850 (2009)

10 D Stampoulis S K Sinha and J C White Assay-dependent phy-totoxicity of nanoparticles to plants Environ Sci Technol 43 9473(2009)

11 Y H Ma L L Kuang X He W Bai Y Y Ding Z Y ZhangY L Zhao and Z F Chai Effects of rare earth oxide nanoparticleson root elongation of plants Chemosphere 78 273 (2010)

12 M L Lopez-Moreno G de la Rosa J A Hernandez-Viezcas J RPeralta-Videa and J L Gardea-Torresdey X-ray absorption spec-troscopy (XAS) corroboration of the uptake and storage of CeO2

nanoparticles and assessment of their differential toxicity in fouredible plant species J Agr Food Chem 58 3689 (2010)

13 J Kurepa T Paunesku S Vogt H Arora B M Rabatic J JLu M B Wanzer G E Woloschak and J A Smalle Uptake anddistribution of ultrasmall anatase TiO2 alizarin red S nanoconjugatesin arabidopsis thaliana Nano Lett 10 2296 (2010)

14 S J Lin J Reppert Q Hu J S Hudson M L Reid T ARatnikova A M Rao H Luo and P C Ke Uptake Transloca-tion and transmission of carbon nanomaterials in rice plants Small5 1128 (2009)

15 R Chen T A Ratnikova M B Stone S Lin M Lard G HuangJ S Hudson and P C Ke Differential uptake of carbon nanoparti-cles by plant and mammalian Cells Small 6 612 (2010)

16 C W Lee S Mahendra K Zodrow D Li Y C Tsai JBraam and P J J Alvarez Developmental phytotoxicity of metaloxide nanoparticles to arabidopsis thaliana Environ Toxicol Chem29 1399 (2010)

17 X M Ma J Geiser-Lee Y Deng and A Kolmakov Interactionsbetween engineered nanoparticles (ENPs) and plants Phytotoxicityuptake and accumulation Sci Total Environ 408 3053 (2010)

18 R Nair S H Varghese B G Nair T Maekawa Y Yoshida andD S Kumar Nanoparticulate material delivery to plants Plant Sci179 154 (2010)

19 L Z Gao J Zhuang L Nie J B Zhang Y Zhang N GuT H Wang J Feng D L Yang S Perrett and X Yan Intrinsicperoxidase-like activity of ferromagnetic nanoparticles Nat Nano-technol 2 577 (2007)

20 S P Singh Multifuncational magnetic quantum dots for cancer ther-anostics J Biomed Nanotechnol 7 95 (2011)

21 J Ding J H Zhao K LCheng G F Liu and D H Xiu In Vivophotodynamic therapy and magnetic resonance imaging of cancer byTSPP-coated Fe3O4 nanoconjugates J Biomed Nanotechnol 6 683(2010)

22 K Men S Zeng M L Gou G Guo Y C Gu F LuoX Zhao Y Q Wei and Z Y Qian Preparation of magnetic micro-spheres based on poly(e-caprolactone)-poly(ethylene glycol)-poly(e-caprolactone) copolymers by modified solvent diffusion methodJ Biomed Nanotechnol 6 287 (2010)

23 A Arkhis A Elaissari T Delair B Verrier and B Mandrand Cap-ture of enveloped viruses using polymer tentacles containing mag-netic latex particles J Biomed Nanotechnol 6 28 (2010)

24 L Tian S Li H N Liu Z F Wang and N Y He An automatedmagstation for high-throughput single nucleotide polymorphismgenotyping and the dual-color hybridization J Biomed Nanotech-nol 5 511 (2009)

25 Z Y Li L He Z Y Shi H Wang S Li H N Liu Y B Dai Z FWang and N Y He Preparation of SiO2(PMMAFe3O4 nanopar-ticles and its application in detecting Ecoli O157H7 using chemi-luminescent immunological method J Biomed Nanotechnol 5 505(2009)

26 Z Y Li L He N Y He Z Y Shi H Wang S Li H N LiuX L Li Y B Dai and Z F Wang Chemiluminescent detect ofEcoli O157H7 using immunological method based on magneticnanoparticles J Nanosci Nanotechnol 10 696 (2010)

27 L He Z Y Li J Fu F Wang C Ma Y Deng Z Y Shi H Wangand N Y He Preparation of SiO2(PMMAFe3O4 nanoparticlesusing linolenic acid as crosslink agent for nucleic acid detectionusing chemiluminescent method J Nanosci Nanotechnol 11 2256(2011)

28 H Zhu J Han J Q Xiao and Y Jin Uptake translocation andaccumulation of manufactured iron oxide nanoparticles by pumpkinplants J Environ Monitor 10 713 (2008)

29 A H Lu E L Salabas and F Schuth Magnetic nanoparticles Syn-thesis protection functionalization and application Angew ChemInt Ed 46 1222 (2007)

30 L H Comas D M Eissenstat and A N Lakso Assessing rootdeath and root system dynamics in a study of grape canopy pruningNew Phytol 147 171 (2000)

Received 10 June 2011 Accepted 20 June 2011

684 J Biomed Nanotechnol 7 677ndash684 2011

Delivered by Ingenta toRice University Fondren Library

IP 12842202150Mon 23 Jul 2012 190458

RESEARCH

ARTIC

LE

Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

of various concentrations Figure 4(A) shows the effect of-Fe2O3 MNPs on the function of the enzyme catalasewhich is found in all plant tissues As seen a nanoparticleaddition in nutrient solution results in a decrease of activ-ity of catalase while the effect of nanoparticles in theseplants showed no significant concentration or size depen-dence A decrease in catalase activity could lead to theobserved accumulation of H2O2 in tissue since there is anincrease in peroxidase activity (Fig 4(B)) to compensatefor the H2O2 removal by catalase even though the activ-ity is still lower than normal levels Taking 20 mg middotLminus1

of -Fe2O3 MNPs solution as an example the catalaseactivity treated by 18 nm MNPs is a litter higher than thattreated by 9 nm (Fig 4(A)) Peroxidase activity is oppo-site to this result (Fig 4(B)) Similar result can also befound in the case of 10 mg Lminus1-Fe2O3 MNPs solutionAs seen from Figure 4(C) the activity of superoxide dis-mutase was inhibited by the suspension of -Fe2O3 MNPsand shows a size and concentration dependence A signifi-cant effect was found at higher concentration (20 mg middotLminus1and larger size (18 nm) Superoxide dismutase (SOD) inplant catalyzes the destruction of the O2minus free radical thedecrease of activity of SOD result in the accumulation ofO2minus free radical in plant leaf Consequently these freeradicals react with H2O2 produced by chloroplast to formOHbull free radicals which might result in the degradation ofchlorophyll However we observed a quite different phe-nomenon from the test of chlorophyl content as shown inFigure 5(A)As can be seen from Figure 5(A) a slight increasing

on the content of chlorophyll was observed after treatedby -Fe2O3 MNPs It is obvious that the introduction ofnanoparticles is contributed to a synthesis of chlorophyllHowever it is not clear whether this is due to a decreasein peroxidase activity intrinsic peroxidase-like activity of-Fe2O3 nanoparticles possess or both Content of soluble

(A) (B) (C)

Fig 5 Physiological parameters in the presence of 9 nm and 18 nm -Fe2O3 magnetic nanoparticles of various concentrations (A) Content ofchlorophyl (B) Content of soluble protein (SP) (C) Content of malondialdehyde (MDA)

protein was highly correlated with the age of plants iecontent of soluble protein decreases with the increasingof the age The results shown in Figure 5(B) reveal that-Fe2O3 MNPs can stimulate the growth of plants espe-cially for those samples treated using 18 nm of 10 mg middotLminus1

nanoparticle suspension Soluble protein was also strongrelative to the Content of chlorophyl of plant leaf With theincrease of content of chlorophyl photosynthesis of plantsincreases resulting in the accumulation of soluble proteinMalondialdehyde (MDA) is an important lipid peroxida-tion product when plant is in stress conditions of agingor injured Its content is closely related to plant senes-cence and stress injury The extent of the damage of plantmembrane system and plant resistance can be known bymeasuring the MDA level of lipid peroxidation The lowcontent of MDA is useful for protecting the structure andfunction of cell membrane As depicted in Figure 5(C)the content of MDA decreases in the present conditionsexcept the treatment using 9 nm of 20 mg middotLminus1 nanopar-ticles indicating that the proxidation of unsaturated fattyacid in cell membrane is weaker than that of the controlThis situation should be due to the increase of the con-tent of SOD CAT and POD in theory It is quite differ-ent from the above mentioned experimental results FromTEM image we are sure that -Fe2O3 MNPs were intro-duced to plant (as will be discussed later) suggesting thatthere may have another mechanism Maybe one of thesethree enzymes or boththree of them waswere replaced by-Fe2O3 MNPs The unusual result obtained under 9 nmof 20 mg middotLminus1 nanoparticles is likely owing to the glomer-ation of small particles Larger particles cannot easily pen-etrate the cell wall and membrane resulting in the lowerutilization efficient and consequently affect the synthesisof chlorophyll and increasing of MDA contentFigure 6 shows TEM (Transmission electron

microscopy JEM-2010 microscope equipped with Oxford

682 J Biomed Nanotechnol 7 677ndash684 2011

Delivered by Ingenta toRice University Fondren Library

IP 12842202150Mon 23 Jul 2012 190458

RESEARCH

ARTIC

LE

Ren et al Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

Fig 6 TEM images of mitochondria in the absence (A) and presence (B) of 9 nm of 20 mg middotLminus1-Fe2O3 MNPs suspension (CndashD) Detection ofnanoparticles in cytoplasm of Chinese mung bean treated with 9 nm of 20 mg middotLminus1-Fe2O3 MNPs suspension

INCA EDS operated at 200 kV accelerating voltage)images of the plant tissues after treating in 9 nm of20 mg middot Lminus1-Fe2O3 MNPs suspension Figure 6(A) isunder treatments of control of epidermal cell of Chinesemung bean root However the cristae of mitochondriabecomes rough and the color of the cytoplasm gets lighter(Fig 6(B)) The tumefaction of mitochondria cannot beobserved indicating that the membrane of mitochondriawas not damaged by the nanoparticles Taking account ofabove mentioned results CAT POD and SOD content ofplant tissue decrease after treating by -Fe2O3 nanoparti-cles this observation demonstrates that the peroxidationof the membrane does not occur Black aggregates werefrequently found in the cytoplasm (Figs 6(C and D))indicating that the sequence of nanoparticle uptakewas from the plant seeds and roots to the stems andleaves

4 CONCLUSIONS

This manuscript reports the first study describing a detailedevidence of impact of -Fe2O3 magnetite nanoparticles

(MNPs) on Chinese mung bean plants by measuring thephysiological parameters such as germination root activ-ity activity of catalase (CAT) peroxidase (POD) andsuperoxide dismutase (SOD) content of chlorophyll sol-uble protein and content of malondialdehyde (MDA) Itshould be noted that although the nanoparticles used inthis study have a diameter size within the nanometrerange aggregates of different sizes were formed in theplant cells Thus it can be concluded that the -Fe2O3

nanoparticles could enter into the tissues or cells fromthe roots Even though more works need further attentionin order to obtain aspects of mechanism including uptakeand translocation and the interactions between the particleswith plant tissue at the cellular level this research never-theless provides convincing evidence that plant uptake isa potential transport pathway of nanoparticles in the envi-ronment Another main significance of this study couldprovide a guideline for the production of so-called sele-nium (Se)-riched rice in China After our inspection sele-nium (Se)-riched rice were produced in two ways One ismanual spraying of Se solution during rice growing andthen through bio-transformation the inorganic selenium

J Biomed Nanotechnol 7 677ndash684 2011 683

Delivered by Ingenta toRice University Fondren Library

IP 12842202150Mon 23 Jul 2012 190458

RESEARCH

ARTIC

LE

Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

into organic selenium and stored in the rice so absorbedby the body Another is the local soil rich in selenium con-tent the rice produced natural selenium Our results willhelp answer the question on how significant physiologicalchanges of plant occur

Acknowledgments X-J Huang thanks ldquoOne HundredPerson Projectrdquo of the Chinese Academy of SciencesChina and N Gu thanks State Key Development Programfor Basic Research of China (Grant No 2006CB933200)and National Natural Science Funds for DistinguishedYoung Scholar (NSFC-60725101) for their financialsupports

References and Notes

1 H X Ren X Chen J H Liu N Gu and X J Huang Toxicity ofsingle-walled carbon nanotube How we were wrong Mater Today13 6 (2010)

2 H Ren and X J Huang Polyacrylate nanoparticles Toxicity or newnanomedicine Eur Respir J 36 218 (2010)

3 S G Xing Y B Jun Z W Hau and L Y Liang Higher accumu-lation of gamma-aminobutyric acid induced by salt stress throughstimulating the activity of diarnine oxidases in Glycine max (L)Merr roots Plant Physiol Biochem 45 560 (2007)

4 D H Lin and B S Xing Root uptake and phytotoxicity of ZnOnanoparticles Environ Sci Technol 42 5580 (2008)

5 D H Lin and B S Xing Phytotoxicity of nanoparticles Inhibi-tion of seed germination and root growth Environ Pollut 150 243(2007)

6 W M Lee Y J An H Yoon and H S Kweon Toxicity andbioavailability of copper nanoparticles to the terrestrial plants mungbean (Phaseolus radiatus) and wheat (Triticum aestivum) Plantagar test for water-insoluble nanoparticles Environ Toxicol Chem27 1915 (2008)

7 V Shah and I Belozerova Influence of metal nanoparticles on thesoil microbial community and germination of Lettuce Seeds WaterAir Soil Poll 197 143 (2009)

8 E Seeger A Baun M Kastner and S Trapp Insignificant acutetoxicity of TiO2 nanoparticles to willow trees J Soil Sediment 9 46(2009)

9 R Barrena E Casals J Colon X Font A Sanchez and V PuntesEvaluation of the ecotoxicity of model nanoparticles Chemosphere75 850 (2009)

10 D Stampoulis S K Sinha and J C White Assay-dependent phy-totoxicity of nanoparticles to plants Environ Sci Technol 43 9473(2009)

11 Y H Ma L L Kuang X He W Bai Y Y Ding Z Y ZhangY L Zhao and Z F Chai Effects of rare earth oxide nanoparticleson root elongation of plants Chemosphere 78 273 (2010)

12 M L Lopez-Moreno G de la Rosa J A Hernandez-Viezcas J RPeralta-Videa and J L Gardea-Torresdey X-ray absorption spec-troscopy (XAS) corroboration of the uptake and storage of CeO2

nanoparticles and assessment of their differential toxicity in fouredible plant species J Agr Food Chem 58 3689 (2010)

13 J Kurepa T Paunesku S Vogt H Arora B M Rabatic J JLu M B Wanzer G E Woloschak and J A Smalle Uptake anddistribution of ultrasmall anatase TiO2 alizarin red S nanoconjugatesin arabidopsis thaliana Nano Lett 10 2296 (2010)

14 S J Lin J Reppert Q Hu J S Hudson M L Reid T ARatnikova A M Rao H Luo and P C Ke Uptake Transloca-tion and transmission of carbon nanomaterials in rice plants Small5 1128 (2009)

15 R Chen T A Ratnikova M B Stone S Lin M Lard G HuangJ S Hudson and P C Ke Differential uptake of carbon nanoparti-cles by plant and mammalian Cells Small 6 612 (2010)

16 C W Lee S Mahendra K Zodrow D Li Y C Tsai JBraam and P J J Alvarez Developmental phytotoxicity of metaloxide nanoparticles to arabidopsis thaliana Environ Toxicol Chem29 1399 (2010)

17 X M Ma J Geiser-Lee Y Deng and A Kolmakov Interactionsbetween engineered nanoparticles (ENPs) and plants Phytotoxicityuptake and accumulation Sci Total Environ 408 3053 (2010)

18 R Nair S H Varghese B G Nair T Maekawa Y Yoshida andD S Kumar Nanoparticulate material delivery to plants Plant Sci179 154 (2010)

19 L Z Gao J Zhuang L Nie J B Zhang Y Zhang N GuT H Wang J Feng D L Yang S Perrett and X Yan Intrinsicperoxidase-like activity of ferromagnetic nanoparticles Nat Nano-technol 2 577 (2007)

20 S P Singh Multifuncational magnetic quantum dots for cancer ther-anostics J Biomed Nanotechnol 7 95 (2011)

21 J Ding J H Zhao K LCheng G F Liu and D H Xiu In Vivophotodynamic therapy and magnetic resonance imaging of cancer byTSPP-coated Fe3O4 nanoconjugates J Biomed Nanotechnol 6 683(2010)

22 K Men S Zeng M L Gou G Guo Y C Gu F LuoX Zhao Y Q Wei and Z Y Qian Preparation of magnetic micro-spheres based on poly(e-caprolactone)-poly(ethylene glycol)-poly(e-caprolactone) copolymers by modified solvent diffusion methodJ Biomed Nanotechnol 6 287 (2010)

23 A Arkhis A Elaissari T Delair B Verrier and B Mandrand Cap-ture of enveloped viruses using polymer tentacles containing mag-netic latex particles J Biomed Nanotechnol 6 28 (2010)

24 L Tian S Li H N Liu Z F Wang and N Y He An automatedmagstation for high-throughput single nucleotide polymorphismgenotyping and the dual-color hybridization J Biomed Nanotech-nol 5 511 (2009)

25 Z Y Li L He Z Y Shi H Wang S Li H N Liu Y B Dai Z FWang and N Y He Preparation of SiO2(PMMAFe3O4 nanopar-ticles and its application in detecting Ecoli O157H7 using chemi-luminescent immunological method J Biomed Nanotechnol 5 505(2009)

26 Z Y Li L He N Y He Z Y Shi H Wang S Li H N LiuX L Li Y B Dai and Z F Wang Chemiluminescent detect ofEcoli O157H7 using immunological method based on magneticnanoparticles J Nanosci Nanotechnol 10 696 (2010)

27 L He Z Y Li J Fu F Wang C Ma Y Deng Z Y Shi H Wangand N Y He Preparation of SiO2(PMMAFe3O4 nanoparticlesusing linolenic acid as crosslink agent for nucleic acid detectionusing chemiluminescent method J Nanosci Nanotechnol 11 2256(2011)

28 H Zhu J Han J Q Xiao and Y Jin Uptake translocation andaccumulation of manufactured iron oxide nanoparticles by pumpkinplants J Environ Monitor 10 713 (2008)

29 A H Lu E L Salabas and F Schuth Magnetic nanoparticles Syn-thesis protection functionalization and application Angew ChemInt Ed 46 1222 (2007)

30 L H Comas D M Eissenstat and A N Lakso Assessing rootdeath and root system dynamics in a study of grape canopy pruningNew Phytol 147 171 (2000)

Received 10 June 2011 Accepted 20 June 2011

684 J Biomed Nanotechnol 7 677ndash684 2011

Delivered by Ingenta toRice University Fondren Library

IP 12842202150Mon 23 Jul 2012 190458

RESEARCH

ARTIC

LE

Ren et al Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean

Fig 6 TEM images of mitochondria in the absence (A) and presence (B) of 9 nm of 20 mg middotLminus1-Fe2O3 MNPs suspension (CndashD) Detection ofnanoparticles in cytoplasm of Chinese mung bean treated with 9 nm of 20 mg middotLminus1-Fe2O3 MNPs suspension

INCA EDS operated at 200 kV accelerating voltage)images of the plant tissues after treating in 9 nm of20 mg middot Lminus1-Fe2O3 MNPs suspension Figure 6(A) isunder treatments of control of epidermal cell of Chinesemung bean root However the cristae of mitochondriabecomes rough and the color of the cytoplasm gets lighter(Fig 6(B)) The tumefaction of mitochondria cannot beobserved indicating that the membrane of mitochondriawas not damaged by the nanoparticles Taking account ofabove mentioned results CAT POD and SOD content ofplant tissue decrease after treating by -Fe2O3 nanoparti-cles this observation demonstrates that the peroxidationof the membrane does not occur Black aggregates werefrequently found in the cytoplasm (Figs 6(C and D))indicating that the sequence of nanoparticle uptakewas from the plant seeds and roots to the stems andleaves

4 CONCLUSIONS

This manuscript reports the first study describing a detailedevidence of impact of -Fe2O3 magnetite nanoparticles

(MNPs) on Chinese mung bean plants by measuring thephysiological parameters such as germination root activ-ity activity of catalase (CAT) peroxidase (POD) andsuperoxide dismutase (SOD) content of chlorophyll sol-uble protein and content of malondialdehyde (MDA) Itshould be noted that although the nanoparticles used inthis study have a diameter size within the nanometrerange aggregates of different sizes were formed in theplant cells Thus it can be concluded that the -Fe2O3

nanoparticles could enter into the tissues or cells fromthe roots Even though more works need further attentionin order to obtain aspects of mechanism including uptakeand translocation and the interactions between the particleswith plant tissue at the cellular level this research never-theless provides convincing evidence that plant uptake isa potential transport pathway of nanoparticles in the envi-ronment Another main significance of this study couldprovide a guideline for the production of so-called sele-nium (Se)-riched rice in China After our inspection sele-nium (Se)-riched rice were produced in two ways One ismanual spraying of Se solution during rice growing andthen through bio-transformation the inorganic selenium

J Biomed Nanotechnol 7 677ndash684 2011 683

Delivered by Ingenta toRice University Fondren Library

IP 12842202150Mon 23 Jul 2012 190458

RESEARCH

ARTIC

LE

Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

into organic selenium and stored in the rice so absorbedby the body Another is the local soil rich in selenium con-tent the rice produced natural selenium Our results willhelp answer the question on how significant physiologicalchanges of plant occur

Acknowledgments X-J Huang thanks ldquoOne HundredPerson Projectrdquo of the Chinese Academy of SciencesChina and N Gu thanks State Key Development Programfor Basic Research of China (Grant No 2006CB933200)and National Natural Science Funds for DistinguishedYoung Scholar (NSFC-60725101) for their financialsupports

References and Notes

1 H X Ren X Chen J H Liu N Gu and X J Huang Toxicity ofsingle-walled carbon nanotube How we were wrong Mater Today13 6 (2010)

2 H Ren and X J Huang Polyacrylate nanoparticles Toxicity or newnanomedicine Eur Respir J 36 218 (2010)

3 S G Xing Y B Jun Z W Hau and L Y Liang Higher accumu-lation of gamma-aminobutyric acid induced by salt stress throughstimulating the activity of diarnine oxidases in Glycine max (L)Merr roots Plant Physiol Biochem 45 560 (2007)

4 D H Lin and B S Xing Root uptake and phytotoxicity of ZnOnanoparticles Environ Sci Technol 42 5580 (2008)

5 D H Lin and B S Xing Phytotoxicity of nanoparticles Inhibi-tion of seed germination and root growth Environ Pollut 150 243(2007)

6 W M Lee Y J An H Yoon and H S Kweon Toxicity andbioavailability of copper nanoparticles to the terrestrial plants mungbean (Phaseolus radiatus) and wheat (Triticum aestivum) Plantagar test for water-insoluble nanoparticles Environ Toxicol Chem27 1915 (2008)

7 V Shah and I Belozerova Influence of metal nanoparticles on thesoil microbial community and germination of Lettuce Seeds WaterAir Soil Poll 197 143 (2009)

8 E Seeger A Baun M Kastner and S Trapp Insignificant acutetoxicity of TiO2 nanoparticles to willow trees J Soil Sediment 9 46(2009)

9 R Barrena E Casals J Colon X Font A Sanchez and V PuntesEvaluation of the ecotoxicity of model nanoparticles Chemosphere75 850 (2009)

10 D Stampoulis S K Sinha and J C White Assay-dependent phy-totoxicity of nanoparticles to plants Environ Sci Technol 43 9473(2009)

11 Y H Ma L L Kuang X He W Bai Y Y Ding Z Y ZhangY L Zhao and Z F Chai Effects of rare earth oxide nanoparticleson root elongation of plants Chemosphere 78 273 (2010)

12 M L Lopez-Moreno G de la Rosa J A Hernandez-Viezcas J RPeralta-Videa and J L Gardea-Torresdey X-ray absorption spec-troscopy (XAS) corroboration of the uptake and storage of CeO2

nanoparticles and assessment of their differential toxicity in fouredible plant species J Agr Food Chem 58 3689 (2010)

13 J Kurepa T Paunesku S Vogt H Arora B M Rabatic J JLu M B Wanzer G E Woloschak and J A Smalle Uptake anddistribution of ultrasmall anatase TiO2 alizarin red S nanoconjugatesin arabidopsis thaliana Nano Lett 10 2296 (2010)

14 S J Lin J Reppert Q Hu J S Hudson M L Reid T ARatnikova A M Rao H Luo and P C Ke Uptake Transloca-tion and transmission of carbon nanomaterials in rice plants Small5 1128 (2009)

15 R Chen T A Ratnikova M B Stone S Lin M Lard G HuangJ S Hudson and P C Ke Differential uptake of carbon nanoparti-cles by plant and mammalian Cells Small 6 612 (2010)

16 C W Lee S Mahendra K Zodrow D Li Y C Tsai JBraam and P J J Alvarez Developmental phytotoxicity of metaloxide nanoparticles to arabidopsis thaliana Environ Toxicol Chem29 1399 (2010)

17 X M Ma J Geiser-Lee Y Deng and A Kolmakov Interactionsbetween engineered nanoparticles (ENPs) and plants Phytotoxicityuptake and accumulation Sci Total Environ 408 3053 (2010)

18 R Nair S H Varghese B G Nair T Maekawa Y Yoshida andD S Kumar Nanoparticulate material delivery to plants Plant Sci179 154 (2010)

19 L Z Gao J Zhuang L Nie J B Zhang Y Zhang N GuT H Wang J Feng D L Yang S Perrett and X Yan Intrinsicperoxidase-like activity of ferromagnetic nanoparticles Nat Nano-technol 2 577 (2007)

20 S P Singh Multifuncational magnetic quantum dots for cancer ther-anostics J Biomed Nanotechnol 7 95 (2011)

21 J Ding J H Zhao K LCheng G F Liu and D H Xiu In Vivophotodynamic therapy and magnetic resonance imaging of cancer byTSPP-coated Fe3O4 nanoconjugates J Biomed Nanotechnol 6 683(2010)

22 K Men S Zeng M L Gou G Guo Y C Gu F LuoX Zhao Y Q Wei and Z Y Qian Preparation of magnetic micro-spheres based on poly(e-caprolactone)-poly(ethylene glycol)-poly(e-caprolactone) copolymers by modified solvent diffusion methodJ Biomed Nanotechnol 6 287 (2010)

23 A Arkhis A Elaissari T Delair B Verrier and B Mandrand Cap-ture of enveloped viruses using polymer tentacles containing mag-netic latex particles J Biomed Nanotechnol 6 28 (2010)

24 L Tian S Li H N Liu Z F Wang and N Y He An automatedmagstation for high-throughput single nucleotide polymorphismgenotyping and the dual-color hybridization J Biomed Nanotech-nol 5 511 (2009)

25 Z Y Li L He Z Y Shi H Wang S Li H N Liu Y B Dai Z FWang and N Y He Preparation of SiO2(PMMAFe3O4 nanopar-ticles and its application in detecting Ecoli O157H7 using chemi-luminescent immunological method J Biomed Nanotechnol 5 505(2009)

26 Z Y Li L He N Y He Z Y Shi H Wang S Li H N LiuX L Li Y B Dai and Z F Wang Chemiluminescent detect ofEcoli O157H7 using immunological method based on magneticnanoparticles J Nanosci Nanotechnol 10 696 (2010)

27 L He Z Y Li J Fu F Wang C Ma Y Deng Z Y Shi H Wangand N Y He Preparation of SiO2(PMMAFe3O4 nanoparticlesusing linolenic acid as crosslink agent for nucleic acid detectionusing chemiluminescent method J Nanosci Nanotechnol 11 2256(2011)

28 H Zhu J Han J Q Xiao and Y Jin Uptake translocation andaccumulation of manufactured iron oxide nanoparticles by pumpkinplants J Environ Monitor 10 713 (2008)

29 A H Lu E L Salabas and F Schuth Magnetic nanoparticles Syn-thesis protection functionalization and application Angew ChemInt Ed 46 1222 (2007)

30 L H Comas D M Eissenstat and A N Lakso Assessing rootdeath and root system dynamics in a study of grape canopy pruningNew Phytol 147 171 (2000)

Received 10 June 2011 Accepted 20 June 2011

684 J Biomed Nanotechnol 7 677ndash684 2011

Delivered by Ingenta toRice University Fondren Library

IP 12842202150Mon 23 Jul 2012 190458

RESEARCH

ARTIC

LE

Physiological Investigation of Magnetic Iron Oxide Nanoparticles Towards Chinese Mung Bean Ren et al

into organic selenium and stored in the rice so absorbedby the body Another is the local soil rich in selenium con-tent the rice produced natural selenium Our results willhelp answer the question on how significant physiologicalchanges of plant occur

Acknowledgments X-J Huang thanks ldquoOne HundredPerson Projectrdquo of the Chinese Academy of SciencesChina and N Gu thanks State Key Development Programfor Basic Research of China (Grant No 2006CB933200)and National Natural Science Funds for DistinguishedYoung Scholar (NSFC-60725101) for their financialsupports

References and Notes

1 H X Ren X Chen J H Liu N Gu and X J Huang Toxicity ofsingle-walled carbon nanotube How we were wrong Mater Today13 6 (2010)

2 H Ren and X J Huang Polyacrylate nanoparticles Toxicity or newnanomedicine Eur Respir J 36 218 (2010)

3 S G Xing Y B Jun Z W Hau and L Y Liang Higher accumu-lation of gamma-aminobutyric acid induced by salt stress throughstimulating the activity of diarnine oxidases in Glycine max (L)Merr roots Plant Physiol Biochem 45 560 (2007)

4 D H Lin and B S Xing Root uptake and phytotoxicity of ZnOnanoparticles Environ Sci Technol 42 5580 (2008)

5 D H Lin and B S Xing Phytotoxicity of nanoparticles Inhibi-tion of seed germination and root growth Environ Pollut 150 243(2007)

6 W M Lee Y J An H Yoon and H S Kweon Toxicity andbioavailability of copper nanoparticles to the terrestrial plants mungbean (Phaseolus radiatus) and wheat (Triticum aestivum) Plantagar test for water-insoluble nanoparticles Environ Toxicol Chem27 1915 (2008)

7 V Shah and I Belozerova Influence of metal nanoparticles on thesoil microbial community and germination of Lettuce Seeds WaterAir Soil Poll 197 143 (2009)

8 E Seeger A Baun M Kastner and S Trapp Insignificant acutetoxicity of TiO2 nanoparticles to willow trees J Soil Sediment 9 46(2009)

9 R Barrena E Casals J Colon X Font A Sanchez and V PuntesEvaluation of the ecotoxicity of model nanoparticles Chemosphere75 850 (2009)

10 D Stampoulis S K Sinha and J C White Assay-dependent phy-totoxicity of nanoparticles to plants Environ Sci Technol 43 9473(2009)

11 Y H Ma L L Kuang X He W Bai Y Y Ding Z Y ZhangY L Zhao and Z F Chai Effects of rare earth oxide nanoparticleson root elongation of plants Chemosphere 78 273 (2010)

12 M L Lopez-Moreno G de la Rosa J A Hernandez-Viezcas J RPeralta-Videa and J L Gardea-Torresdey X-ray absorption spec-troscopy (XAS) corroboration of the uptake and storage of CeO2

nanoparticles and assessment of their differential toxicity in fouredible plant species J Agr Food Chem 58 3689 (2010)

13 J Kurepa T Paunesku S Vogt H Arora B M Rabatic J JLu M B Wanzer G E Woloschak and J A Smalle Uptake anddistribution of ultrasmall anatase TiO2 alizarin red S nanoconjugatesin arabidopsis thaliana Nano Lett 10 2296 (2010)

14 S J Lin J Reppert Q Hu J S Hudson M L Reid T ARatnikova A M Rao H Luo and P C Ke Uptake Transloca-tion and transmission of carbon nanomaterials in rice plants Small5 1128 (2009)

15 R Chen T A Ratnikova M B Stone S Lin M Lard G HuangJ S Hudson and P C Ke Differential uptake of carbon nanoparti-cles by plant and mammalian Cells Small 6 612 (2010)

16 C W Lee S Mahendra K Zodrow D Li Y C Tsai JBraam and P J J Alvarez Developmental phytotoxicity of metaloxide nanoparticles to arabidopsis thaliana Environ Toxicol Chem29 1399 (2010)

17 X M Ma J Geiser-Lee Y Deng and A Kolmakov Interactionsbetween engineered nanoparticles (ENPs) and plants Phytotoxicityuptake and accumulation Sci Total Environ 408 3053 (2010)

18 R Nair S H Varghese B G Nair T Maekawa Y Yoshida andD S Kumar Nanoparticulate material delivery to plants Plant Sci179 154 (2010)

19 L Z Gao J Zhuang L Nie J B Zhang Y Zhang N GuT H Wang J Feng D L Yang S Perrett and X Yan Intrinsicperoxidase-like activity of ferromagnetic nanoparticles Nat Nano-technol 2 577 (2007)

20 S P Singh Multifuncational magnetic quantum dots for cancer ther-anostics J Biomed Nanotechnol 7 95 (2011)

21 J Ding J H Zhao K LCheng G F Liu and D H Xiu In Vivophotodynamic therapy and magnetic resonance imaging of cancer byTSPP-coated Fe3O4 nanoconjugates J Biomed Nanotechnol 6 683(2010)

22 K Men S Zeng M L Gou G Guo Y C Gu F LuoX Zhao Y Q Wei and Z Y Qian Preparation of magnetic micro-spheres based on poly(e-caprolactone)-poly(ethylene glycol)-poly(e-caprolactone) copolymers by modified solvent diffusion methodJ Biomed Nanotechnol 6 287 (2010)

23 A Arkhis A Elaissari T Delair B Verrier and B Mandrand Cap-ture of enveloped viruses using polymer tentacles containing mag-netic latex particles J Biomed Nanotechnol 6 28 (2010)

24 L Tian S Li H N Liu Z F Wang and N Y He An automatedmagstation for high-throughput single nucleotide polymorphismgenotyping and the dual-color hybridization J Biomed Nanotech-nol 5 511 (2009)

25 Z Y Li L He Z Y Shi H Wang S Li H N Liu Y B Dai Z FWang and N Y He Preparation of SiO2(PMMAFe3O4 nanopar-ticles and its application in detecting Ecoli O157H7 using chemi-luminescent immunological method J Biomed Nanotechnol 5 505(2009)

26 Z Y Li L He N Y He Z Y Shi H Wang S Li H N LiuX L Li Y B Dai and Z F Wang Chemiluminescent detect ofEcoli O157H7 using immunological method based on magneticnanoparticles J Nanosci Nanotechnol 10 696 (2010)

27 L He Z Y Li J Fu F Wang C Ma Y Deng Z Y Shi H Wangand N Y He Preparation of SiO2(PMMAFe3O4 nanoparticlesusing linolenic acid as crosslink agent for nucleic acid detectionusing chemiluminescent method J Nanosci Nanotechnol 11 2256(2011)

28 H Zhu J Han J Q Xiao and Y Jin Uptake translocation andaccumulation of manufactured iron oxide nanoparticles by pumpkinplants J Environ Monitor 10 713 (2008)

29 A H Lu E L Salabas and F Schuth Magnetic nanoparticles Syn-thesis protection functionalization and application Angew ChemInt Ed 46 1222 (2007)

30 L H Comas D M Eissenstat and A N Lakso Assessing rootdeath and root system dynamics in a study of grape canopy pruningNew Phytol 147 171 (2000)

Received 10 June 2011 Accepted 20 June 2011

684 J Biomed Nanotechnol 7 677ndash684 2011