journal of biomaterials applications magnetic, fluorescent, and … · 2015-07-08 · biomedical...

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Original Article

Magnetic, fluorescent, andthermo-responsivepoly(MMA-NIPAM-Tb(AA)3Phen)/Fe3O4

multifunctional nanospheres prepared byemulsifier-free emulsion polymerization

Ying Gong1, Jingwen Dai2, Huan Li2, Xin Wang1,Haoran Xiong1, Quanyuan Zhang1, Penghui Li3, Changfeng Yi1,Zushun Xu1,3, Haibo Xu2 and Paul K Chu3

Abstract

Magnetic, luminescent, and thermoresponsive multifunctional nanospheres composed of modified Fe3O4 nanoparticles as

the core and rare earth complex Tb(AA)3Phen as the shell are synthesized by emulsifier-free emulsion polymerization.

The core–shell spherical structure has a size between 140 and 220 nm and exhibits strong green fluorescence of the rare

earth complex Tb(AA)3Phen. In the R2 relaxivity and in vivo MRI studies, the R2 relaxivity of the nanospheres is

562.56 mM–1 s–1 and enhanced T2-weighted images are observed from the nanospheres in the liver and spleen after

injection as a contrast agent. The excellent superparamagnetic, thermosensitive, and fluorescent properties render the

nanospheres useful in biomedical engineering and optical imaging.

Keywords

Luminescence, magnetic properties, nanostructure, rare earth

Introduction

Stimuli-responsive polymer microspheres, which havethe ability to change the physical–chemical and col-loidal properties in response to small external stimulifrom the external environment such as temperature,pH, chemicals, light, electrical field, magnetic field,mechanical stress, etc., have promising applications inbiomedical and biotechnological fields such as deliveryof therapeutic agents, separation of biomacromole-cules, cell labeling and sorting, bioimaging, as well assimultaneous diagnosis and therapy.1–5 Magnetic, lumi-nescent, and thermosensitive nanomaterials are of greatscientific and technological interest6–8 and there hasbeen considerable effort to integrate various functionalnanomaterials with multiple discrete function-related components into a single entity for appli-cation to the multimodality biomedical area. Forinstance, Deng et al.9 have prepared novelthermoresponsive polymer magnetic microspheresbased on cross-linked poly(N-isopropylacrylamide)

and with silica-coated magnetic nanoparticlesmodified with 3-(tirmethoxysily)propyl methacrylate(MPS) as the core. These dual-responsive nanospherespossess superparamagnetic and thermoresponsiveproperties, which make them attractive to applicationssuch as target drug delivery and separation of

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DOI: 10.1177/0885328215575761

jba.sagepub.com

1Hubei Collaborative Innovation Center for Advanced Organic Chemical

Materials, Ministry of Education Key Laboratory for The Green

Preparation and Application of Functional Material, Hubei University,

Wuhan, China2Department of Radiology at Union Hospital Tongji Medical College of

Huazhong University of Science and Technology, Wuhan, China3Department of Physics and Materials Science, City University of Hong

Kong, Tat Chee Avenue, Kowloon, Hong Kong, China

Corresponding author:

Zushun Xu, Hubei Collaborative Innovation Center for Advanced

Organic Chemical Materials, Ministry of Education Key Laboratory for

The Green Preparation and Application of Functional Material, Hubei

University, Wuhan, Hubei 430062, China.

Email: [email protected]

http://jba.sagepub.com/


biomacromolecules. Using fluorescence isothiocyanate(FITC)-labeled magnetic silica as the core and cross-linked poly(N-isopropylacrylamide) as the shell, theyhave also produced multi-stimuli-responsive nano-spheres with well-defined core–shell structures.10 CdTequantum dots (QDs) are used as the luminescencesource instead of the organic dye FITC, and lumines-cent/magnetic composite microspheres with MPS mod-ified silica-coated Fe3O4@SiO2@CdTe microspheres asthe core and cross-linked thermosensitive poly(N-iso-propylacrylamide) have been produced.11 The favor-able properties of these magnetic fluorescentnanomaterials enable many potential biomedical appli-cations pertaining to bioimaging, diagnostics, andtherapeutics.12,13

Unfortunately, organic dyes suffer from poor photo-chemical stability, photobleaching, short lifetime, andpotential toxicity14 and the heavy metals used in thesynthesis of the QDs are toxic15 consequently hamper-ing in vivo applications. Fluorescent lanthanide mater-ials are useful as luminescent probes due to theirexcellent chemical and photostability, large Stokesshift, narrow line-width emission bands, high quantumyields, long lifetime, and generally low toxicity.16

However, the combined use of magnetic nanoparticleswith luminescent materials usually leads to fluorescencequenching effects due to the Fe3O4 NPs.17 In our pre-vious work, we introduced a thermoresponsive polymerlayer on Fe3O4 NPs to produce the outer fluorescentlayer18 and direct contact between the magnetic NPsand luminescent polymer was avoided by inserting anonluminescent polymer layer between the Fe3O4 coreand fluorescent polymer shell, which acted as an inertlayer to sustain fluorescence. By further functionaliza-tion with a thermoresponsive polymer, the multi-sti-muli-responsive nanospheres could be used inextended domains. In the work presented here, tofurther endow the multi-stimuli-responsive polymernanospheres with additional favorable properties, weprepared poly(MMA-NIPAM-Tb(AA)3Phen)/Fe3O4

polymer nanospheres with fluorescent, magnetic, andthermosensitive stimuli-responsive properties by emul-sifier-free emulsion polymerization. Emulsifier-freeemulsion polymerization does not require surfactantsand so clean and monodispersed polymer nanospherescould be readily prepared. The lanthanide complexesTb(AA)3Phen have excellent luminescent propertiesdue to the antenna effect of the ligands and f–f electrontransition of Tb3þ. They exhibit characteristic greenfluorescence at �¼ 345 nm. The nanospheres couldexhibit obvious green fluorescence under the irradiationof UV ultraviolet lamp. Compared to other colors,green fluorescent is more easy to be distinguished byhuman eyes. Most importantly, the intensities ofTb(AA)3phen remain almost constant after reaching

the maximum. The photoluminescence stability offersnot only a wide window for practical considerationfor potential applications but also an insight intonanoparticles design of advanced systems.19 The super-paramagnetic, thermosensitive, luminescent, and cyto-compatible properties of the nanospheres aredetermined and in vivo MRI is demonstrated. Ourresults suggest that the nanospheres can be used in bio-medical applications after dialysis treatment whileavoiding the multistep purification processes.

Materials and methods

Materials

Terbium oxide (Tb4O7, 99%) was purchased fromShanghai Yuelong Nonferrous Metals and used withoutfurther purification.Methylmethacrylate (MMA, 98%),acrylic acid (AA, 98%) was purified by distillation underreduced and stored at 5�C, N-isopropylarylamide(NIPAM, 99%) was purchased from Acros Organicsand used as received. Potassium peroxydisulfate (KPS)was purified by recrystallization in distilled water anddried under vacuum. Iron chloride hexahydrate, ironchloride tetrahydrate, ammonium hydroxide(NH3

.H2O, 25–28%), oleic acid (OA), hydrogen perox-ide (H2O2), absolute ethanol, anhydrous sodiumhydrox-ide, undecylenic acid, hydrochloric acid (HCl, 36–38%),and 1,10-phenanthroline (Phen) were obtainedfrom Sinopharm Chemical Reagent Co. Ltd. China.

Preparation of the magnetic fluid (OA/NaUA Fe3O4

particles)

The magnetic fluids were prepared according to themethod reported previously.20 The Fe3O4 particleswere prepared by coprecipitation method. A total of26.115 g FeCl3 � 6H2O and 13.31 g FeCl2 � 4H2O weremixed in 100ml with deoxygenated water, put into a250ml three-necked flask, and 70ml of NH3 � H2O wasadded in order to produce black precipitates. The solu-tion was heated to 75�C for 1 h and then the productFe3O4 particles were washed with distilled water forseveral times to remove the excess NH3 �H2O until apH value of 7 was obtained. The Fe3O4 particles weredispersed in a 250ml three-necked flask with a mixtureof 50ml ethanol and 50ml distilled water under nitro-gen with vigorous stirring, and 5 g OA was added andheated to 80�C for 1 h. After the product OA/Fe3O4

particles were cooled to room temperature, OA/Fe3O4

particles were washed with ethanol three times and dis-persed in 30ml CHCl3, and the mixture was added intoa 250ml three-necked flask with 50ml aqueous solutioncontaining 8 g NaUA. The solution was stirred at roomtemperature for 1 h. The final product OA/NaUA

2 Journal of Biomaterials Applications 0(0)



Fe3O4 particles were under stirring at room tempera-ture until the weight became constant. The solid con-tent in the magnetic fluid was 10.1wt%.

Preparation of rare earth complex Tb(AA)3Phen

Appropriate amounts of Tb4O7 were dissolved in a min-imum amount of nitric acid (HNO3) and hydrogen per-oxide (H2O2) and evaporated to dryness. The dry nitratewas dissolved in ethanol to form the terbium nitratesolution. Afterward, an aqueous solution containingAA (1.08 g), Phen (0.99 g), and ethanol (30ml) wasadded to a round-bottle flask and the Tb(NO3)3 solution(20mmol) was added drop by drop. The mixturewas heated to 60�C for 6 h while magnetically stirredand the final white powder was washed by ethanolseveral times and dried under vacuum at 40�C for laterusing.

Preparation of Poly(MMA-NIPAM-Tb(AA)3Phen)/Fe3O4 nanospheres

The nanospheres were produced by emulsifier-freepolymerization. The magnetic fluid, MMA, NIPAM,and distilled water were introduced into a four-neckedflask under nitrogen gas with vigorous stirring at roomtemperature for 20min. The mixture was heated to75�C and KPS was added. After reacting for 30min,an aqueous solution of Tb(AA)3Phen was added

dropwise in 20min and the reaction proceeded for 4more hours. All the samples were purified by dialysisfor five days and the dry solid after demulsification wasdried under vacuum for two days and stored for lateruse. The procedures to prepare the nanospheres areillustrated in Scheme 1 and the initial quantities ofthe reactants are listed in Table 1.

Characterization

Fourier transform infrared spectroscopy (FTIR) wasperformed on the Perkin Elmer Spectrum OneTransform Infrared Spectrometer (USA) after thedried samples were pressed with KBr into compact pel-lets. The emission and excitation spectra of the nano-spheres were acquired on the F-2500 spectrometer(Hitachi High Technologies Corporation, Japan). Thepolymer emulsion was dispersed in distilled water at0.25mg/ml and put into a quartz cuvette. The structureof the OA/NaUA Fe3O4 particles and nanosphere pow-ders was determined by X-ray diffraction (XRD,X’PertPro, Philips Corp. Nederland) with Cu Ka radi-ation (�¼ 0.15418 nm) at a scanning rate of 5�/min.The structure of the nanospheres was characterized bytransmission electron microscopy (TEM, Tecnai G20,FEI Corp. USA) at an accelerating voltage of 200 kV.The TEM samples were prepared by placing one dropof the diluted microsphere solution on a carbon-coatedcopper grid and allowing the solvent to evaporateslowly at room temperature. The morphology wasassessed by scanning electron microscopy (SEM,JSM6510LV, JEOL, Japan). The SEM samples wereprepared by putting the diluted nanosphere solutionon glass slides, dried at room temperature, and goldcoated. The magnetic properties were studied on avibrating sample magnetometer (VSM, HH-15,China) at 298K under an applied magnetic field. Thehydrodynamic diameter of the nanospheres was deter-mined by dynamic light scattering (DLS, AutosizeLoc-Fc-963, Malvern Instrument). Thermogravimetricanalysis (TGA) was performed with the dried powdersamples on the Perkin-Elmer TGA-7 from room tem-perature to 750�C at a heating rate of 10�/min. Theconfocal laser scanning microscopy (CLSM) imagesof the slices were acquired on the Spectra Physics

OA NaUA

Fe3O4

KPS75°C

MMANIPAM

MMANIPAM

Tb(AA)3Phen

Scheme 1. Schematic illustration of the process to prepare the

multifunctional core-shell poly(MMA-NIPAM-Tb(AA)3Phen)/

Fe3O4 nanospheres.

Table 1. Initial quantities of the reactants.

Sample code MMA (g) NIPAM (g) Tb(AA)3Phen (g) Magnetic fluid (g) KPS (g) H2O (ml)

S1 2 0.7 0.08 1.5 0.07 50

S2 2 0.7 0.08 3.0 0.07 50

S3 2 0.7 0.08 4.5 0.07 50

S4 2 0.7 0.08 6.0 0.07 50

Gong et al. 3



MaiTai HP tunable 2-photon (690–1040 nm) laser con-focal microscopy (Carl Zeiss LSM710). The R2 mapimages measure and in vivo MRI experiments were per-formed at 25�C on a 3T whole-body MR scanner(MAGNETOM Trio, A Tim System 3T, Siemens,Munich, Germany) with a wrist joint coil. In thein vitro MRI experiments, the parameters were asfollows: field of view (FOV)¼ 120mm, slice thick-ness¼ 2.0mm, base resolution¼ 384� 384, multipleecho times (TE)¼ 40, 80, 120, 160, 200, 240, 280ms,repetition time (TR)¼ 2890ms, and scanning time-¼ 13–14min. In the in vivo MRI experiments, theFOV was 100mm, base resolution was 192� 192,slice thickness was 2.0mm, multiple TE was 62ms,TR was 2890ms, and flip angle was 120�.

Iron content determination

The iron content in the dialyzed nanospheres latexeswas measured by inductively coupled plasma atomicemission spectrometry. The nanospheres latex (1 g)and nitric acid (5ml) were added to 100ml dual-usebottles and heated at 120�C until the latex dissolvedcompletely. After dilution to 50ml with distilledwater, the solution was transferred to a centrifugetube for measurement. The iron content in the solutionwas determined at the specific Fe absorption wave-length of 248.3 nm.

R2 relaxivity measurement

The suspension of the nanospheres with different Feconcentrations was diluted with distilled water to theFe concentration range between 0 and 0.035mmol. Thesamples were added to 0.5ml plastic test tubes and puton 96-well plates in a clinical 3T MR scanner. The T2

relaxation rates (1/T2) versus Fe concentration rela-tionship and T2 relaxivity were determined based on alinear fit.

In vivo T2 MR image observation

The T2 MR images were acquired on the 3T MR scan-ner with 190 g SD mice as the animal model. The micewere anesthetized by 1ml of 10% chloral hydrate andthe MRI solution (S3) was injected into the mousethrough the tail vein at a dose of 4mg of Fe per kgbody weight. The mice were scanned before and afteradministration of the contrast agent and the imageswere taken at different times of 10min, 30min,1, 2, 3, and 8 h after injecting the nanospheres solution.The animals were treated according to the protocols ofthe Institutional Animal Care and Use Committee atHubei University as approved by the InstitutionalAnimal Care.

Histological analysis and optical imaging

The mice were sacrificed 8 h after injection with thenanospheres solution. The liver, spleen, kidney, andlung were fixed in 4% paraformaldehyde for 24 h andtransferred to 30% sucrose in the PBS buffer. The tis-sues were prepared by Prussian blue staining for histo-logical analysis. The luminescence in vivo analysis andthe slides of liver and spleen were prepared for two-photon 740 nm CLSM.

Results and discussion

Chemical structure and morphology

The FTIR spectrum of Tb(AA)3Phen is depicted inFigure 1. The absorption peaks at 850 and 669 cm–1 cor-respond to the d(C–H) in Phen and those at 1538 and1428 cm–1 are associated with the vas(–COO–) and va (–COO–) in AA. Disappearance of the characteristicabsorption peak in AA v(C¼O) at 1700 cm–1 showsAA coordination with Tb3þ. The chemical structure ofthe OA/NaUA Fe3O4 particles and nanospheres is eval-uated by FTIR (Figure 2). The absorption peak at570 cm–1 corresponds to Fe–O vibration and those at2922, 2848, and 1558 cm–1 peaks are assigned to C–Cvibration in NaUA and –CH3, –CH2 stretchingvibration, indicating modification of Fe3O4 withOA and NaUA. The peak at 1731 cm–1 is related to the–COO– in PMMAand those at 1639 and 1551 cm–1 stemfrom C¼O stretching vibration and N–H bendingvibration of PNIPAM, respectively. The FTIR spectraindicate that microspherical nanospheres poly(MMA-NIPAM-Tb(AA)3Phen)/Fe3O4 have been prepared.

Thermal gravimetric (TG) analysis is performed onthe unextracted powder of the nanospheres and the TGcurves are shown in Figure 3. The 61.88% weight lossof OA/NaUA Fe3O4 particles at 650�C corresponds

100

80

60

40

20

04000 3500 3000 2500 2000 1500 1000 500

Wavelength (cm–1)

Tran

smitt

ance

%

Figure 1. FTIR spectrum of Tb(AA)3Phen.




to the OA- and NaUA-modified magnetic fluid.The residual weight percentages of S1, S3, and S4 at650�C are 2.89, 10.15, and 13.85%, respectively. Sincethe thermal analysis is performed in a N2 atmosphere,Fe3O4 oxidation can be neglected. As shown by thedata, the weight loss occur in two stages, the firststage at a temperature below 400�C resulting from thenanospheres on the surface of the modified OA andNaUA and the second stage ending at a temperatureof 650�C due to organic decomposition. The remainingmaterials are composed of mainly Fe3O4 particles andrare earth Tb.

The phase composition of the nanospheres is deter-mined by XRD. As shown in Figure 4, the XRD pat-terns of the prepared OA/NaUA Fe3O4 particlesindicate a cubic structure of Fe3O4 and there are nocharacteristic diffraction peaks from other impurities.According to the JCPDS 19-629 (JCPDS: JointCommittee on Powder Diffraction Standards), the

broad peaks at 2y¼ 30.25�, 35.70�, 43.16�, 53.94�,57.42�, and 62.83� are ascribed to the (220), (311),(400), (422), (511), (440) planes of Fe3O4,respectively.21,22

The morphology of the nanospheres is examined bySEM and TEM (Figure 5). The as-prepared nano-spheres consist of monodispersed spheres with a meanparticle size ranging from 140 to 220 nm and smoothsurface. These nanospheres have narrow size distribu-tion. The core–shell structures can be clearly distin-guished because of the different color contrastbetween the cores and shells and almost every core–shell composite nanoparticles contain several Fe3O4

cores.

Photoluminescent properties

In order to examine the fluorescent properties of thenanospheres, the polymer emulsion is dispersed inwater at a concentration of 0.25mg/ml. The broad exci-tation bands from �¼ 250 to 450 nm is observed using amonitoring wavelength of �¼ 547 nm. The peak at�¼ 345 nm related to the p!p* electron transition ofthe ligands can be identified. As shown in Figure 6, thecharacteristic emission peaks of Tb3þ excited by�¼ 345 nm belong to the transitions of 5D4!

5F6

(492 nm), 5D4!5F5 (547 nm), 7D4!

5F4 (587 nm),and 5D4!

7F3 (623 nm), with the predominant5D4!

5F5 hypersensitive transition at �¼ 547 nm.The nanospheres solution shows the characteristicemission of Tb3þ ion in liquid solution, most import-ant, we can easily distinguish the green fluorescence byhuman eyes. To study the potential of the nanospheresused as optical probes, the fluorescence intensities fromliver and spleen are monitored in vivo to confirm accu-mulation of nanospheres solution. As shown inFigure 7, there is not any fluorescence observed from

Inte

nsity

(A

.U.)

Nanospheres311

220

OA/ NaUA Fe3O4

400

10 20 30 40 50 60 70 802 θ (degree)

511

533

Figure 4. XRD patterns of the OA/NaUA Fe3O4 particles and

nanospheres.

100

80

60

S1

S3

S4

OA/NaUA Fe3O4

Temperature (°C)

40

400 600

20

2000

Wei

ght (

%)

Figure 3. TG curves of the OA/NaUA Fe3O4 particles, S1, S3,

and S4.

Tran

smitt

ance

%

4000 3500 3000

Nanospheres

OA/NauA Fe3O4

2500 2000 1500 1000 500

Wavelength (cm–1)

Figure 2. FTIR spectra of the OA/NaUA Fe3O4 particles and

composite nanospheres.

Gong et al. 5



the liver (Figure 7(b)) and spleen (Figure 7(d)) withoutinjection of the nanospheres solution. In contrast, greendots are observed from slices of the liver Figure 7(a))and spleen (Figure 7(c)) after injection with nano-spheres solution, and the color maps of fluorescentimages show more details about the organ signals.Due to nanospheres solution considerable accumula-tion in liver and spleen, these studies provide clear evi-dence that nanospheres solution can be used as opticalprobes in mice. The integration of the fluorescence with

superparamagnetic property in the prepared nano-spheres, making them more attractively be used asdual-modality optical/MRI probes for diagnosing ofcancer cells in liver and spleen in clinical application.

Magnetic properties

The typical hysteresis loops for Fe3O4 NPs, saturationmagnetization are displayed in Figure 8 and the satur-ation magnetization values are listed in Table 2. It is

(a) (b)

(c) (d)

Figure 5. TEM images: (a) S1, (b) S3, (c) S4, SEM image (d) S2.

5000

4000

3000

2000

1000

0

6000

5000

4000

3000

2000

1000

0

250 300

345

492

547

587

623

Inte

nsity

(a.

u.)

Inte

nsity

(a.

u.)

350Wavelength (nm) Wavelength (nm)

400 500450 450 600550 650

(a) (b)

Figure 6. (a) PL excitation spectra of the nanospheres (�¼ 547 nm); (b) emission spectra of the nanospheres (�¼ 345 nm).




well known that the saturation magnetization of mag-netic composite materials depends on the mass percent-age of the magnetic substance.23,24 The saturationmagnetization of the nanospheres goes up with the

amount of OA/NaUA Fe3O4 particles introduced intothe core. The saturation magnetization values of theOA/Fe3O4 particles, OA/NaUA Fe3O4 particles, S2and S3, are 28.34, 17.84, 3.51, and 7.34 emug–1, respect-ively. The saturation magnetization value of S2 is smal-ler than that of S3 due to the smaller concentration ofOA/NaUA Fe3O4 particles in the nanospheres. Thesamples exhibit a magnetic behavior with negligiblecoercivity or remanences, indicating the as-preparednanospheres are suitable for applications in drug deliv-ery or separation.

(a) (b)

(c) (d)

Figure 7. CLSM images of tissue slices: (a) liver slice and (c) spleen slice after injection with nanospheres solution; (c) liver slice and

(d) spleen slice without injection of nanospheres solution.

30

20

10

–10

–20

–30

–15000 15000–10000 10000–5000 5000

S3

S2

0

OA/Fe3O4

OA/NaUA Fe3O4

Magnetic field (Oe)

Mag

netiz

atio

n (e

um/g

))

0

Figure 8. Magnetization curves of the OA/Fe3O4 particles,

OA/NaUA Fe3O4 particles, S2, and S3 at room temperature.

Table 2. Saturation magnetization (Ms) of the Fe3O4/OA

particles and nanospheres containing different mass ratios of

Fe3O4 particles.

Sample Ms (emug–1)

OA/Fe3O4 particles 28.34

OA/NaUA Fe3O4 particles 17.84

S2 3.51

S3 7.34

Gong et al. 7



Thermosensitive properties

The thermosensitive properties of nanospheres areexamined by DLS. The particle diameter changesfrom large to small because of the excellent temperatureresponsive of PNIPAM. When the materials are heatedto a temperature higher than the volume phase transi-tion temperature, the microgels undergo coil-to-globuletransition by expelling water thus resulting in dramaticsize reduction.25 The hydrodynamic size and PDI of theS1 and S2 are shown in Figure 9(a) and (b) and thenanospheres have a narrow size distribution. Asshown in Figure 9(c), the particle size decreases whenthe temperature increases from 25 to 45�C, because ofthe multiphase behavior with changing temperature forthe PNIPAM thermosensitive materials.

Cell viability

The biocompatibility is assessed and the relative cellviability of HEK293T human kidney cells incubatedwith different concentration of the nanospheres solu-tion (1200, 1000, 800, 600, 400, and 200 mg/ml) in thecomplete medium is presented in Figure 10. Each con-centration of sample was repeated five times. The cellsare plated on 96-well plates at a density of 1� 104/well,grown for 24 h at 37�C and 5% CO2 in DMEMmedium, washed with PBS (pH¼ 7.4) and incubated

Inte

nsity

(%

)

Inte

nsity

(%

)Size (nm) Size (nm)

10 100

S1

220

S2S1S2S3S4

210

200

190

180

170

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150

140

130

120

25 30 35 45

Temperature (°C)40

Hyd

rody

nam

ic D

iam

eter

(nm

)

1000 10 100 1000

PDI:0.008Z-A:215.8

PDI:0.015Z-A:151.5

(a) (b) (c)

Figure 9. (a) Hydrodynamic diameter and size distribution of S1; (b) hydrodynamic diameter and size distribution of S2;

(c) hydrodynamic diameter of S1, S2, S3, S4 as a function of temperature.

100

80

60

40

20

0Control 200 400 600 800 1000 1200

Concentration of nanospheres (ug/mL)

Cel

l via

bilit

y (%

)

Figure 10. Cell viability test of HEK293T human kidney cells at

different nanospheres concentrations.

Fe (mM) 0

30

25

20

15

10

5

00.000 0.005 0.010 0.015 0.020 0.025 0.030 0.040

Fe (mM)

T2–1

(s–1

)

r2=562.56mM–1s–1

0.035

0.010 0.015 0.020 0.025 0.030 0.035 0.040(a)

(b)

Figure 11. (a)T2-weighted MR images of distilled water

and nanospheres solution with different Fe concentrations.

(b) Relaxation rate (1/T2, S–1) as a function of Fe concentration

(mM) in the nanospheres solution.




with different concentrations of nanospheres solution(dose diluted by complete medium, 200–1200 ug/ml)for another 24 h. Afterward, the supernatants wereremoved and cells were washed with PBS for severaltimes. 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetra-zolium bromide (MTT) solution (10 ml/well) was addedinto each well and incubated for 4 h and the culturemedium was discarded. Each well the supernatant wasreplaced with 150�l dimethyl sulfoxide (DMSO) to dis-solve the crystals by agitation for 10min. The survivingcells convert MTT to formazan generating a blue-purple color when dissolved in DMSO. The intensityof formazan is measured at a wavelength of 565 nm ona plate reader for enzyme-linked immunosorbentassays. The following formula is used to calculate theinhibition of cell growth: cell viability (%) ¼ (meanAbs. value of treatment group / mean Abs. value ofcontrol) �100%. The viability of the HEK293Thuman kidney cells is over 90% in the range of 200–1200 mg/ml and the results demonstrate goodcytocompatibility.

R2 relaxivity and in vivo T2 MR image measurement

The R2 map images and T2 relaxation times are mea-sured on a 3T MRI instrument equipped with a wristcoil. Seven aqueous solutions with different concentra-tions [0, 0.010, 0.015, 0.020, 0.025, 0.030, and 0.035(Fe)mmol] are prepared by diluting the MRI solutionwith distilled water. The images and relaxivity are mea-sured. The Fe3O4 particles shorten the spin–spin relax-ation time (T2), resulting in a decreased MRI signalintensity. As the Fe concentration in the nanospheresincreases, the MR contrast signal intensity drops pro-portionally (darkening of the images) due to shorteningof T2 by the embedded Fe3O4 particles. The R2 relax-ivity is then estimated from the slope in the plots of1/T2 versus Fe concentration and r2 is calculated tobe 562.56mM–1 s–1.

Magnetic metal oxide NPs have been very widelystudied as contrast agents in MRI26,27 which is a power-ful medical imaging technique because of its virtuallyunlimited tissue penetration depth and the NPs can be

Before 10 min 30 min

3h2h

100

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20

0Before 10 30 60 120 180

LiverSpleen

Time after administeration (min)480

Rel

ativ

e si

gnal

inte

nsity

(%

)

1h

8h

Figure 12. T2-weigthed MR images of the mouse acquired before and at different time points after injection of the nanospheres

solution and relative MRI signal intensity of the liver and spleen before and after injection of the nanospheres solution.

Gong et al. 9



detected anywhere in the body.28 In particular, ironoxide NPs have received significant attention due tothe proven biocompatibility and biodegradability.Iron from degraded NPs is used in the body such ashemoglobin in red blood cells.29,30 The electron spinmagnetic moment can efficiently induce transversewater proton relaxation because a slow electron spinmotion closely matches slow water proton relaxation.31

In MRI, the superparamagnetic NPs generate localinhomogeneity in the magnetic field decreasing thesignal. Therefore, the regions in the body with ironoxide NPs appear darker in the MR images as aresult of the negative contrast. The T2-weighted MRimages acquired after intravenous injection of thenanospheres show obvious darkening effects in theliver and spleen. The images of Prussian blue stainedtissue slices further confirm accumulation of the nano-spheres in the tissues.32 Figure 12 depicts a series ofMRI images of the mouse spleen and liver after intra-venous injection of the nanospheres solution. Thespleen and liver tissues are selectively enhanced inT2-weighted MRI because the nanospheres are deliv-ered and accumulate in the spleen and liver, as shownin Figure 12. The most significant reduction in the rela-tive of MRI signal from the spleen and liver is 91.8% at6 h and 96.8% at 3 h after injection.

To further investigate accumulation of nanospheresin the mouse, tissue slices are taken from the liver,spleen, kidney, and lung 8 h after injection and stainedwith Prussian blue. As shown in Figure 13, the bluedots in the liver and spleen confirm the existence ofiron oxide in these organs, but there is not any blue

dot in kidney and lung. The results show that the nano-spheres can serve as negative (T2) MR contrast agentsin the liver and spleen.

Conclusion

Fluorescent, magnetic, and thermosensitive nano-spheres are prepared and investigated as a T2 MRIcontrast agent and fluorescence probe. The nano-spheres exhibit superparamagnetism, thermosensitivity,and characteristic green fluorescence of rare earthTb3þ. There is a clear negative contrast enhancementin the T2 images and R2 reaches 562.56mM–1 s–1 asexpected from the moderate r2. This new class of T2

MRI contrast agent is useful in MR imaging relatedto biomedical research.

Authors’ contribution

Ying Gong and Jingwen Dai contributed equally to this

project.

Funding

The work was supported by the National Natural Science

Foundation of China (Grant No. 51273058). This work wassupported, in part, by Nation Nature Science Foundation ofChina (NSFC, 81372369, 81171386), National Basic ResearchProgram of China (973 Program, 2011CB933103), Hubei Key

Laboratory Foundation of Molecular Imaging (grantnumber: 208-69), Hong Kong Research Grants Council(RGC) General Research Funds (GRF) No. CityU 112212,

and City University of Hong Kong Applied Research Grant(ARG) No. 9667085.

(a) (b)

(c) (d)

Figure 13. Prussian blue staining images of (a) liver, (b) spleen, (c) kidney, and (d) lung tissue slices excised from the mice 8 h after tail

vein injection of the nanospheres solution.




Declaration of conflicting interests

None declared.

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