dual-targeting superparamagnetic iron oxide nanoprobes with high andlow target density for brain...

7/24/2019 Dual-targeting Superparamagnetic Iron Oxide Nanoprobes With High Andlow target density for brain glioma imaging

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Dual-targeting superparamagnetic iron oxide nanoprobes with high and

low target density for brain glioma imaging

Juan Zhang a,1, Ning Chen b,1, Hao Wang c, Wei Gu a, Kang Liu a, Penghui Ai d, Changxiang Yan d,,Ling Ye a,

a School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR Chinab Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, PR Chinac Department of Anatomy, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, PR Chinad Department of Neurosurgery, Beijing Sanbo Brain Hospital, Capital Medical University, Beijing 100093, PR China

h i g h l i g h t s

The FA-c(RGDyK) dual-targeting,Cy5.5 labeled Fe3O4nanoprobes wereprepared.

The dual-targeting nanoprobes wereapplied for MR/NIR imaging ofgliomas.

The synergistic targeting ability ofdual-targeting nanoprobes wasdemonstrated.

The density of dual-target plays animportant role in targeting specificity.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:

Received 16 December 2015Revised 30 January 2016Accepted 2 February 2016Available online 2 February 2016

Keywords:

FA-c(RGDyK) dual-targetIron oxide nanoparticles

MR/NIR imagingBrain gliomas

a b s t r a c t

A major limit of superparamagnetic iron oxide nanoparticles (SPIONs) as a magnetic resonance (MR)imaging nanoprobe in clinical applications is that the SPIONs are unable to reach sufficient concentra-tions at the tumor site by passive targeting to produce an obvious contrast effect for tumor imaging.Single-targeting SPIONs systems have been applied to improve the contrast effect. However, they stillsuffer from a lack of efficiency and specificity of the SPIONs to tumors. Herein, we developed folic acid(FA) and cyclic Arg-Gly-Asp-D-Tyr-Lys (c(RGDyK)) dual-targeting nanoprobes based on Cy5.5 labeledFe3O4nanoparticles (NPs). The synergistic targeting ability of the dual-targeting Fe3O4NPs and the effectof the dual-target density on targeting specificity were investigated in brain glioma-bearing mice.In vivo

T2-weighted MR imaging of brain glioma-bearing mice and ex vivo near-infrared imaging of brainsharboring gliomas suggested that the combination of dual-target increased the uptake of NPs by glioma,consequently, enhanced the contrast effect. Moreover, it was revealed that the density of dual-targetplays an important role in targeting specificity.

2016 Published by Elsevier Inc.

1. Introduction

Superparamagnetic iron oxide nanoparticles (SPIONs), such asmagnetite (Fe3O4) nanoparticles (NPs), are the first T2 magnetic

http://dx.doi.org/10.1016/j.jcis.2016.02.004

0021-9797/2016 Published by Elsevier Inc.

Corresponding authors.

E-mail addresses:[email protected](C. Yan), [email protected](L. Ye).1 These authors contribute equally to this work.

Journal of Colloid and Interface Science 469 (2016) 8692

Contents lists available at ScienceDirect

Journal of Colloid and Interface Science

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j c i s
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resonance (MR) nanoprobe used for clinical diagnosis of solidtumors [14]. Nevertheless, SPIONs have limited extravasationability and therefore are subject to uptake by the reticuloendothe-lial system (RES) [5], which makes SPIONs less efficient for MRimaging of tumors. Various targeting molecules have beenattached to the surface of SPIONs to enhance the specific targetingand increase the uptake of SPIONs in tumor by taking advantage oftheir high affinity to cell surface receptors that are overexpressedin tumor cells [610]. For instances, monoclonal antibodies,transferrin (Tf) protein, cyclic arginine-glycine-aspartic acid(cRGD), and folic acid (FA) are commonly used for the design oftumor-targeted MR nanoprobes [11]. However, single-targetingSPIONs still suffer from a lack of efficiency and specificity totumors and exhibit insufficient MR imaging contrast effects dueto receptor saturation[12].

Considering the fact that multiple types of receptors are typi-cally overexpressed on the surface of tumor cells[1315], severaldual-targeting systems have been developed to enhance thetargeting specificity and increase the uptake of NPs in tumors[1620]. Improved efficacy in cancer therapy and imaging has beendemonstrated with combinations of folic acid (FA) and mAb225monoclonal antibody[21], angiopep-2 peptides (ANG) and cyclicarginine-glycine-aspartic acid (cRGD) [22], transferrin (Tf) andwheat germ agglutinin (WGA)[23], and Tf and RGD [24]. It hasbeen demonstrated that in single targeting systems, the targetingefficiency not only depends on the target specificity but also onthe target density [25]. Understanding the effect of dual-targetdensity on targeting specificity is therefore of great importancebut rarely studied.

In this study, we developed FA and cyclic Arg-Gly-Asp-D-Tyr-Lys (c(RGDyK)) dual-targeting Fe3O4 nanoprobes to enhance thetargeting specificity toward brain glioma. FA and c(RGDyK) wereadopted because c(RGDyK) is able to target avb3integrin receptorsoverexpressed in endothelial cells in brain tumors [26,27], whereasFA targets the folate receptor (FR) that is overexpressed in bothbrain capillary endothelial cells and brain glioma cells [28].

Moreover, in comparison with antibody targets, FA and c(RGDyK)could decrease the steric hindrance of nanoprobes across BBBand increase the binding efficiency of the targeting molecules tothe target sites[29]. Therefore, the FA and c(RGDyK) dual-targetcould not only enable Fe3O4nanoprobes to cross the BBB but alsoincrease their uptake by glioma cells [30], which would conse-quently enhance the MR contrast effect between glioma and nor-mal tissue. Meanwhile, the incorporation of near infrared (NIR)fluorescent dye Cy5.5 into the nanoprobes provides additionalNIR imaging mode. The synergistic targeting ability of dual-targetand the effect of dual-target density on targeting specificity towardgliomas were investigated in brain glioma-bearing mice byT2-weighted MR and NIR imaging.

2. Experimental

2.1. Materials

Iron(III) acetylacetonate (Fe(acac)3), benzyl ether,1,2-hexadecanediol, and oleylamine were purchased fromSigmaAldrich (USA). Oleic acid (OA, >90%) was obtained from AlfaAesar (Johnson Matthey, UK). N-(Trimethoxysilylpropyl) ethylenediamine triacetic acid, trisodium salt (TETT, 45% in water) wasprovided by Gelest Inc (USA). Bi-functional polyethylene glycol(H2N-PEG3500-COOH), folic acid conjugated poly(ethylene glycol)(FA-PEG3500-NH2) and c(RGDyK) were received from JenKem

Technology Co. Ltd. (Beijing, China). Other chemicals were ofanalytical grade and used as received.

2.2. Synthesis of oleate-capped Fe3O4 (Fe3O4-OA) NPs

The Fe3O4-OA NPs were synthesized according to the reportedmethod [31]. Briefly, 2 mmol of Fe (acac)3, 10mmol of 1,2-hexadecanediol, 6 mmol of oleic acid, 6 mmol of oleylamine, and20 mL of benzyl ether were mixed and heated at 100 C for 1 hunder vacuum, 200 C for 2 h and 300 C for 1 h under a nitrogenflow. After cooling to room temperature, the mixture was precipi-tated with ethanol, separated by centrifugation. The precipitatewas dissolved in hexane containing small amount of oleic acidand oleylamine and the undissolved impurity was removed by cen-trifugation. The Fe3O4-OA NPs were obtained by the precipitationwith ethanol and then centrifugation and dispersed in hexane forfurther use.

2.3. Synthesis of TETT-modified Fe3O4(Fe3O4-TETT) NPs

The synthesis of TETT-modified Fe3O4was performed accordingto the reported method with minor modification[32]. Typically,100 mg of Fe3O4-OA NPs, 60 mL of anhydrous toluene, and 60 lLof acetic acid were mixed and sonicated for 15 min. Subsequently,3 mL of TETT was added and the mixture was stirred at 70 C for48 h. Then, the precipitate was collected by centrifugation, washedwith toluene and ethanol, and dialyzed against deionized water,finally lyophilized to obtain the powder product.

2.4. Synthesis of dual-targeting, Cy5.5-labeled Fe3O4 NPs

Prior to synthesize the dual-targeting nanoprobes, the Cy5.5was labeled to Fe3O4 NPs. In brief, 1.11 mg of Cy5.5-NHS and6.99 mg of H2N-PEG2000-NH2 at molar ratio of 1:4 (pH = 8) weremixed at room temperature to produce Cy5.5-PEG2000-NH2. Then,100 mg of Fe3O4-TETT NPs was activated by adding 0.25 mg ofEDC and 0.38mg of NHS in 10 mL ofPBS(0.1 M, pH= 6.0), followedby reacting with Cy5.5-PEG2000-NH2(pH = 8) at room temperaturefor 12 h. The resulting product was purified using a centrifugal fil-

ter (MWCO = 5000) and lyophilized to yield Fe3O4-TETT-Cy5.5 NPs.To synthesis dual-targeting nanoprobes, 100 mg of Fe3O4-TETT-

Cy5.5 NPs was activated by EDC and NHS, followed by adding53mg of H2N-PEG3500-RGD and 45.2 mg of H2N-PEG3500-FA. Thereaction was proceeded at room temperature for 12 h (pH = 8) toobtain the high density dual-targeting nanoprobes (Fe3O4-PEG-RGD-FAh). Meanwhile, the reaction of activated Fe3O4-TETT-Cy5.5NPs with 13.3mg of H2N-PEG3500-RGD, 11.3 mg of H2N-PEG3500-FA,and 67.7 mg of H2N-PEG3500-COOH produced the low densitydual-targeting nanoprobes (Fe3O4-PEG-RGD-FAl). For comparisonpurpose, single-targeting (Fe3O4-PEG-RGD) and non-targeting(Fe3O4-PEG) nanoprobes were prepared using the same procedure.Specifically, 53 mg of H2N-PEG3500-RGD and 45.2 mg of H2N-PEG3500-COOH were used to yield the single-targeting nanoprobes

while 90.3 mg of H2N-PEG3500-COOH was added to obtain the non-targeting nanoprobes.

2.5. Characterization

Transmission electron microscopy (TEM) images were acquiredon a JEM-2100F (JEOL, Tokyo, Japan) microscope at an operatingvoltage of 120 kV. X-ray diffraction (XRD) patterns were obtainedon a PANalytical Xpert Pro MPD diffractometer (PANalytical,Holland) using Cu Karadiation (k= 1.54056 , 40 kV, 40 mA) with2h scanning mode. The magnetic properties of NPs weredetermined on a SQUID MPMSXL-7 (Quantum Design, USA). TheUVvis absorption spectra of NPs with Fe concentration of160lM were recorded on a UV-2600 spectrophotometer

(Shimadzu, Japan). The fluorescence emission spectra of NPs withFe concentration of 800 lM was collected on an F-2500

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fluorescence spectrophotometer (Hitachi, Japan). The content of Fewas determined on an inductively coupled plasma optical emissionspectrometry (ICP-OES, Varian 710-ES, USA).

2.6. Relaxivity measurements

Relaxivity measurements were conducted on a 7TMR scanner

(Bruker Pharmascan, Germany) using the RARE-T1+ T2-mapsequence. The measurement parameters were set as follows: repe-tition times (TR) = 3000 ms, multiple echo time (TE) = 11 ms,33 ms, 55 ms, 77 ms, 99 ms, matrix size = 256 mm 256 mm, fieldof view(FOV) = 4.0 4.0 cm2, flip angle (FA) = 180and slice thick-ness = 1 mm. The T2 relaxation times and corresponding MR T2mappings of NPs at various concentrations were acquired. Therelaxivity value of r2 was calculated from the slope of the linearplot of 1/T2versus the Fe concentration.

2.7. Brain glioma model

All animal experiments were performed according to the guide-lines of Capital Medical University animal committee. Mice C6

brain glioma model was established according to the methodreported in literature[33]. Briefly, ICR mice were fixed in a stereo-tactic frame after anesthetized with 6% chloral hydrate(0.10 mL/20 g). 5lL of suspension containing 5 105 C6 gliomacells were implanted into the burr hole on the skull. The injectionwas done slowly over 5 min, stay 10 min and the needle was with-drawn after another 10 min. The burrhole was filled with bone waxand the skin was closed with nonmagnetic structures.

2.8. In vivo MR imaging

MRT2-weighted images of the mice brain bearing glioma beforeand after the intravenous injection of 0.2 mL of nanoprobes at adosage of 10 mg Fe kg1 body were acquired on a 7TMR scanner(Bruker Pharmascan, Germany) using the RARE sequence: TR/TE = 3000/45 ms, matrix size = 256 256, field of view= 2.52.5 cm2, flip angle = 180, slice thickness = 1 mm and number ofaverages = 4. The contrast to noise ratio (CNR) was calculated asfollows: CNR = (St Sb)/rn, whereStandSbare the averaged signal

intensities within the tumor and a normal brain region respec-tively and rn is the standard deviation of noise measured fromthe background noise in the same slice. The DCNR was calculatedfrom |CNRpost CNRpre|/CNRpre[34].

2.9. NIR and confocal imaging

Theex vivo images of the brains harboring gliomas were cap-tured on an optical imaging system (NightOWL II LB983, Germany)with a 630 nm excitation filter and a 680 nm emission band-passfilter set. Afterwards, the brains were fixed in 4% paraformalde-hyde, dehydrated with 30% sucrose solution, sliced in 20lmthickness, stained with DAPI (100 ng/ml), and imaged on a LEICATCSSP5 confocal microscope.

2.10. Prussian blue staining

Fixed brain slices were stained with a 1:1 mixture of 2% potas-sium ferrocyanide (II) trihydrate solution and 2% HCl for 15 min,washed in distilled water, counterstained with nuclear fast redfor 10 min, and examined under an optical microscope.

3. Results and discussion

The Fe3O4-OA NPs were synthesized by thermal decompositionof Fe(acac)3 precursors to obtain high crystallinity, purity, andreproducibility. As water-dispersible and colloidal stability arethe perquisites for bio-imaging applications of NPs, carboxylicsilane TETT were used to replace OA on the surface of Fe3O4 NPs.Next, the Fe3O4-TETT NPs were labeled with Cy5.5 to ensureequivalent fluorescence intensity. Finally, PEG, c(RGDyK), andFA-c(RGDyK) were conjugated onto Fe3O4-TETT-Cy5.5 to obtainnon-targeting (Fe3O4-PEG), single-targeting (Fe3O4-PEG-RGD),low density dual-targeting (Fe3O4-PEG-RGD-FAl) and high densitydual-targeting (Fe3O4-PEG-RGD-FAh) nanoprobes, respectively(Scheme 1). The use of PEG not only confers the nanoprobesadditional solubility and stability in aqueous solutions but alsoprolongs the blood circulation time, which, to some extent,enhances the chance for the nanoprobes to cross the BBB.

Scheme 1. Schematic illustration of the non-targeting, single-targeting, high density dual-targeting, and low density dual-targeting nanoprobes.

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Fig. 1 shows the TEM images of Fe3O4-OA, Fe3O4-TETT,Fe3O4-PEG, Fe3O4-PEG-RGD, Fe3O4-PEG-RGD-FAl and Fe3O4-PEG-RGD-FAh NPs, respectively. The NPs were nearly spherical andmonodispersed with an average diameter of 8 nm as calculated

from 150 individual NPs. All of the nanoprobes exhibit highcolloidal stability after being dispersed in water for at least one

month (Fig. 1BF, insets). The color of the dispersions changedfrom yellow (Fig. 1B, inset) to2 green (Fig. 1CE, inset), implyingthe successful conjugation of Cy5.5.

Fig. 1. TEM images of Fe3O4-OA (A), Fe3O4-TETT (B), Fe3O4-PEG (C), Fe3O4-PEG-RGD(D), Fe3O4-PEG-RGD-FAl(E) and Fe3O4-PEG-RGD-FAh(F). Scale bar= 50 nm. The insetsarethe photographs of corresponding NPs dispersed in hexane (A) or distilled water (BF).

Fig. 2. XRD patterns of Fe3O4-OA and Fe3O4-TETT NPs (A), the fielddependent magnetization curve (MH curve) (B), the inset displays the temperature-dependence of ZFCand FC magnetization at a magnetic field of 100 Oe, and UVvis absorbance (C) and fluorescence emission (D) spectra of various NPs.

2 For interpretation of color in Fig. 1, the reader is referred to the web version ofthis article.

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The crystal structure of Fe3O4-OA NPs was characterized by XRD

(Fig. 2A). For Fe3O4-OA, six characteristic diffraction peaks at 30.1,35.4, 43.1, 56.9, 62.5 and 74.0 can be indexed as a cubicspinel-structured Fe3O4-OA (JCPDS card No. 19-0629). These char-acteristic diffraction peaks also observed in the XRD pattern ofFe3O4-TETT NPs, indicating that the TETT replaced the OA on thesurface of Fe3O4-OA NPs without affecting the crystalline structure.The additional broad diffraction peak observed between 22 and27 was assigned to the amorphous TETT. Furthermore, the mag-netic properties of Fe3O4-OA were studied using a SQUID. As showninFig. 2B, the fielddependent magnetization curve (MH curve)revealed no coercivity and remanence at 300 K, suggesting thesuperparamagnetic behavior of Fe3O4-OA NPs. The temperature-dependent magnetization under zero-field cooling (ZFC) and fieldcooling (FC) measurements (Fig. 2B, inset) gave the estimated

blocking temperature of about 50 K for Fe3O4-OA NPs. The MHcurve indicated that the saturation magnetization (Ms) ofFe3O4-OA NPs was approximately 85.5 emu g

1 at 300 K.The conjugation of FA-c(RGDyK) dual-target onto NPs was ver-

ified by UVvis spectra (Fig. S1). As can be seen, FA possesses acharacteristic absorbance peak at 280 nm while c(RGDyK) presentsa peak at 260 nm. These two peaks were simultaneously presentedin the UVvis spectra of both Fe3O4-PEG-RGD-FAl and Fe3O4-PEG-RGD-FAh dual-targeting nanoprobes, confirming the successfulconjugation of FA and c(RGDyK). Conjugation of Cy5.5 was alsoconfirmed by the absorption peak at 675 nm in the UVvis spectraand the emission peak at 700 nm in the fluorescence spectra of NPs(Fig. 2C).

Next, the relaxivity property of nanoprobes was evaluated by

acquiring the T2 relaxation time and the corresponding MRT2map-ping on a 7TMR scanner. The relaxivityr2was obtained from the

slope of linear fitting the inverse relaxation time (1/T2) versusthe Fe concentration (measured by ICP-OES) (Fig. 3A). It was foundthat the r2value of Fe3O4-PEG NPs was 232.6 mM

1 s1. Upon con-jugating the dual-target, the r2 decreased slightly (in the rangebetween 199.6 and 214.6 mM1 s1), probably due to the shieldeffect of FA and RGD. Nevertheless, theser2values are comparableto the reported value[35]and are suitable forin vivoMRT2imag-ing. Consistently, MRT2mapping of nanoprobes dispersed in waterbecame progressively darker with the increase of Fe concentration(Fig. 3B).

The targeting specificity of the dual-targeting Fe3O4nanoprobes

with a low and high FA-c(RGDyK) density (Fe3O4-PEG-RGD-FAlandFe3O4-PEG-RGD-FAh) was first evaluated by in vivoMR imaging of

Fig. 3. The linear plotting of relaxation rate R 2 versus Fe concentration (A) andcorresponding MRT2mappings (B).

Fig. 4. MR T2-weighted and pseudo-colored MR T2-weighted images of mousebrains bearing gliomas before and after i.v. injection of nanoprobes (A), andcorresponding quantification of the signals on the MR images of brain gliomas byDCNR (B). DCNR = |CNRpost CNRpre|/CNRpre. (For interpretation of the refer-ences to colour in this figure legend, thereader is referred to theweb version of this

article.)

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brain glioma-bearing mice on a 7T MR scanner. The acquiredT2-weighted MR images of glioma-bearing brains before and after

injection of nanoprobes are presented inFig. 4A. Notably, bothdual-targeting nanoprobes led to a darkened contrast effect sur-rounding the tumors. On the contrary, less contrast enhancementeffect was detected after injection of either the single-targetingor non-targeting nanoprobes at the equivalent dosage. To bettervisualize the negative enhancement effect, pseudo-coloredT2-weighted MR images are provided. The color of the MR imagesof brain glioma treated with Fe3O4-PEG-RGD-FAh appeared bluerthan those of images of brain glioma treated with Fe3O4-PEG-RGD-FAl, signifying the synergistic targeting ability toward glioma.The corresponding quantitative analysis of MR signal enhance-ment, which was performed and compared via DCNR [34], isshown in Fig. 5B. It was found that there were approximately54% and 31% increases in DCNR for Fe3O4-PEG-RGD-FAh and

Fe3O4-PEG-RGD-FAl, respectively, suggesting that a greater dual-target density results in a higher targeting specificity. However,due to the fact that an increased number of target moieties wouldlead to greater steric hindrance that impedes the uptake of thenanoprobes by tumor, a detailed study to fully clarification therelationship between dual-target density and the targeting speci-ficity is required.

Additionally, ex vivo NIR fluorescence imagingof brain harboringgliomas was conducted. As can be seen fromFig. 5A, the order offluorescence intensity from weak to strong is non-targeting,single-targeting, low- and high-density dual-targeting NPs, sug-gesting that the high density dual-targeting nanoprobes have thehighest accumulation in gliomas due to the high specificity towardglioma. This was further supported by Prussian blue staining of

brain tissue sections (Fig. 5B). In addition, the synergistic targetingability of dual-targeting nanoprobes toward glioma was investi-

gatedby CLSM.As shownin Fig.5C, onlyweak red fluorescenceorig-inating from Cy5.5 was observed around the glioma treated with

non-targeting nanoprobes. Although single-targeting (Fe3O4-PEG-RGD) nanoprobes resulted in slightly higher fluorescence intensity,the much stronger fluorescence signals were observed from brainslices treated with either Fe3O4-PEG-RGD-FAl or Fe3O4-PEG-RGD-FAh, which further proves the enhanced uptake of dual-targetingnanoprobes by glioma due to an improved targeting specificity.Moreover, it was noted that a higher dual-target density induced agreater accumulation of nanoprobes in the glioma region (Fig. S2).

4. Conclusions

In sum, the dual-targeting iron oxide nanoprobes with low andhigh FA-c(RGDyK) dual-target density were prepared and charac-terized. The in vivo MR imaging of brain glioma-bearing mice

demonstrated that both dual-targeting nanoprobes exhibited animproved targeting specificity toward glioma due to the synergistictargeting ability. This consequently led to an enhanced MR nega-tive contrast. More importantly, it was disclosed that the densityof dual-target plays an important role in targeting specificity. Onthe one hand, the targeting specificity elevates with the increasingof dual-target density. On the other hand, an increased dual-targetdensity might cause a steric hindrance that impedes the uptake ofnanoprobes by glioma. Therefore, optimization the density of dual-target is needed to further improve the targeting specificity ofdual-targeting nanoprobes toward gliomas.

Acknowledgements

The authors gratefully acknowledge the financial supports fromNational Natural Science Foundation of China (81271639),

Fig. 5. (A) Ex vivo fluorescence images of theexcised brain harboring gliomas after injectionof (a) Fe3O4-PEG, (b)Fe3O4-PEG-RGD, (c) Fe3O4-PEG-RGD-FAl, and (d) Fe3O4-PEG-RGD-FAhnanoprobes at an equivalent dosage. (B) Prussian blue staining of mouse brain sections harboring gliomas upon injection of (a) Fe 3O4-PEG, (b) Fe3O4-PEG-RGD, (c)Fe3O4-PEG-RGD-FAl, and (d) Fe3O4-PEG-RGD-FAhnanoprobes. Scale bar = 100lm. (C) CLSM images of the brain slices bearing C6 glioma after i.v. administration of differentnanoprobes. Blue: cell nuclei, Red: Cy5.5 labeled NPs. Whitearrowpoints to the glioma region. Scale bar= 250 lm.(For interpretation of thereferencesto colour in this figurelegend, the reader is referred to the web version of this article.)

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National Key Technology Research and Development Program ofthe Ministry of Science and Technology of China (2014BAI04B01),and the Basic-clinical Key Research Grant (13JL02, 15JL07) fromCapital Medical University.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jcis.2016.02.004.

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