Accepted Manuscript Cy5.5 Conjugated MnO Nanoparticles for Magnetic Resonance/Near-infrared Fluorescence Dual-modal Imaging of Brain Gliomas Ning Chen, Chen Shao, Shuai Li, Zihao Wang, Yanming Qu, Wei Gu, Chunjiang Yu, Ling Ye PII: DOI: Reference:

S0021-9797(15)30016-3 YJCIS 20552

To appear in:

Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

8 May 2015 28 June 2015 29 June 2015

Please cite this article as: N. Chen, C. Shao, S. Li, Z. Wang, Y. Qu, W. Gu, C. Yu, L. Ye, Cy5.5 Conjugated MnO Nanoparticles for Magnetic Resonance/Near-infrared Fluorescence Dual-modal Imaging of Brain Gliomas, Journal of Colloid and Interface Science (2015), doi:

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Cy5.5 Conjugated MnO Nanoparticles for Magnetic Resonance/Near-infrared Fluorescence Dual-modal Imaging of Brain Gliomas

Ning Chen‡#, Chen Shao†#, Shuai Li†, Zihao Wang$, Yanming Qu£, Wei Gu† *, Chunjiang Yu£ *, and Ling Ye† *

School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing

100069, P. R. China ‡

Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing,

100050, P. R. China £

Department of Neurosurgery, Beijing Sanbo Brain Hospital, Capital Medical University, Beijing

100093, P. R. China $

School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, P. R. China


These authors contributed equally to this work

* Corresponding authors. E-mail: [email protected], [email protected], [email protected]


Highlights 

MnO NPs were prepared by a thermal decomposition method.

Water dispersible MnO NPs were obtained by carboxyl silane modification.

PEG-Cy5.5 conjugated MnO NPs were fabricated as a dual-modal nanoprobe.

MnO-PEG-Cy5.5 nanoprobe enables a better detection of brain gliomas.


ABSTRACT The fusion of molecular and anatomical modalities facilitates more reliable and accurate detection of tumors. Herein, we prepared the PEG-Cy5.5 conjugated MnO nanoparticles (MnO-PEG-Cy5.5 NPs) with magnetic resonance (MR) and near-infrared fluorescence (NIRF) imaging modalities. The applicability of MnO-PEG-Cy5.5 NPs as a dual-modal (MR/NIRF) imaging nanoprobe for the detection of brain gliomas was investigated. In vivo MR contrast enhancement of the MnO-PEG-Cy5.5 nanoprobe in the tumor region was demonstrated. Meanwhile, whole-body NIRF imaging of glioma bearing nude mouse exhibited distinct tumor localization upon injection of MnO-PEG-Cy5.5 NPs. Moreover, ex vivo CLSM imaging of the brain slice hosting glioma indicated the preferential accumulation of MnO-PEG-Cy5.5 NPs in the glioma region. Our results therefore demonstrated the potential of MnO-PEG-Cy5.5 NPs as a dual-modal (MR/NIRF) imaging nanoprobe in improving the diagnostic efficacy by simultaneously providing anatomical information from deep inside the body and more sensitive information at the cellular level.

Keywords: Cy5.5 conjugated MnO nanoparticles, dual-modal nanoprobe, magnetic resonance (MR) imaging, near-infrared fluorescence (NIR) imaging, brain gliomas


1. Introduction Magnetic resonance (MR) imaging produces high-resolution images of soft tissues down to 1 mm at clinical field strengths and therefore provides abundant information about anatomical structure and physiological conditions.1 This together with the lack of ionizing radiation makes it the most used non-invasive diagnostic modality. The introduction of MRI contrast agents could accelerate the relaxivity of water and consequently further boost the contrast.2 Currently, gadolinium(Gd)-based complexes are dominant contrast agents in clinics, which induce the local relaxation change of the nearby water protons and reduce longitudinal (T1) relaxation time, resulting in a positive contrast (bright signal) on the T1-weighted MR image.3 However, the association of Gd ions with nephrogenic systemic fibrosis (NSF) in patients with severe renal or kidney disease demands alternative T1 contrast agents.4-5 Manganese (Mn) ions have favored properties as T1 contrast agents including high spin number, labile water exchange and long electronic relaxation time, natural prevalence, and known human biochemistry. 6-9 As a matter of fact, Mn ions was one of the earliest reported examples of paramagnetic contrast material for MR imaging. For example, MnCl2 is used in gastrointestinal imaging.10 However, the use of Mn2+-based T1 contrast agents has been limited due to cardiovascular toxicity.11 Recently, Mn-based nanoparticles (NPs), e.g. manganese oxide (MnO) NPs with different sizes and morphologies, have been emerging as promising T1 contrast agents to overcome the drawbacks of Mn2+-based T1 contrast agents, such as toxicity, short circulation time, and low efficiency of cellular accumulation.11-14 In vivo MR imaging


studies proved that MnO NPs could lead to an increased contrast of entire organs.15-17 For instance, clear T1-weighted images of brain structures, depicting fine anatomic features, were obtained upon injection of MnO NPs.16 These findings suggest that MnO NPs can be utilized not only for the basic neuroscience research but also for the diagnosis of clinical neurological diseases. Despite the aid of contrast agents, the sensitivity of MR imaging is relatively low in comparison to other biomedical imaging techniques. In some cases, the contrast difference between tissues (e.g., normal vs. cancer) might be too subtle to provide precise imaging information.18 Moreover, molecular and/or biological information on the region of interest is hardly provided by MR imaging. Therefore, the integration of other imaging modalities with high sensitivity, such as optical imaging, is highly desirable to offer specific insight into cellular events and real-time intraoperative monitoring of tumor tissue.19-20 To this end, near-infrared fluorescence (NIRF) imaging is the most relevant imaging modality in line with MR imaging, not only because of the higher sensitivity but also due to the weaker fluorescence absorption of biological tissues in the 750-1000 nm region, which ensures a deeper optical penetration with attenuated cytotoxicity and high feature fidelity.21-22 One of the advantages of nanoparticles-based probes is their flexibility when conjugated with a variety of functional moieties.23 On this occasion, MnO NPs offer excellent matrix to accommodate optical imaging modality by conjugation NIR dyes. As such, we prepared the MnO-based nanoprobe with MR and NIRF imaging modalities. Specifically, high crystalline MnO NPs were prepared by the thermal


decomposition of manganese oleate. Water dispersible and colloidal stable MnO NPs were then obtained by exchanging oleate with carboxyl silane. The carboxyl functional groups allow the conjugation with PEG-Cy5.5 to MnO-NPs to yield the MnO-PEG-Cy5.5 NPs. The potential of these MnO-PEG-Cy5.5 NPs as an MR/NIRF dual-modal imaging nanoprobe was explored in a glioma-bearing nude mouse model.

2. Materials and methods 2.1 Materials. Manganese chloride tetrahydrate, sodium oleate, 1-octadecene, and Cy5.5 were purchased from Sigma. N-(trimethoxysilypropyl) ethylene diamine triacetic acid, trisodium salt (TETT silane) was supplied by Gelest. NH2-PEG500-NH2 was









N-hydroxysuccinimide (NHS) and Ethyl(dimethylaminopropyl)-carbodiimide (EDC) were obtained from Acros Oganics (Geel, Belgium). All other chemicals are of analytical grade and used as received 2.2 Preparation of Mn-oleate complex. To a solvent composed of 40 ml of ethanol, 30 ml of distilled water, and 70 ml of n-hexane, 3.96 g of manganese chloride tetrahydrate and 12.17 g of sodium oleate were added and heated at 70°C with vigorous stirring for 4 h. The final solution was transferred to a separatory funnel, and the upper organic layer containing the Mn-oleate complex was retained and washed with distilled water three times. The pink-colored Mn-oleate complex was then obtained after removing the hexane solvent. 2.3 Preparation of oleate capped MnO (MnO-OA) NPs. The as-prepared


Mn-oleate complex (2.468 g) was dispersed in 50 ml of 1-octadecene and then heated at 100 °C for 15 min with N2 purging. Next, the mixture was sequentially heated at 200 °C for 20 min and 280 °C for 10 min. After cooling to room temperature and addition of acetone, the precipitate was collected by centrifugation and re-dispersed in n-hexane. The dispersion was centrifuged again and the supernatant was retained. Upon addition of methanol, the MnO-OA NPs was obtained by centrifugation. 2.4 Exchange of oleate with TETT silane. To confer MnO NPs water dispersible and colloidal stability, the oleate was replaced by TETT silane. 24 Typically, 50 mg of MnO-OA NPs were suspended in 60 ml of anhydrous toluene with 30 μl of acetic acid. After sonication for 15 min, 200 μl of TETT silane was added. Then, the suspension was heated at 70 °C with vigorous stirring for 48 h, during which precipitation occurred. The precipitated MnO-TETT were collected and washed with toluene and methanol respectively for three times. Next, the MnO-TETT NPs were dispersed in deionized water and dialyzed against deionized water through cellulose membrane bag (MWCO = 8000-14000 Da). The purified MnO-TETT NPs were obtained by lyophilization. 2.5 Conjugation of PEG-Cy5.5 onto MnO-TETT NPs. PEG-Cy5.5 was first synthesized by reacting 7.1 mg of NH2-PEG500-NH2 with 1 mg of Cy5.5-NHS at room temperature in 10 ml PBS (pH 8.0) for 24 h. Next, 80 mg of MnO-TETT NPs were dispersed in 10 ml of PBS (pH 4.0), followed by addition of 1 mg of EDC and 1.5 mg of NHS for activation. Then, 5 ml of PEG-Cy5.5 was added and the pH was adjusted to 8.0. The reaction was allowed to proceed under stirring for 24 h at room


temperature. Then, the product was purified by dialysis against deionized water in a cellulose dialysis bag (MWCO = 3500Da), followed by lyophilization to obtain MnO-PEG-Cy5.5 NPs. 2.6 Characterization. Transmission electron microscopy (TEM) images were obtained on a JEM-2100F (JEOL, Japan) microscope at an operating voltage of 100 kV. High-resolution TEM (HRTEM) image was acquired on a JEM-2100F (JEOL, Japan) electron microscope operated at 200 kV. For TEM or HRTEM imaging, the species were prepared by spreading a drop of NPs dispersion on the copper grid and dried under vacuum. X-ray photoelectron spectroscopy (XPS) analysis was performed on an axis ultra-spectrometer (Kratos, UK) by using mono-Al Ka line (1486.71 eV) radiation at a power of 225 W. X-ray diffraction (XRD) pattern was collected on a PANalytical X’pert Pro diffractometer (PANalytical, Holland), using Cu Kα radiation (λ = 1.5405 Å) at a voltage of 40 kV and a current of 40 mA with 2θ scanning mode. The content of Mn was determined on an inductively coupled plasma optical emission spectrometry (ICP-OES, Varian 710-ES, USA). UV-Vis absorption spectra were obtained on a UV-2550 spectrophotometer (Shimadzu, Japan). Fluorescence emission spectra were acquired on an F-2500 fluorescence spectrophotometer (Hitachi, Japan) equipped with a Xe lamp. Magnetic hysteresis loop of MnO-OA NPs was analyzed on a vibrating sample magnetometer at room temperature. 2.7 Relaxivity measurement. The MnO-PEG-Cy5.5 NPs were dispersed in distilled water with Mn concentrations ranging from 0 to 0.5 mM. The T1 relaxation times and corresponding T1-weighted images of MnO-PEG-Cy5.5 NPs at different Mn


concentrations were acquired on a 7.0 T MRI scanner (Bruker Pharmascan, Germany) with the following sequence: repetition times (TR) = 200, 400, 800, 1500, 3000, 5000 ms, echo time (TE) = 11.00 ms, field of views (FOV) = 4.0×4.0 cm2, flip angle (FA) = 180.0 deg, and slice thickness = 1 mm. Relaxivity value of r1 was calculated through the curve fitting of 1/T1 relaxation time (s-1) versus Mn concentration (mM). 2.8 In Vitro Cytotoxicity. The cytotoxicity of MnO-PEG-Cy5.5 NPs against C6 cells was evaluated via an MTT ((3-(4,5-dimethylth-iazolyl-2)-2,5-diphenyltetrazolium bromide) assay. C6 cells were seeded in a 96-well plate at a density of 1×104 cells per well and cultured in Dulbecco’s modified Eagle’s medium (DMEM). After 24 h of incubation, the cells were treated the MnO-PEG-Cy5.5 NPs with different Mn concentrations of 0, 6.25, 12.5, 25, and 50 μM−1, followed by another 24 h of incubation. Then, 100 µl of MTT (0.5 mg mL-1) was added to each well and subsequently incubated for 4 h. Finally, the absorbance at 570 nm was measured using a Multiskan Spectrum microplate reader (Thermo Electron Corpration, USA). The cell viability was calculated as a percentage compared to the control and expressed as mean ± standard deviations (SD) from three independent experiments. 2.9 In vivo toxicity. The care and handling of all experimental animals were conducted in compliance with the Chinese Animal Welfare Act and the Guidance for Animal Experimentation approved by the Ethics Committee of Capital Medical University. Furthermore, efforts were made to minimize the number of studied animals and their sufferings. The ICR mice were sacrificed 28 days after tail-vein injection of MnO-PEG-Cy 5.5 NPs at a dosage of 30.0 mg Mn kg -1. The mice were


anesthetized with 6 % chloral hydrate (i.p., 0.15 ml / 20 g) and fixed on the operating table, and transcardially perfused with 0.9 % saline solution followed by 4 % paraformadehyde in 0.1 M sodium phosphate buffer. The major organs (brain, heart, liver, spleen, lung, and kidney) were collected and fixed in 10 % formalin for 72 h. Then, the tissues were embedded in paraffin, cryosectioned into 5 μm slices, and stained with Hematoxylin and Eosin (H&E) according to standard clinical pathology protocols. The stained sections were examined with a light microscope by two pathologists, respectively. 2.10 Glioma model. Male nude mice were anesthetized with 6 % chloral hydrate (i.p., 0.15 ml / 20 g) and mounted in a stereotaxic frame. C6 glioma-bearing mice were prepared according to previously described technique. 25 Briefly, 5×106 green flurorescence protein (GFP) transfected C6 cells suspended in DMEM (5 μL) were injected into the left striatum using parameters as follows, anteroposterior (A/P) = 0.5 mm, mediolateral (M/P) = -2.0 mm, dorsoventral (D/V) = 4.0 mm. 2.11 In vivo MR imaging. The glioma-bearing nude mice were anesthetized with 6 % chloral hydrate (i.p., 0.15 ml / 20 g). In vivo T1-weighted MR images were acquired before and after the injection of MnO-PEG-Cy 5.5 NPs on a 7.0 T MRI scanner (Bruker Pharmascan, Germany) using following parameters: TR = 250 ms, TE = 8 ms, FA = 144.4 deg, matrix = 256×256, FOV = 3.0×3.0 cm2, and slice thickness = 1 mm. 2.12 In vivo NIRF imaging. The glioma-bearing nude mice were anesthetized with 6 % chloral hydrate (i.p., 0.15 ml / 20 g). NIRF images were collected using a optical imaging system (NightOWL Ⅱ LB983, Germany) with a 630 nm laser diode for


excitation and 680 nm band path filter for emission before and 1 h after the administration of MnO-PEG-Cy 5.5 NPs through tail vein. 2.13 Confocal imaging of glioma-bearing brain slices. Nude mice bearing GFP-expressing brain gliomas were injected by tail vein with MnO-PEG-Cy5.5 NPs at a dosage of 10 mg Mn kg-1. One hour after injection, the mice were sacrificed and the brain was removed, fixed in 4 % paraformaldehyde for 48 h and dehydrated sequentially with 30% sucrose solution for 24 h. Then, brains were dissected and cut at a thickness of 20 μm using a freezing microtome. Subsequently, the brain slices were stained with DAPI (4',6-diamidino-2-phenylindole,100 ng/ml) for 10 min and examined with a LEICA TCS SP5 laser-scanning confocal microscopy. The fluorescence signals from DAPI (nucleus), GFP (tumor), and conjugated Cy5.5 were observed by selecting the appropriate excitation and emission setting, respectively.

3. Results and Discussion To ensure high crystallinity, MnO NPs was prepared by the thermal decomposition method.26 As the oleate coating prevents aggregation and flocculation, the produced oleate capped MnO NPs could be readily dispersed in nonpolar solvents (e.g., hexanes, toluene, and chloroform) and form a black dispersion. TEM image (Figure 1A) verified that the MnO-OA NPs were well-separated with a cubic-like shape and the mean size of MnO-OA NPs was 18.59 ± 1.44 nm (Figure 1B) by statistical analysis of 100 MnO-OA NPs. The crystalline nature of MnO-OA NPs was confirmed by HRTEM image (Figure 1C), which showing well-resolved lattice lines. The spacing


of lattice was 0.263 nm, corresponding to the interplanar distance between (111) planes. This was also supported by the SAED pattern (Figure 1D). The structure and phase purity of MnO-OA NPs was further characterized by XRD. As displayed in Figure 1E, the diffraction peaks at 34.9°, 40.5°, 58.7°, 70.2°, 73.8° could be well indexed as the (111), (200), (220), (311), and (222) reflections of cubic rock salt structure of MnO (JCPDS no. 75-0257). Additionally, EDX spectrum (Figure 1F) confirmed the presence of Mn, O and C elements in MnO-OA NPs.




Frequency (%)








size (nm)


D 0.263 nm




E (111)


(200) (220) (311)

O Mn



JCPDS # 75-0257












2 (degree)


4 6 Energy (keV)



Figure 1 TEM image (A) and size distribution (B) of MnO-OA NPs, (C) HRTEM imaging of an individual MnO-OA, (D) SAED pattern, and (E) EDX spectrum of MnO-OA NPs.


For bioimaging purpose, it is critical to impart MnO NPs water dispersiblity and colloidal stability. This was accomplished by replacing oleate with TETT silane. The magnetic characterization of MnO-TETT NPs was performed and the field dependent magnetization curve of MnO-TETT NPs at room temperature was obtained. As presented in Figure S1, the curve showed no remanence coercivity at zero field, indicating the paramagnetic nature of MnO-TETT NPs.17 On the other hand, the carboxyl groups of TETT silane enable the further conjugation of PEG-Cy5.5 via well-known EDC/NHS chemistry between the -COOH groups of TETT and -NH2 groups of PEG-Cy5.5 as described in the experimental section. In this way, PEG-Cy5.5 was strongly attached to the MnO-TETT NPs through the covalent bond. As revealed by TEM (Figure 2A), the morphology of as prepared MnO-PEG-Cy5.5 NPs resembled with that of MnO-OA NPs. However, the surface of MnO-PEG-Cy5.5 NPs appeared much rougher, probably due to partially dissolution of MnO during the surface modifications. Moreover, MnO-PEG-Cy5.5 NPs exhibited an increased size of 20.93 ± 3.94 nm due to the attachment of PEG-Cy5.5 (Figure 2B). Meanwhile, conjugation of PEG-Cy5.5 onto the MnO-TETT NPs could be visually evidenced by the formation of blue-colored dispersion (inset, Figure 2A). Note that the dispersion of MnO-PEG-Cy5.5 NPs could remain stable for several weeks.



B Frequence (%)

20 15 10 5 0





Size (nm)

Figure 2 TEM image (A) and size distribution (B) of MnO-PEG-Cy5.5 NPs. Inset shows the digital picture of MnO-PEG-Cy5.5 dispersed in water.

Next, the optical properties rendered by conjugated PEG-Cy5.5 were examined and compared with that of TETT silane modified MnO (MnO-TETT) NPs. As expected, two absorption peaks (630 and 675 nm) derived from the conjugated PEG-Cy5.5 appeared in the UV-vis absorption spectrum of MnO-PEG-Cy5.5 NPs, whereas MnO-TETT NPs showed no absorption (Figure S2A). On the other hand, the fluorescence emission spectrum of MnO-PEG-Cy5.5 NPs presented a NIR peak centered at 710 nm (excited at 675 nm). In contrast, no fluorescence emission peak was found for MnO-TETT NPs when excited at the same wavelength (Figure S2B). This NIR fluorescence confers MnO NPs the capability of visualizing tumors in deeper tissues with a low background signal. Furthermore, XPS was used to analyze the elemental composition of MnO-PEG-Cy5.5 NPs. As shown in Figure 3A, the XPS survey spectrum exhibited peaks of C 1s, O 1s and Mn 2p, confirming the presence of carbon, oxygen and manganese elements in MnO-PEG-Cy5.5 NPs. Moreover, the XPS Mn 2p spectrum 15

(Figure 3B) displayed two peaks at 653 and 641 eV, which were assigned to Mn 2p1/2 and Mn 2p3/2, respectively. These values are in good agreement with literature values

O 1s

of Mn2+ in MnO. 27

Mn 2p3/2



Mn 2p1/2

Mn 3p

Mn 2p




C 1s


1200 1000













Binding Energy (eV)

Binging Energy (eV)

Figure 3 XPS (A) survey and (B) Mn 2p spectra of MnO-PEG-Cy5.5 NPs.

Relaxivity refers to the ability to increase the relaxation rate. Essentially, a nanoprobe with high relaxivity can be detected at lower concentrations. Therefore, the longitudinal (r1) relaxivity of MnO-PEG-Cy 5.5 NPs was determined to evaluate the potential of MnO-PEG-Cy5.5 NPs as a T1 contrast agent. As plotted in Figure 4, the r1 of MnO-PEG-Cy5.5 NPs was 5.73 mM-1 s-1 derived from the linear fitting of inverse relaxation time (1/T1) against the Mn concentration. The r2 of MnO-PEG-Cy5.5 was measured to be 40.16 mM-1 s-1 (Figure S3), which gives an r2/r1 ratio of about 7. Technically, an r1/r2 ratio close to unit is desirable for a positive contrast agent. Nevertheless, this r2/r1 ratio is lower than that reported in literature. 28 Additionally, the corresponding T1-weighted images of MnO-PEG-Cy5.5 NPs demonstrated a continuous increase in brightness as increasing of Mn concentration (inset, Figure 4). 16

This together with the high r1 manifests the applicability of MnO-PEG-Cy5.5 NPs as an efficient T1 nanoprobe.


3 -1 -1

r1= 5.73 mM s -1

R1 (s )







5 0.



5 12 0.

06 0.










Mn (mM)

Figure 4 r1 relaxivity measurement and T1-weighted images of MnO-PEG-Cy5.5 NPs. The r1 relaxivity was determined by the linear fitting of the R1 against the Mn concentration.

Prior to in vivo imaging, the biocompatibility of MnO-PEG-Cy5.5 NPs, which is critical for the bio-imaging applications, was investigated. The cytotoxic effect of MnO-PEG-Cy5.5 NPs was first evaluated via the MTT assay. Figure S4 shows the proliferation of C6 cells after 24 h exposure to different concentrations of MnO-PEG-Cy5.5 NPs. It was found that the cell proliferation was dose-dependent. However, the cell viability of MnO-PEG-Cy5.5 was higher than 90 % at all tested Mn concentration (up to 50 μM), suggesting that MnO-PEG-Cy 5.5 NPs induced no significant cytotoxicity. Next, we assessed the in vivo toxicity of MnO-PEG-Cy5.5


NPs. The ICR mice was sacrificed 28 days after injection of MnO-PEG-Cy5.5 at a dosage of 30 mg Mn kg -1 body, which was three times higher than that used for the following MR and NIR imaging. Major organs (brain, heart, liver, spleen, lung, and kidney) were sliced and stained by H&E for histological examination. As compared to the control group, the ICR mice receiving MnO-PEG-Cy5.5 NPs showed no discernible tissue injury or necrosis (Figure S5). For instance, normal hepatic architecture, hepatocytes and portal triad were found in the liver, and norm renal cortex, glomerular tufts and tubules, and normal renal papilla were evidenced in the kidney. Meanwhile, normal splenic architecture, lymphoid follicles and sinuses were detected in the spleen. Thus, histological analysis, which showed no different histological changes, affirmed the biocompatibility of MnO-PEG-Cy5.5 NPs to vital organs. On the basis of high r1 and good biocompatibility, we investigated the feasibility of MnO-PEG-Cy5.5 NPs as an MR contrast agent for glioma imaging. Serial MR T1-weighted images of the transverse section of brain glioma were acquired pre-injection and post-injection of MnO-PEG-Cy5.5 NPs at a dosage of 10 mg Mn kg-1 body. As illustrated in Figure 5, MnO-PEG-Cy5.5 NPs produced a contrast enhancement in both tiny- and large-volume gliomas as early as 10 min post-injection and the contrast enhancement became more obvious as time passing. Nevertheless, a ring-enhancing lesion and higher contrast enhancement were observed for the large-volume glioma, whereas for tiny-volume one, the enhancement was relatively uniform and weaker. The higher contrast enhancement is


most likely due to the increased accumulation of NPs resulting from the destructed blood brain barrier in the large-volume glioma model, while the ring-enhancing lesion is because the necrosis inside tumor region prevented the efficient accumulation of MnO-PEG-Cy5.5 NPs in the deeper region of large-volume glioma. The different enhancement patterns between the large- and tiny-volume gliomas could be correlated with histology and may be potentially valuable. Furthermore, to differential the T1 contrast enhancement induced by MnO-PEG-Cy5.5 NPs between the large and the tiny gliomas, quantitative analysis of MR signal intensities (SI) in the regions of interest (ROI) was performed by setting the SI of the pre-injection as 100 %. As shown in Figure S6, an increase in SI post-injection of MnO-PEG-Cy5.5 NPs was found in both large and tiny gliomas. However, SI in the large glioma is higher than that in the tiny glioma at all scanning time points.

Figure 5 Dynamic MRI in brain glioma-bearing nude mice (A) large-volume and (B) tiny-volume pre-contrast and at various time points after tail-vein injection of MnO-PEG-Cy5.5 NPs. Arrow points to the glioma region.


In additional to serving as a MR contrast agent, the conjugated Cy5.5 confers MnO NPs the NIRF imaging modality. The whole-body NIRF images of nude mouse bearing tiny-volume brain glioma before and after tail-vein injection of MnO-PEG-Cy5.5 NPs are illustrated in Figure 6. It was noted that the intensive fluorescence signal was predominately detected in kidney at 1 h post-injection, suggesting the renal clearance of the NPs. More importantly, an obvious fluorescence signal was found in the brain, showing distinct tumor localization. In addition, ex vivo CLSM imaging of the brain slice hosting glioma (excised 1 h post-injection) was performed to investigate the distribution of MnO-PEG-Cy5.5 NPs in glioma-bearing brain (Figure 7). To visually guide the delineation of the tumor region (on the right side) from the normal tissue (on the left side), a yellow contour line was added. Apparently, the red fluorescent dots of MnO-PEG-Cy5.5 NPs preferentially resided in the GFP-expressing glioma region and only few red spots were found in the normal brain region (Figure. 7A), indicating that MnO-PEG-Cy 5.5 NPs could penetrate and be retained in tumor tissue. Furthermore, the magnified CLSM image of glioma revealed that MnO-PEG-Cy 5.5 NPs were indeed internalized by glioma cells and mainly distributed in the cytoplasm (Figure 7B). Such MR/NIRF dual-modal nanoprobes thus make it feasible to obtain both anatomical and physiological information from deep inside the body and simultaneously acquire more sensitive information at the cellular level.



1 h post-injection

Figure 6 In vivo NIRF images of glioma-bearing nude mice at pre-injection and 1 h post-injection of MnO-PEG-Cy5.5 NPs at a dosage of 10 mg Mn/kg. Yellow arrow points to the glioma region.

Figure 7 CLSM images of the brain slices excised from glioma-bearing nude mice 1 h after i.v. administration of MnO-PEG-Cy5.5 NPs. Blue: DAPI stained nucleus. Green: GFP-transfected glioma cells. Red: MnO-PEG-Cy5.5 NPs. Yellow arrow points to the glioma region. Scale bar = 100 μm. (A) Magnification: 100×, Scale bars = 500 μm. (B) Magnification: 600×, Scale bars = 25 μm. 21

4. Conclusions In summary, we prepared and characterized water-dispersible PEG-Cy5.5 labelled MnO NPs. These MnO-PEG-Cy5.5 NPs possessed a high r1 relaxivity and showed a good biocompatibility. In vivo MR imaging suggested that the MnO-PEG-Cy5.5 NPs enhanced the tumor contrast. Whole-body NIRF imaging demonstrated that the brain tumor could be visually detected by MnO-PEG-Cy5.5 NPs due to their preferential accumulation in the glioma region, which was revealed by ex vivo CLSM imaging at the cellular level. Therefore, our MnO-PEG-Cy5.5 NPs provide a promising platform for developing an MR/NIRF dual-modal nanoprobe towards better detection of gliomas.

Acknowledgements The authors gratefully acknowledge the financial supports from National Natural Science Foundation of China (81271639, 81372694), Beijing Sanbo Brain Hospital Advance research program of innovation (2012SJ005), and the Basic-clinical Key Research Grant (13JL02, 15JL07) from Capital Medical University.

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Graphical abstract





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near-infrared fluorescence dual-modal imaging of brain gliomas.

The fusion of molecular and anatomical modalities facilitates more reliable and accurate detection of tumors. Herein, we prepared the PEG-Cy5.5 conjug...
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