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MR/SPECT Imaging Guided Photothermal Therapy of Tumor-Targeting Fe@Fe3O4 Nanoparticles in Vivo with Low Mononuclear Phagocyte Uptake Jing Wang, Heng Zhao, Zhiguo Zhou, Ping Zhou, Yuping Yan, Ming-Wei Wang, Hong Yang, Yingjian Zhang, and Shi-Ping Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04639 • Publication Date (Web): 18 Jul 2016 Downloaded from http://pubs.acs.org on July 20, 2016

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ACS Applied Materials & Interfaces

MR/SPECT Imaging Guided Photothermal Therapy of Tumor-Targeting Fe@Fe3O4 Nanoparticles in Vivo with Low Mononuclear Phagocyte Uptake Jing Wang†, Heng Zhao†, Zhiguo Zhou†*, Ping Zhou†, Yuping Yan†, Mingwei Wang§*, Hong Yang†, Yingjian Zhang§ and Shiping Yang†* †

The Key Laboratory of Resource Chemistry of Ministry of Education, Shanghai Key

Laboratory of Rare Earth Functional Materials, and Shanghai Municipal Education Committee Key Laboratory of Molecular Imaging Probes and Sensors, Shanghai Normal University, Shanghai 200234, China. §

Department of Nuclear Medicine, Shanghai Cancer Center & Department of

Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China.

KEYWORDS. Fe@Fe3O4 Nanoparticles, Magnetic Resonance Imaging, Single Photon Emission Computed Tomography, Targeting Photothermal Therapy, in Vivo

ABSTRACT. The

125

I-c(RGDyK) peptide PEGylated Fe@Fe3O4 nanoparticles

(125I-RGD-PEG-MNPs) with the average hydrodynamic diameter of ~ 40 nm as a novel multifunctional platform were developed for tumor-targeting MR/SPECT imaging guided photothermal therapy in vivo. On the αvβ3-positive U87MG

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glioblastoma xenograft model, the signals of tumor from T2-weighted MR and SPECT imaging were much higher than those in the blocking group at 6 h post injection (p.i.) of RGD-PEG-MNPs and

125

I-RGD-PEG-MNPs intravenously, respectively. The

pharmacokinetics and biodistribution were analyzed quantitatively by gamma counter ex vivo. The fact suggested that RGD-PEG-MNPs exhibited the excellent targeting property 125

and

low

mononuclear

phagocyte

uptake.

At

6

h

p.i.

for

I-RGD-PEG-MNPs, the maximum uptake of 6.75 ± 1.24% of the percentage

injected dose per gram (ID/g) was accumulated in tumor. At 48 h p.i., only 1.11 ± 0.21% and 0.16 ± 0.09% ID/g were accumulated in liver and spleen, respectively. With the guidance of MR/SPECT imaging, the multifunctional nanoparticles achieved a good photothermal therapeutic efficacy in vivo. INTRODUCTION

With the development of the personalized medicine, multimodality imaging by use of the combination of the different molecular imaging technologies, has been extensively adopted in clinical diagnosis to overcome limitations of the single imaging mode.1-6 Among the biomedical imaging techniques, magnetic resonance (MR) imaging

has the high spatial resolution (several tens of micrometers) and soft

tissue contrast, but shows the limited sensitivity. SPECT imaging is highly sensitive but is low spatial resolution and deficient in the anatomical information. It has been routinely applied used for quantitative in vivo monitoring of living subjects.7-12 The combination of MR and SPECT imaging can provide the complementary information with the high sensitivity and high resolution to visualize the molecular events more 2


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accurately in vivo.13-17 The use of MR/SPECT contrast agents should help to detect tumor more accurately by enhancing the contrast between cancerous and normal tissues particularly on the early stage of cancer development. Therefore, MR/SPECT contrast agents such as radiolabeling of superparamagnetic iron oxide nanoparticles (SPIONs), silica coated SPIONs, gold nanoprobes, and gadolinium chelated dieth-ylenetriaminopentaacetic acid (Gd-DTPA) have been attracted extensive research efforts.13,14,16-18

Generally, the use of the strategy of PEGylated nanoparticles can target tumors effectively with enhancing permeability and retention (EPR) effect for imaging and therapy in vivo to some extent.4,19-21 However, nanoparticles are often rapidly recognized and sequestered by circulating macrophages and Kupffer cells of the mononuclear phagocyte system (MPS) organs (liver, spleen, etc.) to cause the long-term toxicity. Up to date, it is still a challenge to develop a multifunctional nanoplatform with the integration of diagnosis and therapy for the efficient renal clearance and low accumulation in MPS organs.

Owing to the good biocompatibility, high magnetization value and high photothermal conversion efficiency, PEGylated Fe-based nanoparticles have been applied for MR imaging with the high sensitivity, near-infrared photothermal therapy, and magnetic hyperthermia therapy.22-27 However, up to date, no research focused on the pharmacokinetics and biodistribution of Fe@Fe3O4 nanoparticles (MNPs) though it is one of the most important aspects for the clinic translation. In this work, we

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reported

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I-c(RGDyK) peptide conjugated PEGylated Fe@Fe3O4 nanoparticles

(125I-RGD-PEG-MNPs)

to

acquire

a

tumor-targeting

multifunctional

nanotheranostic agent (Scheme 1). Due to the small hydrodynamic diameter (HD) of ~ 40 nm, high-density PEG chains, radioactivity, and the binding property to integrin αvβ3 protein, the high effecive tumor tageting property in vivo was confirmed by MR and SEPCT imaging on the U87MG glioblastoma xenograft model. The in vivo metabolic pathway has been clarified by the the pharmacokinetics and biodistribution of

125

I-RGD-PEG-MNPs with the quantitative analysis. The excellent targeting

photothermal therapy effect has been performed under the guidance of the MR/SEPCT imaging.

EXPERIMENTAL SECTION

Materials. Fe(CO)5 was purchased from Development of Beijing Chemical Technology Co., Ltd. Branch. Oleylamine (70%), 1-octadecene (ODE, 90%), hexadecylamine (HDA, 90%) were purchased from Sigma Aldrich. Chloramine-T (99%) and sodium metabisulfite (Na2S2O5, 97%) was purchased from J&K Chemical. The integrin αvβ3 targeting peptide cyclic c(RGDyK) was purchased from Peptide International, Inc. DSPE-PEG2000 and DSPE-PEG2000-NHS were purchased from Nanocs Inc. Na125I was obtained from Shanghai GMS Pharmaceutical Co., Ltd.

Characterization. TEM was performed using a JEOL JEM-2100 transmission electron microscope. XRD was measured on a Rigaku D/MAX 2250 diffractometer

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with Cu Kα radiation. FT-IR spectra were recorded on a Nicolet Avatar 370. Zeta potential and hydradynamic diameter were carried on a Malvern Zetasizer Nano ZS model ZEN3600. The hysteresis loop was detected on a Quantum Design SQUID magnetometer. Photothermal imaging and radio TLC chromatogram were recorded on a FLIR A300 camara and AR-2000 radio-TLC scanner, respectively. The photothermal therapy was performed under the irradiation of an 808 nm laser from Shanghai Xilong Optoelectronics Technology Co., Ltd.

Preparation of PEG-MNPs. Fe@Fe3O4 nanoparticles (MNPs) was synthesized according to the previous report.28 0.5 mL of oleic acid-coated MNPs in chloroform (10 mg/mL) was mixed with 2 mL of DSPE-PEG2000 in chloroform (5 mg/mL) in a glass vial. After the solution was shaken for overnight, the solvent was evaporated completely. Subsequently, water (5 mL) was added. The excess DSPE-PEG2000 was dialyzed with the molecular weight cut-off of 14000 (MWCO) for 48 h. After dialysis, the dispersion was filtered using the 0.1 µm cellulose acetate syringe filter.

Preparation

of

RGD-PEG-MNPs.

In

a

glass

DSPE-PEG2000-NHS (10 mg) and the cyclic c(RGDyK)

vial,

the

mixture

of

peptide (1 mg) in 2 mL of

phosphate-buffer solution (PBS, pH=8.5) was stirred magnetically for 5 h at 25 °C, then lyophilized. Subsequently, DSPE-PEG2000 (10 mg) in chloroform (2 mL) was added to the above mixture and sonicated to dissolve. After 0.5 mL of oleic acid-coated MNPs in chloroform (10 mg/mL) was added to the above mixture and shaken for overnight. The solvent was evaporated completely, then water (5 mL) was

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added. The obtained nanoparticles were purified by dialysis (molecular weight cut-off, MWCO 14000) for 48 h. After dialysis, the dispersion was fitered using the 0.1 µm cellulose acetate syringe filter. 125

I Labeling of RGD-PEG-MNPs. The RGD-PEG-MNPs was labeled by using

the chloramine-T method. In a polypropylene vial, a mixture of 0.2 mL RGD-PEG-MNPs (1 mg/mL), 1.2 mCi Na125I and 100 µL of chloramine-T in PBS (pH=7.4) with the concentration of 4 mg/mL was shaken for 4 min, then the iodination was quenched with 200 µL of Na2S2O5 in PBS (pH=7.4) with the concentration of 5 mg/mL. The excess

125

I was completely removed through the

centrifugation filtration and washed with water for 5 times until the filtration solution was no detachable gamma activity. The radiolabeling yield was measuerd by the radiochromatography using a TLC scanner.

125

I-RGD-PEG-MNPs were dispersed in

the saline solution.

Radiolabeling Stability of 125

125

I Labeling in Solution. An approximate 10 µL

I-RGD-PEG-MNPs (1 mg/mL) with 500 µL of saline solution and RMPI with 10%

FBS was incubated at 37 °C in a water bath for two weeks, respectively. The radioactivity of

125

I-RGD-PEG-MNPs was monitored by the radiochromatography

using a TLC scanner to determine the percentage of 125I on the 125I-RGD-PEG-MNPs.

Photothermal Evaluation in Solution. The aqueous solution (1 mL, 50 µg/mL) of RGD-PEG-MNPs in a quartz cuvette was exposed to an 808 nm laser (1 W/cm2) for 10 min. The increase of temperature was monitored by an IR thermal camera. The 6


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photothermal conversion efficiency of RGD-PEG-MNPs was calculated according to the literature.29

MTT Assay. Human glioblastoma (U87MG) and human breast cancer (MCF-7) cells were obtained from Shanghai Cancer Center & Department of Oncology, Shanghai, China. U87MG and MCF-7 cells were seeded into a 96-well cell culture plate at 5×104 cells/well in DMEM, with 10% FBS and 1% penicillin-streptomycin at 37 °C with 5% CO2 for 24 h, folllowed by the treatment with different concentrations of RGD-PEG-MNPs (0, 5, 10, 20, 50, 100 and 200 µg/mL in DMEM, respectively) for 12 h and 24 h, respectively. 20 µL MTT (5 mg/mL ) solution was added to each well, then incubated for 4 h under the similar incubation condition. After the supernatant was washed, the purple formazan crystal was lysed with DMSO (150 µL). The optical absorption value was measured at 490 nm using an Multiskan MK3 enzyme-linked immunosorbent assay reader with the absorbance at 690 nm as a reference.

T2-weighted MRI in Vitro. To optimize the incubation time, U87MG and MCF-7 cells were incubated with RGD-PEG-MNPs (100 µg/mL) for different time (0, 0.5, 1, 2 and 4 h, respectively) at 37 °C with 5% CO2. Then the cells were washed for three times with PBS, collected and resuspended in PBS with 0.5% xanthan gum for MR imaging. To investigate the receptor-mediated uptake of RGD-PEG-MNPs, U87MG cells were incubated with PEG-MNPs (100 µg/mL) and RGD-PEG-MNPs (100 µg/mL) for 2 h under the similar incubation condition. All MR imaging were

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performed at a 0.5-T MR imaging system. T2-weighted MR images were measured using a traditional spin-echo sequence: TR = 6000 ms, TE = 200 ms, RG1 = 25 db, DRG1 = 3, SW = 100 kHz. The ∆T2/T2 value in cells (%) = (T2 value of only cells − T2 value of cells incubated with NPs) /T2 value of only cells* 100%.

Photothermal Ablation in Vitro. After U87MG and MCF-7 cells were incubated with PEG-MNPs (100 µg/mL) and RGD-PEG-MNPs (100 µg/mL) for 2 h, the excess nanoparticles were removed by washing with PBS. After the 808 nm laser (1 W/cm2) irradiation for 10 min, a standard MTT assay was adopted to measure the cell viability. The blocking experiment and the negative control experiment were investigated under the similar condition.

Tumor Model. Specific pathogen-free 5-6 weeks old BALB/c nude mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Animal care and handling procedures were approved by the guidelines of the Regional Ethics Committee for Animal Experiments. U87MG cells (1×106) were grafted into the BALB/c nude mice. After three weeks, the tumor volume reached about 200 mm3 for further experiments.

Blood Analysis and Histology Examination. The healthy BALB/c nude mice (5-6 weeks old) were divided into four groups randomly (n = 5 per group). Two groups of mice were intravenously injected with saline solution (200 µL) and RGD-PEG-MNPs (10 mg/kg of body weight), and then sacrificed at 24 h intravenous injection (i.v.). The other two groups of mice were injected intravenously with the similar condition, 8


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and then sacrificed at 30th day i.v. Before the mice was euthanatized, the blood of each mice was collected for blood analysis. The heart, liver, spleen, lung and kidney from those mice were collected, and then stained with H&E.

MR Imaging in Vivo. U87MG tumor-bearing mice were intravenously injected with RGD-PEG-MNPs (10 mg/kg of body weight) with or without free RGD (2.5 mg/kg of body weight). In vivo MR imaging was performed on a 7 T Siemens Magnetom Trio system and imaged with the different time (0, 1 , 3 , 6 , 12 and 24 h, respectively). T2-weigehted MR imaging by a fast spin-echo sequence: TR/TE =1500/32 ms, matrix size = 256 × 256，FOV = 30 × 30 mm, slice thickness = 1.5 mm, scan time ~15 min. The T2 relaxation time of the tumor area before and after the injection of RGD-PEG-MNPs using a T2 map multi-slice multi-echo sequence (TR/TE = 1500/32 ms, matrix size = 256 × 256，11 echoes, FOV = 30 × 30 mm, slice thickness = 1.5 mm). To select a region of interest (ROI) within the phantom area, the software can automatically calculate the mean T2 value. The ∆T2/T2 value in tumor (%) = ( T2 value of pre-injection − T2 value of post-injection) /T2 value of pre-injection * 100%.

SPECT Imaging in Vivo. U87MG tumor-bearing were injected intravenously with 125

I-RGD-PEG-MNPs solution (10 mg/kg of body weight, 500 µCi) with or without

free RGD (2.5 mg/kg of body weight), and then imaged with a SPECT imaging system (Bioscan) at 1, 3, 6, 12, 24 and 48 h i.v., respectively. Fusion and 3D reconstruction were performed with InVivoscope software.

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Biodistribution of

125

I-RGD-PEG-MNPs. U87MG tumor-bearing mice were

intravenously injected with 125I-RGD-PEG-MNPs (0.4 mg/kg of body weight, 20 µCi). The blood clearance profile was calculated by collecting blood (10 µL) in heparinized capillaries with the different time (1, 6, 12, 24 and 48 h, respectively) after injection. The half-life time was obtained by a single exponential fitting. The heart, liver, spleen, lung, kidney, stomach, brain, intestine, skin, muscle, bone, tumor, bladder, urine and feces were collected with the different time (1, 6, 12, 24 and 48 h, respectively) after post injection. Samples were weighted. The radioactivity was measured by a gamma counter. %ID/g = organ (tissue) count / injection count / weight of organ (tissue) * 100%.

Photothermal Therapy in Vivo. U87MG tumor-bearing were divided into four groups (n=5 per group) as follows. control group a: mice injected saline only (200 µL); control group b: mice injected RGD-PEG-MNPs only (10 mg/kg of body weight); control group c: mice injected saline (200 µL) + 808 nm laser irradiation; photothermal therapy group d: mice injected RGD-PEG-MNPs (10 mg/kg of body weight) + 808 nm laser irradiation. Tumors in control group c and photothermal therapy group d were covered with an 808 nm laser (0.5 W/cm2) for 5 min at 6 h i.v.. Mice were injected with RGD-PEG-MNPs for three times (for every other 48 h) and treated for three times (at 6 h i.v.). The tumor sizes were measured every other day during the therapy process. The tumor volume was calculated using the formula: V = ab2 /2 (a = length, b = width). After the photothermal therapy, mice were sacrificed. The tumors were removed for H&E and TUNEL stain. 10


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RESULTS AND DISCUSSION

Synthesis and Characterizations of RGD-PEG-MNPs. Fe@Fe3O4 nanoparticles (MNPs) were synthesized according to the previous report.28 The representative TEM images were presented in Figure 1a. The average diameter was 11.5 ± 1.4 nm (see Supporting Information, Figure S1a). The XRD

pattern of as-synthesized MNPs

showed the (110) and (200) diffraction peaks of the body centred cubic iron (see Supporting Information, Figure S1b). No Fe3O4 peaks can be obviously observed because of the peak broadening of their small crystal domains.30 At 300 K, the magnetization value was ~100 emu/g at 5 KOe field (see Supporting Information, Figure S1c), which denmonstrated a higher magnetization compared to Fe3O4 nanoparticles.31 In order to solubilized the oleic acid-coated MNPs in the aqueous media, the hydrophobic MNPs were encapsulated with PEG-phospholipids via the Vander Waals interaction to form PEGylated MNPs (PEG-MNPs).32 To further perform the targeting property of MNPs, the activated carboxyl functional PEG-phospholipids was used to conjugate RGD to form RGD modified PEG-phospholipids, then PEG-phospholipids and RGD modified PEG-phospholipids (w:w = 2:1) were both coated on the surface of MNPs to form RGD modified MNPs (RGD-PEG-MNPs). T2-weighted MR imaging of RGD-PEG-MNPs by a 7.0T MR imaging system revealed a concentration-dependent negative contrast, with the transverse relaxivity (r2) of ∼ 259.8 mM-1 s-1, demonstrating their great potential for MR imaging (see Supporting Information, Figure S2). As shown by TEM (see Supporting Information, Figure S3a 11



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and Figure 1b), the average diameter of PEG-MNPs and RGD-PEG-MNPs was 12.0 ± 0.5 nm (see Supporting Information, Figure S3b) and 12.0 ± 1.1 nm (see Supporting Information, Figure S1d), respectively. The HD was 24.5 ± 0.5 nm for MNPs (see Supporting Information, Figure S3c), 38.5 ± 0.4 nm for PEG-MNPs (see Supporting Information, Figure S3d) and 38.6 ± 0.1 nm for RGD-PEG-MNPs (Figure 1c), respectively, which was larger than that shown from TEM due to the PEG-phospholipid layer on the surface of MNPs and a little aggregation. The zeta potential increased from -22.5 ± 0.1 to -14.8 ± 0.6 mV before and after the RGD conjugation due to the positive charge of RGD, respectively (see Supporting Information, Figure S4). The vibration bands located at 1104 cm−1 (νC–O–C) and 1640 cm−1 (amide I) were designated to the PEG units and the amide groups, respectively (see Supporting Information, Figure S5), denmonstrating the successful coating of PEG and RGD on the surface of MNPs.33,34 To the further application of nanoparticles in the biological system, their colloidal stability is one of the most important properties. Therefore, the stability of RGD-PEG-MNPs was evaluated in water, PBS and RMPI with 10% FBS (simulation of in vivo plasma) by the change of HDs as a function of time (Figure 1d). In the above three media, RGD-PEG-MNPs showed the similar HD and stability. Especially, in the simulation of in vivo plasma, the HD was only ∼35 nm. More importantly, after 48 h, no obvious change was observed. The property of such small HD and good stability in the simulated physiological environment provided the great possibility for the further investigation in vivo.

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Targeting Property of RGD-PEG-MNPs in Vitro. Prior to the application of RGD-PEG-MNPs in vivo, the in vitro cytotoxicity was performed on MCF-7 (low integrin αvβ3 expression) and U87MG (high integrin αvβ3 expression) cells via a standard methylthiazolyl tetrazolium (MTT) assay (Figure 2a). It was clear that the cell viability was higher than ~85% within the concentration of 200 µg/mL after 12 and 24 h incubation, respectively, indicating that RGD-PEG-MNPs have a very low cytotoxicity. To identify the optimal incubation time for tumor cells by MR imaging, the T2 value and T2-weighted MR images were obtained after MCF-7 and U87MG cells were incubated with RGD-PEG-MNPs (100 µg/mL) with different times (0.5, 1, 2 and 4 h, respectively) on a 0.5 T MR system. After the incubation for 2 h, the T2-weighted MR image gradually turned darker in U87MG cells compared to that in MCF-7 cells (Figure 2b1). The quantitative analysis of the ∆T2/T2 value further confirmed this point (Figure 2b2). After MCF-7 and U87MG cells were incubated for 2 h, respectively, the ∆T2/T2 value of U87MG cells was ~1.7 times higher than that of MCF-7 cells. However, the ∆T2/T2 value showed no remarkable difference bewteen MCF-7 and U87MG cells when the incubation time was below 2 h, which might be no sufficient binding between the integrin αvβ3 protein and RGD-PEG-MNPs within such short time. After the incubation for 4 h, no substantial difference of the ∆T2/T2 value for MCF-7 and U87MG cells was observed which may be due to the saturated binding of RGD-PEG-MNPs to the integrin αvβ3 protein on the surface of U87MG cells.35 Therefore, the optimal incubation time of RGD-PEG-MNPs was selected as 2 h for further T2-weighted MR imaging investigation.

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To further confirm the targeting property of RGD-PEG-MNPs, the T2 values and T2-weighted MR images were collected after MCF-7 and U87MG cells were incubated with PEG-MNPs and RGD-PEG-MNPs (100 µg/mL) for 2 h, respecctively. After the incubation with RGD-PEG-MNPs, the T2-weighted MR image of U87MG cells showed a significantly signal decrease (Figure 2c1), and the ∆T2/T2 value of U87MG cells was ~1.5 times higher than that of MCF-7 cells (Figure 2c2). Furthermore, the ∆T2/T2 value of U87MG cells incubated with RGD-PEG-MNPs was ~1.8 times higher that of U87MG cells incubated with PEG-MNPs. The difference of the MR signal should be due to the specific interaction between the integrin αvβ3 protein U87MG cells and RGD on the surface of MNPs. The blocking experiment was designed to further verify their targeting property. U87MG cells were pre-incubated with RGD (50 µg/mL) for 30 min, then treated with RGD-PEG-MNPs for another 2 h, the ∆T2/T2 value of U87MG cells incubated with RGD-PEG-MNPs was ~2.9 times higher than that of U87MG cells pre-incubated with RGD. All these results suggested that RGD-PEG-MNPs can specifically affect T2-weighted MR imaging through the integrin αvβ3 mediated cellular binding and uptake.36

Targeting Photothermal Therapy in Vitro. Considering the near-infrared absorption property of RGD-PEG-MNPs (50 µg/mL) (see Supporting Information, Figure S6a), the RGD-PEG-MNPs showed an obvious photothermal conversion property under the 808 nm laser irradiation (1 W/cm2) with the photothermal conversion efficiency of ~ 25.4% (see Supporting Information, Figure S6b, S6c and S6d), which was similar to the value reported by our previous report.23 Based on the 14


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targeting property of RGD-PEG-MNPs, the targeting photothermal effect were further investigated by a standard MTT assay on MCF-7 and U87MG cells incubuted for 2 h. As presented in Figure 2d, MCF-7 and U87MG cells were incubated with RGD-PEG-MNPs (100 µg/mL), respectively, then exposed to an 808 nm laser (1 W/cm2) for 10 min. The viability were ~26.0% for U87-MG cells and ~ 54.2% for MCF-7 cells, respectively. However, for the control MCF-7 and U87MG cells only incubated with RGD-PEG-MNPs, the viability was ~88.6% MCF-7 cells and ~89.3% for U87MG cells, respectively. For another control group of U87MG cells incubated with PEG-MNPs (100 µg/mL), then exposed to an 808 nm laser for 10 min, the viability was ~ 63.9%. For U87MG cells and MCF-7 cells only exposed to an 808 nm laser, the viability was ~80.4% and ~78.5%, respectively. For the blocking experiment group, after U87MG cells were pre-incubated with RGD (50 µg/mL) for 30 min, incubated with RGD-PEG-MNPs for another 2 h. Under the same irradiation, the viability was ~74.0%. All these data confirmed that the combination of RGD-PEG-MNPs and the 808 nm laser irradiation was able to significantly kill targeting cancer cells.

In Vivo Toxicity. To further investigate the long-term in vivo toxicity of RGD-PEG-MNPs,

healthy

ICR

mice

were

injected

intravenously

with

RGD-PEG-MNPs saline solution (10 mg/kg of body weight). After the i.v. for 24 h and 30 days, respectively, the mice blood and organs (heart, liver, spleen, lung and kidney) were collected for blood analysis and histological examination. The organ slices were stained by H&E staining. Compared to the control group, the organs still 15



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remained the similar morphology for the experimental group (Figure 3a). The liver function (AST, ALP and ALT) and kidney function (UA, BUN and CREA) both remained the normal level (Figure 3b and Figure 3c), suggesting no obvious toxicity to liver and kidney, and there no noticeable changes of the other blood index. Under our experimental condition, the results clearly demonstrated that RGD-PEG-MNPs have very low toxicity in vivo.

MR/SPECT Imaging in Vivo. Encouraged by the good transverse relaxation porperty of Fe@Fe3O4 nanoparticles,23 T2-weighted targeting MR imaging in vivo after the i.v. of RGD-PEG-MNPs (10 mg/kg of body weight) was obtained on the U87MG tumor-bearing mice on a 7-T MR imaging scanner (Figure 4a and 4b). As shown in Figure 4c, at 6 h p.i., the ∆T2/T2 value of tumor increased ~50.1% compared with that of pre-injection. However, in the case of the blocking group, the ∆T2/T2 value in tumor only increased ~8.0%. The ∆T2/T2 value in tumor in the experiment group was ~6.2 times higher than that in the blocking group.

To investigate the possibility of the application of RGD-PEG-MNPs for SPECT imaging, RGD-PEG-MNPs was labeled with

125

I with a standard chloramine-T

method (see Supporting Information, Figure S7).37 The radiolabeling yield was determined to be ~60% by the radiochromatography using a TLC scanner (see Supporting Information, Figure S8a). After the purification, the purity of 125

I-RGD-PEG-MNPs was higher than 99% (see Supporting Information, Figure S8b).

The radiolabeling stability of

125

I-RGD-PEG-MNPs was tested in saline solution and

16


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RMPI with 10% FBS at 37 °C for 14 days. More than 88% of 125I was still bounded to the RGD-PEG-MNPs, which demonstrated their high radiolabeling stability (see Supporting Information, Figure S9 and Figure S10). For SPECT imaging in vivo, the U87MG 125

tumor-bearing

mice

(n

=

3)

were

injected

intravenously

with

I-RGD-PEG-MNPs saline solution (10 mg/kg of body weight, 500 µCi). For the

blocking experiment, free RGD (2.5 mg/kg of body weight) was pre-injected intravenously. After 30 min,

125

I-RGD-PEG-MNPs were injected intravenously with

the same dose. The small animal SPECT imaging in vivo was acquired at 1, 3, 6, 12, 24, and 48 h i.v., respectively (Figure 4d). From 1 to 6 h i.v., the radioactive signal from

125

I-RGD-PEG-MNPs gradually increased. Afterward, the radioactive signal

gradually weakened and almost was invisible at 48 h i.v.. However, for the blocking group, the radioactive signal in tumor was almost undetectable within 48 h (Figure 4e). These results demonstrated that 125I-RGD-PEG-MNPs entered into the tumor with the specific targeting property via the integrin αvβ3-receptor mediated endocytosis.

Pharmacokinetics and Biodistribution. To investigated the pharmacokinetics, biodistribution and the targeting ability of RGD-PEG-MNPs to tumor, the U87MG tumor-bearing mice (n = 5 per group) were injected intravenously with 125

I-RGD-PEG-MNPs saline solution (0.4 mg/kg of body weight, 20 µCi). The blood,

major organs, and tissues were examined at different time (1, 6, 12, 24 and 48 h, respectively, Figure 5a). The

125

I-RGD-PEG-MNPs displayed the long blood

circulation time (t1/2 = 10.8 h) with a single exponential fitting (Figure 5c), which was available for the active targeting and enhanced permeability and retention (EPR) 17



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property to tumor.38 As show in Figure 5b, the tumor uptake of 125I-RGD-PEG-MNPs gradually accumulated in the tumor from 1 to 6 h i.v.. At 6 h i.v., the tumor maximum uptake (6.75 ± 1.24 % of the injected dose per gram (ID/g)) was mainly attributed to the tumor specific binding affinity of RGD-PEG-MNPs.39 From 6 to 48 h the tumor accumulation of RGD-PEG-MNPs decreased. At 48 h i.v., the tumor uptake was determined to be only 0.14 ± 0.05% ID/g. Once the tumor uptake of 125

I-RGD-PEG-MNPs reached its maximum, they re-entered into the blood slowly

and diffused in normal tissue.40 The biodistribution of

125

I-RGD-PEG-MNPs was

further quantitatively analyzed to provide the insight on the clearance pathways. As shown in Figure 5c, at 1 h i.v., the uptake of

125

I-RGD-PEG-MNPs in the kidney

reached the maximum value of 2.32 ± 0.24 ID/g, then gradually decreased to 0.28 ± 0.09% ID/g at 48 h i.v., which was in aggrement with the clearance of 125

I-RGD-PEG-MNPs from blood, indicating MNPs could be execreted partially from

the urine system.41 The decrease of the relative signal intensity analysis of MR imaigng in the kidney (see Supporting Information, Figure S11a and Figure S11c) further confirm this point. The highest uptake of

125

I-RGD-PEG-MNPs in the liver

and spleen was obtained at 1 h i.v., then gradually decreased. From 1 to 48 h i.v., the accumulation of the

125

I-RGD-PEG-MNPs in the liver decreased from 8.47 ± 1.81 to

1.11 ± 0.21% ID/g, and reduced from 1.87 ± 0.63 to 0.16 ± 0.09% ID/g in the spleen, respectively (Figure 5d), suggesting the clearance of

125

I-RGD-PEG-MNPs through

the hepatic route.42 The decrease of the relative signal intensity analysis of MR imaging in the liver (see Supporting Information, Figure S11a and Figure S11d) also

18


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provided the additional information. From 1 to 48 h i.v., the uptake level of 125

I-RGD-PEG-MNPs in small intestine and large intestine decreased from 1.36 ±

0.39 to 0.04 ± 0.01% ID/g, and from 1.16 ± 0.13 to 0.04 ± 0.02% ID/g (see Supporting Information, Table S1), respectively, suggesting that a small amount of 125

I-RGD-PEG-MNPs could be cleared out from the body through the metabolism

way 125

besides

the

urinary

and

hepatic

system.

The

accumulation

of

I-RGD-PEG-MNPs in skin, muscle, and lung also decreased with the time,

indicating that 125I-RGD-PEG-MNPs were distributed temporally to these organs and tissues and could be efficiently cleared out within 48 h without the increase amount uptaken by the liver and spleen.42 As a result, the efficient renal and hepatic clearance of 125I-RGD-PEG-MNPs were attributed to the small HD and high-density PEG units on the surface of MNPs, which enabled the 125I-RGD-PEG-MNPs highly stable in the physiological environment.

MR/SPECT Imaging Guided in Vivo Photothermal Therapy. On the basis of the promising in vitro photothermal therapy result, photothermal imaging was conducted to measure the temperature in tumor. U87MG tumor-bearing mice were intravenously injected with RGD-PEG-MNPs (10 mg/kg of body weight). Considering that RGD-PEG-MNPs were accumulated in the tumor with a maximum at 6 h i.v. resulted from the MR/SPECT imaging and quantitative analysis, the tumor was irradiated with an 808 nm laser (0.5 W/cm2) for 5 min at 6 h i.v.. As presented in Figure 6a, the temperature of tumor increased ~8.2 °C. For a control group, the mouse was injected intravenously with the saline solution (200 µL), the temperature of tumor increased 19



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only ~1.7 °C under the same condition. For the blocking group, the mouse was intravenously injected with free RGD (2.5 mg/kg of body weight). After 30 min, RGD-PEG-MNPs were injected intravenously with the same dose, the temperature of tumor increased only ~3.5 °C under the same condition. The fact further confirmed the targeting property of RGD-PEG-MNPs and would be benefit for the following photothermal therapy in vivo.

Under the guidance of MR/SPECT and phototheral imaging, we further investigated photothermal therapy in vivo. Twenty U87MG tumor-bearing mice were divided into four groups as follows. Control group a: mice injected saline only; control group b: mice injected RGD-PEG-MNPs only; control group c: mice injected saline plus the laser irradiation; photothermal therapy group d: mice injected RGD-PEG-MNPs plus the laser irradiation. The changes of the body weight and relative tumor volume of each group were recorded as a function of time. No obvious body weight loss (see Supporting Information, Figure S12) and abnormal behavior of mice was observed for each group, indicating the low toxic side effects of the photothermal treatment. As shown in Figure 6b, the relative tumor volumes in control group b and c were similar to that in control group a. While the relative tumor volumes at the last day for all the control groups (a, b, and c) were about ~8.0 times lager than those at the first day, the tumor in the photothermal therapy group d nearly disappeared on the 8.5 day. As shown in Figure 6c1-c4 and 6d1-d4, the tumors were almost completely destroyed after sixteen days of the photothermal treatment. After the photothermal treatment, the mice were sacrificed and tumors were collected. The photographs of tumor in each 20


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group were shown in Figure 6g. The tumor growth in photothermal therapy group was almost completely inhibited. TUNEL staining showed that tumor cells in photothermal therapy group were extensively stained brown compared to three control groups (Figure 6e1-e4). As shown in Figure 6h, 24.1 ± 1.9% and 29.9 ± 2.3% positive cells were found only intravenously injected with RGD-PEG-MNPs and saline plus the laser irradiation, respectively. Howerer, 47.7 ± 2.4% TUNEL positive cellswere observed in photothermal therapy group, indicating a significant cancer therapy effect in such a short therapy time. Similarly, from the results of H&E staining (Figure 6f1-f4), nuclear shrinkage and fragmentation was obviously observed in photothermal therapy group. Our results all demonstrated that RGD-PEG-MNPs have a potential agent for highly effective targeting photothermal therapy.

CONCLUSION

In summary, we have successfully developed Fe@Fe3O4 NPs

125

I-c(RGDyK) peptide PEGylated

with a small HD of ~ 40 nm. By use of their good transverse

relaxation and radioactive property,

125

I-RGD-PEG-MNPs have been developed for

MR and SPECT imaging in vivo tumor. The pharmacokinetics and biodistribution in vivo confirmed the excellent targeting property to tumor and low mononuclear phagocyte uptake of MNPs. Furthermaore, due to the high photothermal conversion efficiency, the nanoparticles could be rendered as a multifunctional nanotheranostic agent for MR/SPECT imaging guided the highly effective photothermal therapy in

21



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Page 22 of 38

vivo. Our work provided a feasible approach to be a novel theranostic agent for the clinical transformation of Fe@Fe3O4 nanoparticles in the future.

ASSOCIATED CONTENT

Supporting Information.

The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

TEM size distribution of MNPs and RGD-PEG-MNPs. XRD pattern and magnetic hysteresis loop of MNPs. T2-weighted MR images and T2 relaxation rates (r2) of RGD-PEG-MNPs. TEM images and TEM size distribution of PEG-MNPs. Hydrodynamic diameter of MNPs and PEG-MNPs. Zeta potentials of PEG-MNPs and RGD-PEG-MNPs. FT-IR spectra of MNPs, PEG-MNPs and RGD-PEG-MNPs. UV-vis-NIR spectral, temperature elevated curve and photothermal effect of RGD-PEG-MNPs.

125

I Labeling of RGD-PEG-MNPs. Radio TLC chromatogram of

125

I-RGD-PEG-MNPs.

125

I-RGD-PEG-MNPs. Radio TLC chromatogram of

The

curves

of

the

radiolabeling 125

stability

of

I-RGD-PEG-MNPs on the

first day and 14th day. In vivo T2-weighted MR images of tumor mice at the different time points. The biodistribution data of

125

I-RGD-PEG-MNPs. The weight of tumor

mice during the photothermal therapy.

AUTHOR INFORMATION Corresponding Author

22


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*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was partially supported by National Natural Science Foundation of China (Nos. 21271130, 21371122, and 21571130) and International Joint Laboratory on Resource Chemistry of Ministry of Education (IJLRC).

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Scheme 1. The schematic illustration of the synthetic process of RGD-PEG-MNPs and 125I-RGD-PEG-MNPs.

Figure 1. TEM images of MNPs (a) and RGD-PEG-MNPs (b), respectively. (c) The hydrodynamic diameter of RGD-PEG-MNPs. (d) The changes of the hydrodynamic

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diameters as a function of time and photographs (inset) of RGD-PEG-MNPs in the different media (water, PBS, and RMPI with 10% FBS).

Figure 2. (a) In vitro viability of U87MG and MCF-7 cells incubated with RGD-PEG-MNPs with different concentrations for 12 h and 24 h at 37 °C, respectively. T2-weighted MR images (b1, from left to right, the incubation time from 0 to 4 h) and the ∆T2/T2 value (b2) of RGD-PEG-MNPs in U87MG cells and MCF-7 incubated for different times (0.5, 1, 2, and 4 h, respectively) at 37 °C on a 0.5T MR system. The incubated concentration was 100 µg/mL. T2-weighted MR images (c1, from left to right, the group order corresponding to that of c2 ) and the ∆T2/T2 value (c2) of RGD-PEG-MNPs in U87MG and MCF-7 cells at 37 °C. (d) In vitro viability of

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U87MG and MCF-7 cells incubated with RGD-PEG-MNPs and PEG-MNPs at 37 °C under the 808 nm laser irradiation (1 W/cm2) for 10 min, respectively. U87MG and MCF-7 cells as the control groups; Free RGD + U87MG (+): U87MG cells pretreated with free RGD (50 µg/mL), then incubated RGD-PEG-MNPs; U87MG (-): U87MG cells incubated with PEG-MNPs; MCF-7 (-): MCF-7 cells incubated with PEG-MNPs; MCF-7 (+): MCF-7 cells incubated RGD-PEG-MNPs; U87MG (+): U87MG cells incubated RGD-PEG-MNPs. In Figure c and d, the incubated concentration, time and temperature were 100 µg/mL, 2 h and 37˚C. (***p

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