Geometrical Confinement of Gadolinium Oxide Nanoparticles in Poly

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Biological and Medical Applications of Materials and Interfaces

Geometrical confinement of gadolinium oxide nanoparticles in PEG/RGD-modified mesoporous carbon nanospheres as an enhanced T MRI contrast agent 1

Ye Kuang, Yi Cao, Min Liu, Guangyue Zu, Yajie Zhang, Ye Zhang, and Renjun Pei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09709 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 18, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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ACS Applied Materials & Interfaces

Geometrical confinement of gadolinium oxide nanoparticles in PEG/RGD-modified mesoporous carbon nanospheres as an enhanced T1 MRI contrast agent

Ye Kuang1, Yi Cao1, Min Liu1, Guangyue Zu1, Yajie Zhang1, Ye Zhang1, Renjun Pei1,2,*

1

CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and

Nano-Bionics, Chinese Academy of Sciences, Suzhou, 215123, China 2

School of Nano Technology and Nano Bionics, University of Science and

Technology of China, Hefei, 230026, China.

KEYWORDS: geometrical confinement, Gd2O3 nanoparticles, mesoporous carbon nanoparticles, T1 contrast agent, tumor-targeting imaging

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ABSTRACT: A new strategy for designing contrast agents (CAs) based on geometrical confinement will become an competent way to improve the relaxivity of CAs. Herein, a magnetic resonance imaging (MRI) nanoconstruct is fabricated through loading Gd2O3 nanoparticles into mesoporous carbon nanospheres, followed by conjugation of poly(ethylene glycol) (PEG) and c(RGDyK) peptide (Gd2O3@OMCN-PEG-RGD), which could prolong the blood circulation half-life as well as improve the tumor-targeting ability. As a result, the Gd2O3@OMCN-PEG-RGD exhibits an outstandingly high relaxivity (r1=68.02 mM-1 s-1), which is ~5.3 times higher than Gd2O3 nanoparticles (r1=12.74 mM-1 s-1). Afterward, both MTT test and H&E staining show the Gd2O3@OMCN-PEG-RGD has wonderful biocompatibility in vitro and in vivo. Moreover, the in vivo MR images indicate that the Gd2O3@OMCN-PEG-RGD could accumulate in the tumor region more rapidly than that of Gd2O3@OMCN-PEG. This study presents a facile method to fabricate a MRI CA with excellent T1 contrast ability based on geometrical confinement and excellent biocompatibility, which could act as an optimal contender for sensitive in vivo tumor imaging with outstanding targeting ability.


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1. INTRODUCTION In order to obtain desirable images of different tissues and internal structures without radiated damage, MRI is an effective imaging technique through applying magnetic field as well as radio-frequency pulses.1-3 Many of its sensitivity, specificity, and accuracy rely on the utilization of contrast agents (CAs), which are used to short proton relaxation process of surrounding water protons and improve the signal intensity of tumor areas to enhance diagnostic efficiency.4 There are two kinds of MRI CAs according to the two different relaxation modes: T1 CAs (positive CAs) for enhancing longitudinal relaxivities and T2 CAs (negative CAs) for improving transverse relaxivities. T1 CAs has attracted more attention because T1 CAs can provide a brighter image of the interest region, faster scanning and a higher signal-to-noise ratio.5,6 However, Gd(III)-DTPA and the corresponding derivatives as most frequently used T1 contrast agents in clinic display low r1 value (about 4.0 mM-1 s-1), inferior tissue specificity and insufficient signal-enhancing effects.7-9 Thus, there is an urgent requirement for fabrication of novel MRI CAs with superior T1 relaxivity and tissue specificity. Based on the Solomon–Bloembergen–Morgan (SBM) theory,10 both the inner-sphere (IS) and the outer-sphere (OS) play a role in improving the paramagnetic relaxation enhancement of Gd-base CAs.11,12 The whole longitudinal relaxivity (r1) consist of the relaxivity of IS (r1IS) and that of OS (r1OS). For r1IS, the amount of water molecules (q), the residence lifetime of water protons (τm), the space between the metal ion and the surrounding water protons (rGdH), and the rotational correlation time



(τR) of the CAs are the dominant contributors. For r1OS, the most influential parameter is the diffusion correlation time (τD) of the water molecules surrounding the CA. Thus, the fabrication of novel contrast agents with excellent longitudinal relaxivity could be based on the increase of these parameters.9 A general method for enhancing relaxivity by confining contrast agents (Magnevist, gadofullerenes, or gadonanotubes) into the nanoporous of mesoporous silicon nanoparticles was reported.1 The rotation of these CAs and the diffusion of surrounding water molecules around these CAs could be limited by geometrical confinement, thus leading to an increase of both τR and τD. Furthermore, by loading Magnevist into silicon nanoparticles with various sizes of pores, the r1 value of the as-prepared CAs changed with the pore size, for example, r1~24 mM-1 s-1 from a pore size (5-10 nm), ~10 mM-1 s-1 from a pore size (30-40 nm), and r1~4 mM-1 s-1 from a larger pore size.9 Due to the large pore size or particle size of these mesoporous silicon nanoparticles, it was restricted to act as an effective enhanced MRI CA because of the mismatch between the size of CAs and pore size. Moreover, Ni et al. has confined the Gd2O3 nanoparticles into the channels of mesoporous silica nanoparticles and the as-prepared CAs exhibited a higher r1 relaxivity (45.08 mM-1s-1) at 0.5 T.13 More recently, Gd2O3 nanoparticles have been also encapsulated into the PAMAM dendrimer and showed r1 relaxivity of 53.9 mM-1 s-1 at 7 T.7 Unfortunately, there is lack of the adequate studies on in vivo tumor MR images in those prior works. The targeted MR imaging of tumor tissue in vivo is necessary for providing the bright tumor signals, which can be utilized for locating tumor and image-guided tumor therapy. These enhanced r1 contrast agents through


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geometrical confinement need to explore their potential in the targeted MR images for in vivo applications. Base on the large pore volume, superior surface area, and uniform mesoporous structures, mesoporous carbon nanospheres with the nanoporous structure have attracted

more and more attention in

the

fields of

biotechnology

and

nanomedicine.14-17 It was reported that mesoporous carbon nanoparticles (MCN) exhibited lower cytotoxicity, higher loading capacity, and more advance functions than mesoporous silica nanospheres (MSN) -based nanocarriers.18-20 Compared with carbon nanotubes and graphene, a relatively smooth edge of MCN is favorable to the minimal damage in vitro.19 Moreover, various species (such as drugs, imaging agents) can be transported between the outside and the inside due to the special pore structure. Finally, MCN possesses the enhanced loading of nanoparticles and can effectively deliver cargo through the cell membrane, which make them a perfect nanoparticles-transport platform.21 In this work, Gd2O3 nanoparticles were loaded into the oxidized MCN to fabricate a CAs with high relaxivity, followed by conjugation with PEG and RGD peptide onto the surface of OMCN, which could prolong the blood circulation half-life as well as improve the tumor-targeting ability.22,23 Afterward, the characterization of the Gd2O3@OMCN-PEG-RGD NPs were carried out through TEM, STEM-HAADF, DLS, FT-IR spectroscopy, BET and relaxivity measurements. Furthermore, the biocompatibility of Gd2O3@OMCN-PEG-RGD NPs was evaluated via MTT test and H&E staining, and the MRI capability were assessed by



T1-weighted MR imaging in vitro and in vivo through U87MG cells and nude mice bearing U87MG tumors.

2. MATERIALS AND METHODS 2.1. Materials. Pluronic F127, N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride

(EDC) and diethylene

glycol (DEG) was purchased from

Sigma-Aldrich. GL Biochem (Shanghai) Ltd provided c(RGDyK) peptide (cyclo (Arg-Gly-Asp-D-Tyr-Lys)). The GIBCO Life Technologies supplied Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS). All the rest of reagents were obtained from domestic suppliers.

2.2. Fabrication of the oxidized mesoporous carbon nanospheres (OMCN). Mesoporous carbon nanospheres were fabricated according to a pervious method.20,24 In order to get low-molecular-weight phernolic resols, phenol (0.6 g), formalin solution (37 wt %, 2.1 mL) and NaOH solution (0.1 M, 15 mL) were mixed and stirred at 70 °C for 0.5 h. Then, Pluronic F127 (0.96 g) dissolved in 15 mL of H2O was put in and the blend was agitated at 66 °C at a speed (340±40 rpm) for 2 h. Afterward, 50 mL H2O was used to dilute this solution. During this reaction, the color of the solution was changed from colorless transparent to pink and turned to crimson at the end. After stirring for another 18 h, the reaction was halted when the deposit was appeared. Next, the acquired solution and 56 mL H2O was put into an autoclave and heated at 130 °C for 24 h. After centrifugation and wash with distilled water, the


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product was obtained. Furthermore, the MCN was collected after heating under a highly pure N2 atmosphere at 550 °C for 5 h. Finally, MCN was added into a hybrid acid solution of H2SO4 and HNO3 (3:1), sonicated for 3 h and then stirred at 60 °C for 4 h. The excessive acid was washed and the OMCN was obtained through freeze-dried.

2.3. Preparation of Gd2O3 nanoparticles. Synthesis of Gd2O3 nanoparticles was performed according to the previous method with minor modification.6,13 In detail, gadolinium chloride hexahydrate (1.15 g) was added in 20 mL of DEG. After stirring for 24 h at 60 °C, NaOH solution (0.75 mL, 3 mM) was put into the above solution quickly. The solution was heated to 140 °C and stirred for 1 h, and then remained at 180 °C for 4 h. Finally, the transparent colloidal product was preserved at 4 °C.

2.4. Synthesis of Gd2O3@OMCN-PEG-RGD. The as-prepared OMCN solution (10 mL, 50 mg mL−1) were mixed with Gd2O3 solution (10 mL) and sonicated for last 4 h. The product (Gd2O3@OMCN) was obtained by centrifugation (14 000 rpm, 10 min), washed with ethanol repeatedly, and dispersed in 5 mL H2O. Afterward, 50 mg EDC was mixed with 5 mL Gd2O3@OMCN for stirring 15 min. The mixture was centrifuged at 14 000 rpm again for 10 min to eradicate the redundant EDC and subsequently dispersed in 5 mL solution containing NH2-PEG-COOH (MW=2000, 5 mg mL-1). After stirring overnight, Gd2O3@OMCN-PEG-COOH was washed with PBS through centrifugation at 14 000 rpm (10 min). Finally, 50 mg EDC was mixed



with the above solution for stirring 15 min and then c(RGDyK) (0.5 mL, 1 mg mL-1) was added. This mixture was stirred overnight and purified by ultrafiltration (MWCO = 100 kDa, Millipore). The Gd2O3@OMCN-PEG-RGD was obtained and stored at 4 °C for following experiments.

2.5. Characterization of Gd2O3@OMCN-PEG-RGD. The morphology of Gd2O3@OMCN-PEG-RGD was characterized by TEM (HT7700, Hitachi) and the size and zeta potential was measured by dynamic light scattering (DLS, Malvern), respectively. Scanning transmission electron microscopy high-angle annular dark-field (STEM-HAADF) images and element mapping images were acquired on a Tecnai G2 F20 S-Twin microscope. Micromeritics ASAP 2000 system was used to determine nitrogen adsorption-desorption isotherms at 77 K. FT-IR were acquired by Thermo Fisher FTIR spectrometer, and the products were pelletized with KBr before measurement. Gadolinium ion concentrations were detected by inductively coupled plasma atomic emission spectroscopy (ICP-AES).

2.6. Stability of Gd2O3@OMCN-PEG-RGD and release studies. 5 mL solution of Gd2O3@OMCN-PEG was loaded in a dialysis bag (MWCO = 7000 Da) against 100 mL deionized water. After dialysis for 3, 6, 24 and 48 h, 1 mL dialysate was took out from the flask to measure the gadolinium ion concentrations by ICP-AES and 1 mL fresh H2O was immediately put in to recover the volume. The size of Gd2O3@OMCN-PEG-RGD in medium was measured using DLS after stirring 0, 3, 6,


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24 and 48 h.

2.7. Measurement of MRI of Gd2O3@OMCN-PEG-RGD. To measure the T1 relaxivity, the as-prepared Magnevist, Gd2O3, Gd2O3@OMCN, Gd2O3@OMCN-PEG, and Gd2O3@OMCN-PEG-RGD with different gadolinium ion concentrations were dispersed in water. A 0.5 T NMR-analyzer (GYPNMR-10) was used to measure relaxivity and obtained T1-weighted MR images at 35 °C. The parameters for T1-weighted images included TE = 8.6 ms and TR = 100 ms. The r1 relaxivity was analyzed from the curve fitting of 1/T1 (s−1) versus the gadolinium ion concentration (mM).

In

addition,

the

measurement

of

Gd2O3@OMCN-PEG

and

Gd2O3@OMCN-PEG-RGD in 1.5 T (magnetic field, most used clinically) was also carried out.

2.8.

In

vitro

cytotoxicity

evaluation.

The

biocytotoxicity

of

Gd2O3,

Gd2O3@OMCN-PEG, and Gd2O3@OMCN-PEG-RGD against U87MG cells was performed using MTT assay. When cells reached 50-60% confluence after incubation for 24 h, 100 µL of sample in DMEM with different concentrations was added to each well and placed into the incubator for another 24 h. And then, the solution was removed

and

replaced

with

DMEM

(100

µL)

and

10

µL

of

3-(4,

5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 5 mg mL-1, 10 µL) was subsequently added and incubated for further 4 h. Afterward, the remaining medium was eradicated and the intracellular blue-violet formazan crystals was



dissolved with 150 µL DMSO. The optical density (OD) was counted using a cell imaging microplate reader (Cytation 3, BioTek) at 570 nm wavelength. the cell viability (%) was calculated from following equation: (ODsample - ODblank/ODcontrol ODblank) × 100%.

2.9. In vitro MRI study. U87MG cells were seeded in 10 mL culture dishes at a cells density of 1 × 105. When the cells reached to 50-60% confluency, the medium was removed and 10 mL medium without FBS containing (a) PBS, (b) free Gd2O3, (c) Gd2O3@OMCN-PEG, (d) Gd2O3@OMCN-PEG-RGD was added. In addition, MCF-7 without integrin expression was chosen as a control to test the targeting efficiency of Gd2O3@OMCN-PEG-RGD. The concentration of Gd was maintained at 0.1 mM in each group. After incubating for 2 h, the cells were harvested and washed thrice with PBS. Finally, the resulting cells were added to 200 µL Eppendorf tubes and then a compact pellet was acquired after centrifugation at 1000 rpm at the tube bottom and immediately detected for MRI. The according parameters were adjusted as follows: NS (number of scans) = 1, TE (echo time) = 8.6 ms, and TR (repetition time) = 100.0 ms.

2.10. In vivo MRI, biodistribution, and toxicity study. Nanjing Sikerui Biological Technology Co. LTD provide female athymic nude mice (4 weeks old, 20 g), which were acclimated for 1 week. All animal experiments were carried out under the guide of the relevant laws and institutional guidelines. To model a tumor, 1 × 106 U87MG


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cells were dispersed in 100 µL of PBS and injected subcutaneously into the armpit of nude mice. After raising for 10 days, the tumor size achieved around 5 mm. Then 100 µL of 24% urethane solution was used to anesthetize the tumor-bearing mice by intraperitoneal injection. All the mice were treated with Gd2O3, Gd2O3@OMCN-PEG, Gd2O3@OMCN-PEG-RGD, and Magnevist, respectively. In all group, 200 µL solutions in physiological saline were injected through tail-vein and the Gd concentration was remained at 0.03 mmol kg-1. Finally, the mice were inserted into an animal handing system, and then placed in the 1.5 T imaging systems (35 °C). MR images were acquired at 2, 6, 12, 24, and 48 h after injection. Spin echo T1-weigthed imaging was carried out with the following parameters: matrix = 512 × 256 mm, TR / TE = 100 / 14.26 ms, FOV = 80 × 45 mm, , slice thickness = 0.8 mm. The tumor-bearing mice (n=10) were adopted for biodistribution study of Gd2O3@OMCN-PEG and Gd2O3@OMCN-PEG-RGD through tail-vein injection. The mice were sacrificed at 24 h post-injection (5 with Gd2O3@OMCN-PEG and 5 with Gd2O3@OMCN-PEG-RGD, and the Gd concentration was remained at 0.03mmol kg-1). The heart, liver, spleen, lung, kidney and tumor were collected and weighed. The Gd content in various origins was detected by ICP-AES. %ID g-1 of nanocomposites in the organ was calculated by the following equation: %ID g-1 = ([Gd] in organ) / (([Gd] in injected solution) x (volume of injected) x (wet weight of organ)). The

in

vivo

toxicity

of

Gd2O3@OMCN-PEG-RGD

was

tested

by

Hematoxylin-Eosin (H&E) staining. 200 µL of Gd2O3@OMCN-PEG-RGD solution



(Gd3+ concentration of 0.03 mmol kg-1) was injected into mice through tail-vein, and 200 µL of physiological saline was as a control through tail-vein injection. After rising for 2 days, organs were collected, including heart, liver, spleen, lung, and kidney for H&E staining.

2.11. Statistical analysis. Quantitative data were showed as mean ± SD. In order to calculate the statistical significance for multigroups, one way analysis of variance (ANOVA) was performed.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of Gd2O3@OMCN-PEG-RGD. In present work, a facile method was offered for fabricating MRI CAs with high r1 relaxivity. Briefly, Gd2O3 nanoparticles were loaded into OMCN, followed by conjugation of PEG and RGD. Mesoporous carbon nanospheres (MCN) were synthesized according to a pervious method.20,24 As shown in the Scheme 1, OMCN was obtained by oxidation of MCN using a mixed acid solution of H2SO4 and HNO3. After oxidation, the surface of OMCN is full of hydrophilic carboxyl groups and easy for the further surface modification.25 Then, the prepared Gd2O3 nanoparticle (~ 2.6 nm) was mixed with OMCN under ultrasound. Thereafter, EDC was added in the solution of Gd2O3@OMCN for activating the carboxyl group and the redundant EDC was discarded via centrifugation. Afterward, NH2-PEG-COOH was mixed with the above solution and conjugated onto the surface of Gd2O3@OMCN via EDC chemistry


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(Gd2O3@OMCN-PEG). Meanwhile, a portion of Gd2O3@OMCN-PEG was labelled with

RGD

according

to

the

above

procedure.

Finally,

the

obtained

Gd2O3@OMCN-PEG-RGD was utilized for the targeted T1-weighted MR imaging of tumor tissue through tail-vein injection.

Scheme 1. Schematic illustration for targeted T1-weighted MR imaging of Gd2O3@OMCN-PEG-RGD.

To enhance the water dispersion and modification sites, the surface of MCN was formed hydrophilic carboxyl groups through oxidization.19,21 As shown in Figure 1A, the MCN exihibited as the uniform and monodispesre nanospheres (around 90~100 nm). The morphology and size of OMCN was maintained after oxidation (Figure 1B), which indicated OMCN can be used as a carrier to encapsulate nanoparticles. As shown in Figure 1E, nitrogen adsorption/desorption isotherm measurements illustrate that the OMCN presented relatively excellent specific surface areas (1131.71 m2 g-1)



and mesoporous size of mean 2.8 nm. Moreover, the Gd2O3 nanoparticles are monodisperse (Figure S1A) and the average size is about 2.6 nm through the result of size distribution (Figure S1B).9 Finally, in orde to vertify the successful confinement of Gd2O3 into the pores of the OMCN after ultrasonic treatment, STEM-HAADF image and elemental mapping image of Gd2O3@OMCN are presented in Figure S2. Elemental mapping image shows the significant signal of Gd, which demonstrates that Gd2O3 was successfully loaded in the Gd2O3@OMCN nanocomposites.7,13 For the better applications in vivo, the Gd2O3@OMCN was coated with a layer of PEG, which could obviously improve the blood circulation half-life, and then, the PEGlyted Gd2O3@OMCN was further modified with a targeting ligand to enhance the accumulation of OMCN in the tumor region. From the Figure 1C and Figure 1D, the morphology of Gd2O3@OMCN-PEG and Gd2O3@OMCN-PEG-RGD both remain intact, which is favor for the stability and safety. The data of DLS shows that the mean size of MCN, OMCN, Gd2O3@OMCN-PEG, and Gd2O3@OMCN-PEG-RGD is 90.2 nm, 93.6 nm, 93.8 nm, and 96.9 nm, respectively (Figure 1F). The mean size of these nanospheres increased with the according surface modification. As shwon in Figure 1G, the peaks (black arrow) at 1734 cm-1 and 1353 cm-1 in OMCN are attributed to the C=O and C-O of surface carboxyls in OMCN, which illustrats that MCN was successfully oxidized as OMCN and the zeta potential is changed from -18.1 mV (MCN) to -37.7 mV (OMCN) as shown in Figure 1H. In the spectrum of the Gd2O3@OMCN-PEG-RGD, an increase in intensity of the characteristic peak (1530 cm-1) of amine group is observed as compared with Gd2O3@OMCN-PEG and OMCN.


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It indicates that the amine group appeared through modifying the RGD peptide with the primary amine groups.26,27 This result is agreement with the change of zeta potential (from Gd2O3@OMCN-PEG of -25.1 mV to Gd2O3@OMCN-PEG-RGD of 5.37 mV), suggesting that the RGD peptide was conjugated successfully onto the surface

of

Gd2O3@OMCN-PEG.

There

was

no

precipitation

of

the

Gd2O3@OMCN-PEG and the Gd2O3@OMCN-PEG-RGD for a month, which is due to modifying PEG on the outer side of these nanospheres. But for Gd2O3@OMCN, a large number of sedimentation appeared during this period. It demonstrates that PEG on the nanosystem surface is favorable for the stability in the solution. To vertify the stability of the Gd2O3@OMCN-PEG-RGD nanosystem, the release of Gd ion from the nanoconstruct was detected at various time intervals (3, 6, 24, and 48 h). However, no Gd3+ ions were detected via ICP-AES (the detection limit: 0.007 mg L-1) at each time interval.9,28 As shown in Figure S3, the size of Gd2O3@OMCN-PEG-RGD in medium after stirring 0, 3, 6, 24 and 48 h remains constant. This result indicates that the toxic Gd3+ ions would not release from Gd2O3@OMCN-PEG-RGD and the stability of Gd2O3@OMCN-PEG-RGD is good in medium, which makes it suitable for the following experiments in vitro and in vivo.



Figure 1. TEM images of MCN (A), OMCN (B), Gd2O3@OMCN-PEG (C), and Gd2O3@OMCN-PEG-cRGD (D). Nitrogen sorption isotherms of as-synthesized


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OMCN and pore diameter distribution curve (inset) (E). The mean size measured at each step (F). (G) FT-IR spectra of MCN (a), OMCN (b), Gd2O3@OMCN-PEG (c), and Gd2O3@OMCN-PEG-cRGD (d). (H) The corresponding zeta potential measured at each step.

3.2. Longitudinal Relaxivity (r1) and MRI of Gd2O3@OMCN-PEG-RGD in Vitro. To verify the potential of Gd2O3@OMCN-PEG-RGD as T1 MRI CA, longitudinal relaxivity (r1) was detected using a 0.5 T MRI scanner at 35 °C. The r1 value was measured from the slope of curves. As shown in Figure 2A, the r1 relaxivity of Magnevist is 4.05 mM-1 s-1, while 12.74 mM-1 s-1 for free Gd2O3. After confining the Gd2O3 into

the

pores

of

OMCN,

the

r1

value

of

Gd2O3@OMCN,

Gd2O3@OMCN-PEG, and Gd2O3@OMCN-PEG-RGD increased to 68.77, 68.01, and 68.02 mM-1 s-1, respectively. This result shows that the surface modification of PEG or PEG-RGD does not affect the r1 value. This is because PEG and RGD on the surface of Gd2O3@OMCN have little effect on the free exchange of water protons with the bulk.1 The r1 values of Gd2O3@OMCN-PEG and Gd2O3@OMCN-PEG-RGD in the higher magnetic field (1.5 T) present 59.24 and 58.96 mM-1 s-1, which reveals a minor reduction because of the raise of magnetic intensity.13,29 On the other hand, Gd2O3@OMCN, Gd2O3@OMCN-PEG, and Gd2O3@OMCN-PEG-RGD provided the significant brighter images than free Gd2O3 and Magnevist through the following studies using T1-weighted images (Figure 2B) at the same gadolinium concentration. Up to 5.3 times enhancement of r1 value was attributed to the geometrical



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confinement of Gd2O3 into the pores of OMCN. According to the BSM theory, prolonged tumbling time and limited diffusion of water molecules will induce an increase of both τR and τD and then lead to an enhancement of r1 value.1,9 As shown in Table S1, free Gd2O3 with different shapes or coatings exhibits the different r1 value, with the highest as 28 mM-1 s-1; meanwhile, free Gd2O3 loading into dendrimer or porous silica exhibited a much higher r1 value (53.9 mM-1 s-1).29-31 However, all r1 values of these CAs showed much lower than the CAs prepared in this work (68.02 mM-1 s-1). This is because the pore size of OMCN (2.8 nm) is suitable for restricting the rotation of ultrasmall Gd2O3 nanoparticles (size is 2.6 nm) and the diffusion of water molecules.9 Therefore, confinement of free Gd2O3 into the pores of OMCN could be an efficient way to prepare MRI CAs with high T1 relaxivity.

Figure 2. Relaxivity measurements (A) and T1-weighted imagings (B) in vitro of different Gd concentrations (0.1 mM, 0.05 mM, 0.025 mM, 0.0125 mM, and 0.00625 mM)

for

Magnevist,

Gd2O3,

Gd2O3@OMCN,

Gd2O3@OMCN-PEG,

and

Gd2O3@OMCN-PEG-RGD in physiological saline. The measurements were carried out using a 0.5 T MRI scanner at 35 °C.


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3.3. In Vitro Toxicity Assay and Cellular MRI Study. As a MRI CA, biotoxicity is another important parameter needed to be considered. In this work, U87MG cells were utilized to assess the biocytotoxicity of three nanopatrticle systems (free Gd2O3, Gd2O3@OMCN-PEG, and Gd2O3@OMCN-PEG-RGD) with various concentrations through the standard MTT assay. As shown in Figure 3A, free Gd2O3 possessed certain toxicity against U87MG cells with increasing Gd concentration. In detail, when the concentration of Gd was increased from 88.9 µM to 200 µM, the cell vitality of U87MG cells was declined from 88.81% to 81.46%. However, even at the Gd concentration of 200 µM, the cell vitality for Gd2O3@OMCN-PEG and Gd2O3@OMCN-PEG-RGD is 93.46% and 94.56%, respectively. This is due to PEG on the surface of Gd2O3@OMCN which could inhibit the leakage of Gd2O3.7 Therefore, the result suggests that Gd2O3@OMCN-PEG-RGD with excellent biocompatibility could be a potential MRI CA. The cellular MRI images of Gd2O3@OMCN-PEG-RGD were evaluated on U87MG and MCF-7 cells. Briefly, U87MG cells were incubated with physiological saline, Gd2O3, Gd2O3@OMCN-PEG and Gd2O3@OMCN-PEG-RGD, respectively. As a control, MCF-7 cells were incubated with Gd2O3@OMCN-PEG-RGD under the same condition. As shown in Figure 3B, the Gd2O3 group provides a slight enhancement of brightness in comparison with the cells incubated with physiological saline, while the cells incubated with Gd2O3@OMCN-PEG exhibites much brighter image than that of Gd2O3 group, which is assigned to the high T1 contrast ability of



Gd2O3@OMCN-PEG

in

comparison

with

free

Page 20 of 33

Gd2O3.

Moreover,

the

Gd2O3@OMCN-PEG-RGD exhibits the brightest image of U87MG cells (MCF-7 cells present a relatively dark image) because the targeting affinity between RGD and U87MG cells (overexpressing RGD receptors).32,33 In order to analyze the difference of MR signal quantitatively, the intensity of image signal was estimated.29 When the blank group is set as a baseline (100%), the signal intensities of free Gd2O3, Gd2O3@OMCN-PEG, and Gd2O3@OMCN-PEG-RGD are enhanced to 289.32%, 576.52%, and 735.94%, respectively. By comparison, MCF-7 cells incubated with Gd2O3@OMCN-PEG-RGD show a relative low signal intensity (565.98%). This result illustrates that Gd2O3@OMCN-PEG-RGD could target to the U87MG cells which overexpress αvβ3 integrin.

Figure 3. (A) Cell viability of U87MG cells incubated with free Gd2O3, Gd2O3@OMCN-PEG and Gd2O3@OMCN-PEG-RGD at various Gd3+ concentrations for 24 h. (B) MR images and signal intensity ratio of U87MG cells treated by free Gd2O3, Gd2O3@OMCN-PEG, and Gd2O3@OMCN-PEG-RGD. MCF-7 cells as a


Page 21 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


control were incubated with Gd2O3@OMCN-PEG-RGD. Cells were incubated with samples (Gd concentration of 0.1 mM) for 2 h, and the same volume of physiological saline was utilized as the blank control (set as 100%). Signal intensity presents a statistically significant difference (∗, p < 0.05; n = 3).

3.4. In Vivo MRI on Tumor-Bearing Mice. The MRI efficiency of Gd2O3, Gd2O3@OMCN-PEG, Gd2O3@OMCN-PEG-RGD against U87MG tumor-bearing mice was acquired using a 1.5 T MRI scanner. After injection, the MR signal intensity of tumor areas by Magnevist showed no apparent change, while the Gd2O3 group exhibited a slight signal enhancement since Gd2O3 possesses higher T1 contrast ability than Magnevist (shown in Figure 4A). In detail, the tumor MR images of Gd2O3 group appeared a little bright after 2 h injection and got its brightest signal after 6 h. Moreover, Gd2O3@OMCN-PEG group exhibited its brightest signal after 24 h injection due to the enhanced blood circulation time based on the increase size. Furthermore, the Gd2O3@OMCN-PEG-RGD group presented the brightest images in all these groups. In depth, a small bright spot appeared in tumor areas after 2 h injection and then the boundary of tumor areas could be preliminarily observed after 6 h. Finally, tumor tissue could be clearly imaged after 24 h and the signal began to reduce after 48 h. The results illustrate that Gd2O3@OMCN-PEG-RGD possesses the favorable targeting ability for enhancing MRI signal at tumor region.32,34 Relative intensity (RI, based on gray intensity estimated by ImageJ Software) of signals in tumor issue was quantitatively analyzed.29 As shown in Figure 4B, the



Page 22 of 33

tumors of mice treated with Magnevist present relative low RI and the values of RI are

kept

around

100%.

However,

for

the

mice

treated

with

Gd2O3,

Gd2O3@OMCN-PEG or Gd2O3@OMCN-PEG-RGD, the signal intensity is obviously improved. Briefly, the Gd2O3 group presents its highest RI (116.25% ± 10%) after 6 h injection. By comparison, the Gd2O3@OMCN-PEG group shows 116 ± 16% increase after 12 h injection and its largest enhancement is 136 ± 17% after 24 h. Afterward, the value declines to 114 ± 15% in 48 h. In contrast, the more obvious increase of intensity is found for the mice in the Gd2O3@OMCN-PEG-RGD group. After 2 h injection, the value of IIR increases to 155 ± 15% and then continues to enhance as time goes on. The maximum value (288 ± 20%) appears after 24 h injection and the RI is kept at 253 ± 16% even after 48 h. The high RI value of 288 ± 20% is sufficient to detect the location of tumor based on the published results.35 Compared to the Magnevist, Gd2O3, and Gd2O3@OMCN-PEG groups, the prominent contrast performance of Gd2O3@OMCN-PEG-RGD could ascribe to the targeting ability of RGD moiety.


Page 23 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. (A) Sagittal MR Images of U87MG tumor-bearing mice (n=5) injected with Magnevist, Gd2O3, Gd2O3@OMCN-PEG, and Gd2O3@OMCN-PEG-RGD at Gd dose of 0.03 mmol kg-1. The images of preinjection were also acquired to estimate the signal intensity increasing rates. The red dotted circle shows the location of tumor. (B) Intensity increasing rate of MR signal for U87MG tumor bearing mice (n = 5) injected

with

Magnevist,

Gd2O3,

Gd2O3@OMCN-PEG,

and

Gd2O3@OMCN-PEG-RGD at Gd dose of 0.03 mmol kg-1. Intensity increasing rate



Page 24 of 33

shows a statistically significant difference (∗, p < 0.05; n = 5).

The biodistribution of Gd2O3@OMCN-PEG and Gd2O3@OMCN-PEG-RGD in various origans at 24 h injection were showed in Figure S4. Gd2O3@OMCN-PEG exhibits a tumor uptake of about 3.5% injected dose (ID) g-1. However, a higher of tumor uptake about 14.8% for Gd2O3@OMCN-PEG-RGD demonstrates the RGD-targeted efficiency, which is consistent with the outcomes of the In Vivo MR images. In addition, both groups displayed prominent uptake in the liver and spleen and low uptake in the heart, lung, and kidney. This is because these two nanocomposites undergo RES uptake in the liver and spleen.36 In order to further estimate the in vivo toxicity of Gd2O3@OMCN-PEG-RGD, histological toxicity assessment was conducted. As shown in Figure 5, for the Gd2O3@OMCN-PEG-RGD treatment group, no sign of inflammatory response is appeared in the samples, no alter appears in the kidney morphology, no pulmonary fibrosis is found in the lung, and no necrosis is observed.37 Generally, all investigated organs present negligible histological changes, and nearly all of the tissues are found similar

compared

to

the

untreated

mice.

In

conclusion,

the

Gd2O3@OMCN-PEG-RGD is a potential tumor-targeted MRI CA with excellent targeting performance and negligible toxicity for U87MG tumor-bearing mice.


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Figure 5. the tissue sections of H&E staining from mice injected with Gd2O3@OMCN-PEG-RGD at the concentration of Gd3+ (0.03 mmol kg-1) and mice injected with PBS.

4. CONCLUSION In summary, Gd2O3@OMCN-PEG-RGD was fabricated for targeted T1-weighted MR imaging of cancer cells in vitro and in vivo. TEM images and BET results show that the mesoporous size of OMCN is mean 2.8 nm (diameter around 90~100 nm) and the average size of Gd2O3 is about 2.6 nm. The enhanced r1 value of Gd2O3@OMCN-PEG-RGD is attributed to confine ultrasmall Gd2O3 nanoparticles into the pores of MCN, which limits the tumble of Gd2O3 and the diffusion of water molecules.

Compared

with

free

Gd2O3

(r1=12.74

mM-1

s-1,

0.5T),

Gd2O3@OMCN-PEG-RGD possesses 5.3 times increase and presents r1=68.02 mM-1 s-1.

Gd2O3@OMCN-PEG-RGD

performs

excellent

biocompatibility

through

evaluating the cytotoxicity against U87MG cells and presents obvious brightness of cells than free Gd2O3 and Gd2O3@OMCN-PEG. Base on the superior targeting efficiency

of

RGD,

the

tumor

tissue

could

be


obviously

detected

in


Gd2O3@OMCN-PEG-RGD group. Hence, Gd2O3@OMCN-PEG-RGD possesses excellent biocompatibility and high T1 contrast ability based on geometrical confinement, which has great potential to make up for deficiencies of the commercial CAs in clinic.

ASSOCIATED CONTENT Supporting Information TEM Image and HR-TEM (inset) of Gd2O3, STEM-HAADF Image and Gd Elemental Mapping Image of Gd2O3@OMCN, Size Change of Gd2O3@OMCN-PEG-RGD in Mediums, Biodistribution of Gd2O3@OMCN-PEG and Gd2O3@OMCN-PEG-RGD, and Relaxivity Values (r1) of Related Gd-Containing Nanoparticles; This material is

available free of charge via the Internet at http://pubs.asc.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], Tel: 86-512-62872776 Notes These authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the Natural Science Foundation of China


Page 26 of 33

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(21775160),

the

National

Key

Research

and

Development

program

(2016YFA0101500) and the CAS/SAFEA International Innovation Teams program.

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TOC graphic


Geometrical Confinement of Gadolinium Oxide Nanoparticles in Poly

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