An Effectively pH-activated Theranostic Platform for Synchronous

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Biological and Medical Applications of Materials and Interfaces

An Effectively pH-activated Theranostic Platform for Synchronous Magnetic Resonance Imaging Diagnosis and Chemotherapy Dan Wang, Haiyan Lin, Guilong Zhang, Yuanchun Si, Hongyi Yang, Guo Bai, Chi Yang, Kai Zhong, Dongqing Cai, Zhengyan Wu, Renfei Wang, and Duohong Zou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11408 • Publication Date (Web): 24 Aug 2018 Downloaded from http://pubs.acs.org on August 26, 2018

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ACS Applied Materials & Interfaces

An Effectively pH-Activated Theranostic Platform for Synchronous Magnetic Resonance Imaging Diagnosis and Chemotherapy Dan Wang†,||, Haiyan Lin‡,||, Guilong Zhang⊥,||, Yuanchun Si†, Hongyi Yang#, Guo Bai§, Chi Yang§, Kai Zhong#, Dongqing Cai⊥, Zhengyan Wu⊥, Renfei Wang ‡,*, Duohong Zou†,§,*

†

Department of Dental Implant Center, Stomatologic Hospital & College, Key

Laboratory of Oral Diseases Research of Anhui Province, Anhui Medical University, Hefei 230032, People’s Republic of China. ‡

Hangzhou Stomatological Hospital, University of Chinese Academy of Sciences,

Hangzhou 310002, People’s Republic of China. §

Second Dental Clinic, Ninth People’s Hospital, Shanghai Jiao Tong University

School of Medicine, Shanghai Key Laboratory of Stomatology, National Clinical Research Center of Stomatology, Shanghai 200001, People’s Republic of China ⊥

Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei

Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, People’s Republic of China #

High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese

Academy of Sciences, Hefei, Anhui 230031, People’s Republic of China

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ABSTRACT: Current magnetic resonance imaging (MRI) guided pH-switching therapeutic platforms have encountered problems, such as low relaxation rates, poor pH-switching efficiencies, and a lag in the drug release behind the MRI. Herein, we designed a nanoplatform with tunable pore size which could match the size of drug molecules for pH-switching MRI and chemotherapy via ultrasmall manganese oxide-capped mesoporous silica nanoparticles (USMO@MSNs). USMO@MSN could quickly dissolve under weakly acidic conditions and leach abundant Mn2+ ions (leaching ratio: 76%), enhancing the MR contrast. The longitudinal relaxation rate (r1) of USMO@MSNs significantly increased from 0.65 to 5.61 mM-1s-1 as the pH decreased from 7.4 to 4.5, showing an ultrahigh-efficiency pH-switching T1-weighted MR contrast ability for in vivo tumor. Meanwhile, the matching pore structure allowed effective loading of doxorubicin (DOX) on USMO@MSNs to form smartly therapeutic system (USMO@MSNs-DOX). The DOX release rate was strongly proportional to the pH-switching MRI signal of USMO@MSNs-DOX, allowing the release of DOX to be efficiently monitored by MRI. Confocal observations indicated that USMO@MSNs-DOX could be effectively internalized by HSC3 cells, and the entire system showed a good pH-switching theranostic performance for HSC3 cells. Therefore, this simple pH-switching system provides a new avenue for timely cancer diagnosis and personalized therapy. KEYWORDS: pH-activated, synchronous, MRI, theranostic platform, tumor chemotherapy

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1. INTRODUCTION In recent years, mesoporous silica nanoparticles (MSNs) have been widely investigated as efficient drug carriers because of their intrinsic large specific surface and high pore volume.1-3 Meanwhile, MSNs that possess abundant surface active sites could be used to easily fabricate controlled drug release systems by nanoparticle surface modification, which would be beneficial to enhance the efficacy of drug delivery and decrease drug side effects for the body.4-7 Currently, many smart delivery systems, such as pH,8 enzyme,9 temperature,10 redox,11 light12 and magnetic field-response systems,13 based on MSNs have been developed and have shown good adaptability and responsiveness to a variety of external environmental stimuli. However, these smart drug delivery systems (SDDSs) have been difficult to be monitored, causing difficulty in assessing the in vivo therapy process, which could negatively affect a patient’s next treatment decision.14,15 Therefore, it was necessary to develop trackable SDDSs via combining imaging agents into SDDSs to fabricate integrated theranostic systems. Magnetic resonance imaging (MRI) is a powerful technique for early disease detection because of the absence of radiation and the non-invasive acquisition of 3D tomographic images with exquisite soft tissue contrast and anatomical detail.16-18 To further improve the imaging signal and resolution, approximately 50% of all MR examinations involve the use of contrast agents (CAs).19-21 MR contrast agents (MRCAs) can be broadly categorized into two types: paramagnetic metal ions (such as Gd3+ and Mn2+) and superparamagnetic iron oxide nanoparticles (SPIOs).22 For 3



paramagnetic metal ions, spin-lattice relaxation time (T1) of water protons are reduced, which increases the signal intensity and causes a brightened T1-weighted image. In contrast, SPIOs are used to shorten the spin-spin relaxation time (T2) of water, leading to a negative signal intensity and dark images. The paramagnetic Gd cHSC3tes as T1 CAs have shown good clinical use for tumor detection.23-25 However, some studies have indicated that gadolinium-based agents have a potential risk for kidney damage because of nephrogenic systemic fibrosis,26-28 thereby leading to new concerns over the safety of gadolinium CAs for clinical use. Mn-based CAs that also have effectively positive contrast enhancement have attracted increasingly more attention because of their non-toxicity and in vivo safety.29,30 Generally, free Mn2+

has a better longitudinal relaxation rate than

Mn-based nanoCAs.31 However, free Mn2+ suffers from short blood retention time in vivo, thus leading to low accumulation and poor performance for disease contrast. Therefore, the development of stimuli-responsive Mn-based CA is urgent to ensure that more Mn2+ remains at the tumor site for effective contrast. The microenvironment of tumor cells is weakly acidic,32-35 therefore, increasing the accumulation of Mn2+ in tumor tissue was an effective strategy to enhance MR imaging effect through the design of pH-switching Mn-based CAs. Nevertheless, the Mn2+ leaching amount from most pH-activated Mn-based CAs was less than 25% under certain acid conditions,36,37 which severely limited their T1-weighted contrast efficiency for tumor diagnosis. Therefore, constructing a smart theranostic system with both high relaxivity and excellent response to the microenvironment of a tumor for accurate 4


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MRI and drug delivery is challenging and shows important scientific significance. In this work, a core-shell structured nanoplatform was fabricated by ultrasmall manganese oxide (USMO)-capped mesoporous silica nanoparticles (USMO@MSNs), and applied for diagnosis and therapy of tumor. As shown in Scheme 1, USMO coated onto the pore channels of MSNs could be effectively and quickly dissolved under weakly acidic conditions because of ultrasmall size and high reactivity, which facilitates the release of Mn2+ in the site of interest and realizes pH-switching MRI tumor diagnosis. In addition, doxorubicin (DOX) could be effectively loaded into pore channels of USMO@MSNs (USMO@MSN-DOX) via the perfect matching between pore structure (mainly 1.42 nm) of USMO@MSNs and the size of DOX molecules, so that DOX could be synchronously released with the dissolution of Mn2+. More importantly, USMO@MSNs had ultra-high response ability for weakly acidic solution and showed a higher release rate for Mn2+ and DOX in comparison with traditional Mn-based theranostic system, allowing the timely observation of DOX chemotherapy through MRI processes. In addition, this nanoplatform showed good biocompatibility for normal cells with neutral or weakly alkaline microenvironment, which would significantly reduce the side effects of DOX. By integrating high-efficiency chemotherapy and pH-switching MRI, this smart nanotheranostic agent shows broad prospects for additional clinical applications. 2. EXPERIMENTAL SECTION 2.1. Materials. All chemical reagents were used as received without further purification. Diethanolamine (98%, DEA), MnSO4 (99.9%), tetraethyl orthosilicate 5



(TEOS), cetyltrimethylammonium chloride (98%, CTAC) were purchased from Sinopharm Co. (Shanghai, China). DOX·HCl, PEG, Hoechst 33342, propidium iodide (PI), and trypan blue were purchased from Sigma-Aldrich Co. (USA). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo (Japan). Other chemicals of analytical grade were received from Sinopharm Co. (Shanghai, China). 2. 2. Preparation of USMO@MSN. First, MSN was fabricated via a classical soft template method.38 Briefly, 1 g of CTAC was dissolved into the mixed solution containing distilled water (80 mL) and ethanol (20 mL), and the solution was stirred for 30 min under 60 °C. Subsequently, 1.0 mL of DEA was added into the resulting solution and continuously stirred for 30 min at the same temperature. Then, 5 mL of a TEOS solution containing TEOS (1 mL), ethanol (1.5 mL), and distilled water (2.5 mL) was dropwisely added into the resulting solution and stirred for 6 h. Afterward, a MnSO4 solution (25 mg/mL, 2 mL) was quickly injected into the mixed solution and continuously reacted for 12 h under N2 gas fluid protection. Then, the white powder was collected via centrifugation, and it was washed and vacuum dried. Finally, the white powder was further treated via calcination at high temperature under N2 gas protection to obtain USMO@MSNs. 2.3. The loading and release of drug. USMO@MSN (10 mg) was dispersed in a DOX solution (4 mg/mL, 3 mL) via sonication treatment, and the new solution was shaken at a rate of 200 rpm/min for 24 h at room temperature. Subsequently, DOX was loaded into the pore channels of USMO@MSNs, and the loading capacity (LC) of DOX was calculated via monitoring the change in the DOX concentration in the 6


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supernatant before and after adding USMO@MSNs. The equation was as follows: LC(%)=(Cpre-Cpost)×V/M0×100%, Cpre and Cpost were the concentration of DOX in the supernatant before and after absorption, respectively, V was the volume of the solution, and M0 was the amount of USMO@MSNs added. In addition, the release behavior of DOX from USMO@MSNs-DOX was investigated via the following methods: 5 mg of USMO@MSNs-DOX was first dispersed indifferent pH values (pH 4.0, 5.5, 6.5, and 7.4) of simulated body fluid (SBF, 6 mL) for 5 h under shaking at a rate of 200 rpm/min. The concentration of DOX in the supernatant was collected at different time intervals. 2.4. MR experiment. A certain amount of USMO@MSNs was dispersed in PBS of different pH values for 4 h. Subsequently, USMO@MSN solutions with different pH values were fixed in 600 µL tubes using 1% agarose gel, and the tubes were scanned to measure the relaxation rate of the samples. Meanwhile, the cell MR experiment samples were also prepared via the following process: the amount of HSC3 cells 0was first incubated to a density of 5×107 cells/dish. Then, the HSC3 cells were treated with 0, 5, 20, and 40 µg/mL of USMO@MSNs for 4 h under pH 7.4 and 5.5. Next, HSC3 cells were washed twice with PBS and then harvested. Finally, the HSC3 cells were centrifuged at 500 rpm/min. The cell tubes were carefully scanned under a 9.4 T MR spectrometer. All MR studies were conducted on a 9.4 T/400 mm wide bore scanner (Agilent Technologies, Inc., Santa Clara, CA, USA) using a volume RF coil (with an inner diameter of 40 mm). For in vitro or in cell phantom MR experiments, the scanning 7



procedure began with a localizer, and a series of inversion-prepared fast spin-echo images were acquired for longitudinal relaxation time (T1) measurements. The series was identical in all aspects (TR: 6000 ms, effective TE: 5.6 ms, BW: 25 kHz, slice thickness: 1 mm, 96×96 matrix, 1 average) except for 20 different inversion times (TIs) that were varied linearly from 10 to 2500 ms. The relationship between signal intensity (SI) versus TI was fit by nonlinear least-squares regression to the following exponential T1 decay model: SI(TI)=A1*exp(-TI/T1)+SI(0). For the in vivo MR studies, the mice used in the experiment were treated in accordance with the Ethics Committee Guidelines of Hefei Institutes of Physical Science, Chinese Academy of Sciences. The cancer model was established through direct subcutaneous injection of 5×106 HeLa cells into the left hind leg of BALB/c nude mice. After two to three weeks, tumor volume was apparent. Next, tumor bearing nude mice were anesthetized with isoflurane (3.5 % induction, 1.0-1.5 % maintenance) in air/O2 (2:1) for the duration of the scan. The animals were placed in a prone position on a specially designed cradle and inserted into the magnet. The respiratory rate and rectal temperature were monitored throughout the experiment with a physiologic monitoring unit (model 1030; SA Instruments, Inc., Stony Brook, NY). For the duration of the experiment, the animal’s body temperature was maintained at 36.5 °C with a homemade heating pad. Following the acquisition of a tripilot scan, T1-weighted MR images were acquired, typically along the coronal orientation, using a spin-echo sequence. For the mouse studies, the following acquisition parameters were chosen: repetition time (TR) 8


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= 370 ms, echo time (TE) = 11.6 ms, field of view (FOV) = 40 mm × 40 mm, matrix size = 192 × 192, slice thickness = 1 mm (12 slices, gap = 0), 1 average, and bandwidth (BW) = 50 kHz. For the rat studies, the following acquisition parameters were chosen: TR = 590 ms, TE = 13 ms, FOV = 40 mm × 60 mm, matrix size = 256 × 256, slice thickness = 1 mm (30 slices, gap = 0), 1 average, and bandwidth (BW) = 50 kHz. We obtained a series of pre-injection baseline T1-weighted MR images prior to CA injections via tail vein. Post-injection scans were obtained 15 min, 30 min, 60 min, 90 min and 120 min after the injection. Throughout the scanning sessions, precise measurements and markers were used to ensure consistent placement of the animal’s tumor in the animal holder and of the animal’s tumor within the magnet. Pulse oximeter triggering was used for MRI acquisition to reduce artifacts arising from respiratory movement. Dynamic MRA images of rats were acquired using a 3D-CEMRA sequence with parameters as follows: T1-weighted fast field echo (T1FFE), TR = 7 ms, TE = 3 ms, FOV = 100 mm×100 mm, slices = 60, slice thickness = 1 mm, and flip angle = 30°. 2.5. In vitro cytotoxicity. First, HSC3 cells were cultured in a Dulbecco’s modified Eagle’s medium (DMEM)/high-glucose medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% penicillin-streptomycin at 37 °C in a humidified atmosphere with 5% CO2. The viability of the HSC3 cells was investigated by a standard CCK-8 assay, and the specific process was as follows: HSC3 cells were placed into 96-well plates and adjusted to a density of 1×104 cells/well. The cells were then treated with different concentrations of USMO@MSNs 9



and USMO@MSNs-DOX for 12 and 24 h. Next, the culture medium was removed and incubated in fresh medium containing 120 µL of 10% CCK-8 at 37 °C for 2 h. The number of viable cells was measured at a wavelength of 450 nm with a Fluster Optima microplate reader (BMG Lab technologies, Germany). Measurements were made in eight independent experiments (n=6, where n indicates the number of wells in a plate for each experimental condition). 2.6. FITC accumulation assay. FITC were rationally conjugated to USMO@MSNs-DOX according to a previously reported method.39 HSC3 cells were placed in 6-well plates and adjusted to a cell density of 2×105 cells/well. Subsequently, the different concentrations of FITC-labeled USMO@MSNs were used to incubate the HSC3 cells for 1, 2, and 4 h at 37°C. Next, extracellular particles were washed twice using PBS, and the cells were trypsinized, washed three times using ice-cold pH 7.4 PBS, and resuspended in 300 µL PBS. Finally, about one thousand cells were chosen for analysis using flow cytometry (BD FACS can flow cytometer, BD Biosciences). CellQuest software was used to calculate the fluorescence intensity of the HSC3 cells. 2.7. Confocal microscopy. HSC3 cells were seeded in 24-well plates and adjusted to a cell density of 5×104 cells/well. Then, different concentrations of FITC-labeled USMO@MSNs-DOX were used to culture the HSC3 cells for 1, 2, and 4 h. In addition, HSC3 cells were washed three times using ice-cold PBS and fixed using 4% paraformaldehyde for 30 min, followed by staining with DAPI for an additional 10 min at 37 °C in the dark. Finally, a confocal microscope (Zeiss LSM710 10


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NLO, Germany) was used to observe the cells. 2.8. Characterization. The morphology and structure were investigated using a transmission electron microscope (TEM) (JEM-ARM200F, JEOL Co., Japan). The composition was analyzed on a Fourier transform infrared (FT-IR) spectrometer (iS10, Nicolet Co., USA). The amount of organic matter was characterized by a thermogravimetric analyzer (Q600, TA Co., USA). The surface potential and hydrodynamic size of the particles were measured by a zetasizer (Nanotrac Wave II, MicrotracCo., USA). The content of Mn2+ ions was determined via inductively coupled plasma-optical emission spectrometry (ICP-OES) (Optima 7200plus, Thermo Fisher Scientific Co., USA). The interaction between the drug and the particles was analyzed by X-ray photoelectron spectroscopy (XPS, ESCALAB250, Thermo-VG Scientific Co., USA). 2. RESULTS AND DISCUSSION 2.1. Synthesis and characterization of USMO@MSNs. MSNs were first synthesized via a classical method based on diethanolamine (DEA)-controlled hydrolysis of tetraethyl orthosilicate. To construct the ordered porous structures, CTAC micelles were used as templates under a reaction process. Meanwhile, the size of the MSNs could be adjusted via varying the amount of DEA added. In this work, MSNs ranging in size from 30 to 50 nm were prepared because the suitable size could facilitate passive accumulation of particles in the tumor site (Figure 1a). Subsequently, MSNs were further treated via adding a manganese source of a certain concentration as a reactant to cap the pore channels of the MSNs, forming core-shell structured 11



manganese hydroxide capped MSNs (HUSMO@MSNs, Figure 1b). In addition, to obtain a high-porosity and ultrasmall manganese oxide (USMO), HUSMO@MSNs were heated at a high temperature of 800 K with the protection of N2 gas fluid. As shown in Figure 1c, obvious pore channels were formed, and an apparent core-shell structure appeared, indicating that organic matter in the pore channels was combusted and HUSMO transformed into USMO nanocrystals. High-resolution TEM (HRTEM) further confirmed the existence of USMO crystals on the surface of MSNs, and USMO showed significant lattice fringes and an ultrasmall size of ~3 nm (red arrow in Figure 1d,), implying that high activity USMO shell was successfully prepared. Dynamic light scattering (DLS) experiments were performed to analyze the hydrodynamic size of the particles. Figure 1S showed narrow peaks for MSNs, HUSMO@MSNs, and USMO@MSNs, indicating their good colloidal dispersion in the solution. Interestingly, USMO@MSNs had no any modification and kept an excellent stability at different media, which might be ascribed to the electrostatic repulsion interaction between particles that had the relatively high charge at neutral condition. Additionally, the surface potential of USMO@MSNs gradually increased with decreasing pHs, which might be because the content of Mn2+ ions surrounded by USMO@MSNs gradually increased with decreasing pHs (Figure 2c). USMO@MSNs displayed a similar size in comparison with HUSMO@MSNs and were larger than MSNs because of the USMO coating. X-ray diffraction (XRD) analysis showed a change in crystallinity from MSNs to USMO@MSNs (Figure 2a). For MSNs, a typical broad peak occurred from 15 to 35 degrees, indicating that MSNs had no 12


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obvious crystal lattice and were amorphous. After coating with HUSMO, new diffraction peaks appeared, but the peaks were broad and weak, suggesting a low degree

of

crystallinity

for

HUSMO.

After

high-temperature

treatment,

USMO@MSNs showed significant diffraction peaks assigned to body centered cubic crystals of MnO, implying that the effectively pH-switching nanoplatform was successfully prepared due to quick dissolution of MnO under acid condition. In the FT-IR spectra (Figure 2b), HUSMO@MSNs displayed new peaks at 575, and 2852 and 2894 cm-1 compared to the spectra of MSNs, which were assigned to the Mn-O stretching vibration and C-H stretching vibration, respectively. These results suggested that Mn sources were successfully coated onto MSNs and organic templates have not been removed. In the USMO@MSN curve, the peaks at 2852 and 2894 cm−1 disappeared, implying that the CTAC templates were successfully removed, creating many pore channels for chemotherapy drug loading. Subsequently, USMO@MSN-DOX offered new peaks at 1581, 1620, and 1725 cm-1, suggesting that DOX

could

be

effectively

loaded

into

USMO@MSNs.

The

nitrogen

adsorption-desorption isotherms illustrate the ultrahigh porosity and specific surface area (962 m2/g) of MSNs (Figure 2d), but the porosity and specific surface area of HUSMO@MSNs dramatically decreased mainly because organic templates and HUSMO occupied the pore channels of particles. In addition, USMO@MSNs regained a relatively high porosity and specific surface area (741 m2/g), implying that the organic templates in the pore channels were removed. Furthermore, the USMO@MSN isotherm does not show a hysteresis loop at 0.1-0.8 P/P0, suggesting 13



the ordered mesopore structure of USMO@MSNs. Compared to MSNs, the pore size of USMO@MSNs dramatically decreased to 1.5 nm, which well-matched the size of drug molecules (inset of Figure 2d). In addition, the pore size change of USMO@MSNs was also explored as the content of USMO increased. It could be seen in Figure S2 that the pore size of USMO@MSNs gradually narrowed, and the specific surface area and porosity of USMO@MSNs significantly decreased along with the increasing content of USMO. Moreover, when the content of USMO was 10%, USMO@MSNs showed the optimal pore size which well-matched the size of DOX (Figure 2d). According to the molecular structure characteristic, the perfect matching between pore structure (mainly 1.42 nm) of USMO@MSNs and the size of DOX molecules (1.37 nm) could induce the efficient loading of drug molecules (loading capacity: 456 mg/g), and meanwhile block the release of DOX from the USMO@MSNs. Subsequently, the composition and interaction between USMO@MSNs and DOX were also explored via XPS. The full spectrum of the USMO@MSNs-DOX confirms the presence of C, O, Mn, and Si elements (Figure 3a). Meanwhile, USMO@MSNs-DOX showed a new peak at 399.9 eV assigned to N1s (inset of Figure 3a), indicating that DOX was successfully loaded into USMO@MSNs. Furthermore, for the C1s XPS spectrum of the USMO@MSNs-DOX, the new peaks at 286.4 and 288.2 eV appeared and were ascribed to -C=C- and -C=O, which originated from DOX (Figure 3c). In addition, the Mn2p peak shifted from 641.8 to 642.9 eV after loading DOX (Figure 3b), which might be ascribed to the coordination 14


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interaction between DOX and Mn element. Moreover, the Si2p peak of USMO@MSNs-DOX also appeared to shift from 103.7 to 103.1 eV (Figure 3d), implying the existence of hydrogen-bond interactions between USMO@MSNs and DOX. Therefore, DOX was loaded into USMO@MSNs via physical adsorption, coordination, and hydrogen-bond interactions. 2.2. Magnetic Property Investigation and Release Profile of Mn2+ and DOX. Field-dependent magnetization (M-H) curves show that the magnetization value of USMO@MSNs and HUSMO@MSNs gradually increased with increasing external magnetic field, and no remanence and coercivity were observed at 3 K and 300 K (Figure 4a,b). Meanwhile, ZFC/FC curves showed that USMO@MSNs and HUSMO@MSNs possessed low overlapping temperature (Figure 4c). These results indicated that USMO@MSNs had excellent paramagnetic behavior and exhibited a similar magnetization to that of HUSMO@MSNs at 300 K, but possessed a stronger magnetism at 3 K, implying that USMO@MSNs possessed a better crystal magnetic domain. Subsequently, the longitudinal relaxation rate (r1) of USMO@MSN was calculated via the ratio of 1/T1 to the Mn2+ ion concentration and was 0.68 mM-1s-1 at pH 7.4 (Figure 4d). However, the r1 value gradually increased with decreasing pH to 5.61 mM−1s−1 at pH 4.5, confirming that USMO@MSNs had a good pH-switching T1 MRI contrast ability. To further illustrate the pH-switching behavior of USMO@MSNs, the Mn2+ release behavior was quantitatively evaluated, as shown in Figure 4e. According to the results, the release amount of Mn2+ was negligible at pH 7.4, but gradually increased with decreasing pHs. Furthermore, the amount of Mn 15



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released from USMO@MSNs reached 78% at pH 4.5, showing effective Mn leaching ability. Therefore, according to the above analysis, the Mn2+ release behavior effectively enhanced the pH-switching MRI contrast of USMO@MSNs. More importantly, the release of DOX could be simultaneously triggered during the dissolution of USMO into Mn2+ ions. In addition, the release rate of DOX significantly increased along with Mn2+ release, which might be because the breakage of the coordination interaction between DOX and USMO@MSNs accelerated DOX release. These results implied that this nanoplatform had good ability for timely assessing the diagnosis and therapy effect of disease tissue. Besides, the release profile of DOX further confirmed pH-dependent performance (Figure S3). 2.3. In Vitro and In Vivo MRI Investigation. Next, the MR contrast enhancement

of

HSC3

cells

treated

with

different

concentrations

of

USMO@MSNs-DOX was also investigated at various pH values. As shown in Figure 5a, HSC3 cells gradually brightened as the USMO@MSNs-DOX incubation concentration increased. In addition, USMO@MSNs-DOX at pH 5.5 showed better brightening of the cellular MR images than those at pH 7.4, suggesting good pH-switching imaging ability on HSC3 cells. Furthermore, the corresponding contrast signal enhancements in the HSC3 cell images at pH 5.5 was also significantly higher than that of neutral condition (Figure 5b), further confirming the pH-switching function of this nanoplatform for cancer cell imaging. In vitro experiment, the excellent contrast ability of USMO@MSN response to pH encouraged us to explore in vivo application. In order to assess the advantages of USMO@MSN for in vivo 16


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MRI, pure MnO was also investigated for comparison. T1 images of tumor were acquired pre-injection and 24, 40, 56, 72, and 88 min p.i. of MnO and USMO@MSN at a dose of 2 mg/kg (Figure 5d,e). The images of tumor tissue injected with pure MnO gradually brightened, and showed a contrast enhancement. Nevertheless, the tumor tissue of mice injected with USMO@MSN dramatically brightened in comparison with pure MnO, which might be attributed to the quick dissolution of USMO. In detail, MRI signal intensity of tumor injected with pure MnO only increased from 45.8±3.3 to 80.8±10.7, but USMO@MSNs remarkably increased from 43.9±6.3 to 124±13.3 (Figure 5c). These results further confirmed that USMO@MSNs was an excellent pH-activated Mn-based contrast agent, and had good application for in vivo tumor diagnosis. 2.4. Cell Viability, Uptake, and Localization. Subsequently, the viability of HSC3 cells treated with USMO@MSNs-DOX was investigated to explore the application of USMO@MSNs-DOX in cancer therapy. Firstly, the bio-safety of USMO@MSNs was investigated, and cell viability had no significant decrease for 12 h and 24 h incubation (Figure 6a), suggesting

that USMO@MSNs possessed

excellent biocompatibility. In Figure 6b, USMO@MSNs-DOX showed no notable decrease in cell viability at pH 7.4, which might be due to the inability of USMO@MSNs-DOX to release DOX. However, USMO@MSNs-DOX showed a dose-dependent cytotoxic effect at pH 6.5, which was similar to that observed for free DOX. Moreover, for pH 5.5, USMO@MSNs-DOX showed stronger apoptosis for HSC3 cells compared to free DOX and that at pH 6.5, implying that more DOX was 17



released from USMO@MSNs-DOX. In addition, as shown in Figure 6e, cell apoptosis gradually increased with T1 relaxation time decrease, further confirming that this nanoplatform could be used to real-time monitor cancer chemotherapy by MRI owing to the synchronous release of Mn2+ and DOX. Therefore, according to the above analysis, USMO@MSNs realized excellent performance for pH-switching MR imaging and drug delivery. The intracellular localization of particles in the living cells was observed by confocal laser scanning microscopy (CLSM), and green fluorescent molecules (FITC) were labeled with USMO@MSN-DOX to confirm the location of the particles in the cells. As shown in Figure 7a, HSC3 cells treated with USMO@MSNs-DOX presented green and red fluorescence in the cytomembrane and cytoplasm, indicating that USMO@MSNs-DOX was internalized in the HSC3 cells, and DOX was released in the cytoplasm. Moreover, the green and red fluorescent intensity of the HSC3 cells gradually brightened with increasing USMO@MSN-DOX concentrations, showing a dose-dependent uptake ability. In addition, the cell uptake of USMO@MSNs-DOX was also observed after different incubation time, and the cell uptake was time-dependent. For an incubation time of 1 h, the fluorescence intensity of HSC3 cells was weak, implying that only a few particles crossed the cell membrane and penetrated the cytosolic space. However, with increasing incubation time, increasingly more particles accumulated in the cytoplasm, and abundant DOX molecules were released. These results illustrate that DOX could be effectively delivered into cells via the USMO@MSN-DOX nanoplatform, and a greater contrast 18


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effect could be simultaneously obtained. In addition, flow cytometric measurements were used to quantitatively investigate the uptake of HSC3 for USMO@MSNs-DOX (Figure 7b,c). Only 10.17% of cells incorporated USMO@MSNs-DOX at an incubation concentration of 2 µg/mL. Nevertheless, as the particle concentration increased, the fluorescence intensities of the HSC3 cells gradually increased, and the number of cells internalized by USMO@MSNs-DOX further increased to 64.38% (4 µg/mL) and 99.54% (8 µg/mL). Similarly, with lengthening incubation time, the fluorescence intensity of the HSC3 cells increased, and the percent of HSC3 cells labeled by the particles gradually increased from 37.84% to 68.29% and further to 95.55%, implying that more particles crossed the cytomembrane and penetrated the cytoplasm. The Mn content in cells was determined using inductively coupled plasma mass spectrometry (ICP-MS) to further assess the uptake of nanoplatform. The maximum Mn uptake of HSC3 cells incubated with various concentrations of USMO@MSNs appeared at a dosage of 8 µg/mL (Figure 6d). Furthermore, it could be seen that cells treated with USMO@MSNs had a high Mn content in comparison with untreated cells, and Mn uptake gradually increased with prolonging incubation time. These results presented the consistent information in comparison with Figure 6c. 4. CONCLUSIONS In summary, a highly efficient pH-switching MR CA and drug delivery nanoplatform was developed and applied in cancer diagnosis and therapy. USMO@MSNs-DOX under a weakly acidic condition effectively released abundant Mn2+ ions that efficiently enhanced the MR contrast, and the release rate of Mn2+ ions 19



was up to 76% over 24 h. Meanwhile, the r1 value of the USMO@MSNs increased from 0.68 mM-1s-1 to 5.61 mM-1s-1, showing excellent pH-switching contrast ability. In comparison with pure MnO, the prepared USMO@MSNs presented a better pH-activated T1-weighted MRI for tumor diagnosis in vitro and in vivo. Moreover, USMO@MSNs showed a very high DOX loading capacity (460 mg/g), and DOX could be simultaneously and quickly released from USMO@MSNs-DOX under the USMO dissolution process. Besides, USMO@MSNs-DOX exhibited pH-switching MRI and a therapeutic process for HSC3 cells, showing that cancer therapy could be monitored in real time and improving the effects of cancer therapy. In addition, flow cytometry and CLSM observations revealed that USMO@MSNs-DOX could effectively cross the cytomembrane and enter the cytoplasm and that the uptake of USMO@MSNs-DOX was concentration- and time-dependent. Thus, a pH-activated theranostic platform based on USMO@MSNs will offer a new route for tumor diagnosis and therapy.

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Particle size distribution, N2 sorption isotherms, release profile of DOX. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (R.W.), [email protected] (D.Z.). Author Contributions ||

D.W., H.L., and G.Z. are co-first authors.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The authors acknowledge financial support from the National Natural Science Foundation of China (No. 21407151), the Youth Innovation Promotion Association of Chinese Academy of Sciences (No. 2015385), the Science and Technology Major Project of Anhui Province (No. 17030701051), the Natural Science Foundation of Anhui

Province

(No.

1808085MB38),

Zhejiang

Characteristic

Discipline

(2014-112-01), and Zhejiang Provincial Health Science and Technology Project (2017H011).

21



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Scheme 1. (a) Schematic illustration of the preparation of USMO@MSNs-DOX; (b) USMO@MSN-DOX nanotheranostic agent enters a cancer cell via endocytosis for the pH-switching T1-weighted MRI monitoring of drug release and chemotherapy.

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Figure 1. TEM images of (a) MSNs, (b) HUSMO@MSNs, and (c) USMO@MSNs. (d) High-resolution TEM image of USMO@MSNs.

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Figure 2. (a) XRD spectra and (b) FT-IR spectra of samples. (c) Change in the zeta potential of USMO@MSN with respect to pH value. (d) N2 adsorption and desorption isotherm curves and pore size distribution (inset) of samples.

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Figure 3. (a) Full XPS spectra, (b) Mn2p peaks, (c) C1s peaks, and (d) Si2p peaks before and after loading USMO@MSNs with DOX.

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Figure 4. Hysteresis loops of HUSMO@MSNs and USMO@MSNs at (a) 3 K and (b) 300 K. (c) Temperature dependence of the ZFC and FC magnetization curves for HUSMO@MSN and USMO@MSN samples. (d) Longitudinal relaxation rates of USMO@MSN treated with pH solutions calculated from the ratio of 1/T1 to the concentration of Mn2+. Release behavior of (e) Mn2+ from USMO@MSNs-DOX under different pH conditions. (f) The correlation between DOX release and Mn2+ release of USMO@MSNs-DOX at pH 4.5.

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Figure 5. T1-weighted images (a) and the corresponding T1 values (b) of HSC3 cells treated with different USMO@MSN concentrations under varying pH conditions. (c) MRI signal changes in the tumor of T1-weighted images at transverse planes. T1-weighted MR images of tumor tissue in nude mice treated with (d) pure MnO and (e) USMO@MSN acquired at an injection dose of 2.0 mg/kg. Tumors are marked by a white arrow.

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Figure 6. (a) CCK-8 assay for HSC3 cell viabilities after incubation with different concentrations of USMO@MSN for 12 h and 24 h. (b) The viability of HSC3 cells treated with DOX and USMO@MSN-DOX under different pH conditions for 24 h. (c) Fluorescent microscopy images of HSC3 cells treated with FITC labeled USMO@MSN-DOX. (d) Intracellular Mn uptake by HSC3 cells after different concentrations of USMO@MSNs treatment for 1, 2, and 4 h, respectively. (e) The relation between MR signal of cell treated with USMO@MSN-DOX and apoptosis.

35



Figure 7. (a) CLSM observations of HSC3 cells treated with different concentrations of FITC-labeled USMO@MSNs for 3 h and 4 µg/mL of FITC-labeled USMO@MSNs for 1, 2, and 4 h. For each panel, the images from left to right show DOX in cells (red), FITC fluorescence in cells (green), cell nuclei stained by DAPI (blue), the bright field, and the four left-most images merged into one. All images share the same scale bar. (b) Flow cytometry analysis of HSC3 cells treated with different concentrations of FITC-labeled USMO@MSNs. (c) Flow cytometry analysis of HSC3 cells treated with FITC-labeled USMO@MSNs for 1, 2, and 4 h.

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TOC Graphic

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An Effectively pH-activated Theranostic Platform for Synchronous

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