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Multifunctional Redox-Responsive Mesoporous Silica Nanoparticles for Efficient Targeting Drug Delivery and Magnetic Resonance Imaging Liang Chen, Xiaojun Zhou, Wei Nie, Qianqian Zhang, Weizhong Wang, Yanzhong Zhang, and Chuanglong He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11802 • Publication Date (Web): 18 Nov 2016 Downloaded from http://pubs.acs.org on November 19, 2016

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ACS Applied Materials & Interfaces

Multifunctional Redox-Responsive Mesoporous Silica Nanoparticles for Efficient Targeting Drug Delivery and Magnetic Resonance Imaging Liang Chena, Xiaojun Zhoub, Wei Niea, Qianqian Zhanga, Weizhong Wanga, Yanzhong Zhanga, Chuanglong He*ab a

College of Chemistry, Chemical Engineering and Biotechnology, Donghua

University, Shanghai 201620, China. b

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials,

Donghua University, Shanghai 201620, China.

*Corresponding author.

Professor Chuanglong He

College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, 2999 North Renmin Road, Shanghai 201620, China.

Tel. /fax: +86 021 677 92742

Email address: [email protected] (C.L He)

ACS Paragon Plus Environment


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ABSTRACT The convenient modification of mesoporous silica nanoparticles (MSN) can provide great opportunities for constructing new generation of nanocarriers with multiple functions. In the current study, we fabricated a new multifunctional drug delivery system based on MSN capped by gadolinium-based bovine serum albumin complex (BSA-Gd) and hyaluronic acid (HA) via reductive-cleavable disulfide bond. In this multifunctional nanoparticle (MSN-ss-GHA), BSA-Gd component was prepared by biomineralization and acted as both smart gatekeeper and contrast agent for magnetic resonance (MR) imaging, while HA was served as targeted molecule to improve the specific affinity of MSN-ss-GHA towards cancer cells. The successful fabrication

of

MSN-ss-GHA

was

demonstrated

by

a

series

of

physicochemical characterization. The redox-sensitive drug release behavior of doxorubicin hydrochloride (DOX) loaded MSN-ss-GHA (DOX@MSN-ss-GHA) was also verified. Comparatively, the MSN-ss-GHA exhibited excellent biocompatibility and distinctly enhanced cell uptake by 4T1 cells. More importantly, the improved in vitro MR imaging ability of MSN-ss-GHA than that of Gd-DTPA was also confirmed. The results also suggested that the DOX@MSN-ss-GHA could efficiently deliver DOX into 4T1 cells and showed enhanced cytotoxicity than that of non-targeted nanocarrier. The in vivo experiment also demonstrated the negligible toxicity of MSN-ss-GHA and improved anti-tumor suppression of DOX@MSN-ss-GHA. Thus, this multifunctional MSN-based theranostic agent holds potential for efficient redox-responsive targeting drug delivery and MR imaging.


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KEYWORDS: mesoporous silica nanoparticles; redox-responsive; targeted drug delivery; cancer theranostics; magnetic resonance imaging



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1. INTRODUCTION Despite the flourishing development of innovative therapeutic methods, chemotherapy still remains to be one of the most frequently used approaches for combating against cancer in clinic.1 Unfortunately, the chemotherapeutic drug in free formulation commonly encounters the non-specific absorption and premature degradation, thereby inevitably leading to the occurrence of adverse side effects and low efficiency.2 By discriminating the normal and cancer cells via discrepant microenvironment and specifically releasing chemotherapeutic drugs at tumor site, stimuli-responsive drug delivery systems (DDSs) based on nanocarriers have gained increased attention during the past decade to enhance therapeutic efficiency and impair side effects to healthy organs.3 Although the enhanced permeability and retention (EPR) effect could passively improve the accumulation of nanocarriers in tumor site, further conjugation of active targeted ligands onto nanocarriers surface is more favorable to deeply enhance the cellular internalization of nanocarriers.4 Apart from improving the tumor accumulation and cancer cell endocytosis, the combination of multi-functional moieties into the nanocarriers render them with both therapeutic ability and diagnostic imaging capability, known as “theranostics”, has also attracted growing research interest.5 Consequently, the rational design of sophisticated all-in-one drug delivery system is highly worthwhile to meet the growing demand for accurately destroying cancer and realizing precise therapy.6 Benefiting from their innately superior properties including high loading capacity,7 tunable pore size

8,9

and outstanding biocompatibility,10 mesoporous silica


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nanoparticles (MSN) have drawn burgeoning research interest as one of the most promising nanocarriers for nanomedicine.11 In particular, the easily functionalized surface of MSN offers unprecedented opportunity to generate new nanoformulations for cancer theranostics by combining functional segment possessed imaging capacity or synergistic therapeutic effect with the nanocarriers.12 Recently, theranostic magnetic MSN were developed by Zhang et al for precise nanomedicine.13 After carefully coating magnetic core with mesoporous silica shell, the gatekeeper β-cyclodextrin was grafted onto surface of silica via reductive platinum prodrug linker and peptide ligand was further conjugated into β-cyclodextrin through host-guest interaction for targeting drug delivery. Different from encapsulated the inorganic nanoparticles into mesoporous silica by precisely controlled synthetic process, the employment of functional gatekeepers to cap the pore of MSN for theranostics is probably more convenient for simultaneously stimuli-responsive drug delivery and diagnostic.14, 15 For instance, Lu et al prepared the envelope-type MSN for smart drug release and magnetic resonance (MR) imaging.2 The ultra-small up-converting nanoparticles were immobilized onto the surface of MSN by acid-labile bond, which could confer both pH-responsiveness and MR imaging capacity to the nanocarriers. However, the hazardousness of up-converting nanoparticles should be carefully disposed. In consequence, it’s exceedingly rewarding to exploit a safety and functional gatekeeper to rationally construct multifunctional MSN nanocarriers with responsive drug release behavior and targeting ability as well as diagnostic imaging function for efficient cancer theranostics.



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With this in mind, the multifunctional MSN nanocarrier was developed for both targeting redox-responsive drug delivery and MR imaging in the present work. We first utilized bovine serum albumin (BSA) to synthesis gadolinium-based complex by biomineralization method. Then the functional BSA-Gd complex was acted as capping agent to block MSN via reductive-cleavable disulfide bond linkage. The redox-responsive MSN drug delivery system has been widely investigated because of the obvious discrepancy of glutathione (GSH) concentration between normal condition (~10 µM) and tumor microenvironment (1-10 mM).16 Although a vast number of redox-responsive MSN systems have been developed,17-19 the combination of this BSA-Gd complex with MSN has not been previously reported to the best of our knowledge. Taking advantages of high longitudinal proton relaxivity of BSA-Gd, this nanocarrier could be applied for both redox-responsive drug delivery and MR diagnostic imaging. In addition, hyaluronic acid (HA), an excellent targeted molecule with low immunogenicity,20 was subsequently grafted onto the BSA-Gd through amide bond formation to improve the cancer cell uptake of nanocarrier, denoted as MSN-ss-GHA. The physicochemical properties of the obtained nanoparticles were systematically characterized. The antitumor drug doxorubicin hydrochloride (DOX) was loaded into MSN-ss-GHA (DOX@MSN-ss-GHA) and the responsive drug delivery manner was evaluated. The biocompatibility of MSN-ss-GHA was also evaluated by cytotoxicity and hemolysis assay. Moreover, the MR imaging ability of MSN-ss-GHA was demonstrated in vitro, and the targeted ability of MSN-ss-GHA towards breast cancer cell line (4T1 cells) was investigated in detail. The efficient


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intracellular drug delivery, subcellular distribution and enhanced in vitro therapeutic efficacy of DOX@MSN-ss-GHA were examined. Last, the in vivo tests were also performed to investigate the in vivo toxicity and anti-tumor efficacy of MSN-ss-GHA.

2. MATERIALS AND METHODS 2.1. Materials Cetyltrimethylammonium

bromide

(CTAB),

fluorescein5(6)-isothiocyanate

(FITC) and tetraethyl orthosilicate (TEOS) were supplied by Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). N-Hydroxysuccinimide (NHS) was obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Doxorubicin hydrochloride (DOX) was obtained from the Beijing Huafeng United Technology Co, Ltd. (Beijing, China). Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), trypsin, penicillin and streptomycin were all received from Shanghai Yuanxiang medical equipment Co. 3-mercaptopropyltrimethoxysilane (MPTES), mercaptopropionic acid, glutathione (GSH), Gd(NO3)3·6H2O, 2,2′-dipyridyl disulfide (Py-ss-Py) and N-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Aladdin Chemistry, Co., Ltd. (Shanghai, China). Cell Counting Kit-8 (CCK-8) was provided by Beyotime Institute of Biotechnology (China). Paraformaldehyde and 4,6-Diamidino-2-phenylindole (DAPI) were obtained from Bestbio (Shanghai, China). Hyaluronic acid (HA, Mw ~31000) was bought from Zhenjiang Dong Yuan biotechnology corporation (Zhenjiang, China). Other reagents were commercially available and directly used in the experiment. Deionized (DI) water with resistivity of 18.2 MΩ·cm was provided by our own lab.



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2.2. Synthesis of MSN-ss-COOH First, thiol-functionalized MSN (MSN-SH) were prepared by the previously reported protocol.21 Typically, 1.25 g of CTAB and 0.5 g of NaOH were first added into 600 mL of DI water. The mixture was vigorously stirred at 80 °C for 1 h. Thereafter, 6.0 ml of TEOS and 1.2 mL of MPTES were successively introduced into the above solution. After continuously reacted at 80 °C for another two hours, the nanoparticles were separated by centrifugation and purified by ethanol for several times. The products were dispersed in 160 mL ethanol containing 9 mL concentrated hydrochloric acid and refluxed at 80 °C for 24 h to extract the surfactant. The MSN-SH were harvested by centrifugation and repeatedly purified by ethanol and water. Next, 1 g of MSN-SH were first suspended in methanol (100 mL), into which 1 g of 2,2′-dipyridyl disulfide (Py-ss-Py) was added. The mixture was degassed and protected by nitrogen atmosphere. After that, 4 mL of acetic acid was added via syringe. The mixture was stirred under ambient condition for 24 h and centrifuged at 10000 rpm to obtain MSN-ss-Py, which were also purified by anhydrous ethanol for three times. To prepare MSN-ss-COOH, the obtained MSN-ss-Py nanoparticles were dispersed in 100 mL N,N-dimethylformamide supplemented with 4 mL acetic acid under the nitrogen atmosphere. Afterwards, 1 mL of 3-mercaptopropionic acid was introduced into the solution. The reaction was maintained in oil-bath (40 ºC) for 24 h. Finally, the MSN-ss-COOH were collected by high-speed centrifugation and purified by washing with ethanol.


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The FITC-labeled MSN-ss-COOH were synthesized according to our previously reported protocol.22 By adding 0.6 mL of FITC-APTES solution in the synthetic procedure of MSN-SH before the addition of TEOS and MPTES, the FITC-labeled MSN-SH were prepared. Then FITC-labeled MSN-ss-COOH were synthesized by the abovementioned methods.

2.3. Preparation of BSA-Gd Complex BSA-Gd complex were prepared by a typical procedure reported previously.23 Briefly, BSA (0.25 g) was mixed with 9 mL of DI water at 37 ºC, into which 1 mL Gd(NO3)3 aqueous solution (50 mM) was slowly added. 1 mL of NaOH aqueous solution (2 M) was quickly injected into the mixture after five minutes. The solution was stirred at 37 ºC for 12 h. Thereafter, the solution was poured into dialysis bag (cut-off Mw = 3500) and dialyzed against DI water for 24 h to purify the product. The Gd-based BSA complexes (BSA-Gd) were collected and stored in refrigerator for further study.

2.4. Preparation of Multifunctional MSN-ss-GHA To prepare BSA-Gd modified MSN, 50 mg MSN-ss-COOH were ultrasonically dispersed in 30 mL PBS, and then 25 mg EDC and 18 mg NHS were introduced into the solution to activate the carboxyl group. After stirred for 2 h, 0.5 mL of BSA-Gd solution was added into the above system. The reaction was continued for one day under ambient condition. The MSN-ss-Gd-BSA were harvested and purified by centrifugation. Lastly, the HA was coated onto MSN-ss-Gd-BSA through amide formation. HA



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(25 mg) was dissolved in PBS (10 mL) containing 8 mg EDC and 5 mg NHS and stirred for 2 h. Then the prepared MSN-Gd-BSA dispersed in 20 mL PBS were introduced into the activated HA solution, followed by another 24 h reaction. The final HA modified MSN-ss-Gd-BSA nanoparticles, which denoted as MSN-ss-GHA, were obtained and washed by centrifugation.

2.5. Characterization The morphology and structure of the prepared nanoparticles were examined by transmission electron microscope (TEM) at an acceleration voltage of 200 kV with a LaB6 electron gun (JEM-2100, JEOL Ltd., Japan). Nitrogen adsorption/desorption experiment was tested by using a Micromeritics Tristar II analyzer (Micromeritics, USA). The surface areas and average pore size distributions were calculated by Brunauer−Emmett−Teller

(BET)

and

Barrett−Jyner−Halenda

(BJH)

method,

respectively. Small-angle X-ray diffraction (XRD) patterns were recorded by a D/MAX-2550 PC diffractometer using Cu Kα radiation at 45 kV and 40 mA with 2θ range in 1–10º (Rigaku Inc., Japan). Thermogravimetric (TG) analysis was performed from the room temperature to 900 ºC under nitrogen flow by a TG 209 F1 analyzer (Netzsch, Germany). Fourier transform infrared (FTIR) spectrum was examined by KBr disc technique and tested on a Nexus 670 spectrometer (Thermo Nicolet, USA). Dynamic light scattering (DLS) was conducted to evaluate the average diameter and size distribution of as-prepared nanoparticles using a BI-200SM multi-angle dynamic/static laser scattering instrument (Brookhaven, USA). Zeta-potential was determined by a Zetasizer Nano ZS apparatus (Malvern Instruments, UK). The


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content of Si element was determined by Leeman Prodigy Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES, Hudson, NH03051, USA).

2.6. The Load and Release behavior of DOX Anticancer drug DOX was loaded into the multifunctional nanocarriers for investigating their drug delivery capability. Briefly, 5 mL of MSN-ss-GHA dispersed in PBS (2 mg/mL) was blended with 1 mL of DOX solution (1 mg/mL) with the assistance of bath ultrasonic. The mixture was sealed and stirred 24 h at room temperature. Thereafter, the DOX-loaded hybrid nanoparticles, denoted as DOX@MSN-ss-GHA, were obtained by centrifugation. Then DOX@MSN-ss-GHA nanoparticles were washed by PBS for several times to remove unbounded DOX. The drug loading efficacy was calculated by the similar method reported previously.25 Next, in vitro release behavior of drug was investigated. DOX@MSN-ss-GHA nanoparticles (5 mg) were dispersed in 1 mL of corresponding buffer solution, which was sealed in a dialysis bag (cut-off Mw = 3500). The dialysis bag was soaked in 9 mL of corresponding solution (pH 5.0 or pH 7.4) and placed in shaker at 37 ºC. For the redox responsive drug release, 10 mM GSH was introduced into the PBS solution to simulate the tumor microenvironment. At predetermined time intervals, the sample solutions were taken out from the solution for analysis and equivalent fresh PBS solutions were supplemented. The amount of released DOX was measured by absorption value at wavelength of 480 nm.

2.7. In Vitro MR Imaging Capacity For the measurement of transverse proton relaxation times (T1) and T1-weighted



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MR imaging of MSN-ss-GHA, the MSN-ss-GHA aqueous solution with different Gd concentrations were analyzed by an NMI20-Analyst NMR Analyzing and Imaging system (Shanghai Niumag Corporation, Shanghai, China). The commercial T1 contrast agent of MR imaging Gd-diethylenetriaminepentaacetic acid (Gd-DTPA) was tested as control. The instrumental parameters were listed as follows: TR = 625 ms, TE = 100 ms, number of excitation is 1, 0.5 T of magnet, CPMG sequence, point resolution set as 156 mm × 156 mm, 0.6 mm of section thickness. The transverse relaxivity (r1) was calculated by linear fitting the inverse T1 relaxation time (1/T1) as a function of Gd concentration.

2.8. Cell Lines and Culture Murine breast cancer cell line (4T1 cells), human cervical carcinoma cell line (HeLa cells) and human umbilical vein endothelial cell line (HUVEC cells) were provided by Chinese Academy of Sciences, Shanghai Institute of Cell Biology (Shanghai, China). Both 4T1 cells and HUVEC cells were grown in RPMI 1640 complete medium, into which 10% FBS, 100 U mL–1 penicillin and 100 µg·mL–1 streptomycin was added. HeLa cells were cultured in DMEM supplemented with 10% FBS and 100 U penicillin/streptomycin. The cells were cultured at 37 ºC in a humidified atmosphere with 5 % CO2.

2.9. In Vitro Assessment of Cytotoxicity The cytotoxicity of MSN-ss-GHA was assessed by CCK-8 assay. Typically, 4T1 and HUVEC cells were cultured in 96-well plate with density of 1 × 104 cells/well. After fully adhesion of cells, the new medium containing different concentrations of


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MSN-ss-GHA were added. After co-cultured for 24 h, the medium were extracted and the cells were washed by PBS. Lastly, the cells were incubated with 100 µL of serum-free medium and 10 µL CCK-8 solution at 37 ºC for another 2 h. The absorption value at 450 nm was determined by a microplate reader (Multiskan MK3, Thermo). The cells treated with complete medium were set as blank group. The relative cell viability was determined by using the absorption value of treated group with respect to the control group, and four parallel experiments were conducted for each group.

2.10. Hemolysis Assay The hemolysis assay was carried out by similar protocol in our previous report.24 First, 1 mL of fresh blood was harvested from ICR mice and washed with PBS by centrifugation at 3000 rpm to isolate red blood cells (RBCs). The obtained RBCs were purified more than three times by PBS and then diluted with PBS. Afterwards, 1.2 mL of MSN-ss-GHA dispersion at different concentrations were mixed with 0.3 mL of RBCs solutions and maintained at 37 ºC for three hours. Then the mixture was centrifuged and the supernatant was measured by microplate reader. The hemolysis percentage was calculated through the same equation in our previous report.24

2.11. Targeted Cell Uptake of Silica Nanoparticles The targeted cell uptake of silica nanoparticles was also thoroughly investigated. 4T1 cells were cultured in 6-well plate at density of 2 × 105 cells per well. Then the cells were incubated with FITC-labeled MSN-ss-COOH and MSN-ss-GHA at different concentrations (50 and 100 µg/mL) for 4 h. After carefully remove the silica



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nanoparticles, the cells were detached, collected and repeatedly washed with PBS by centrifugation. After filtered through 400-mesh sieves, the cells suspended in PBS were analyzed by flow cytometry (BectonDickinson, USA) with Cell Quest software. To visualize the cell uptake of two kinds of silica nanoparticles, the 4T1 cells were first allowed to incubate in culture dishes with 20 mm glass bottom for fully adhesion. Then, the medium were discarded and fresh medium containing FITC-labeled silica nanoparticles (100 µg/mL) were added. The cells were maintained at culture condition for 4 h. The cells were then slightly rinsed with PBS and fixed by 4% paraformaldehyde for 0.5 h. After that, the cells were stained with DAPI and observed by confocal laser scanning microscope (Carl Zeiss LSM 700). For the competitive inhibition experiment, the 4T1 cells were pre-incubated with 5 mg/mL of HA solution for 2 h before the silica nanoparticles were added. Moreover, the endocytosis of silica nanoparticles was also studied by bio-TEM. The cells were seeded in 6-well plate with 2 × 105 cells per well and cultured for fully adhesion. After incubated with corresponding silica nanoparticles for 24 h, the cells were rinsed by PBS and incubated with different silica nanoparticles for another 24 h. Subsequently, the cells were fixed by 2.5% glutaraldehyde. The ultrathin sections of TEM samples were prepared by the same procedure as described in our previously report

25

and observed by JEM-2100. On the other hand, the amount of silicon

element in 4T1 cells was also evaluated. After incubated with silica nanoparticles for 24 h, the cells were trypsinized and repeatedly washed by PBS. Afterwards, the cells were lyophilized, dissolved in HF solution and diluted by 2 wt% HNO3 aqueous


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solutions. Finally, the transparent solution was directly subjected to ICP-AES to measure the silicon concentration.

2.12. Intracellular Uptake and Distribution of DOX@MSN-ss-GHA CLSM was used to investigate the intracellular uptake and intracellular distribution of free drug and drug-loaded nanocarriers. In detail, 4T1 cells were seeded and cultured in culture dishes with 20 mm glass bottom (105 cells per dish) for cell attachment. Subsequently, the cells were further co-cultured with free DOX, DOX@MSN-ss-COOH or DOX@MSN-ss-GHA (5 µg/mL of DOX concentration) for 4 h. Subsequently, the cells were slightly washed by buffer solution and fixed by paraformaldehyde. The cell nuclei was stained with DAPI and directly observed by CLSM. To detect the subcellular localization, the cells were incubated with DOX@MSN-ss-GHA or DOX for 4 h. After removing the cell medium, the cells were incubated with Lysotracker Green DND-26 (Molecular Probes, Invitrogen, USA) for another 15 min. The fluorescence imaging was immediately conducted using CLSM.

2.13. The cytotoxicity of DOX-loaded MSN-ss-GHA For in vitro drug efficacy evaluation, the 4T1 cells or HeLa cells (104 per well) were cultured in 96-well plate for 24 h. Next, the cells was washed by PBS and incubated with free DOX, DOX@MSN-ss-COOH or DOX@MSN-ss-GHA at different DOX concentrations for 24 h. The cell viability of each group was determined by CCK-8 test and calculated as the above mentioned procedure.

2.14. In vivo experiments



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All animal experiments were conducted in compliance with the instructions of Institutional Animal Care and Use Committee (IACUC) to ensure that all the animals were hospitably treated during the studies. Balb/c female mice and nude Balb/c female mice were commercially available from Slac laboratory animal Co. Ltd (Shanghai, China). Prior to evaluate the in vivo therapeutic efficacy of the hybrid nanoparticles, the in vivo toxicity of MSN-ss-GHA was first assessed by histological section. The 6-8 weeks Balb/c female mice were anesthetized by 1% pentobarbital PBS solution. Then MSN-ss-GHA dispersed in PBS (2 mg/mL) was intravenously injected into the mouse at a dosage of 20 mg/kg. The healthy mouse was used as control. The mice were raised for one week and then euthanized to remove the major organs including heart, liver, spleen, lung and kidney. The organs were fixed, embedded, sectioned and stained with hematoxylin and eosin for analysis. For in vivo therapy, the tumor model was established on female Balb/c mice by subcutaneously injection of 0.1 mL 4T1 cell suspension (~ 2 × 107 cells/mL). After the tumor diameter reached 4 mm, the mice were randomly divided into four groups (n= 4 per group): PBS, free DOX, DOX@MSN-ss-COOH and DOX@MSN-ss-GHA. For the treatment, the mice were injected with free DOX or DOX-loaded nanoparticles via trail vein. The injection was performed every two days with DOX dosage of 3 mg/kg. The tumor volume and body weight of mice were recorded every other day until the end of treatment. The tumor volume was determined by the equation: tumor volume = a × b2/2, where a and b represented the length and width of


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tumor, respectively. After the treatment, all nude mice were euthanized and the tumors were excised and photographed by digital camera.

2.15. Statistical Analysis Three parallel experiments were performed for all experiments. Statistical analysis was conducted using the one-way analysis of variance (ANOVA) and the Scheffe's post hoc test. The criterion of statistical significance was *p < 0.05 and **p < 0.01.

3. RESULTS AND DISCUSSION 3.1. Preparation of Multifunctional MSN To construct versatile mesoporous silica nanoparticles, the MSN-SH were first synthesized via one-pot reaction and successively modified by those functional organic compounds as schematically illustrated in Scheme 1. The MSN-SH were first treated with 2,2-dithiodipyridine and 3-mercaptopropionic acid to achieve MSN-ss-COOH with disulfide bond, which could be broken by relatively high level of GSH in tumor cells. Subsequently, BSA-Gd complex was covalently conjugated onto the surface of MSN-ss-COOH through carbodiimide-catalyzed amidation reaction. Finally, the HA was coated onto hybrid MSN to obtain the versatile silica nanoparticles. From the TEM images in Figure 1, it was observed that the as-prepared MSN-SH displayed spherical morphology with diameter around 200 nm. Clearly, the typical ordered mesoporous network of MSN also appeared in the magnified image (Figure 1B). After the modifications, the TEM images showed that the mesoporous channels were partly obscured by those functional organic compounds and the surface



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of hybrid silica nanoparticles became rough. The DLS results also confirm the successful functionalization as the hydrodynamic diameter was increased from 286.7 nm for MSN-SH to 302.2 nm for MSN-ss-GHA (Figure S1), even though the diameter were all understandably bigger than the size measured by TEM images.26 Furthermore, the other physiochemical properties of silica nanoparticles were also systematically investigated. As shown in Figure 2A, the mesoporous structure of MSN-SH was confirmed by the characteristic diffraction peaks (100), (110) and (200) in XRD pattern,27 which deservedly disappeared after the surface functionalization. The variation of surface area and average pore size during the process of modification were also monitored by N2 adsorption-desorption isotherms. The bare MSN-SH exhibited the typical type IV isotherm (Figure 2B), implying their well-ordered mesoporous channels. Specifically, accompany of disappearance of characteristic isotherm,28 the BET surface area was gradually dropped from 1018.1 cm3/g of MSN-SH to 718.7 cm3/g of MSN-ss-COOH and 341.6 cm3/g of MSN-ss-GHA, suggesting that the pores of MSN-SH were definitely covered with those functional macromolecules. Furthermore, the weight loss of the nanoparticles was determined by thermogravimetric analysis. As depicted in Figure 2C, the weight loss of MSN-SH was calculated to be 18.21%, while it increased to 21.3% after the carboxylation of MSN-SH. Accordingly, the value gradually rose up to 37.09% and 53.51% for BSA-Gd coated MSN and MSN-ss-GHA, respectively. Obviously, the content of organic compounds in the nanoparticles was increased along with the process of the modification, consistently implying the success of functionalization. Also, the


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compositions of those nanoparticles were characterized by FTIR spectra (Figure S2). Compared with the bare MSN-SH, the stretching vibration of carbonyl groups around 1710 cm-1 was observed in the spectrum of MSN-ss-COOH, which confirmed the appearance of carboxyl groups on the silica nanoparticles. Moreover, considering the stretching vibration band of S-H on MSN-SH was too weak to detect by FTIR spectroscopy,29 the SH groups were verified via the Raman signal at 2579 cm–1, which expectedly disappeared after the interposition of carboxyl groups (Figure S3). Subsequently, after the conjugation of BSA-Gd, the strong absorption around 1650 and 1541 cm–1 belongs to the characteristic amide I and amide II bands of BSA,30 suggesting the successful attachment of BSA-Gd on MSN-ss-COOH. It was noticed that there are no new peaks after the covalent grafting of HA on hybrid nanoparticles, which was explainable as the characteristic absorption peaks of HA may be overlapped with the BSA.31 Lastly, the zeta potential of the nanoparticles were also measured and showed in Figure 2D. The zeta potential decreased from –18.1 mV of MSN-SH to –43.4 mV of MSN-ss-COOH because of the highly negative charge of carboxyl groups.32 Both the BSA-Gd coated MSN and MSN-ss-GHA were highly negative-charged, which was beneficial for the sufficient stability of the hybrid nanoparticles.20

3.2. In Vitro DOX Release Behavior After validating the successful construction of multifunctional hybrid MSN nanoparticles, the drug loading and smart releasing capability of MSN-ss-GHA were investigated. Certainly, anticancer drug DOX can be successfully loaded into the



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mesoporous channel of MSN-ss-GHA. At the studied condition, the drug encapsulation efficacy and loading efficacy was calculated to be 92.13% and 8.42%, respectively. Afterwards, the release manner of DOX from MSN-ss-GHA was monitored at different conditions. Figure 3 showed that a small amount of DOX (11.77%) was released from MSN-ss-GHA within the first 4 h, after which only 5% of DOX was leaked out, suggesting the good capping efficacy of organic component. By contrast, the released amount of DOX reached up to 51.11% at the first 12 h when 10 mM GSH was added. Since GSH is a widely reported reducing agent, we deduced that the disulfide linkage in MSN-ss-GHA could be cleaved when exposed to high concentration of GSH within cancer cellular environment, thus triggering a faster DOX releasing rate. The effective redox-responsive release manner was also evidenced by the elevated amount of releasing DOX at pH 5.0, which increased from 53.02% in the absence of GSH to 67.12% under 10 mM GSH. More than redox-responsiveness, the pH-dependent release profile was also observed since the DOX was diffused much quicker at pH 5.0 than pH 7.4 under same conditions. The faster release rate under acidic condition was probably attributed to the improved solubility of DOX and electrostatic interaction. It has been previously reported that the subdued electrostatic interaction between DOX and BSA at acidic pH values would facilitate the release of DOX from nanoparticles.33 Therefore, the grafting of BSA-Gd complex onto MSN surface via disulfide linkage could possibly render the carrier with dual-responsive drug delivery capability, which is advantageous for


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improving therapeutic efficacy of nanocarriers.

3.3. Assessment of Biocompatibility It’s prerequisite to assess the biocompatibility of MSN-ss-GHA prior to the biomedical application of the nanoparticles. So the in vitro cytotoxicity of MSN-ss-GHA was evaluated by using the standard CCK-8 assay. As shown in Figure 4, the influence of MSN-ss-GHA on the viability of two kinds of cells was studied at a series of concentrations (7.5, 15, 30, 62.5, 125, 250 and 500 µg/mL). It was found that the prepared MSN-ss-GHA exhibited no obvious cytotoxicity against the two kinds of cells. Concretely, the viability of 4T1 cells treated with MSN-ss-GHA for 48 h was 91.3% at a high concentration of 500 µg/mL. At same concentration, the viability of HUVEC cells also reached up to 85.4% after 48 h incubation with MSN-ss-GHA. Since the outstanding cytocompatibility of MSN has been proven,10 we deduced that the incorporation of the functional complex and HA has no negative effect on the biocompatibility of MSN. Also, the hemolysis assay was conducted to preliminarily assess the hemocompatibility of MSN-ss-GHA. The UV-Vis spectra of supernatant solution were showed in Figure 4C. Apparently, negligible hemolytic activity of MSN-ss-GHA was detected in the range of our studied concentration. The hemolytic percentage of MSN-ss-GHA was also calculated by the absorbance at 541 nm. The result manifested that the hemolysis percentage of MSN-ss-GHA were all lower than 3% regardless of the concentrations (Figure 4D), which clearly demonstrated the favorable hemocompatibility of MSN-ss-GHA.



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3.4. Targeted Cell Uptake of Hybrid Nanoparticles Apart from the applicable biocompatibility, the specific uptake of nanocarriers by cancer cells is also particularly beneficial for the efficient anti-tumor drug delivery. Therefore, the targeted cell uptake of MSN-ss-GHA by 4T1 cells was systematically studied and MSN-ss-COOH was used as control. First, the cell uptake of nanoparticles was quantitatively evaluated by analyzing the intracellular fluorescent intensity of 4T1 cells treated with MSN-ss-COOH and MSN-ss-GHA. As depicted in Figure 5A, both the cell endocytosis of MSN-ss-COOH and MSN-ss-GHA were concentration-dependent. However, at the same concentration, MSN-ss-GHA all displayed a significant increase of fluorescent intensity compared with the MSN-ss-COOH (Figure 5B). Besides, it could be observed in Figure 5C that the percentage of cells internalized with MSN-ss-GHA was also significantly higher than that for MSN-ss-COOH under same concentration, indicating the enhanced cell uptake efficacy of MSN-ss-GHA. Furthermore, the targeted cell uptake of nanoparticles was intuitively validated by CLSM. After incubated with MSN-ss-COOH and MSN-ss-GHA for 4 h, the fluorescence images (Figure S4) revealed that a large amount of green fluorescent MSN-ss-GHA were emerged in 4T1 cells, while the MSN-ss-COOH were rarely internalized by cells through nonspecific adsorptive endocytosis mechanism.34 In general, it was perceived that the enhanced endocytosis of MSN-ssGHA was mainly resulted from the specific binding efficacy of HA with the overexpressed CD44 receptor on 4T1 cell surface.35 To further confirm this specific interaction, a


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competitive study was performed by pre-treating 4T1 cells with free HA. It can be seen that the presence of HA could partly prevent the MSN-ss-GHA from specific binding to CD44 receptors, as evidenced by the reduced green fluorescent in 4T1 cells. The above results revealed that the CD44 receptor-mediated endocytosis strongly facilitated the sufficient targeting cell uptake of MSN-ss-GHA. Additionally, the different internalization of the nanoparticles into cells was convinced by bio-TEM images in Figure 6. Obviously, the MSN-ss-COOH nanoparticles were encapsulated into vesicular compartments and still maintained the spherical morphology, which demonstrated that the MSN-ss-COOH could be taken up by the 4T1 cell via endocytosis. Distinctly, more amounts of MSN-ss-GHA nanoparticles were observed in the vesicles of 4T1 cells compared to the MSN-ss-COOH. In addition, some of the internalized MSN-ss-GHA nanoparticles were sporadically located in the cytoplasm rather than the vesicles, indicating that the MSN-ss-GHA may be transported outside the endosomes/lysosomes.36 To quantitatively clarify the different internalization of these nanoparticles, the ICP-AES was performed to quantify the amount of Si element in 4T1 cells. It was also found that the Si amount in 4T1 cells for MSN-ss-GHA was significantly higher than that of MSN-ss-COOH at the same conditions (Figure S5), which was in agree with the above outcomes. Therefore, we concluded that the incorporation of HA could improve the internalization of MSN-ss-GHA into 4T1 cells via receptor-mediated endocytosis.

3.5. The MR Imaging Capability of MSN-ss-GHA



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Since the BSA-Gd component in MSN-ss-GHA could be acted as positive contrast agent in MR imaging,37-39 we next explored the feasibility of using the as-prepared hybrid nanoparticles for T1-weighted MR imaging. The relaxivity values of the MSN-ss-GHA were determined by line fitting of 1/T1 as a function of Gd concentration, while a commercial contrast agent Gd-DTPA was used as control. Figure 7B shows that the r1 value of MSN-ss-GHA was 17.38 mM-1s-1, which was much higher than most of the reported Gd-based nanoparticles systems.37 Moreover, almost four-times increase of r1 value compared with that of Gd-DTPA (4.39 mM-1s-1) was also observed. The reason for this ultra-high r1 value probably resulted from the decrease of tumbling rate of the paramagnetic metal complexes and the improved interaction between water and the Gd complexes by grafting them onto the surface of nanoparticles.40 Furthermore, the T1-weighted MR images of MSN-ss-GHA and Gd– DTPA at different concentrations of Gd were also shown in Figure 7A. As expected, the MR signal intensity of hybrid MSN nanoparticles was apparently brighter than that of Gd-DTPA at the same Gd concentration. These results implied that the high relativity could not only make MSN-ss-GHA a promising MR imaging contrast, but is also specifically beneficial for dose reduction, thereby minimizing the side effect towards kidney.

3.6.

The

Internalization

and

Therapeutic

Effect

of

DOX@MSN-ss-GHA To demonstrate the intracellular drug delivery ability of MSN-ss-GHA, the cell internalization of DOX loaded MSN was monitored by CLSM. As seen from Figure 8,


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the free DOX quickly diffused through 4T1 cell membrane into nucleus, which was evidenced by the superposed fluorescent of red DOX and blue DAPI. In contrast, as nanocarriers, both DOX@MSN-ss-COOH and DOX@MSN-ss-GHA were first localized in cytoplasm, suggesting the MSN were internalized by receptor-mediated endocytosis. Those drug-loaded MSN detained in cytoplasm are expected to slowly release DOX to destroy cancer cells.41 It can be also noticed that the stronger red fluorescence was observed in cells incubated with MSN-ss-GHA than that of MSN-ss-COOH. It corroborated that more MSN-ss-GHA were taken up by 4T1 cells through active targeting effect, which is in accordance with the aforementioned results. Furthermore, the intracellular distribution of DOX and DOX@MSN-ss-GHA was also detected. Lysotracker dye was applied to stain the lysosomal compartments of 4T1 cells. As seen in Figure 9, despite a portion of the red fluorescence was co-localized in the lysosomes, the red of free DOX was dominantly overlapped with the blue fluorescence of nuclei. Whereas the red spots of MSN-ss-GHA were primarily overlapped with the green spots of lysosomes in the cells, signifying that the MSN-ss-GHA were endocytosed and mainly distributed in lysosomes. Having demonstrated the internalization experiments, the therapeutic efficacy of free DOX and DOX-loaded nanoparticles towards 4T1 cells were evaluated by CCK-8 assay. Figure 10 showed the cell viability of 4T1 cells treated by free DOX and DOX-loaded nanoparticles at a series of DOX concentrations. Particularly, the DOX@MSN-ss-COOH exhibited the lowest drug cytotoxicity among those three groups at various DOX concentrations. The viability of cells treated by



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DOX@MSN-ss-COOH at DOX concentration of 10 µg/mL was 41.95%, while the viability of the treated cells was 24.08% and 20.09% for MSN-ss-GHA and free DOX, respectively. In comparison to DOX@MSN-ss-COOH, the significantly enhanced drug cytotoxicity of DOX@MSN-ss-GHA principally arose from the improved cellular uptake of MSN-ss-GHA through the above proved CD44 receptor-mediated endocytosis. The similar results were also received from another CD44-overexpressed cancer cell line (HeLa cells), in which the DOX@MSN-ss-GHA exhibited stronger toxicity than that of non-targeted DOX@MSN-ss-COOH at equivalent DOX concentration

(Figure

S6).

Hence,

the

improved

therapeutic

efficacy

of

DOX@MSN-ss-GHA towards CD44-overexpressed cancer cell marked the satisfying targeted ability of the as-prepared multifunctional nanocarriers.

3.7. In vivo assessment The positive results of in vitro experiments indeed inspired us to assess the in vivo anti-tumor efficacy of our multifunctional nanoparticles. First, the in vivo toxicity of our nanocarriers was explored by histological assessment after one week postinjection. Figure 11A showed the representative slices of major organs of Balb/c mice treated with or without MSN-ss-GHA. Clearly, no tissue lesion or inflammation was detected in the treated group for its similar histological patterns with the control mice, verifying the well-documented favorable biocompatibility of MSN.10 Further, the anti-tumor efficacy of DOX@MSN-ss-GHA was primarily investigated by monitoring the 4T1 tumor volume of mice treated with different therapeutic approaches in two weeks. Encouragingly, the improved efficacy of


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DOX@MSN-ss-GHA was also reflected by the in vivo therapy against tumor model. As presented in Figure 11B, the tumor was strongly inhibited by the DOX@MSN-ss-GHA in comparison to control mice and mice treated with DOX@MSN-ss-COOH and free DOX. Meanwhile, it can be clearly seen that the smallest tumor was excised from mice treated with DOX@MSN-ss-GHA (Figure 11C) at the end of treatment. The mice underwent treatment showed no significantly body weight loss throughout the experiment (Figure 11D), implying our treatments were well-tolerated by tested mice. These data demonstrated that the DOX@MSN-ss-GHA hold more efficient potency to suppress the growth of tumor compared with non-targeted DOX@MSN-ss-COOH, which probably benefited from their desirable targeted ability and enhanced drug delivery capacity.

4. CONCLUSION Overall, the multifunctional MSN nanocarriers were successfully prepared by successively conjugated functional BSA-Gd complex and HA onto surface of MSN through cleavable disulfide linkage. The as-prepared nanocarriers could not only realize the redox-responsive manner of drug delivery, but also displayed remarkably enhanced cell uptake of 4T1 cells. More importantly, the BSA-Gd also endows these nanocarriers with favorable MR imaging property. In addition, the targeted ability of MSN-ss-GHA nanoparticles were further evidenced by the improved cytotoxicity of DOX@MSN-ss-GHA than non-targeted nanocarriers. The in vivo evaluations also demonstrated the good biocompatibility of MSN-ss-GHA and efficient tumor suppression of DOX@MSN-ss-GHA. Hence, we concluded that our design ensures



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the obtained MSN nanocarriers with stimuli-responsiveness, targeting ability and MR imaging function for multi-purpose cancer theranostics. Given that the BSA could also mediate the biominerilized preparation of other functional nanomateirals, such as gold nanocluster, semiconductor nanomaterials (CuS, Bi2S3, etc), it is also encouraging to graft other functional complexes onto MSN surface via cleavable bond for multifunctional fluorescence imaging or chemo-photothermal combined cancer theranostics.

ASSOCIATED CONTENT Supporting information The average diameter of bare MSN-SH and MSN-ss-GHA tested by DLS. The FTIR spectra of nanoparticles in the process of preparation of multifunctional MSN. The Raman spectra of MSN-SH and MSN-ss-COOH. The CLSM images of 4T1 cells treated by MSN-ss-COOH and MSN-ss-GHA for four hours. The amount of Si element internalized in 4T1 measured by ICP-AES after treated with MSN-ss-COOH and MSN-ss-GHA. The in vitro therapeutic efficacy of DOX@MSN-ss-GHA against HeLa cells.

AUTHOR INFORMATION Corresponding Author *Professor

Chuanglong He

Tel. /fax: +86 21 677 92742. Email address: [email protected].


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Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT We greatly appreciate the financial support provided by Open Foundation of State Key Laboratory for Modification of Chemical Fibers and Polymer Materials (LK1416),

Chinese

Universities

Scientific

Fund

(CUSF-DH-D-2015043),

International Cooperation Fund of the Science and Technology Commission of Shanghai Municipality (15540723400) and the National Natural Science Foundation of China (31271028, 31570984).

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Radiotherapy Sensitization. Biomaterials 2015, 37, 447-455.


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FIGURES

Scheme 1. The schematic diagram of synthetic process of multifunctional mesoporous silica nanoparticles for redox-responsive targeting drug delivery and MR imaging.



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Figure 1. TEM images and the corresponding enlarged images of (A, B) MSN-SH and (C, D) MSN-ss-GHA nanoparticles.


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Figure 2. (A) XRD patterns; (B) N2 absorption-desorption isotherm (inset: pore size distribution); (C) Thermogravimetric analysis and (D) Zeta potential of nanoparticles in the process of preparation of MSN-ss-GHA, a, b, c, d in D represented MSN-SH, MSN-ss-COOH, MSN-ss-Gd-BSA and MSN-ss-GHA, respectively.



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Figure 3. The in vitro DOX cumulative releasing profile at different conditions.


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Figure 4. The cell viability of (A) 4T1 cells and (B) HUVEC cells treated by different concentrations of MSN-ss-GHA (7.5, 15, 30, 62.5, 125, 250, 500 µg/mL) for 24 h. (C) The UV-Vis of supernatant solutions of RBCs after incubated with different concentrations of MSN-ss-GHA; (D) Hemolytic percentage of RBCs treated with MSN-ss-GHA at different concentrations. RBCs treated with PBS and deionized water was set as negative and positive group, respectively. The inset image represented the digital pictures of hemolysis results.



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Figure 5. (A) The flow cytometry results of 4T1 cells treated by MSN-ss-COOH and MSN-ss-GHA. (B) fluorescence intensity of 4T1 cells incubated with MSN-ss-COOH and MSN-ss-GHA at different concentration. (C) The percentage of 4T1 cells internalized with MSN-ss-COOH and MSN-ss-GHA at different concentraion.


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Figure 6. (A, C) TEM images and (B, D) magnified images of 4T1 cells treated by (A, B) MSN-ss-COOH and (C, D) MSN-ss-GHA for one day. The red arrows represent the MSN-ss-GHA out of the vesicles.



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Figure 7. In vitro MR imaging properties of MSN-ss-GHA and commercial Gd-DTPA. (A) The corresponding T1-weighted MR images of MSN-ss-GHA solution and Gd-DTPA at various Gd concentrations. (B) linear fitting of 1/T1 as function of Gd concentration.


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Figure 8. CLSM images of 4T1 cells treated by DOX, DOX@MSN-ss-COOH and DOX@MSN-ss-GHA with DOX concentration of 5 µg/mL for four hours.



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Figure 9. CLSM images of 4T1 cells after treated by DOX and MSN-ss-GHA for 4 h, verifying the intracellular location of free drug and drug carrier. The red fluorescent represented free DOX and DOX@MSN-ss-GHA, green fluorescent represented lysosomes.


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Figure 10. The in vitro therapeutic effect of free DOX and DOX-loaded MSN: cell viability of 4T1 cells after treated by free DOX, DOX@MSN-ss-COOH and DOX@MSN-ss-GHA at different DOX concentrations for one day.



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Figure 11. (A) The H&E stained histologic section of major organs of mice after injected with the as-prepared MSN-ss-GHA for one week at a dose of 20 mg/kg. (B) The tumor volume of 4T1-tumor-bearing mice subjected to different treatments. (C) Representative images of tumor issue after the treatments. (D) The body weight changes of tumor-bearing mice treated with different methods.


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Table of Contents Multifunctional Mesoporous Silica Nanoparticles for Redox-Responsive Targeting Drug Delivery and Efficient Magnetic Resonance Imaging.


Multifunctional Redox-Responsive Mesoporous ... - ACS Publications

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