Self-Decomposable Mesoporous Doxorubicin@Silica

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Self-Decomposable Mesoporous Doxorubicin@Silica Nanocomposites for Nuclear Targeted Chemo-Photodynamic Combination Therapy Jie Wang, Dajun Xu, Tao Deng, Yunyan Li, Le Xue, Tong Yan, Dechun Huang, and Dawei Deng ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00486 • Publication Date (Web): 30 Mar 2018 Downloaded from http://pubs.acs.org on March 31, 2018

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ACS Applied Nano Materials

Self-Decomposable Mesoporous Doxorubicin@Silica Nanocomposites for Nuclear Targeted Chemo-Photodynamic Combination Therapy Jie Wang,a Dajun Xu,a Tao Deng,a Yunyan Li,a Le Xue,a Tong Yan,b Dechun Huang,*,a and Dawei Deng*,a,b a

Department of Pharmaceutical Engineering and b Department of Biomedical Engineering,

School of Engineering, China Pharmaceutical University, Nanjing 210009, P. R. China.

KEYWORDS Self-decomposition, Doxorubicin@silica, Radial mesopores, Nuclear targeting, Combination therapy

ABSTRACT

Concerns associated with the non-degradability of silica (SiO2)-based nanoplatforms have been hindering their potential clinical translation as drug carriers. Hence, in this work, by embedding drug (doxorubicin (DOX) or methylene blue (MB), etc.) molecules into SiO2 nanoparticles (NPs), self-decomposable drug-embedded SiO2 NPs were prepared firstly. Importantly, we found that the intermediate morphology during the decomposition depends on the type of the embedded

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drug molecules, (e.g., DOX, mesoporous nanostructures; MB, center-hollowed nanoshells). Secondly, different from previous studies, here, the intermediate mesoporous DOX-embedded SiO2 (mDOX@SiO2) NPs with radial mesopores were modified with nuclear localization signal peptide to achieve nuclear targeted DOX delivery upon the fragmentation of NPs. Meanwhile, MB (a widely used photosensitizer) was further uploaded into the mesopores to realize chemophotodynamic combination therapy. At last, in vitro and in vivo antitumor efficacy and toxicity of the as-designed drug delivery system were evaluated. The results showed that compared with the non-targeting and chemo-only systems, the self-decomposable NPs with nuclear targeting capability and MB loading exhibited enhanced therapeutic efficacy, and no noticeable systemic toxicity was observed, indicating that the present system should be a promising paradigm in the design of SiO2-based drug carriers.

1. INTRODUCTION

Designing a drug delivery system should lay emphasis on at least three features, i.e., high drugloading efficiency, controlled drug release, and safe decomposition of drug carriers.1–3 In recent decades, the advancement of nanotechnology has inspired constant attempts to design new drug carriers that were expected to meet these criterions.4–6 A large variety of nanomaterials have been used, including carbon, gold, silica, magnetic and semiconductor NPs due to the unique properties not found in their bulk counterparts.6–11 Among them, low toxic mesoporous materials (e.g., mesoporous SiO2 NPs, iron diselenide hedgehog-like NPs, etc.) have emerged as one of the most promising drug carrier systems because of their high cargo-loading capacity.7,12,13 Many mesoporous SiO2 NP-based drug delivery systems have been reported and considerable progress has been made, especially regarding the controllable release when diverse physical and chemical


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stimulus were employed as triggers.14–16 For example, light-operated mechanized SiO2 NPs that are able to retain drug molecules and then release them upon exposure to UV light have been reported.17

However, few of these configurations achieved the degradability of SiO2 matrix.8,18 Although SiO2 NPs is generally accepted as nontoxic, it has been reported that long term retention in vivo would cause hemolysis, and the potential toxicity of the organosilane precursors (e.g., 3-aminopropyl-triethoxysilane and 3-mercaptopropyl-triethoxysilane) used for surface modification has not been comprehensively studied.19–21 Thus, the excretion of SiO2 scaffolds upon decomposition is needed. Recently, the self-decomposition of hollow mesoporous SiO2 NPs has been reported and ascribed to the thin pore wall, large pores and high surface area.22 However, the pore size of these NPs, which was determined by the diameter of the rod-shaped micelles formed from the self-assembly of CTAC (the used template),23,24 could not be tuned and thus was not suitable for uploading large cargos such as bio-macromolecules. Besides, the decomposition rate could not be controlled, while higher decomposition rate is favorable for repeated administration in drug delivery. Currently the most widely adopted method to realize the decomposition of SiO2-based drug carriers is by preparing organo-bridged SiO2 NPs, that is, introducing various organic groups into the SiO2 framework.18,25,26 For instance, disulfide bond was introduced to generate thioether-bridged framework, and fragmentation of the NPs was observed due to the reduction of disulfide bond by intracellular glutathione.26 However, the synthesis of organo-bridged SiO2 NPs is relatively intricate, and their degradation is stimuli intensity-dependent. Recently, as an alternative strategy, drug-embedded SiO2 NPs were prepared, either by synthesizing drug-containing alkoxysilanes27–29 or by simply introducing drug molecules into NP growth media,30 in which drug molecules served as the “organic bridge”


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to generate incompletely cross-linked SiO2 matrix. Particularly, the latter approach was considered better in terms of ease of preparation and the retaining of chemical integrity of drugs, and the resulting NPs could decompose simultaneously with the release of drug molecules.30 However, in both configurations, the drug-loading efficiency of the resulting nonporous NPs was rather limited. In addition to degradability of drug carriers, site-specific drug delivery is also highly needed for improving therapeutic efficacy and reducing systemic toxicity.31,32 Passive targeting mechanism based on the enhanced permeability and retention effect of tumor vasculature has been widely adopted in cancer therapy.33,34 However, for cell- and organelle-specific delivery, which is necessary in some cases to maximize the therapeutic effect, active targeting is needed.35,36 For example, for the delivery of a typical anticancer drug DOX that functions mainly in nuclei, nucleus-specific delivery is highly desired to reach a peak (effective) concentration at a reduced administration dose.37,38 To date, mesoporous SiO2 NP-based drug carriers that can be used for nuclear targeted delivery, namely, with a diameter smaller than about 30 nm to enter the nuclear pore complexes, has rarely been reported. A pioneering work was done by preparing small mesoporous SiO2 NPs (~25 nm in diameter) and then modifying them with nuclear targeting peptide, and the resulting NPs showed favorable nuclear accumulation.39 Unfortunately, these NPs were not decomposable. In this study, we firstly prepared self-decomposable drug-embedded SiO2 NPs by introducing DOX or MB molecules into the SiO2 NP growth media, and observed that the intermediate morphology (mesoporous nanostructures or nanoshells) during subsequent decomposition depends on the embedded drug molecule. Secondly, based on the intermediate mesoporous nanostructures, a self-decomposable nuclear targeted multi-drug loaded drug


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delivery system was fabricated further, specifically, by modifying NLS peptide onto mDOX@SiO2 NPs to achieve nuclear targeted DOX delivery upon the thorough fragmentation of NPs; by uploading MB into the mesopores to realize chemo-photodynamic combination therapy. In vitro and in vivo experiment results showed improved antitumor effect compared with the non-targeting and chemo-only systems, and the systemic toxicity was acceptable. Thus, the as-prepared self-decomposable mDOX@SiO2 NPs should be a promising improvement to the current SiO2-based drug carriers. 2. EXPERIMENTAL SECTION 2.1 Materials Tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTES), methylene blue (MB), citraconic anhydride, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) were purchased from Aladdin Chemical Reagent Co. Doxorubicin hydrochloride (DOX·HCl) and fluorescein sodium were purchased from Sinopharm Chemical Reagent Co. NLS peptide (KKKRK) were purchased from GL Biochem Ltd (Shanghai, China). MPA was synthesized in our laboratory. Human malignant glioma cell line (U87MG) was purchased from American Type Culture Collection (ATCC). BALB/c mice were purchased from Qinglongshan Co. Ltd. (Nanjing China). BALB/c mice bearing U87MG tumor were purchased from KeyGen Biotech. Co. Ltd. (Nanjing, China). The in vivo experiments on mice were approved by the Department of Science and Technology of Jiangsu Province and Jiangsu Association for Laboratory Animal. 2.2 Syntheses of DOX@SiO2 NPs and mDOX@SiO2 NPs


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In a typical synthesis, 15 mg of DOX·HCl was dissolved into 35 mL absolute ethanol, and then 1.6 mL 25% aqueous ammonia and 50 µL TEOS were sequentially added. The resulting mixture was stirred at room temperature in dark. 24 h later, DOX@SiO2 NPs were obtained via centrifugation at 10000 rpm for 3 min and repeated washing with absolute ethanol. The DOX concentration in NPs was calculated by measuring the concentration difference in the initial reaction solution and the supernatant after separation of NPs. Varying the initial amount of DOX could tune the DOX concentration of NPs. For the preparation of mDOX@SiO2 NPs, 20 mg of DOX was used. The synthetic procedures of MB- and fluorescein-embedded SiO2 NPs were similar with that described above, except the MB or fluorescein sodium was used to replace DOX·HCl. 2.3 Synthesis of NLS-mDOX@SiO2/MB NPs NLS modification: mDOX@SiO2 NPs obtained after 3 days of incubation was used for subsequent conjugations. Firstly, 20 mg of mDOX@SiO2 NPs was dissolved into 60 mL of absolute ethanol, and then 40 µL of APTES and 2 mL of 25% aqueous ammonia were subsequently added. The mixture was stirred at room temperature in dark for 12 h, and then the NPs were collected via centrifugation at 10000 rpm for 3 min and repeated washing with water. Meanwhile, 30 mg of EDCI and 18 mg of NHS were added into NLS peptide (60 mg) aqueous solution, and after 2 h of stirring, the solution was mixed with NP solution. The resulting mixture was stirred at room temperature under N2 atmosphere for 4 h, followed by dialysis against distilled water (molecular weight cutoff: 1500). For citraconic anhydride modification, the purified NPs were dissolved in ethanol, and then 0.1 mL of citraconic anhydride was added and


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stirred overnight. The solution was then centrifuged and the sediment was washed with water and then stored at 4 ºC for further use. Loading of MB molecules: 10 mg of the as-prepared NLS-mDOX@SiO2 NPs was dissolved in 20 mL distilled water, into which 5 mg of MB was added. The mixture was stirred at room temperature for 4 h before collected via centrifugation. The sample was further washed with water several times until the supernatant showed no obvious MB-characteristic (blue) absorption. The amount of MB loaded onto NPs was calculated by measuring the concentration difference between the solutions before and after adsorption. The co-loading of MPA and MB was performed with similar procedures. 2.4 Characterization UV-Vis absorption spectra were taken using a Shimadzu UV-2550 spectrophotometer. PL emission spectra were obtained using an Edinburgh FS5 spectrophotometer. A HT7700 transmission electron microscope (Hitachi, Japan) with an acceleration voltage of 100 kV was employed to acquire TEM images. The aqueous solution of NPs was dripped onto carbon-coated copper grids to deposit NPs onto the films. The surface area and distribution of pore diameter were measured using N2 adsorption/desorption measurements in a V-Sorb 2800 surface analyzer system (Gold-APP Instrument, China). 2.5 In vitro cell and in vivo mouse imaging U87MG cells were firstly seeded into confocal Petri dishes containing DMEM medium with 10% (v/v) fetal bovine serum, and incubated at 37 ºC in a humidified atmosphere containing 5% CO2. After 24 h of incubation, 100 µL NP solution (about 0.5 mg/mL) was added. After


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incubated for another 2 h, 50 µL Hoechst 33342 solution was added. 15 min later, cells were washed with PBS (pH 7.4) for three times. Fluorescence images of cells were obtained using a laser confocal scanning microscope (LCSM, Olympus Fluoview 1000). In vivo optical imaging was performed using a homemade small animal NIR imaging system. Specifically, about 100 µL (0.5 mg/mL) of NP solution was administrated into mice bearing U87MG tumor via intravenous injection. The in vivo distribution of the NPs in mice was monitored at scheduled time points. 2.6 Measurement of in vivo Si distribution using ICP-MS Briefly, 200 µL of 3 mg/mL of mDOX@SiO2/MB-NLS NPs was intravenously injected into tumor bearing mice. At different time points (8, 24 or 48 h) post injection, mice were sacrificed and major organs including tumors were collected and weighted. A mouse without NP treatment was used as control. A certain amount of each organ was weighted and digested under ultrasonication at 50 ºC in the mixture of concentrated HNO3 and H2O2. The yellow solution was then neutralized with (NH4)2CO3, and then NaOH solution was added to dissolve the NPs at about 80 ºC.

Before

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A 7700x quadrupole ICP-MS (Agilent) was used to obtain ICP-MS results. 2.7 In vitro evaluation of cell viability MTT assays were conducted to evaluation the antitumor efficacy in vitro. Briefly, U87MG cells were seeded onto 96-well plate, and then 100 µL of different samples, as described in the main text, were added into each well. After about 30 h of co-incubation, each well was washed three times with PBS (pH 7.4) before addition of 15 µL of MTT solution, and the cells were cultured for another 4 h. The medium was then removed and 150 µL of DMSO was added into each well. After gentle shaking for 15 min at room temperature, the optical density of each well at 570 nm


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was measured. These procedures were repeated four times. The cells for photodynamic therapy was irradiated with a 660 nm laser at a power of 100 mW/cm2 for 5 min after 18 h of coincubation. 2.8 In vivo evaluation of antitumor efficacy and toxicity In a typical therapeutic procedure, 100 µL of different samples (0.5 mg NP/mL, 0.06 mg DOX/mL) was injected through tail vein into U87MG tumor-bearing mice, which were randomly divided into six groups. 18 h later, mice were irradiated with 660 nm light at a power of 100 mW/cm2 for 5 min. This was repeated every 2 days in the following two weeks. The tumor size and body weight of each mouse were recorded. At last, the mice of each group were scarificed, and the tumors and major organs were collected for the immunohistochemistry assay, in which tissues were stained with hematoxylin and eosin (H&E) and examined using a microscopy. 3. RESULTS AND DISCUSSION As shown in Figure 1, in this work, i) we firstly prepared self-decomposable DOX@SiO2 NPs with radial mesopores, and ii) then explored their potential in nuclear targeted chemophotodynamic combination therapy by conjugating NLS peptide and uploading MB molecules.


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Figure 1. Schematic illustration of the synthesis of NLS-mDOX@SiO2/MB NPs and their application in nuclear targeted chemo-photodynamic combination therapy. 3.1 Synthesis of mDOX@SiO2 NPs and Mechanistic Insight Firstly, DOX-embedded SiO2 (DOX@SiO2) NPs were prepared by introducing DOX into SiO2 NP growth media. The DOX-characteristic absorption and PL spectra of the resulting NPs shown in Figure 2a and 2b suggest the successful incorporation of DOX molecules into NPs. In addition, unlike the pure SiO2 NPs mixed with free DOX molecules, the absorption and PL properties of DOX@SiO2 NPs were found to be basically independent on solution pH (Figure S1). Considering the pH dependence of absorption and PL properties of DOX molecules (Figure S2),40 these results further suggest the successful incorporation of DOX into SiO2 NPs, in which DOX molecules were “grown inside” rather than adsorbed on NP surface. The DOX concentration in NPs was calculated to be about 100 µg/mg under this synthetic concentration (see Experimental for details). These DOX@SiO2 NPs were spherical and uniform in diameter


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(Figure 2c and 2d). The diameter could be tuned at least in the range of about 80–300 nm (Figure S3). Incubating these DOX@SiO2 NPs in pH 5 PBS allowed the release of DOX molecules (Figure S4, the release rate in pH 5 PBS was found to be faster than in pH 7.4 PBS or deionized water), during which the PL intensity of the incubating solution increased. This PL enhancement is consistent with the fact that free DOX fluorescence in pH 5 PBS is stronger than those embedded in DOX@SiO2 NPs (Figure S1), and indicates the chemical integrity of the released DOX molecules (further evidence from high performance liquid chromatography can be found in Figure S5). 1.2x106

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Figure 2. (a) Absorption and (b) PL spectra of aqueous solutions (pH 6.3) of DOX, SiO2 NPs and DOX@SiO2 NPs. (c) Representative TEM image and (d) size distribution of DOX@SiO2 NPs. Next, we tuned the DOX concentration in DOX@SiO2 NPs and studied their decomposition using TEM. As shown in Figure 3, (i) at low DOX concentration (Figure 3a, 70 µg/mg), the NPs


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remained compact during the examined period, although small inner cavities could be distinguished after 7 d of incubation in pH 5 PBS. Increasing the DOX concentration lead to decomposition of NPs (Figure 3b and 3c). (ii) At the highest examined DOX concentration (120 µg/mg), radial mesopores extending from the NP center to the surface were observed from the freshly prepared NPs (Figure 3c, left). After 3 d of incubation, the pore size increased (Figure 3c, middle). These mesoporous DOX@SiO2 (mDOX@SiO2) NPs exhibited characteristic Type IV nitrogen adsorption-desorption isotherms consistent with the presence of radial mesopores (Figure S6). The calculated surface areas of the freshly prepared and incubated (for 3 d) mesoporous NPs were about 288 m2/g and 420 m2/g, respectively, and the average pore diameters were about 2 nm and 5–10 nm, respectively.

Figure 3. TEM images of DOX@SiO2 NPs with different DOX concentrations (a, 70 µg/mg; b, 100 µg/mg; c, 120 µg/mg) before and after incubated in pH 5 PBS for different periods of time.


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It was noticed that the formation of mDOX@SiO2 NPs was distinct from previous reports, in which center-hollowed nanoshells were formed with the release of drug (i.e., MB) molecules from MB-embedded SiO2 NPs.30 The possible driving forces leading to this difference can be found in Figure S7. One of the advantages of mesoporous NPs over nanoshells is the higher drug loading efficacy (here, the intermediate nanoshells were found to be difficult to be uploaded with drugs into the cavity). Therefore, the mDOX@SiO2 NPs with large radial mesopores were further explored as desirable drug carriers in nuclear-targeted combination therapy. 3.2 Synthesis of NLS-mDOX@SiO2/MB NPs for Nuclear Targeted Chemo-Photodynamic Combination Therapy In this work, a nuclear localization signal (NLS) peptide (sequence KKKRK) that has been reported to mediate nuclear localization41 was modified onto the mDOX@SiO2 NPs to maximize the therapeutic effect of DOX. The NPs were firstly functionalized with 3-aminopropyltriethoxysilane and then NLS peptide was conjugated (endosomal pH-degradable citraconic anhydride was further conjugated to lower the positive surface charge caused by the amino functionalization and polyamine nature of NLS, Figure S8).42 To further enhance the therapeutic efficacy, MB, a commonly used photosensitizer in photodynamic therapy and has also been recognized as a sensitizer to DOX,43,44 was uploaded into the mesopores to achieve chemophotodynamic combination therapy (Figure 4a). The MB uploading efficiency was high (about 240 µg/mg), likely owing to the electrostatic interaction between positively charged MB and the negatively charged NPs and the presence of mesopores. Incubating the resulting NLS-modified MB-loaded mesoporous NPs (NLS-mDOX@SiO2/MB NPs) in water lead to fragmentation of NPs, as indicated by dynamic light scattering and TEM measurements (Figure 4b and 4c). NPs


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loaded with MB were found to decompose faster than those without MB loading, probably because of the pulling force given from MB to SiO2 matrix due to the opposite charge.

mDOX@SiO2 NPs (1) NLS-mDOX@SiO2 NPs (2) NLS-mDOX@SiO2/MB NPs (3) MB

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Figure 4. (a) Absorption spectra of mDOX@SiO2 NPs, NLS-mDOX@SiO2 NPs, NLSmDOX@SiO2/MB NPs and MB. Inset is the photograph of the corresponding samples (upper) before and (bottom) after centrifugation. (b) Dynamic light scattering diameters and (c) TEM images of NLS-mDOX@SiO2/MB NPs after incubated in water for different durations. It has been recognized that NLS peptide could associate with importins α and β sequentially to mediate nuclear localization of the conjugated cargo, and consequently nuclear targeted DOX delivery were expected upon the fragmentation of NLS-mDOX@SiO2/MB NPs (Figure 5a). Figure 5b presents the cellular accumulation of NLS-mDOX@SiO2/MB NPs and mDOX@SiO2MB NPs without NLS modification in U87MG cells after co-incubated for different periods of time. As shown, after 12 h of co-incubation, both groups showed similar cellular localization and


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NPs mainly accumulated in cytoplasm, which should be ascribed to the limited DOX release. However, after 24 h of co-incubation, the nuclear accumulation in the NLS-modified group was much more distinct than the mDOX@SiO2/MB group (cellular uptake and distribution of mDOX@SiO2/MB NPs were similar with DOX@SiO2 NPs, Figure S9). These results suggest the nuclear targeting capability of the NLS-mDOX@SiO2/MB NPs upon fragmentation, which should be useful to achieve desirable therapeutic outcomes at a limited DOX concentration. Next, the in vitro cancer cell killing efficacy of these NPs was then studied. In a typical therapeutic procedure, U87MG cells were co-incubated with NLS-mDOX@SiO2/MB NPs at a final concentration of 50 µg/mL for 18 h and then irradiated using a 660 nm laser at a power of 100 mW/cm2 for 5 min, followed by another 12 h of incubation. The generation of reactive oxygen species (ROS) upon MB loading and light irradiation was confirmed using 2,7dichlorodihydrofluorescein diacetate (Figure 5c), which could be hydrolyzed by intracellular esterase and then oxidized by ROS into fluorescent 2,7-dichlorofluorescein (DCF). Standard methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the cancer cell killing efficacy. DOX, mDOX@SiO2 NPs, mDOX@SiO2/MB NPs without NLS modification and NLSmDOX@SiO2 NPs without MB loading were also co-incubated with cells as comparison. As can be seen in Figure 5d, compared with the chemo-only and non-targeted therapy, the nuclear targeted combination treatment using NLS-mDOX@SiO2/MB NPs with light irradiation showed the most effective cancer cell killing efficacy.


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Figure 5. (a) Schematic illustration of the nuclear targeted delivery of the fragmented NLSmDOX@SiO2/MB NPs. (b) Representative LCSM images of U87MG cells after co-incubated with (i) mDOX@SiO2/MB NPs or (ii) NLS-mDOX@SiO2/MB NPs for different durations. (c) Representative LCSM images of U87MG cells after co-incubated with (i) pure MB, (ii) NLSmDOX@SiO2 NPs or (iii) NLS-mDOX@SiO2/MB NPs for 2 h and then irradiated with a 660 nm laser for 5 min. (d) U87MG cell viability after treated with different therapeutic agents at different concentrations for 30 h. Next, MPA, a near-infrared fluorescent dye was co-loaded with MB into NLSmDOX@SiO2 NPs to evaluate the in vivo distribution. Absorption and PL spectra of the coloaded NPs are provided in Figure S10. After injected into U87MG tumor-bearing mice, the NPs showed desirable tumor accumulation 4 h post injection (Figure 6a), and more distinct


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localization at tumor site over other organs was observed 8 h post injection. Ex vivo imaging of the major organs and tumor collected 24 h post injection further indicated the high tumorous accumulation of the injected NPs. Partial dissociation of MPA from NPs was noticed from the urocystic distribution 10 min and 4 h post injection. Thus, to demonstrate that the PL signal at tumor site came from the co-loaded NPs (rather than free MPA), fluorescent histological sections of mouse organs were further performed using DOX fluorescence as the intrinsic signal of NPs, and the results indicated favorable tumor accumulation of the co-loaded NLS-mDOX@SiO2 NPs (Figure S11). Quantitative biodistribution of Si were also determined using ICP-MS (Figure S12). The results showed that the Si concentration in tumor was higher than in other major organs (heart, spleen, kidney, lung and liver) at all tested time points (8 h, 24h and 48 h), with the highest concentration observed to be about 120 ng/mg 8 h post injection. Liver was found to be the second mostly accumulated organ with the concentration ranging from 30–55 ng/mg. These results are in line with previously reported in vivo Si distribution determined using ICPMS,45 in which the superior tumorous accumulation was observed and ascribed to the enhanced permeability and retention effect.


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Figure 6. (a) Dynamic distribution of MPA and MB co-loaded NLS-mDOX@SiO2 NPs intravenously injected in U87MG tumor-bearing mice (tumor highlighted by white ciecle), and the corresponding ex vivo imaging of major organs and tumor 24 h post injection. (b) Body weight and (c) tumor volume of each group of mice during the treatment period. Tumor volume was calculated as width2 × length × 0.5. The presented volume was normalized by the initial volume of each group. (d) Photograph of tumors collected from each group after the treatment. (e) Representative histopathology sections of tumor from each group. Encouraged by the high tumor accumulation, in vivo antitumor efficacy of NLSmDOX@SiO2/MB NPs (without MPA) was further explored. U87MG tumor-bearing mice were randomly divided into six groups, and each group received different intravenous adminstration.


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For the groups given light irradiation, a 660 nm laser at a power of 200 mW/cm2 was applied to irradiate the tumor site for 5 min. During the treatment, the body weights and tumor volumes were measured every 2 days (Figure 6b and 6c). After two weeks of treatment, the mice were sacrificed and the tumors were collected (Figure 6d) and analyzed via histopathology analysis (Figure 6e). The major organs were also subjected to histopathology analysis to evaluate the systemic toxicity (Figure 7). The results suggest that free DOX showed no significant inhibiting effect on tumor growth, likely due to the insufficient tumor accumulation of DOX molecules at a limited dose. In mice treated with NPs without NLS modification or MB loading, the growth of mice tumor was partially delayed compared with the saline group. However, when treated with NLS-mDOX@SiO2/MB NPs, that is, given chemo and photodynamic combined therapy, the mice showed efficiently inhibited tumor growth. Furthermore, the HE stained tumor slices also showed that the tumor tissues were severely damaged when treated with NLS-mDOX@SiO2/MB NPs (Figure 6e), further demonstrating the favorable therapeutic effect of nuclear targeted chemo-photodynamic combination therapy using NLS-mDOX@SiO2/MB NPs. During the treatment period, no adverse symptoms (such as change in food intake) or body weight loss was observed (Figure 6b), and histopathology analysis showed that similar with the saline group, no obvious organ lesion or abnormality was observed in the group treated with NLSmDOX@SiO2/MB NPs (Figure 7), suggesting that the systemic toxicity of our treatment was acceptable.


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Figure 7. Representative histopathology sections of major organs harvested from mice after 2 weeks of treatment with (upper) saline and (bottom) NLS-mDOX@SiO2/MB NPs. 4. CONCLUSIONS In summary, self-decomposable drug-embedded SiO2 NPs were prepared, and the intermediate morphology during decomposition depends on the drug molecules. Furthermore, by using the intermediate mDOX@SiO2 NPs with radial mesopores, a multifunctional drug delivery system with nuclear targeting and chemo-photodynamic combination therapy capability was fabricated by conjugating NLS peptide and uploading MB molecules. Based on in vitro and in vivo antitumor and toxicity evaluations, these as-fabricated drug carriers exhibited enhanced therapeutic efficacy without noticeable systemic toxicity. These results suggest that our selfdecomposable multifunctional drug delivery system should be a promising improvement to the present SiO2-based drug carriers. ASSOCIATED CONTENT Supporting Information. Additional characterizations of DOX@SiO2 NPs, DOX molecules, mDOX@SiO2 NPs, NLS-mDOX@SiO2/MB NPs, fluorescein-embedded SiO2 NPs, MPA/MB


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co-loaded NLS-mDOX@SiO2 NPs, high performance liquid chromatography results, fluorescent histological sections of mouse organs. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (D.D.). *E-mail: [email protected] (D.H.). Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (81371627 and 81220108012), and the Program for New Century Excellent Talents (NCET-120974) in the University of the Ministry of Education of China. REFERENCES (1) Petros, R. A.; DeSimone, J. M. Strategies in the Design of Nanoparticles for Therapeutic Applications. Nat. Rev. Drug Discov. 2010, 9, 615–627. (2) Ferrari, M. Cancer Nanotechnology: Opportunities and Challenges. Nat. Rev. Cancer 2005, 5, 161– 171. (3) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery. Nat. Mater. 2013, 12, 991–1003.


21


Page 22 of 26

(4) Cho, K.; Wang, X.; Nie, S.; Chen, Z.; Shin, D. M. Therapeutic Nanoparticles for Drug Delivery in Cancer. Clin. Cancer Res. 2008, 14, 1310–1316. (5) Farokhzad, O. C.; Langer, R. Impact of Nanotechnology on Drug Delivery. ACS Nano 2009, 3, 16–20. (6) Prato, M.; Kostarelos, K.; Bianco, A. Functionalized Carbon Nanotubes in Drug Design and Discovery. Acc. Chem. Res. 2008, 41, 60–68. (7) Dykman, L.; Khlebtsov, N. Gold Nanoparticles in Biomedical Applications: Recent Advances and Perspectives. Chem. Soc. Rev. 2012, 41, 2256–2282. (8) Slowing, I.; Viveroescoto, J.; Wu, C.; Lin, V. Mesoporous Silica Nanoparticles as Controlled Release Drug Delivery and Gene Transfection Carriers. Adv. Drug Deliv. Rev. 2008, 60, 1278–1288. (9) Reddy, L. H.; Arias, J. L.; Nicolas, J.; Couvreur, P. Magnetic Nanoparticles: Design and Characterization, Toxicity and Biocompatibility, Pharmaceutical and Biomedical Applications. Chem. Rev. 2012, 112, 5818–5878. (10) Deng, D.; Chen, Y.; Cao, J.; Tian, J.; Qian, Z.; Achilefu, S.; Gu, Y. High-Quality CuInS2/ZnS Quantum Dots for In Vitro and In Vivo Bioimaging. Chem. Mater. 2012, 24, 3029–3037. (11) Zhou, J.; Yang, Y.; Zhang, C. Toward Biocompatible Semiconductor Quantum Dots: From Biosynthesis and Bioconjugation to Biomedical Application. Chem. Rev. 2015, 115, 11669–11717. (12) Yang, P.; Gai, S.; Lin, J. Functionalized Mesoporous Silica Materials for Controlled Drug Delivery. Chem. Soc. Rev. 2012, 41, 3679–3698. (13) Deng, D.; Hao, C.; Sen, S.; Xu, C.; Kral, P.; Kotov, N. A. Template-Free Hierarchical Self-Assembly of Iron Diselenide Nanoparticles into Mesoscale Hedgehogs. J. Am. Chem. Soc. 2017, 139, 16630–16639. (14) Coll, C.; Bernardos, A.; Martínez-Máñez, R.; Sancenón, F. Gated Silica Mesoporous Supports for Controlled Release and Signaling Applications. Acc. Chem. Res. 2013, 46, 339–349. (15) Mackowiak, S. A.; Schmidt, A.; Weiss, V.; Argyo, C.; von Schirnding, C.; Bein, T.; Bräuchle, C. Targeted Drug Delivery in Cancer Cells with Red-Light Photoactivated Mesoporous Silica Nanoparticles. Nano Lett. 2013, 13, 2576–2583.


22

Page 23 of 26 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


(16) Blum, A. P.; Kammeyer, J. K.; Rush, A. M.; Callmann, C. E.; Hahn, M. E.; Gianneschi, N. C. Stimuli-Responsive Nanomaterials for Biomedical Applications. J. Am. Chem. Soc. 2015, 137, 2140– 2154. (17) Ferris, D. P.; Zhao, Y.-L.; Khashab, N. M.; Khatib, H. A.; Stoddart, J. F.; Zink, J. I. Light-Operated Mechanized Nanoparticles. J. Am. Chem. Soc. 2009, 131, 1686–1688. (18) Du, X.; Li, X.; Xiong, L.; Zhang, X.; Kleitz, F.; Qiao, S. Z. Mesoporous Silica Nanoparticles with Organo-Bridged Silsesquioxane Framework as Innovative Platforms for Bioimaging and Therapeutic Agent Delivery. Biomaterials 2016, 91, 90–127. (19) Lin, W.; Huang, Y.; Zhou, X.-D.; Ma, Y. In Vitro Toxicity of Silica Nanoparticles in Human Lung Cancer Cells. Toxicol. Appl. Pharmacol. 2006, 217, 252–259. (20) Croissant, J. G.; Fatieiev, Y.; Khashab, N. M. Degradability and Clearance of Silicon, Organosilica, Silsesquioxane, Silica Mixed Oxide, and Mesoporous Silica Nanoparticles. Adv. Mater. 2017, 29, 1604634. (21) Park, M. V. D. Z.; Neigh, A. M.; Vermeulen, J. P.; de la Fonteyne, L. J. J.; Verharen, H. W.; Briedé, J. J.; van Loveren, H.; de Jong, W. H. The Effect of Particle Size on the Cytotoxicity, Inflammation, Developmental Toxicity and Genotoxicity of Silver Nanoparticles. Biomaterials 2011, 32, 9810–9817. (22) Kong, M.; Tang, J.; Qiao, Q.; Wu, T.; Qi, Y.; Tan, S.; Gao, X.; Zhang, Z. Biodegradable Hollow Mesoporous Silica Nanoparticles for Regulating Tumor Microenvironment and Enhancing Antitumor Efficiency. Theranostics 2017, 7 (13), 3276–3292. (23) Hoffmann, F.; Cornelius, M.; Morell, J.; Fröba, M. Silica-Based Mesoporous Organic-Inorganic Hybrid Materials. Angew. Chemie Int. Ed. 2006, 45 (20), 3216–3251. (24) Wang, W.; Wang, P.; Tang, X.; Elzatahry, A. A.; Wang, S.; Al-Dahyan, D.; Zhao, M.; Yao, C.; Hung, C. Te; Zhu, X.; Zhao, T.; Li, X.; Zhang, F.; Zhao, D. Facile Synthesis of Uniform Virus-like Mesoporous Silica Nanoparticles for Enhanced Cellular Internalization. ACS Cent. Sci. 2017, 3 (8), 839– 846.


23


Page 24 of 26

(25) Croissant, J.; Cattoën, X.; Man, M. W. C.; Gallud, A.; Raehm, L.; Trens, P.; Maynadier, M.; Durand, J.-O. Biodegradable Ethylene-Bis(Propyl)Disulfide-Based Periodic Mesoporous Organosilica Nanorods and Nanospheres for Efficient In-Vitro Drug Delivery. Adv. Mater. 2014, 26, 6174–6180. (26) Chen, Y.; Meng, Q.; Wu, M.; Wang, S.; Xu, P.; Chen, H.; Li, Y.; Zhang, L.; Wang, L.; Shi, J. Hollow Mesoporous Organosilica Nanoparticles: A Generic Intelligent Framework-Hybridization Approach for Biomedicine. J. Am. Chem. Soc. 2014, 136, 16326–16334. (27) Tang, L.; Fan, T. M.; Borst, L. B.; Cheng, J. Synthesis and Biological Response of Size-Specific, Monodisperse Drug-Silica Nanoconjugates. ACS Nano 2012, 6, 3954–3966. (28) Vivero-Escoto, J. L.; Rieter, W. J.; Lau, H.; Huxford-Phillips, R. C.; Lin, W. Biodegradable Polysilsesquioxane Nanoparticles as Efficient Contrast Agents for Magnetic Resonance Imaging. Small 2013, 9, 3523–3531. (29) Della Rocca, J.; Huxford, R. C.; Comstock-Duggan, E.; Lin, W. Polysilsesquioxane Nanoparticles for Targeted Platin-Based Cancer Chemotherapy by Triggered Release. Angew. Chemie Int. Ed. 2011, 50, 10330–10334. (30) Zhang, S.; Chu, Z.; Yin, C.; Zhang, C.; Lin, G.; Li, Q. Controllable Drug Release and Simultaneously Carrier Decomposition of SiO2-Drug Composite Nanoparticles. J. Am. Chem. Soc. 2013, 135, 5709–5716. (31) Brannon-Peppas, L.; Blanchette, J. O. Nanoparticle and Targeted Systems for Cancer Therapy. Adv. Drug Deliv. Rev. 2004, 56, 1649–1659. (32) Nicolas, J.; Mura, S.; Brambilla, D.; Mackiewicz, N.; Couvreur, P. Design, Functionalization Strategies and Biomedical Applications of Targeted Biodegradable/biocompatible Polymer-Based Nanocarriers for Drug Delivery. Chem. Soc. Rev. 2013, 42, 1147–1235. (33) Fang, J.; Nakamura, H.; Maeda, H. The EPR Effect: Unique Features of Tumor Blood Vessels for Drug Delivery, Factors Involved, and Limitations and Augmentation of the Effect. Adv. Drug Deliv. Rev. 2011, 63, 136–151.


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(34) Prabhakar, U.; Maeda, H.; Jain, R. K.; Sevick-Muraca, E. M.; Zamboni, W.; Farokhzad, O. C.; Barry, S. T.; Gabizon, A.; Grodzinski, P.; Blakey, D. C. Challenges and Key Considerations of the Enhanced Permeability and Retention Effect for Nanomedicine Drug Delivery in Oncology. Cancer Res. 2013, 73, 2412–2417. (35) Danhier, F.; Feron, O.; Préat, V. To Exploit the Tumor Microenvironment: Passive and Active Tumor Targeting of Nanocarriers for Anti-Cancer Drug Delivery. J. Control. Release 2010, 148, 135– 146. (36) Du, C.; Deng, D.; Shan, L.; Wan, S.; Cao, J.; Tian, J.; Achilefu, S.; Gu, Y. A pH-Sensitive Doxorubicin Prodrug Based on Folate-Conjugated BSA for Tumor-Targeted Drug Delivery. Biomaterials 2013, 34, 3087–3097. (37) K. C., R. B.; Thapa, B.; Xu, P. pH and Redox Dual Responsive Nanoparticle for Nuclear Targeted Drug Delivery. Mol. Pharm. 2012, 9, 2719–2729. (38) Anajafi, T.; Scott, M. D.; You, S.; Yang, X.; Choi, Y.; Qian, S. Y.; Mallik, S. Acridine Orange Conjugated Polymersomes for Simultaneous Nuclear Delivery of Gemcitabine and Doxorubicin to Pancreatic Cancer Cells. Bioconjug. Chem. 2016, 27, 762–771. (39) Pan, L.; He, Q.; Liu, J.; Chen, Y.; Ma, M.; Zhang, L.; Shi, J. Nuclear-Targeted Drug Delivery of TAT Peptide-Conjugated Monodisperse Mesoporous Silica Nanoparticles. J. Am. Chem. Soc. 2012, 134, 5722–5725. (40) Karukstis, K. K.; Thompson, E. H. Z.; Whiles, J. A.; Rosenfeld, R. J. Deciphering the Fluorescence Signature of Daunomycin and Doxorubicin, Biophys. Chem. 1998, 73, 249–263. (41) Kang, B.; Mackey, M. A.; El-Sayed, M. A. Nuclear Targeting of Gold Nanoparticles in Cancer Cells Induces DNA Damage, Causing Cytokinesis Arrest and Apoptosis. J. Am. Chem. Soc. 2010, 132, 1517– 1519. (42) Lee, Y.; Fukushima, S.; Bae, Y.; Hiki, S.; Ishii, T.; Kataoka, K. A Protein Nanocarrier from ChargeConversion Polymer in Response to Endosomal pH. J. Am. Chem. Soc. 2007, 129, 5362–5363.


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Page 26 of 26

(43) Yano, S.; Hirohara, S.; Obata, M.; Hagiya, Y.; Ogura, S.; Ikeda, A.; Kataoka, H.; Tanaka, M.; Joh, T. Current States and Future Views in Photodynamic Therapy. J. Photochem. Photobiol. C Photochem. Rev. 2011, 12, 46–67. (44) Ma, P.; Mumper, R. J. Anthracycline Nano-Delivery Systems to Overcome Multiple Drug Resistance: A Comprehensive Review. Nano Today 2013, 8, 313–331. (45) Lu, J.; Liong, M.; Li, Z.; Zink, J. I.; Tamanoi, F. Biocompatibility, Biodistribution, and

Drug-Delivery Efficiency of Mesoporous Silica Nanoparticles for Cancer Therapy in Animals. Small 2010, 6 (16), 1794–1805.

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Self-Decomposable Mesoporous Doxorubicin@Silica

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