Metal–Organic Framework-Based Nanoplatform for Intracellular

Sep 4, 2018 - Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, ...
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Biological and Medical Applications of Materials and Interfaces

Metal-Organic-Framework-Based Nanoplatform for Intracellular-Environment Responsive Endo/Lysosomal Escape and Enhanced Cancer Therapy Kai Dong, Zhenzhen Wang, Yan Zhang, Jinsong Ren, and Xiaogang Qu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11972 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 4, 2018

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Metal-Organic-Framework-Based Nanoplatform for Intracellular-Environment Responsive Endo/Lysosomal Escape and Enhanced Cancer Therapy Kai Dong, † Zhenzhen Wang, †,‡ Yan Zhang, †,‡ Jinsong Ren*,† and Xiaogang Qu*,† †

Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization,

Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People’s Republic of China ‡

University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China

KEYWORDS: Metal-organic frameworks, Endo/lysosomal escape, Aptamer, Intracellularenvironment responsive, Cancer therapy

ABSTRACT: Nowadays, the efficient endo/lysosomal escape and the subsequent releasing of drugs in cytosol are the major obstacles for nanoplatform-based cancer therapy. Herein, we firstly reported a metal-organic-framework-based nanoplatform (DOX@ZIF-8@AS1411) for intracellular-environment responsive endo/lysosomal escape and enhanced cancer therapy. In our system, the nanoplatform firstly targeted to the cancer cells. Then, it was entrapped in endo/lysosomes, where occurred pH-responsive decomposition and released abundant Zn ions.

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The released Zn ions could induce an influx of counter ions, promote ROS generation to rupture the endo/lysosomal membrane and accelerate the release of anti-cancer drugs in cytosol. Finally, the released drugs and the generation of ROS could synergistically enhance the cancer therapy. With the excellent biocompatibility, effective endo/lysosomal escape and enhanced therapeutic effect, the novel DDSs are supposed to become a promising anticancer agent for the cancer therapy and bring more opportunities for the biomedical application.

1. INTRODUCTION Currently, cancer is a major health burden throughout the world. Chemotherapy is a significant strategy widely applied in clinic, which uses chemical matters as anticancer drugs to devastate cancers. However, a critical challenge for cancer chemotherapy is that most drugs cannot effectively enter cells, which results in poor therapeutic effect.1-2 In order to overcome the obstacle, the construction of new drug delivery systems (DDSs) with the effective payload and high hydrophilicity have attracted broad attention. 3-4 In recent years, nanoscale DDSs as a great promising method have been widely reported to transport drug molecule into cancer cells.5-12 However, these DDSs generally are internalized by cells via endocytosis, entrapped in endo/lysosomes, reduced drug release in cytosol, resulting in unexpected therapeutic efficacy.1316

Therefore, it is a growing need to develop smart DDSs that can promote the endo/lysosomal

escape, accelerate the release of anti-cancer drugs and enhance cancer therapy. To achieve efficient endo/lysosomal escape, DDSs integrating with peptides, toxins, lipids, polycationic polymers and photosensitizers have been constructed.17-25 The mechanism of these DDSs for endo/lysosomal escape has been recognized, such as pore formation in the endosomal

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membrane, proton sponge effect, membrane fusion and photochemical internalization (PCI).26 Nevertheless, some problems and disadvantages have hindered their application. For example, DDSs modification with peptides, toxins, lipids, polycationic polymers usually showed immunogenicity, toxicity, low stability and inferior targeting. Although PCI could induce the formation of reactive singlet oxygen (ROS) for controllable endo/lysosomal escape, they still remain an issue that some payloads may also be photo-oxidized by ROS due to localization near the activated photosensitizers. Therefore, in order to overcome the above obstacles and achieve more effective drug delivery, the new types of DDSs are urgently needed. Recently, metal-organic frameworks (MOFs) have emerged as one kind of novel porous materials for widely applying in catalysis, gas storage, molecule separation and so on.27-35 As an extensively and deeply studied MOFs, zeolitic imidazolate framework-8 (ZIF-8) has revealed remarkable potential applications in the field of biomedicine due to the simple and rapid synthetic process, homogeneous pore sizes, high surface area, facile modification, excellent dispersibility and favorable biocompatibility.36-45 More importantly, the ZIF-8 has shown the pHresponsive decomposition feature, which could release Zn ions and the encapsulated cargoes under acidic condition. Additionally, it was reported that Zn ions could induce an influx of counter ions and promote ROS generation to rupture the endo/lysosomal membrane.46-48 Inspired by these characteristics, we envision that the ZIF-8 nanoparticles can become a promising candidate for the efficient stimuli-responsive endo/lysosomal escape and release of drug for efficient cancer therapy. Herein, for the first time, we reported a MOFs-based nanoplatform for the intracellularenvironment endo/lysosomal escape, drugs release, and enhanced cancer therapy (Scheme 1). In our system, the ZIF-8 was available for the loading of abundant drug by a facile one-pot process.

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Furthermore, the aptamer (AS1411) was introduced into the DDSs which could enable the system to target the cancer cell and reduce the side effect.49-51 Moreover, the advantage of the pH-responsive decomposition enabled the DDSs to efficiently release the drugs in the acidic endo/lysosomal compartments. More importantly, the DDSs were decomposed in the endo/lysosomal compartments and released an abundant of Zn2+, which could induce the generation of ROS to rupture the endo/lysosomal vesicle and enhance the endo/lysosomal escape. Finally, the released drug and the generation of ROS could synergistically enhance the cancer therapy. 2. EXPERIMENTAL SECTION 2.1 Chemicals Zinc nitrate hexahydrate, 2-methylimidazole were obtained from Sigma-Aldrich. Doxorubicin (DOX)

was

purchased

from

Aladdin.

The

AS1411

GGTGGTGGTGGTTGTGGTGGTGGTGGGTCAAGTAGACCAC-3′)

were

aptamers prepared

(5′by

Sangon Biotechnology. Water in this work was acquired by a Milli-Q water system. 2.2 Preparation of drug delivery nanocarriers (DOX@ZIF-8) 0.2 g Zn(NO3)2·6H2O was dissolved into 7.5 mL H2O. Then, 1 mg DOX was dissolved into the zinc solution with a magnetic stirrer. 10 mL solution containing of 2-methyl imidazole (0.33 g) was added into the aforementioned medium. The solution was kept stirring for 15 min with a magnetic stirrer. The samples were collected by centrifuging at 10000 rpm for 10 min. The asprepared nanoparticles were washed with water and ethanol for completely removing the redundant reactants. Ultimately, the obtained products were dried in a vacuum at 60 °C for 12 h. 2.3 Preparation of aptamer functionalized drug delivery nanocarriers (DOX@ZIF-

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8@AS1411) To produce the aptamer modified drug delivery nanocarriers, G-quadruplex structure of AS1411 was first formed in the PBS buffer. Then, the aptamer (5 µM) was reacted with DOX@ZIF-8 nanoparticles (10 mL, 1 mg/mL) in a 4 oC overnight. The as-prepared product was centrifuged at 10000 rpm for 10 min. The sample was washed by buffer for three times. 2.4 In vitro pH-responsive drug release experiments. In a typical pH-responsive drug release procedure, DOX@ZIF-8@AS1411 nanoparticles were respectively immersed in PBS (5 mM, pH 7.4) and PBS (5 mM, pH 5.0). At appropriate intervals, DDSs was centrifuged (12000 rpm, 10 min) and the supernatant was collected. The UV-vis spectrometer was applied to measure the absorption of DOX in the supernatant, which was used to calculate the cumulative DOX releasing from DDSs. 2.5 Cell cultures HeLa and HEK 293T cells were obtained from ATCC (American Type Culture Collection). The conditions of cell culture are as follows: Dulbecco’s modified Eagle medium (DMEM), 10% fetal bovine serum (FBS) and antibiotic (100 U/mL penicillin, 100 µg/mL streptomycin). Then, cells in the medium were cultured in the incubator (5% CO2, 37 °C). Before seeding in the plates, cells were digested by trypsin and re-suspended in new medium. 2.6 Cellular uptake and location HeLa cells were seeded in 24-well plates and allowed to adhere overnight. Then, the DOX@ZIF8@AS1411 nanoparticles were introduced into the plates containing the cells. After incubation for 4 h, PBS was used to wash cells for three times. Then, Lysosomal fluorescent probe (LysoTracker Green) was applied to stain cells. After staining for 20 min, redundant fluorescent

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probe was removed by PBS and 4% formaldehyde was used to fix cells for 30 min. Following, cell nucleus were stained by Hoechst 33258. After 20 min, cells were washed by PBS for twice and observed by an optical system microscopy (Olympus BX-51). 2.7 Specific targeting for cancer cells Cells were seeded in 24-well plates and cultured overnight. Then, DOX@ZIF-8@AS1411 nanoparticles were introduced with HeLa cells and HEK 293T cells. After incubation for 4 h, PBS was used to wash cells for twice. The specific targeting of cancer cells was evaluated using flow cytometry (BD LSRFortessa) and fluorescence microscopy (Olympus BX-51). 2.8 The integrity of the lysosomal membrane The integrity of the lysosomal membrane was used to evaluate the pH-responsive endo/lysosomal escape of DOX@ZIF-8@AS1411 nanoparticles. Cells were planted in 24-well plates and cultured overnight. Then, ZIF-8@AS1411 nanoparticles were introduced with HeLa cells for culturing 24 h. As the control group, the HeLa cells were cultured for 24 h without nanoparticles. After PBS rinsing, the cells were treated with AO (5 µg mL-1) for 15 min and were then rinsed with PBS. Then, cells were observed under a fluorescence microscopy (Olympus BX-51) and samples were excited at 488 nm. Emission was detected at 537 nm (green) and 615 nm (red). 2.9 Intracellular ROS levels HeLa cells were planted in 6-well plates for 1 day. Then, HeLa cells were treated with 50 µg mL−1 DOX@ZIF-8@AS1411 nanoparticles for 12 h. The treated cells were incubated with 10 µM of the 2,7-dichlorofluorescin diacetate (DCFH-DA), which was the fluorescent probe for

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measuring the intracellular ROS generation. The intracellular ROS generation was monitored using fluorescence microscopy. 2.10 The cytotoxicity of the DOX@ZIF-8@AS1411 nanoparticles against cancer cells Firstly, the MTT assay was employed to assess the cytotoxicity of the DOX@ZIF-8@AS1411 nanoparticles against cancer cells. HeLa cells were planted in 96-well plates (5000 cells per well) for 24 h. Then, a series of nanoparticles with various concentrations were used to incubate with cells. At incubation for 24 h, the DMEM including nanoparticles were eliminated and washed with PBS twice. Then, MTT was added in cells and cultured for another 4 h. Following, the formative formazan crystals were dissolved by DMSO. The absorbance of formazan crystals at 490 nm (calibrating background absorbance at 630 nm) was detected by a microplate reader (Bio-Rad model-680). Five replicates were done in each group. Additionally, Annexin V-FITC Apoptosis detection Kit (BestBio) was applied to evaluate the apoptosis of cells. The operating steps were followed by the kit protocols. Different cell types (intact, early apoptotic, late apoptotic/necrotic and damaged cells) were quantified by a flow cytometry (BD LSRF Fortessa TM X-20). 2.11 Instruments The morphology of the nanocompounds was characterized by field emission scanning electron microscope (Hitachi S4800) and transmission electron microscopy (FEI Tecnai G2 F20). The Rigaku-Dmax 2500 diffractometer was used to evaluate the crystalline structures of nanocompounds. The UV-vis spectrophotometer (Jasco V550) was applied to record the absorbance of samples. Zeta potentials measurements were performed with a vertically polarized He-Ne laser (Nano-ZS90, Malvern).

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3. RESULTS AND DISCUSSION In our work, the anticancer drug@ZIF-8 (DOX@ZIF-8) was first constructed in a one-pot process. In the detailed procedure, organic linkers (2-methylimidazole) assembled with Zn2+ to form ZIF-8. Due to the chelation with Zn2+ and π-π interaction with 2-methylimidazole, DOX was encapsulated into the ZIF-8 in the synthetic process. Then, the targeted aptamer AS1411 was modified with the anticancer drug@ZIF-8 by the electrostatic interaction. As shown in the Figure 1a, the as-prepared DDS were monodispersed and the average size was about 100 nm. The XRD pattern of the DDS was consistent with the ZIF-8 (Figure 1b). The sharp diffraction peaks indicated the DDS had the high crystallinity. The zeta potentials of the synthesized nanoparticles were characterized. Compare with the DOX@ZIF-8, the zeta potential of the DOX@ZIF8@aptamer obviously decreased, which implied the negatively charged aptamer was anchored on the DOX@ZIF-8 (Figure 1c). The DDS was also characterized by the FTIR spectra. As shown in the Figure S1, the absorption peaks at 3138, 2933 and 1580 cm-1 were assigned to the stretching vibration of -CH3, C-H in the imidazole and C=N, respectively. Moreover, the weak peak at 1087 cm-1 was attributed to the phosphodiester in aptamer. The X-ray photoelectron spectra (XPS) analysis demonstrated the DDSs had the C, N, O, P and Zn (Figure S2). These results indicated the DDSs were successfully synthesized. The amount of the attached aptamer on the DDS was calculated by UV-vis absorption spectra (Figure S3), which was approximately 0.3 µmol/g DOX@ZIF-8. Additionally, the loading capacity of DOX was determined to be 0.1 mg/mg DDSs by using UV-vis spectrometry (Figure S4). The DDS were stable and dispersible in the different solution (PBS, FBS, and DMEM), which was significant for following biomedical research. (Figure S5).

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It was widely reported that ZIF-8 could decompose under acidic conditions and stably exist under neutral conditions. Therefore, we explored the pH-responsive drug release characteristics of the DDS. The DDS were immersed in PBS solution (pH 7.4) and PBS solution (pH 5.0), respectively. The cumulative release of DOX from the DDS was monitored by UV-vis absorption spectra. As shown in the Figure S6a, there was few release of DOX (< 5%) from the DDS after 24 h at the neutral PBS (pH 7.4). Nevertheless, 50% of the encapsulated DOX was released within 24 h under acidic conditions (PBS, pH 5.0). These results indicated that the DDS was a hopeful pH-responsive nanoplatform. Furthermore, the pH-responsive release of zinc cions was also measured the supernatants by an inductively coupled plasma optical emission spectrometer (ICP-OES). As shown in the Figure S6b, the release profiles of zinc ions were similar to DOX, which was stable under neutral conditions and gradually released in the acidic medium. The pH-responsive decomposition of the DDS also was demonstrated by the TEM analysis. As shown in the Figure S7, the DDS in the pH 5.0 solution were dissociated, whereas in the neutral PBS the DDS maintained their morphology. These results confirmed that the DDS had a remarkable capacity for pH-triggered controlled drug release. Then, the internalization and targeting ability of the DDS in the cancer cell were evaluated by fluorescence microscopy. To investigate the process of the cell internalization, the cells were first incubated with DDSs. Then, organelle specific fluorescent probes (LysoTracker Green and Hoechst 33258) were used to stain with lysosome and nucleus. As shown in the Figure 2a, the overlap of green fluorescence (lysosome) and red fluorescence (DDSs) were observed, which indicated the DDSs were endocytosed through the lysosome pathway. Additionally, the AS1411 in the DDS has been reported to highly bind to nucleolin, which is overexpressed on the cytomembrane. Thus, we investigated the ability of the DDS for targeted drug delivery. The

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cellular targeting efficiency was investigated by incubating HeLa cells (human cervical cancer cells) and HEK 293T cells (normal cells) with the DDS. The fluorescence images of DDS incubation with HeLa cells revealed stronger luminescence signals whereas weak fluorescence was observed from DDS incubation with HEK 293T cells (Figure 2b). Subsequently, the fluorescence intensity in HeLa cells and HEK 293T cells was quantified by the flow cytometry. Figure 2c exhibited that the fluorescence intensity of HeLa cells incubated with DDSs was more than six-fold increase relative to HEK 293T cells, which indicated that the DDS had excellent targeting effectiveness. These results demonstrated that the DDS could be effectively endocytosed and targeted to the cancer cells. With the outstanding property of pH-triggered controlled drug release and effective endocytosis, we next investigated the pH-responsive endo/lysosomal escape and drug release. The integrity of the lysosomal membranes was revealed by AO staining assay. As shown in the Figure 3a, the cells without incubation the DOX-free DDS have shown the red fluorescent dots, which demonstrated that the lysosomal membranes were integrated. However, the negligible red fluorescent dots were observed in the cell incubating with the DOX-free DDS after 24h, which indicated that the DDS can rupture the lysosomal membrane structure and increase the lysosomal membrane permeation. We inferred that lysosomal membrane damage was due to the reactive oxygen species (ROS) generation in cells causing by the released zinc ions. In order to verify the hypothesis, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was used as ROS fluorescence probe for evaluating the intracellular ROS generation. In the Figure 3b, the ROS generation was noticed in the cell incubating with the DOX-free DDS and the intracellular ROS levels showed a remarkable promotion with the amount of DDSs increased. These results demonstrated that the ROS generation was the main factor in the lysosomal membrane damage. Then, the drug

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unpacking in the cell was studied by the fluorescence microscopy. The fluorescence images revealed that the co-localization DDS in the lysosomes after 3h incubation (Figure 3c). When the cells were incubated with DDS after 6h, the DOX was observed in the cytosol. With incubating time prolonging, more and more DOX was localized in the cytosol and entered the nuclei. Overall, these results suggested that the DDS could effectively escape from the endosomal compartments and release the drug into the cytosol. After having demonstrated the cell-responsive endo/lysosomal escape and drug unpacking, we further estimated the therapeutic efficacy of DDSs in vitro. The cytotoxicity of the DDS, DOX-free DDS, aptamer-free DDS and free DOX was examined by MTT assay. Figure 4a showed that nearly 80% cells survived after treatment with DOX-free DDSs at a concentration of 50 µg/mL, which demonstrated the DOX-free DDSs are biocompatible. For free DOX, the lower toxicity has been shown due to the less efficient cellular uptake. Although aptamer-free DDSs have exhibited some cytotoxicity, the higher cytotoxicity has been shown in the cancer cells incubation with the DDS at the same condition, which was attributed to the high targeting of DDSs to cancer cells. In order to testify this speculation, the toxicity experiments of DDSs on HEK 293T cells (normal cells) as a contrast were also performed. Figure S8 showed the lower cytotoxicity to HEK 293T cells at the same concentration of DDSs compared with the HeLa cells, which verified that the higher cytotoxicity to cancer cells was derived from the targeting of DDSs. Additionally, the cell viability was further identified by co-staining assay with fluorescein-annexin V and propidium iodide (PI). In the Figure 4b, the obvious apoptosis revealed after treatment with DDS. These results were consistent with the cytotoxicity assay. These results demonstrated that the DDS could target delivery drugs to cancer cell, increase the uptake of drugs and enhance cancer therapy.

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4. CONCLUSIONS In conclusion, a new paradigm for intracellular-environment responsive endo/lysosomal escape and drug unpacking of DDSs has been demonstrated. The novel DDSs were able to effectively load abundant drug and target to the cancer cells. Additionally, our studies have shown that the DDSs could effectively escape from the endo/lysosomal compartments due to the pH-triggered zinc ions release and the ROS generation. The pH-responsive endo/lysosomal escape could facilitate the drugs release into the cytosol and entrance into the nuclei, which could give rise to the enhanced cancer therapy. With the excellent biocompatibility, effective endo/lysosomal escape and enhanced therapeutic effect, the novel DDSs are supposed to become a promising anticancer agent for the cancer therapy and bring more opportunities for the biomedical application.

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Scheme 1. Schematic illustration of the construction of the aptamer modified drug@ZIF-8 nanoparticles and the mechanism of nanoparticles for stimuli-responsive endo/lysosomal escape and enhanced cancer therapy.

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Figure 1. Characterization of DOX@ZIF-8@AS1411. (a) TEM micrographs of DOX@ZIF8@AS1411. (b) XRD of ZIF-8 (black) and DOX@ZIF-8@AS1411 (red). (c) Zeta potentials of DOX@ZIF-8 (red) and DOX@ZIF-8@AS1411 (green). Bars represent mean ± SD (n = 3).

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Figure 2. (a) Fluorescence microscopy images of the intracellular distribution of DOX@ZIF8@AS1411 nanoparticles. (b) Fluorescence microscopy images and (c) Flow cytometry analysis of the targeting of DOX@ZIF-8@AS1411 towards HEK 293T cells and HeLa cells.

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Figure 3. (a) Fluorescence microscopy images of permeability change of lysosomal membrane treated with the ZIF-8@AS1411 nanoparticles. (b) Fluorescence microscopy images of induced ROS production of HeLa cells treated with 0 and 50 µg mL−1 DOX@ZIF-8@AS1411 nanoparticles. (c) Fluorescence microscopy images of the intracellular distribution of DOX@ZIF-8@AS1411 nanoparticles for 3h, 6h and 12h.

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Figure 4. (a) MTT assay and (b) flow cytometry analysis for assessment of the cell viability when incubated with free DOX, ZIF-8, DOX@ZIF-8 and DOX@ZIF-8@AS1411 for 24 h.

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ASSOCIATED CONTENT Supporting Information. The FTIR spectrum and XPS spectra of DOX@ZIF-8@AS1411 nanoparticles. Quantification of AS1411 and DOX in the DDSs. Photos of DOX@ZIF8@AS1411 nanoparticles in phosphate buffered saline (PBS), DMEM cell medium, and fetal bovine serum (FBS). The release profiles of DOX and Zn2+ in pH 7.4 and pH 5.0 buffer solution. TEM images of the DOX@ZIF-8@AS1411 nanoparticles immersed in (a) pH 7.4 and (b) pH 5.0 PBS for 3h. The MTT assay of DOX@ZIF-8@AS1411 nanoparticles incubation with HEK 293T cells. These materials are available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We acknowledge financial support by the National Natural Science Foundation of China (Grants 21431007, 21533008, 21601175, 21871249 and 21820102009), Key Program of Frontier of Sciences (CAS Grant QYZDJ-SSW-SLH052). REFERENCES (1) Jain, R. K.; Stylianopoulos, T. Delivering Nanomedicine to Solid Tumors. Nat. Rev. Clin. Oncol. 2010, 7, 653-664.

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(2) Wang, A. Z.; Langer, R.; Farokhzad, O. C. Nanoparticle Delivery of Cancer Drugs. Annu Rev. Med. 2012, 63, 185-198. (3) Kipp, J. E. The Role of Solid Nanoparticle Technology in the Parenteral Delivery of Poorly Water-Soluble Drugs. Int. J. Pharm. 2004, 284, 109-122. (4) Wong, P. T.; Choi, S. K. Mechanisms of Drug Release in Nanotherapeutic Delivery Systems. Chem. Rev. 2015, 115, 3388-3432. (5) Dong, K.; Liu, Z.; Li, Z. H.; Ren, J. S.; Qu, X. G. Hydrophobic Anticancer Drug Delivery by a 980 nm Laser-Driven Photothermal Vehicle for Efficient Synergistic Therapy of Cancer Cells In Vivo. Adv. Mater. 2013, 25, 4452-4458. (6) Chen, C. E.; Geng, J.; Pu, F.; Yang, X. J.; Ren, J. S.; Qu, X. G. Polyvalent Nucleic Acid/Mesoporous Silica Nanoparticle Conjugates: Dual Stimuli-Responsive Vehicles for Intracellular Drug Delivery. Angew. Chem. Int. Ed. 2011, 50, 882-886. (7) Liu, J. A.; Bu, W. B.; Pan, L. M.; Shi, J. L. NIR-Triggered Anticancer Drug Delivery by Upconverting Nanoparticles with Integrated Azobenzene-Modified Mesoporous Silica. Angew. Chem. Int. Ed. 2013, 52, 4375-4379. (8) Liu, J. N.; Bu, J. W.; Bu, W. B.; Zhang, S. J.; Pan, L. M.; Fan, W. P.; Chen, F.; Zhou, L. P.; Peng, W. J.; Zhao, K. L.; Du, J. L.; Shi, J. L. Real-Time In Vivo Quantitative Monitoring of Drug Release by Dual-Mode Magnetic Resonance and Upconverted Luminescence Imaging. Angew. Chem. Int. Ed. 2014, 53, 4551-4555. (9) Xie, J.; Lee, S.; Chen, X. Y. Nanoparticle-Based Theranostic Agents. Adv. Drug Deliver. Rev. 2010, 62, 1064-1079. (10) Liu, Z.; Robinson, J. T.; Sun, X. M.; Dai, H. J. PEGylated Nanographene Oxide for Delivery of Water-Insoluble Cancer Drugs. J. Am. Chem. Soc. 2008, 130, 10876-10877.

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