Photodynamic

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Imaging Guided and Light Triggered Chemo- /Photodynamic/Photothermal Therapy Based on Gd (III) Chelated Mesoporous Silica Hybrid Spheres Dan Yang, Guixin Yang, Shili Gai, Fei He, Ruichan Lv, Yunlu Dai, and Piaoping Yang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00462 • Publication Date (Web): 29 Sep 2016 Downloaded from http://pubs.acs.org on October 6, 2016

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ACS Biomaterials Science & Engineering

Imaging Guided and Light Triggered Chemo/Photodynamic/Photothermal Therapy Based on Gd (III) Chelated Mesoporous Silica Hybrid Spheres Dan Yang, Guixin Yang, ShiliGai, Fei He,* Ruichan Lv, Yunlu Dai, and Piaoping Yang* Key Laboratory of Superlight Materials and Surface Technology, Ministry of Education,College of Material Sciences and Chemical Engineering, Harbin Engineering University, Harbin, 150001, P. R. China KEYWORDS: Photodynamic therapy, Photothermal therapy, Cancer, Silica, Imaging

ABSTRACT: Exploring the combined anti-cancer therapeutic strategy to overcome the limitation of single mode and pursue higher therapeutic efficiency is highly promising in both fundamental and clinical investigations. Herein, a theranostic nanoplatform based on mesoporous silica, which is functionalized by hybrid nanospheres photosensitizer Chlorin e6 (Ce6), photothermal agent carbon dots (CDs), and imaging agent Gd (III) ions has been rationally designed and fabricated. A thermo/pH-coupling sensitive polymer (P(NIPAm-co-MAA)) coated on composite acted as a key “gatekeeper” to control the drug release in the appropriate time and location. Upon light irradiation, two mode synergy therapy effect of photodynamic and photothemal can be achieved by the photoactive Ce6 and CDs. Meanwhile the CDs loaded in the channels of mesoporous silica hybrid spheres can also play a role in the hand of “gatekeeper”-

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polymer to control the drug release process. Combined with the thermo/pH-sensitive drug release induced controllable chemotherapy, this platform shows synergistic therapeutic efficacy better than any single/dual-therapy, which is confirmed evidently by in vivo and in vitro assay. Considering the chelated Gd3+ simultaneous introduced magnetic resonance imaging (MRI) and computed tomography (CT) properties, this multifunctional platform should be of excellent potential in the imaging-guided cancer therapy field.


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INTRODUCTION Cancer has been deemed as one of main thorny challenges to human health. Among the chemotherapeutic agents, doxorubicin (DOX) is a commonly used anticancer drug of cytotoxic agent, which is widely used in clinical trials for multiple human cancers viz., acute leukemia, malignant lymphoma, breast, liver, lung, and ovarian cancer.1–5 However, clinical trials indicate that DOX has significant limitations due to its poor solubility, strong cytotoxicity to normal tissue of human, non-specific drug release.6 To conquer these obstacles, controlled and targeted drug release is major criteria which must be achieved in cancer chemotherapy. Hence, many reports have been found on the synthesis of various materials conjugated with DOX, served as targeted drug release carrier to reduce unwanted toxicity and enhance specificity to tumor cells.7– 12

Thereinto, mesoporous silica-based materials have received substantial attentions because of it

has the prominent physicochemical characteristics such as easily prepared, controllable morphology, large surface area and pore volume for drug loading, and excellent water solubility.4,7,13–16 Recently, a novel yolk-shell mesoporous silica-based material has been prepared by two silica sources of tetraethoxysilane (TEOS) and 2-bis(triethoxysilyl)ethane (BTSE).17 The formation of yolk-shell structure with a kernel inside hollow sphere will give the credit to the presence of the BTSE. The yolk-shell mesoporous silica material possesses all the advantages of the conventional mesoporous silica-based materials prepared by single silica precursor, containing highly uniform and controllable size (380-110 nm), tailorable core diameter and shell thickness, large surface area, high pore volume and interconnected ordered mesochannels. Particularly, the test result show that this yolk-shell mesoporous spheres has hardly any hemolysis activity to human red blood cells (RBCs) even up to high concentration of 2000 µg/mL, which is not only much better than that of conventional mesoporous silica particles


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but also that of non-porous amorphous silica spheres. Especially, the simple and green preparation process does not need multistep coating processes, any sacrificial templates, and poisonous etching agents. It shows great promise in the clinical applications such as intravenous administration and transport. Although mesoporous silica-based materials have a lot of advantages, the drug release system based on this material is only a single classical chemotherapy and also similarly has a noticeable drawback. The premature and under-/over-release of drug can generate drug resistance along the way, arouse toxic side effects, limit therapeutic effect, or cause permanent damage to immune systems. So, it is an urgent need that researchers focused on developing a new generation of cancer treatment methods. Based on new drug carrier systems, some drug control release method, which can achieve high concentration gathering of the drugs at the tumor site to improve the therapy efficiency and decrease the side effect of the drug to human body have been developed. Excitingly, physical stimulus-responsive therapies have shown their abilities to overcome these barriers, reduce unwanted side effects and enhance therapeutic efficacies in many studies.18–21 Physical stimulus-responsive therapy is a kind of responsive to external physical stimuli usually with assistance from nanoscale agents, such as light,22–242 pH value,25–27 magnetic field 28–31 or temperature.32–34 Among them, pH controlled drug release is a type of trigger therapy against cancer and has been extensively exploited.35,36 On the other hand, magnetic field can be utilized to attract the drug release system with magnetism property at the tumor site. Besides, the temperature has always been used to control the tumor-specific delivery utilizing the featured reagent which can generate overheating effect.37–39 However, until now, these control release effect by these physical response method is still limited and new method for release the drug at tumor site to a large extent is still urgent.


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In the past decade, some of the light-induced physical phenomena, like photothermal and photodynamic, have been draw dramatic research focus because of their potential application in cancer phototherapy field. The phototherapy (including PDT and PTT) using laser to destroy cancer is classical therapeutic method with the control of physical stimuli.40–42 All of phototherapy strategies benefited from the process that light is absorbed by photosensitizing agents and then optical energy is converted into either heat or ROS/singlet oxygen to exterminate tumor cells.43 Besides, except the therapy efficacy, the themal effect of the light can also be employed to acieve control drug release at the tumor site. Especialy in the mesoporous silicabased drug release system, the thermal effect upon light irradiation can not only been used to promote the drug release process, but also format the multi-mode therapy effect to improve the therapy efficiency. Because the synergistic system combined several therapy effects should be highly promising due to their enhanced therapeutic efficacy and reduced side effects compared with the individual mode. However, the drug release promotion system under activation of light is still rare, and the multi-functional nano-platform with rational design is needed. Accordingly, in this work, a new nanoplatform with the multi-mode therapy effect (PDT/PTT/chemo) and two-mode imaging functions (magnetic resonance imaging (MRI)/ X-ray computed tomography (CT)) based on mesoporous silica drug delivery system has been rational designed. The as-prepared yolk-shell structured organic-inorganic hybrid silica spheres are modified by amino groups (-NH2), and then chelated with Gd-DTPA, then Ce6 was coupled using the condensation reaction of the amide bonds. Doxorubicin (DOX) was chosen as the anticancer drug and encapsulated into the channel of yolk-shell spheres. Thermo/pH-sensitive polymer poly[(N-isopropylacrylamide)-co-(methacrylic acid)] (named as P(NIPAm-co-MAA)) encapsulates into the pores and outer shell of yolk-shell spheres, which acted as “gatekeeper” to


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control release of the anti-drug in yolk-shell spheres because a reversible phase transition in aqueous solution of the low critical solution temperature of PNIPAm incorporating co-monomer units (such as methacrylic acid) can easily be operated to above normal body temperature (37°C). In addition, CDs are attached as the key in the “gatekeeper” hand because those carbon nanoparticles can absorb the photon energy from 980 nm laser, and then convert into heat to open thermo-sensitive polymer. The systematic strategy is illustrated in Scheme 1. The physicochemical properties, in vitro and in vivo anti-tumor therapeutic efficacy have been investigated in detail.

EXPERIMENTAL SECTION Reagents and Materials. All chemical reagents were used as received without further purification.

Doxorubicin

diethylenetriaminopentaacetic dicyclohexylcarbodiimide

(DOX),

triethylamine,

acid (DCC),

(DTPA), 1,

N,

N-dimethylformamide

N-hydroxysuccinimide

2-Bis(triethoxysilyl)ethane

(BTSE)

(DMF), (NHS), and

tetraethylorthosilicate (TEOS) were purchased from Aladdin Co. Ltd (Shanghai, China). 3Aminopropyltrimethoxysilane (APTS), chlorin e6 (C34H36N4O6, > 95%), 1-(3-dimethy laminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), nhydroxysuccinimide (NHS), 1,3diphenylisobenzofuran (DPBF), cetyltrimethylammonium bromine (CTAB), GdCl3 (99.99%), 34,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 4’,6-diamidino-2-phenylindole (DAPI), calcein AM and propidium iodide (PI) were purchased from Sigma-Aldrich Co. Ltd (USA). Synthesis of Yolk-Shell Spheres. Yolk-shell spheres were synthesized according to previous report with some modifcation.24 Briefly, 0.4 g of CTAB is added to 100 mL of ethanol


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aqueous solution ( the molar ratio is 1:3) including 0.5 mL of concentrated ammonia aqueous solution (25 wt. %). Next, the mixture is directly heated to 35 °C, and two kinds of silane precursors (0.5 mL, BTSE: TEOS = 1: 1) is rapidly added under vigorous stirring. After magnetic stirring for 24 h, the white reaction mixture is centrifugally separated and washed three times with ethanol. Then the obtained product is dispersed in deionized water (160 mL) at 70 °C and maintained at this temperature for 12 h to achieve the morphology of yolk-shell spheres. The product is centrifugally separated and washed three times with ethanol again. Finally, to remove the pore- guiding reagent (CTAB), the above obtain product is homogeneously dissolved into 120 mL of ethanol solution including 0.24 mL of concentrated hydrochloric acid (37%). Then, it is magnetic stirred at 60 °C and maintained at this temperature for 3 h. The surfactants removal step is repeated twice to ensure CTAB completely removal. After removed template and washed the white product with ethanol again, and the precipitate is dried under vacuum. Synthesis of Gd-Si-Ce6 Sample. The as-synthesized yolk-shell spheres are firstly modified by cationic polymer with -NH2. Typically, yolk-shell spheres are homogeneous dispersed in the deionized water and ethanol solution. APTES is added to the mixture and directly heated to 45 °C for 8 h under stirring. This reaction mixture is centrifuged and washed with water and ethanol sequentially. Then as-prepared yolk-shell spheres-NH2 is dissolved into DMSO. DDC, NHS and DTPA are added into the particle suspension successively. After continuously stirring at 80 °C for 30 min, the mixture is cool down to room temperature. Subsequently, the obtained product, yolk-shell spheres-DTPA, is collected under 12000 rpm. The 10 mg of pre-sample is dispersed in GdCl3 solution (25 mg GdCl3 in 10 mL of citrate buffer solution) and stirred for 8 h at room temperature, the yolk-shell spheres-DTPA-Gd sample (Gd-Si) is finally prepared and collected by centrifugation. The standard EDC-NHS reaction is selected to covalent binding of Ce6 to Gd-


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Si. Briefly, 1.5 mg of Ce6 was dissolved in 1 mL of DMSO, followed by the addition of 1 molar equivalent of NHS and EDC for activation for 30 min. After activation of carboxyl groups, 2 mg of Gd-Si in phosphate buffered saline (PBS) (pH = 7.4, 2 mL) is added and the reaction solution is kept at room temperature for 12 h under vigorous stirring. The product is washed several times with distilled water and centrifuged to remove impurity. The resultant is dispersed in PBS buffer (pH = 7.4) for further usage. The final Gd-Si-Ce6 is denoted as GSC. The account of Ce6 loaded on GSC is measured by the Ce6 characteristic absorption peak at 404 nm. To measure the release behavior of Ce6 from GSC, a GSC solution is incubated in PBS (pH = 4, 7.4, and 9) for different periods (0, 3, 7, 12, 24, and 48 h). The concentration of Ce6 grafted on GSC is determined using UV/Vis spectra after removal of disengaged Ce6 by centrifugal separation. Synthesis of Gd-Si-Ce6-P(NIPAm-MAA). The polymer is successful introduced by means of chain polymerization. Typically, 0.02 g of Gd-Si-Ce6 is dissolved in 1 mL of 1, 4-dioxane, then 0.003 g of TPO, 0.125 g of NIPAm, and 7.6 µL of MAA are added with stirring. The mixture solution is ultrasonically treated to receive the gel. All the synthetic process must keep in dark place. Finally, the result solution is exposed to the UV lamp (200 W/cm2) for 5 min. Synthesis of DOX-loaded Gd-Si-Ce6-CDs-P(NIPAm-MAA). In a typical procedure, 20 mg of Gd-Si-Ce6-P(NIPAm-MAA) is dissolved in 4 mL of DOX solution (1 mg/mL) and 5 mL of neutral CDs solution by further shaking (200 rpm) for 12 h at 45 °C to obtain DOX-loaded Gd-Si-Ce6-CDs-P(NIPAm-MAA). The final product is labeled as DOX-GSCCP. A neutral CDs solution was prepared by adding 50 mg multi-wall carbon nanotubes into 50 mL of solution of HNO3/H2SO4 (1/3, volume ratio). The reaction time is prolonged to 24 h at 80 °C with water reflux to cut nanotubes to CDs. The as-prepared CDs solution is diluted to 1:10 and neutralized by NaOH to neutral. Besides, Based on the standard curve of DOX at a wavelength of 480 nm:


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A480nm = 0.0187C + 0.0233 (C is the concentration of DOX and A is the absorbance corresponding concentration of DOX), the loading and control-release account of DOX with time are determined using a UV/Vis spectrophotometer. The loading concentration of anti-drug is 69%, calculated by IE% =

(ெభ ିெమ ) ெభ

× 100% (IE% is the anti-drug loading of carrier, M1 is total

drug weight and M2 is drug weight in supernatant). Detection of Singlet Oxygen. DPBF was employed as 1O2 probe which reacted irreversibly with 1O2 to cause time-dependent photo-degradation of DPBF at a wavelength of 410 nm. The generation of singlet oxygen was determined by the following equation: ∆Abs = ADPBF Asample+DPBF, where A is the absorbance of UV/Vis spectra. Briefly, a DMSO solution of specimen (50 µg/mL) is added into 0.5 mg/mL stock solution of DPBF in DMSO. The DPBFcontaining solution of the sample is irradiated with 808 nm (0.5 W/cm2), or 650 nm laser (0.5 W/cm2) for different times, and the DPBF-containing solution of GSC and Ce6 at different concentrations is exposed to 650 nm laser (0.5 W/cm2) for 10 min. The absorbance is recorded by a UV/Vis spectrophotometer at 410 nm. Intracellular ROS Assay. Intracellular ROS assay of HeLa cancer cells is performed with a confocal laser scanning microscope (CLSM, Leica SP8). Typically, HeLa cells at a density of 5 × 104/well are firstly seeded into 6-well culture plates and then cultured at 37°C and 5% CO2 for 12 h as a monolayer cell. Next, 2 mL of as-prepared materials containing Gd-Si, GSC, GSCCP (500 µg/mL) are added into each well in the dark for 24 h at 37°C and incubated for another 4h. During the treating time, HeLa cells are exposed to a 650 nm laser (0.5 W/cm2) for 10 min. Then, the mixture solution is rinsed three times with PBS solution. After removal of the supernatant, 1 mL of carboxy-DCFH-DA is re-added into the cell plate. Finally, the cell plate is re- washed by PBS solution and measured on CLSM.


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Release Behavior of DOX. In a typical process, 8 mL of PBS solution (pH = 7.4) is slowly added into the cuvette containing DOX-GSCCP nanoparticles. The pH of aforementioned mixture solution is hold at 7.4 for 5 h at 37 °C, and then the PBS solution is adjusted to pH = 5.5 or pH = 4.0. To detect the behavior of control release, 8 mL PBS solution is taken out at certain time intervals to determine the account of DOX in the supernatant by UV/Vis measurements at a wavelength of 480 nm. On the other hand, to test the effects of temperature on the control release, we repeate the above experimental process instead of the temperature adjusted to 50 °C. Beside, to study the enhance effects of laser radiation on DOX release, DOX-GSCCP nanoparticles is incubated in the PBS solution (pH = 4) for 6 h and then exposed to 980 nm NIR radiation at the density of 0.5 W/cm2. The results are also determined using a UV/Vis spectrophotometer at a wavelength of 480 nm. In Vitro and in Vivo CT Imaging. In vitro and in vivo CT imaging measurements are conducted on a Philips 64-slice CT scanner at 120 kV voltages (Philips Medical System). DOXGSCCP are dissolved in PBS with various concentrations of 0.23, 0.12, 0.06, 0.03, 0.015, and 0.007 mg/mL for CT imaging. In vivo CT imaging is detected by subcutaneous and intravenous injection of DOX-GSCCP nanoparticles (0.5 mg/mL, 0.1 mL) into the tumor-bearing Kunming mouse the mouse. In Vitro and in Vivo T1-Weighted MR Imaging. In vitro MRI tests are performed using a 1.2 T MRI instrument (Shanghai Niumai Corporation Ration NM120-Analyst). Typically, DOXGSCCP nanoparticles are dissolved in water with different Gd concentrations. The amount of Gd bound per nanoparticle, which is calculated by ICP-MS measurement, is 108.23 µg/mg. T1weighted MR images are acquired using an inversion recovery sequence. Finally, the r1 relaxivity values are measured by means of the line fitting of 1/T1 relaxation time (s−1) versus the Gd


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concentration (mM) of DOX-GSCCP nanoparticles. In vivo MRI experiment is conducted at a 1.2 T MRI instrument after subcutaneous injection of DOX-GSCCP nanoparticles (1 mg/mL, 0.1 mL). In Vitro Viability of GSCCP. Firstly, L929 fibroblast cells are seeded into a 96-well cell plate with a density of 7000-8000 cells/ well, and maintained at 37 °C for 24 h under 5% CO2 to attach the wells as monolayer cells. Then, the cells are incubated with testing materials with different concentration of 7.8, 15.6, 31.2, 62.5, 125, 250 and 500 µg/mL for another 24 h. The wells with culture only are checked as blank control group in the left. After that, the as-prepared MTT stock solution (0.02 mL/well) is added into the plate and allowed to stand at 37 °C for another 4 h. After the addition of DMSO (0.15 mL/well), the cell plate is shaken for 5 min at 150 rpm to homogeneous mixing formazan and DMSO solvent. Formazan is the reduction product of viable cells and MTT and can be dissolved by DMSO. The absorbance of formazan is measured by a micro-plate reader at a wavelength of 490 nm. Hemolysis Assay. Red blood cells are obtained by washing and centrifuging several times with 1% normal saline until the supernatant is transparent to remove the serum from the human blood stabilized by EDTA.K2. After that, the purified red blood cells are diluted to 10 mL of PBS solution (pH = 7.4). Then, 0.3 mL of diluted red blood cells suspension is intensive mixed with 1.2 mL of PBS solution, 1.2 mL of deionized water, 1.2 mL of yolk-shell spheres suspensions and 1.2 mL of MSN spheres suspensions. The concentration of yolk-shell spheres and MSN spheres suspensions is 15.63, 31.25, 62.5, 125, 250, 500, and 1000 µg/mL, respectively. After the samples tubes are shaken and kept in 37°C stable for 2 h, the mixtures are centrifuged and the absorbance values of the upper supernatants at 490nm is recorded by UV/Vis spectrometer. The percent hemolysis of red blood cells is calculated by the following equation:


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Hemolysis % = (Asample - Acontrol(-)) / (A control(+) - Acontrol(-)), where A is the absorbance of UV/Vis spectra, Acontrol(-) is the absorbance of PBS and RBCs mixture solution deeded as negative control, and Acontrol(+) is the absorbance of RBCs dispersed in ultrapure H2O deeded as the positive control.. Cellular Uptake. Cellular uptake of DOX-GSCCP nanoparticles by HeLa cells is examined using CLSM. HeLa cells are seeded into 6-well plates at a density of 5 × 104 well–1 per well and overnight cultured in the incubator as a monolayer. The following day, free Ce6, Gd-Si, GSCCP, DOX-GSCCP (2 mL, 500 µg/mL) are added to the cells and further incubated for different time (0.5 h, 1 h and 3 h) at 37 °C. After the Hela cells are washed three times with PBS, DAPI solution (20 µg/mL in PBS, 1 mL/well) are added into the cells and further incubated for 10 min at 37 °C to perform nuclei stained. Subsequently, the Hela cells are washed three times with PBS, and they are then fixed with 1 mL of formaldehyde (2.5%, 1 mL /well) for 10 min at 37 °C and further rinsed three times with PBS. Finally, the cells are visualized on Leica TCS SP8. Dye Experiment. HeLa cells are seeded into a 6-well plate at a density of 5 × 104 cells per well and incubated in a humid 5% CO2 atmosphere overnight. Then 2 mL culture medium containing materials (0.5 mg/mL) was added. During the cultivation of 4 h, Hela cells are exposed to the light for 10 min. After the cells are washing three times with PBS, the cells are stained with calcein AM (2 mmol/mL) and ropidium iodide (PI, 5 mmol/mL), and imaged by CLSM. In Vitro and In Vivo Cytotoxicity. The cytotoxicity of DOX-GSCCP is measured similar to the in vitro cell-viability using MTT assay. Except for the difference that the HeLa fibroblast cells are incubated with different concentrations of free DOX, Ce6, GSCCP, GSC and DOXGSCCP without and with lasers irradiation (0.5 W/cm2) for 24 h.


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Female Kunming mice (25-30 g) are purchased from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Harbin, China), and all the mice procedures are approved by The National Regulation of China for Care and Use of Laboratory Animals. Briefly, the tumors-bearing mice are achieved by subcutaneous injecting U14 cells (murine cervical carcinoma cell lines) in the left axilla of each female mouse. When the tumor diameter reached ~ 6-10 mm, the tumor-bearing mouse are segregated into six groups (n = 5, each group) and are treated with tested materials which correspond to that in the toxicity in vitro and dye test. As follow, the first group of mice is taken as blank control group without injected material instead of the same volume of normal saline. Every two days, the mice in the treatment group are injected 0.1 mL material per mouse, and the account of pure DOX is determined by the 69% loading concentration of DOX-GSCCP in the treatment group. For the group exposed to the lasers, the tumor site with 650 nm laser and/or 980 nm laser irradiation (0.5 W/cm2) for 10 min after 1 h of different materials injected. The therapeutic efficacy of each group is estimated by monitoring the tumor sizes and body weights changes of the mice in each group every two days, up to 14 days. Histological Examination. To determine the histological changes of the tumors and main organ, one tumor-bearing mouse in each group is isolated at the 14th day after treatment. Afterwards, the tumors and organs are dehydrated and embedded in liquid paraffin successively. The sliced tumor and organs tissues (3-5 mm) are stained with Hematoxylin and Eosin (H&E) and imaged by a microscope. Characterization. Powder X-ray diffraction (XRD) patterns were obtained on a Rigaku D/max TTR-III diffractometer at a scanning rate of 15°/min in the 2θ range from 10° to 80° or at a scanning rate of 5°/min in the 2θ range from 0.5° to 8°, with graphite monochromatized Cu Kα


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radiation (λ = 0.15405 nm). Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) were carried out on FEI Tecnai G2 S-Twin. Fourier transform infrared spectroscopy (FT-IR) spectra were measured on a Perkin-Elmer 580B IR spectrophotometer using the KBr pellet technique. N2 adsorption/desorption isotherms were obtained on a Micromeritics ASAP Tristar II 3020 apparatus. Pore size distribution was calculated by the Barrete-Jonere-Halenda (BJH) method. DOX concentration is detected by UV1601 UV/Vis spectrophotometer.

RESULTS AND DISCUSSION Yolk-shell spheres were synthesized in the ethanol aqueous solution by introducing dualsilicon source of TEOS and BTSE as precursors and CTAB as a surfactant. TEM image shows that the sample consists of uniform spheres with homogeneous diameter of approximate 110 nm (Figure S1A). By observing the high magnification TEM image (Figure S1B), the doughnutshape void space with the width of 30 nm, and permeable outer mesopore silica wall with thickness of 20 nm can be clearly confirmed. This unique structure was produced from hydrolysis and condensation of TEOS and BTSE. The formation mechanism of dissolution and reassembly provides an accessible ordered meso-channels (2.7 nm), high pore volume (1.17cm3/g) and large surface area (769cm3/g) for coupling with functional groups. The N2 adsorption/desorption isotherm of yolk-shell spheres (Figure S1C) presents the typical IV-type isotherm with a wide H2 hysteresis loop at P/P0 > 0.42, which is characteristic of typical mesoporous structure. The low-angle XRD pattern (Figure S1D) presents the uniform mesoporous channel of the as-synthesized sample. As shown in Figure 1A and B, the sample (GSCCP) functionalized by Ce6, Gd(III), CDs and P(NIPAm-co-MAA) maintains the shape and


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size of original yolk-shell spheres. Close observation reveals that the sample has an apparent yolk-shell structure, which is verified by the cross-section compositional profile lines (Figure 1C), especially the line of Si element with three peaks. The unique structure is much different from the traditional mesoporous silica nanospheres (MSN) synthesized using TEOS as single silica source. In addition, the STEM image (Figure 1D) and the elemental mapping images (Figure 1E) of the sample also indicate the yolk-shell structure of yolk-shell spheres and Gd3+ ions are uniform distributed on the surface of yolk-shell spheres. The results are quite fit in with the synthesis mechanism (Figure S4) and cross-section compositional profile lines of Gd element (Figure 1C). This is also confirmed by FT-IR spectra (Figure S2), the absorbance peak at 930 cm–1 for Si-OH vibration on pure MSN is much higher than that of yolk-shell spheres, illustrating the relatively lower silanol density in the presence of the surface of yolk-shell spheres to hinder the modification of –NH2 and P(NIPAm-co-MAA). The peak at around 2900 cm−1 is indicative of the stretch vibrations of the C-H bond in the -CH2-CH2- group. Meanwhile, the absorbance peak at 1414 cm–1 for bending vibration of the C-H bond are used to confirm the BTSE silane condensation in the yolk-shell spheres. By contrast, these vibration peaks are not existed for pure MSN. It is necessary to modify the surface of yolk-shell spheres for potential biological application. For yolk-shell spheres-NH2, characteristic peaks of amide groups at 3440 cm–1 (νN–H) and 1587 cm–1 (δN–H) shows the present of -NH2 groups on the surface of yolk-shell spheres (The symbols of ν and δstand for stretch vibration and deformation vibrations of the N– H bond in the –NH2– group, respectively), which are suitable for further functional modification of DTPA and Ce6 by using condensation reaction of EDC/NHS (Figure S4). The yolk-shell spheres-DTPA presents intense peak at the 1620 cm–1 is used to confirm the covalent conjugation of DTPA based on the present of the carboxylic groups of DTPA hydrolyzate. After


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chelation with Gd3+, no obvious change in peak shape is discovered, except for a slight shift of the vibration peak of C=O. Meanwhile, the change of zeta-potential of the samples further proves the successful modification in each step (Figure S3). The isoelectric point (IEP) of the traditional MSN is around 20 mV, because the existence of Si-OH bond leading to the surface negative charge. By contrast, the IEP is lower than 20 for the pristine yolk-shell spheres. After ammonification, the IEP shifts toward higher pH values close to 5-6 owing to the decolonization of positively charged -NH3+ groups. After the successful covalent conjugation of DTPA, the IEP is close to zero due to the protonation of the carboxylate leads to the surface charge reduction. Similarly, the surface charge modification of Si-Gd and GSC is caused by successful chelation of unpaired electrons of the Gd3+ ions and covalent conjugation of Ce6. Change of the IEP before and after drug release due to the presence and leave of DOX further confirms that the release mechanism. The modification of different functional groups on yolk-shell spheres has been verified by aforementioned results. Then, we characterized GSCCP to make sure if it is potential for biological application. Regarding the photophysical property of Gd-Si-Ce6 (GSC), optical absorption is used to investigate the conjugation efficiency of Ce6 to Gd-Si. The UV/Vis absorbance spectra of Gd-Si, Ce6 and Gd-Si-Ce6 (Figure 2A) indicated that Gd-Si without Ce6 has scarcely any absorption at the range of 300-900 nm. By contrast, a strong Soret absorption at 406 nm and weak Q-bands between 500 and 700 nm are observed in the absorbance of GSC, which is similar to the absorption of Ce6, suggesting that is successfully conjugated and no change in the Ce6 chromophore upon conjugation. Meanwhile, the conjugation stability of Ce6 is estimated using standard curve of Ce6 at 404 nm. As shown (Figure 2B), a good linear relationship between concentration and absorbance value of Ce6 apparently existed, and can be


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describe as the following equation: A404nm = 0.255C - 0.031 (R2 = 0.9945). After Ce6 with varied concentrations was mixed with Si-Gd and then free Ce6 was removed, the concentrations of Ce6 in Si-Gd-Ce6 samples was determined by UV/Vis spectra of Si-Gd-Ce6 at 404 nm after subtraction of absorbance contributed by Si-Gd. We investigated the relationship between the feeding amount of Ce6 and the concentrations of Ce6 finally loaded on Si-Gd by mCe6/mSi-Gd × 100%. The loading amount of Ce6 on Gd-Si reaches a maximum of 20% when Ce6 concentration is above 2 mM (Figure 2C). In addition, in the release curves of Ce6 (Figure 2D), it is apparent that the Ce6 release from Gd-Si is accelerated in basic solution (pH = 9) while Ce6 loaded on Gd-Si is very stable in acidic PBS (pH = 5) and neutral PBS (pH =7), which is attributed to the deprotonation of carboxyl groups in the Ce6 molecules. The ROS generation capability of GSCCP and Ce6 triggered by 650 nm light is estimated by monitoring the decrease absorption degree of DPBF at 410 nm, which is widely used to capture singlet oxygen and reacts irreversibly with ROS to lead to photo-degradation of DPBF. As shown in Figure 3A and B, the ROS production ability by GSC triggered by 808 nm light is weaker than that triggered by 650 nm light due to the characteristic absorption peaks of Ce6 at around 650 nm. However, even if the ROS generation capability of GSCCP is weaker than that of free Ce6 at various concentration the solution, the toxicity efficiency using GSCCP is higher than that of free Ce6 in the cancer cells, which may be caused by the repellent effect of abundant carboxyl groups in free Ce6 molecules with cancer cells and the enhanced permeability and retention (EPR) effect for tumor-target release of anti-cancer drugs of GSCCP. The oxidative stress sensitive dye DCFH-DA is selected as ROS-index probe in the assay of intracellular photodynamic effect. DCFH excited at 520 nm will be oxidized to DCF and presents green fluorescence when ROS is existed. In Figure 3C, HeLa cells dealt with free Ce6 molecules and


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DOX-GSCCP exposed to 650 nm laser irradiation presents inordinately bright green fluorescence, while the sample incubated with Gd-Si emerges sightless color, illustrating that Ce6 has the ability to produce efficient ROS and further implying that DOX-GSCCP has the ability to act as candidate of photodynamic agent. The DOX-GSCCP may have another important contribution-photothermal effect because the existence of carbon dots. To prove our guess, the infrared thermal imaging test in vitro is conducted using HeLa cells incubated with pure Ce6 molecules, pure carbon dots, GSC, GSCP and GSCCP and DOX-GSCCP upon 980 nm and 650 nm laser irradiation with a density of 0.5 W/cm2. As expected, the wells incubated the DOX-GSCCP and GSCCP particles show higher contrast infrared thermal images and obvious temperature elevation (Figure 4A and B, wells c and d) due to coupling of carbon dots with yolk-shell spheres, which is the only heat source in this composite. More importantly, the temperature of HeLa cells is rapidly increased to about 60 °C only after 3 min irradiation. Such temperature is high enough to kill cancer cells, which is higher than cell death temperature (40-60°C).44 Furthermore, the similar effect can also be verified by results in vivo (Figure 4C and D). We further studied the release behavior of DOX-GSCCP as the drug delivery system, wherein P(NIPAm-MAA) acted as the “gatekeeper” to control the release of DOX from yolkshell spheres. Under normal circumstances (37 °C, pH = 7.4), DOX can hardly be released from the system in the PBS solution (Figure 5A). By contrast, the increased account of DOX control release is tested when the pH value is adjusted to 4 or 5.5, and the release efficiency is up to 65% at pH value of 4 after 36 h. It is indicative that the DOX-GSCCP possesses an excellent stability under relevant physical condition in this assay and the control release of DOX from DOXGSCCP is a strictly pH dependent process. Not only intracellular lysosomes and endosomes of


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tumors but their microenvironments of extracellular are usually acidic, thus, the pH sensitive release behavior will allow us to better achieve the intracellular DOX control delivery. DOX released from carrier by diffusion-controlled mechanism is attributed to the reduced electrostatic forces between PMAA and drugs in the acidic environment (pH = 5.5 and pH = 4.0). Meanwhile, when the temperature is changed to 50 °C (Figure 5B), the release amount of DOX is markedly increased in all release processes compared with above results, indicating DOX-GSCCP also has a temperature-dependent release property because the polymer brushes (P(NIPAm-co-MAA)) can easily cause the shrinkage when conditions are much hotter than the average body temperature of 37 degrees. While the pores channels of yolk-shell spheres are forced to open, the anti-drug is therefore released. In addition, it is clear that the release amount of DOX is much higher at acidic conditions than that under normal circumstance. In addition, the continuous 980 nm laser radiation has been demonstrated to effectively cause excessive local heating via trigger the carbon dots which can also be an effective way to control antidrug release. As shown in Figure 5C, an abrupt release of DOX up to 53.32% is detected in the first six hours. By contrast, only 16.49 % DOX is released without irradiation. These results are in good agreement with that aforesaid temperature-dependent release behavior at pH = 4 and show that 980 nm light can enhance drug release. It should be noted a burst release of DOX is observed in the first five hours and then gradually reduces under more acidic environment for the system with polymer modification (Figure S5). In comparison, DOX can hardly be released from the system with polymer modification and without irradiation within 6 h. Once exposed under the 980 nm laser, release efficiency of the system with polymer modification is much higher than that without irradiation within 6 h. The results clearly indicate that the P(NIPAm-MAA) polymer acts as the


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“gatekeeper” to control the release of DOX from the pores of yolk-shell spheres and to prevent premature release of DOX. We detected cellular uptake behavior to study the progress of carrier entering into cells. HeLa cells are first incubated with Gd-Si, free Ce6, GSC and DOX-GSCCP for various times (0.5, 1, and 3 h) at 37 °C and then imaged using a CLSM. As shown in Figure 6, the fluorescence of intracellular Ce6 is clearly enhanced with the time for all the tested materials incubated in the cells, and GSC shows obviously stronger Ce6 fluorescence inside cells than free Ce6 molecules. The fluorescence intensity is an indicator of the amount of Ce6 delivered into cells. We thus can infer GSC can enter into cells more easily than free Ce6, one possible reason that tumor cells with negative charge easily refuse to accept free Ce6 molecules in possession of abundant carboxyl groups, and choose to devour the GSC composite owing to carboxyl groups from coordinate bonds with the amine groups on the yolk-shell spheres surface.39 Before to explore GSCCP served as a dual modality CT/MR imaging contrast reagent and drug carrier system for biomedical application, it is necessary to check their cytocompatibility. On the one hand, in vitro cell viability of GSCCP with different concentrations (7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/mL) on L929 cells were evaluated by MTT assay (Figure 7A). Negligible negative effect on the normal cells can be detected from the bar chart even at concentrations up to1000 µg/mL, illustrating high biocompatibility of the specimens. Moreover, the biocompatibility of GSCCP with red blood cells should also be tested to further ensure the long-term biocompatibility. In this study, we conducted a contrast test to compare traditional MSN and yolk-shell spheres of the hemolytic efficiency. The hemolytic result and digital photographs are given in Figure 7B. Even though all the two samples almost have no side effects and no damage to red blood cells, the reduce of “cell-contactable silanol groups” density and


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acidity lead to the much lower hemolytic efficiency of yolk-shell spheres relative to conventional MSN. Based on above discussed, the cell viability and hemocompatibility of the yolk-shell structure hybrid spheres are excellent, which offers great potential for their applications in biotechnology involving intravenous administration and drug delivery. Before investigating the tumor inhibition effect, the cytotoxicity of the tested materials to HeLa cells in vitro is detected by standard MTT assay. In Figure 7C, the cells viability treated with lasers and GSCCP with the concentration up to 1000 µg/mL doesn’t show any significant downward trend. This further indicates the GSCCP has a good cytocompatibility within wide concentration range and the lasers have no side effect on cells, which is crucial for its further biological application. By contrast, the decrease of cell viability in varying degrees is observed after the cells are incubated with the other materials with various concentrations (7.8-1000 µg/mL) under laser excitation. It is worth noting that the inhibition efficacy of DOX-GSCCP with 650 nm and 980 nm light is strongest at different concentrations. These data show satisfactory results for in vitro cytotoxicity in all dosages for the particles, which can be attributed to the combined photodynamic/photothermal therapy, and thermo/pH responsive DOX release induced thermotherapy. Good cytocompatibility of the GSCCP and excellent inhibition efficacy of the DOX-GSCCP with the two kinds of lights imply that nanoparticles can serve as drug carrier for their further biological applications. The toxicity result in vitro is also verified by the dye experiment. In Figure 7D, it is apparent that the inhibitory effect on HeLa cells in the dye experiment is consistent with the result of cytotoxicity assay in vitro. The cells in the group incubated with the materials or exposed to the laser alone have no obvious cell apoptosis. To investigate the tumor inhibition efficacy of the tested materials, we performed a range of controlled trials in vivo, and the results are presented in Figure 8. Body weight can be served as a


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critical factor to indicate the systemic toxicity of the tested materials to the body. In this assay, the body weights in all groups show obvious increase during the treating times, implying no obvious side effect (Figure 8A). In addition, analogical growth rate of tumor volume are obtained when the diseased region is treated with normal saline, pure 650 nm/980 nm lasers, or only GSCCP composite (Figure 8B). This fact illustrates that short interval NIR irradiation can avoid overheating and ensure the safety of GSCCP composite for human tissue. More importantly, the group treated with multimode synergistic therapy of chemotherapy/PDT/PTT (DOX-GSCCP under 650 nm and 980 nm laser irradiation) exhibits highest tumor inhibition efficacy than those treated only by PDT (GSC under 650 nm laser irradiation) or PTT/chemotherapy (DOX-GSCCP under 980 nm laser). The toxicity results have no different from that in vitro. The representative photograph of tumor-bearing mouse and tumor tissue segregated on the 14th day are given in Figure 8C. It is obvious that the size of tumor under “chemotherapy + PDT + PTT” tri-mode treatments is obviously smaller than those treated with PDT alone or chemotherapy/PTT dual mode treatments. The molecule imaging techniques (MRI, CT, PET, and optical imaging) have their own disadvantages including low plane resolution of MRI, low-contrast of soft tissue of CT, poor spatial resolution of PET, and low penetration depth of optical imaging. To get more accurate information in the clinical prognosis and diagnosis, reasonable integrating the merits of two or more imaging technique in one system to form multimodal imaging is highly desired. Different from other imaging contrast agents, the single gadolinium (Gd) nuclide is capable of playing as MRI and CT contrast agent simultaneously.45–50 Aforementioned theranostics and imagingguided therapy are our target in this study, thus the X-ray CT and MR imaging effect of DOXGSCCP are assessed. First, the HU value of different concentrations of DOX-GSCCP in


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deionized water is determined by X-ray CT, and the water as the black control group. As shown in Figure 9A, a sharp signal enhancement is observed and the CT value called Hounsfield units (HU) linearly enhances with the increase of DOX-GSCCP concentration in the deionized water. To further verify the in vivo effectiveness, the mouse was injected with 0.1 mL of DOX-GSCCP in situ and then imaged using X-ray CT. Compared with the CT signal pre-injection, it is strikingly increased in the tumor site after injection. Moreover, Figure S6 shows that the liver site of CT imaging effect in the intravenous injection condition, it can be seen that the HU intensities of the liver is improved in various degrees at 1 h and 3 h after intravenous injection of the nanoparticles. Besides, the obvious CT single enhancement in the liver may be caused by the presence of the Gd ions in the nanoparticles from the blood to the liver through the pharmacokinetic profile. The CT results in vitro/vivo illustrate that the DOX-GSCCP nanoparticles have a potential of contrast reagent for CT imaging. Due to the excellent spatial resolution and high penetration depth, MRI has been acted as an important diagnostic imaging technique to help researchers to identify and distinguish the nidus from other soft tissues more precisely.51 According to the previously reported “negative-lattice shield effect” (n-LSE), the high Gd ions concentration in the nanoparticles surface is an important factor of the weak longitudinal relaxivity of paramagnetic Gd (III) ions with seven unpaired electrons in shortening T1-weighted relaxation time of water proton. Therefore, in our study, a Gd-DTPA complex is conjugated to amino on the surface of GSCCP to maximize the longitudinal relaxivity. The T1 contrast capability of DOX-GSCCP is explored on a clinical 1.2 T MRI instrument. Concentration-dependent positive contrast enhancement is acquired for DOXGSCCP dispersions and no such signal enhancement is discovered for pure water. As plotted in Figure 9B, a linear relationship is observed when the concentration of the Gd is plotted against


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longitudinal relaxivity. The relaxivity parameter (r1), calculated from the quantitative measurement of the MR signal of the concentration-dependent relaxation rate (1/T1), is 18.05 s–1 mM–1 (Figure 9B), which is obviously higher than that (0.81 s–1 mM–1) of our reported GdOF nanoparticle,2 Gd-doped carbon quantum dots (r1 = 5.88–11.5 s–1 mM–1)52 and Gd2O3 nanoparticles (r1 = 6.9 s–1 mM–1).53 This increase in r1 value is attributed to the higher Gd3+ concentration in the outer shell of the composite and thus exhibits strong water proton spinlattice relaxation effect, indicating that DOX-GSCCP materials have great potential for T1weighted MRI contrast reagent in the future clinical application. Furthermore, the efficiency of T1-weighted MRI is assessed with intratumorally injection of DOX-GSCCP (0.1 mL, 1 mg/mL) into the tumor-bearing mouse (Figure 9B). Comparing the images before and after injection, an apparent signal enhancement on the tumor site is observed after injection, which verifies MR contrast enhancement effect of Gd ions chelated with nanocomposites. To further test if GSCCP materials produce any harmful biological effects on mouse during the treatment process, the following important hematology, kidney functions and liver function markers, is employed for the haematological assessment, including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), blood urea level (BUN), the ratio of albumin and globulin (A/G), red blood cells (RBC), white blood cells (WBC), mean corpuscular volume (MCV), hemoglobin (HGB), mean corpuscular haemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), and hematocrit (HCT). As shown in Table S1, all of the above results of the GSCCP-treated mice ranged in normal reference value range are observed, indicating that GSCCP nanoparticles has scarcely any hepatic toxicity during the healing process. Meanwhile, from the hematoxylin and eosin (H&E) staining images of main organs after 14th day treatment (Figure 10), it is easy to see that those


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tissue still retain their normal morphology and either noticeable pathological changes or obvious apoptosis is not existed. The results further reveal that no significant acute toxicity is introduced by the theranostic agents during its short stay in the body. However, the tumor tissues are inordinately destroyed in the therapeutic groups. In this regard, DOX-GSCCP has sufficient capability to competent for the position of drug delivery carrier in the biological medicine field. In addition, the long-term tissue bio-distribution of Gd ion in the mouse body can be served as an indirect signal of the metabolism and deposition of the GSCCP composites. Based on the results of histopathology, five main organs containing heart, liver, spleen, lung and kidney are marked for the analysis of Gd account. The Gd account in organs is measured using ICP-MS after GSCCP administrated at various times (1 h, 2 h, 3 h, 6 h, 7 h, 1 d and 7 d). It is easily to see that the changes of Gd content in the mice body from one kind of organ to another, as described in Figure S7. GSCCP mainly distributes or accumulates in the tissues and organ enriched with pharmacokinetics, such as the spleen, lung and liver. Gd content reaches maximum in the early stage after the injection and then gradually declines over 1 week, Even if different density of Gd ion accumulated in major organs is detected in the early stage after the injection, the quantity dwindles because the composite can generally be excreted from the body with prolonged time through the body's normal metabolism and organ functions.

CONCLUSIONS In summary, concerning photodynamics therapy (PDT) is not working under the low oxygen partial pressure, we presents a novel imaging-guide chemotherapy/PDT/PTT synergistic anticancer theranostic system to control function with two kinds of lasers, which is composed of mSiO2-Gd-Ce6-Cdots-P(NIPAm-co-MAA) yolk-shell hybrid spheres. The inner inorganic-


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organic hybrid silica materials not only possess all the advantages of the conventional mesoporous silica-based materials. Surprise is that the yolk–shell spheres exhibit hardly any hemolysis activity to human red blood cells (RBCs) even up to high concentration of 1000 µg/mL, which is not only much better than that of conventional mesoporous silica particles but also that of non-porous amorphous silica spheres. Noteworthy, the EPR effect will be significantly enhanced by using non-toxic, high-MW polymer and about 110 nm size mSiO2, which will be of great clinical benefit., Besides, owing to higher Gd3+ concentration in the outer shell of the composite, the MRI/CT signal significantly enhancement, thus achieving the target of multiple imaging-guide therapy to enhance the therapeutic efficiency. Above all, our contribution provides a feasible design and a potential platform for future clinical therapy, which can effectively overcome the drawbacks of the singlet anti-tumor mode.

ASSOCIATED CONTENT Supporting Information. Figure S1-S7 and Table S1 can be seen from the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected] 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.


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ACKNOWLEDGMENTS Financial supports from the National Natural Science Foundation of China (NSFC 21271053, 21401032, 51472058, and 51502050), China Postdoctoral Science Foundation (2014M560248, 2015T80321), Natural Science Foundation of Heilongjiang Province (B201403), Outstanding Youth Foundation of Heilongjiang Province (Grant No. JC2015003), and Heilongjiang Postdoctoral Fund (LBH-Z14052, LBH-TZ0607) are greatly acknowledged.

REFERENCES (1) Han, L.; Tang, C.; Yin, C. Dual-targeting and pH/redox-responsive multi-layered nanocomplexes for smart co-delivery of doxorubicin and siRNA. Biomaterials, 2015, 60, 42-52. (2) Lv, R.; Yang, P.; He, F.; Gai, S.; Li, C.; Dai, Y.; Yang, G.; Lin, J. A Yolk-like Multifunctional Platform for Multimodal Imaging and Synergistic Therapy Triggered by a Single Near-Infrared Light. ACS Nano, 2015, 9, 1630-1647. (3) Lv, R.; Yang, P.; He, F.; Gai, S.; Yang, G.; Dai, Y.; Hou, Z.; Lin, J. An Imaging-Guided Platform for Synergistic Photodynamic/Photothermal/Chemo-Therapy with pH/TemperatureResponsive Drug Release. Biomaterials, 2015, 63, 115-127. (4) Yang, G.; Gong, H.; Liu, T.; Sun, X.; Cheng, L.; Liu, Z. Two-Dimensional Magnetic WS2@Fe3O4 Nanocomposite with Mesoporous Silica Coating for Drug Delivery and ImagingGuided Therapy of Cancer. Biomaterials, 2015, 60, 62-71. (5) Yang, P.; Gai, S.; Lin, J. Functionalized Mesoporous Silica Materials for Controlled Drug Delivery. Chem. Soc. Rev., 2012, 41, 3679-3698. (6) Feng, J.; Zhang, H. Hybrid Materials Based on Lanthanide Organic Complexes: a Review. Chem. Soc. Rev., 2013, 42, 387-410.


27


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

Page 28 of 46

(7) Chen, Y.; Ai, K.; Liu, J.; Sun, G.; Yin, Q.; Lu, L. Multifunctional Envelope-Type Mesoporous Silica Nanoparticles for Ph-Responsive Drug Delivery and Magnetic Resonance Imaging. Biomaterials, 2015, 60, 111-120. (8) Li, Y.;

Shi, J. Hollow-Structured

Mesoporous Materials: Chemical Synthesis,

Functionalization and Applications. Adv. Mater., 2014, 26, 3176-3205. (9) Panyam, J.; Labhasetwar, V. Biodegradable Nanoparticles for Drug and Gene Delivery to Cells and Tissue. Adv. Drug Del. Rev., 2003, 55, 329-347. (10) Sridhar, R.; Lakshminarayanan, R.; Madhaiyan, K.; Barathi, V. A.; Limh, K. H. C.; Ramakrishna, S. Electrosprayed Nanoparticles and Electrospun Nanofibers Based on Natural Materials: Applications in Tissue Regeneration, Drug Delivery and Pharmaceuticals. Chem. Soc. Rev., 2015, 44, 790-814. (11) Sun, T.; Zhang, Y. S.; Pang, B.; Hyun, D. C.; Yang, M.; Xia, Y. Engineered Nanoparticles for Drug Delivery in Cancer Therapy. Angew. Chem. Int. Ed., 2014, 53, 12320-12364. (12) Yang, D.; Ma, P. a.; Hou, Z.; Cheng, Z.; Li, C.; Lin, J. Current Advances in Lanthanide Ion (Ln(3+))-Based Upconversion Nanomaterials for Drug Delivery. Chem. Soc. Rev., 2015, 44, 1416-1448. (13) Deng, Y.; Qi, D.; Deng, C.; Zhang, X.; Zhao, D. Superparamagnetic High-Magnetization Microspheres with an Fe3O4@SiO2 core and Perpendicularly Aligned Mesoporous SiO2 Shell for Removal of Microcystins. J. Am. Chem. Soc. , 2008, 130, 28-29. (14) Tang, F.; Li, L.; Chen, D. Mesoporous Silica Nanoparticles: Synthesis, Biocompatibility and Drug Delivery. Adv. Mater. , 2012, 24, 1504-1534.


28

Page 29 of 46

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


(15) Xu, Z. H.; Gao, Y.; Huang, S. S.; Ma, P. A.; Lin, J.; Fang, J. Y. A Luminescent and Mesoporous

Core-Shell

Structured

Gd2O3:Eu3+@nSiO2@mSiO2 Nanocomposite

as

a Drug Carrier, Dalton Trans., 2011, 40, 4846-4854. (16) Eurov. D. A.; Kurdyukov, D. A.; Kirilenko, D. A.; Kukushkina, J. A.; Nashckekin, A. V.; Smirnov, A. N.; Goluber, V. G. Core-Shell Monodisperse Spherical mSiO2/Gd2O3:Eu3+@mSiO2 Particles as Potential Multifunctional Theranostic Agents. J. Nanopart. Res., 2015, 17, 82. (17) Teng, Z.; Wang, S.; Su, X.; Chen, G.; Liu, Y.; Luo, Z.; Luo, W.; Tang, Y.; Ju, H.; Zhao, D.; Lu, G. Facile Synthesis of Yolk-Shell Structured Inorganic-Organic Hybrid Spheres with Ordered Radial Mesochannels. Adv. Mater. , 2014, 26, 3741-3747. (18) Lopes, J. R.; Santos, G.; Barata, P.; Oliveira, R.; Lopes, C. M. Physical and Chemical Stimuli-Responsive Drug Delivery Systems: Targeted Delivery and Main Routes of Administration. Curr. Pharm. Des., 2013, 19, 7169-7184. (19) Nakayama, M.; Okano, T. Multi-Targeting Cancer Chemotherapy Using TemperatureResponsive Drug Carrier Systems. React. Funct. Polym., 2011, 71, 235-244. (20) Wang, H.; Di, J.; Sun, Y.; Fu, J.; Wei, Z.; Matsui, H.; Alonso, A. d. C.; Zhou, S. Biocompatible PEG-Chitosan@Carbon Dots Hybrid Nanogels for Two-Photon Fluorescence Imaging, Near-Infrared Light/pH Dual-Responsive Drug Carrier, and Synergistic Therapy. Adv. Funct. Mater. , 2015, 25, 5537-5547. (21) Liu, F.; He, X.; Lei, Z.; Liu, L.; Zhang, J.; You, H.; Zhang, H.; Wang, Z. Facile Preparation of Doxorubicin-Loaded Upconversion@Polydopamine Nanoplatforms for Simultaneous In Vivo Multimodality Imaging and Chemophotothermal Synergistic Therapy. Adv. Health. Mater. , 2015, 4, 559-568. (22) Lin, J.; Wang, S.; Huang, P.; Wang, Z.; Chen, S.; Niu, G.; Li, W.; He, J.; Cui, D.; Lu, G.;


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Chen, X.; Nie, Z. Photosensitizer-Loaded Gold Vesicles with Strong Plasmonic Coupling Effect for Imaging-Guided Photothermal/Photodynamic Therapy. ACS Nano, 2013, 7, 5320-5329. (23) Yang, G.; Lv, R.; He, F.; Qu, F.; Gai, S.; Du, S.; Wei, Z.; Yang, P. A Core/Shell/Satellite Anticancer Platform for 808 NIR Light-Driven Multimodal Imaging and Combined Chemo/Photothermal Therapy. Nanoscale, 2015, 7, 13747-13758. (24) Song, X.; Liang, C.; Gong, H.; Chen, Q.; Wang, C.; Liu, Z. Photosensitizer-Conjugated Albumin-Polypyrrole Nanoparticles for Imaging-Guided In Vivo Photodynamic/Photothermal Therapy. Small, 2015, 11, 3932-3941. (25) Guo, M.; Huang, J.; Deng, Y.; Shen, H.; Ma, Y.; Zhang, M.; Zhu, A.; Li, Y.; Hui, H.; Wang, Y.; Yang, X.; Zhang, Z.; Chen, H. pH-Responsive Cyanine-Grafted Graphene Oxide for Fluorescence Resonance Energy Transfer-Enhanced Photothermal Therapy. Adv. Funct. Mater. , 2015, 25, 59-67. (26) Huang, P.; Lin, J.; Li, W.; Rong, P.; Wang, Z.; Wang, S.; Wang, X.; Sun, X.; Aronova, M.; Niu, G.; Leapman, R. D.; Nie, Z.; Chen, X. Biodegradable Gold Nanovesicles with an Ultrastrong Plasmonic Coupling Effect for Photoacoustic Imaging and Photothermal Therapy. Angew. Chem. Int. Ed., 2013, 52, 13958-13964. (27) Sheng, Z.; Hu, D.; Zheng, M.; Zhao, P.; Liu, H.; Gao, D.; Gong, P.; Gao, G.; Zhang, P.; Ma, Y.; Cai, L. Smart Human Serum Albumin-Indocyanine Green Nanoparticles Generated by Programmed Assembly for Dual-Modal Imaging-Guided Cancer Synergistic Phototherapy. ACS Nano, 2014, 8, 12310-12322. (28) Cheng, Z.; Dai, Y.; Kang, X.; Li, C.; Huang, S.; Lian, H.; Hou, Z.; Ma, P.; Lin, J. GelatinEncapsulated Iron Oxide Nanoparticles for Platinum (IV) Prodrug Delivery, Enzyme-Stimulated Release and MRI. Biomaterials, 2014, 35, 6359-6368.


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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


(29) Zhou, Z.; Sun, Y.; Shen, J.; Wei, J.; Yu, C.; Kong, B.; Liu, W.; Yang, H.; Yang, S.; Wang, W. Iron/Iron Oxide Core/Shell Nanoparticles for Magnetic Targeting MRI and Near-Infrared Photothermal Therapy. Biomaterials, 2014, 35, 7470-7478. (30) Wang, C.; Xu, H.; Liang, C.; Liu, Y.; Li, Z.; Yang, G.; Cheng, H.; Li, Y.; Liu, Z. Iron Oxide @Polypyrrole Nanoparticles as a Multifunctional Drug Carrier for Remotely Controlled Cancer Therapy with Synergistic Antitumor Effect. ACS Nano, 2013, 7, 6782-6795. (31) Peng, J.; Qi, T.; Liao, J.; Chu, B.; Yang, Q.; Qu, Y.; Li, W.; Li, H.; Luo, F.; Qian, Z. Mesoporous Magnetic Gold "Nanoclusters" as Theranostic Carrier for Chemo-Photothermal Cotherapy of Breast Cancer. Theranostics, 2014, 4, 678-692. (32) Chi, X.; Ji, X.; Xia, D.; Huang, F. A Dual-Responsive Supra-Amphiphilic Polypseudorotaxane Constructed from a Water-Soluble Pillar 7 arene and an AzobenzeneContaining Random Copolymer. J. Am. Chem. Soc., 2015, 137, 1440-1443. (33) Wang, Y.; Wang, K.; Zhao, J.; Liu, X.; Bu, J.; Yan, X.; Huang, R. Multifunctional Mesoporous Silica-Coated Graphene Nanosheet Used for Chemo-Photothermal Synergistic Targeted Therapy of Glioma. J. Am. Chem. Soc. , 2013, 135, 4799-4804. (34) Li, J.; Hu, Y.; Yang, J.; Wei, P.; Sun, W.; Shen, M.; Zhang, G.; Shi, X. Hyaluronic AcidModified Fe3O4@Au Core/Shell Nanostars for Multimodal Imaging and Photothermal Therapy of Tumors. Biomaterials, 2015, 38, 10-21. (35) Yu, Q.; Wei, Z.; Shi, J.; Guan, S.; Du, N.; Shen, T.; Tang, H.; Jia, B.; Wang, F.; Gan, Z. Polymer-Doxorubicin Conjugate Micelles Based on Poly(ethylene glycol) and Poly(N-(2hydroxypropyl) methacrylamide): Effect of Negative Charge and Molecular Weight on Biodistribution and Blood Clearance. Biomacromolecules, 2015, 16, 2645-2655. (36) Zhang, X.; Yang, P.; Dai, Y.; Ma, P. a.; Li, X.; Cheng, Z.; Hou, Z.; Kang, X.; Li, C.; Lin, J.


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Page 32 of 46

Multifunctional Up-Converting Nanocomposites with Smart Polymer Brushes Gated Mesopores for Cell Imaging and Thermo/pH Dual-Responsive Drug Controlled Release. Adv. Funct. Mater. , 2013, 23, 4067-4078. (37) Gupta, M. K.; Martin, J. R.; Werfel, T. A.; Shen, T.; Page, J. M.; Duvall, C. L. Cell Protective, ABC Triblock Polymer-Based Thermoresponsive Hydrogels with ROS-Triggered Degradation and Drug Release. J. Am. Chem. Soc. , 2014, 136, 14896-14902. (38) Wang, B.; Wang, J.-H.; Liu, Q.; Huang, H.; Chen, M.; Li, K.; Li, C.; Yu, X.-F.; Chu, P. K. Rose-Bengal-Conjugated Gold Nanorods for in vivo Photodynamic and Photothermal Oral Cancer Therapies. Biomaterials, 2014, 35, 1954-1966. (39) Tian, B.; Wang, C.; Zhang, S.; Feng, L.; Liu, Z. Photothermally Enhanced Photodynamic Therapy Delivered by Nano-Graphene Oxide. ACS Nano, 2011, 5, 7000-7009. (40) Guan, Y.; Li, M.; Dong, K.; Ren, J.; Qu, X. NIR-Responsive Upconversion Nanoparticles Stimulate Neurite Outgrowth in PC12 Cells. Small, 2014, 10, 3655-3661. (41) Chen, G.; Agren, H.; Ohulchanskyy, T. Y.; Prasad, P. N. Light Upconverting Core-Shell Nanostructures: Nanophotonic Control for Emerging Applications. Chem. Soc. Rev., 2015, 44, 1680-1713. (42) Tsang, M.-K.; Bai, G.; Hao, J. Stimuli Responsive Upconversion Luminescence Nanomaterials and Films for Various Applications. Chem. Soc. Rev., 2015, 44, 1585-1607. (43) Lu, S.; Tu, D.; Hu, P.; Xu, J.; Li, R.; Wang, M.; Chen, Z.; Huang, M.; Chen, X. Multifunctional Nano-Bioprobes Based on Rattle-Structured Upconverting Luminescent Nanoparticles. Angew. Chem. Int. Ed., 2015, 54, 7915-7919. (44) Tian, Q.; Hu, J.; Zhu, Y.; Zou, R.; Chen, Z.; Yang, S.; Li, R.; Su, Q.; Han, Y.; Liu, X. Sub-10 nm Fe3O4@Cu2-xS Core-Shell Nanoparticles for Dual-Modal Imaging and Photothermal


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Page 33 of 46

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


Therapy. J. Am. Chem. Soc. , 2013, 135, 8571-8577. (45) Zhou, J.; Zhu, X.; Chen, M.; Sun, Y.; Li, F. Water-Stable NaLuF4-Based Upconversion Nanophosphors with Long-Term Validity for Multimodal Lymphatic Imaging. Biomaterials, 2012, 33, 6201-6210. (46) Wen, S.; Li, K.; Cai, H.; Chen, Q.; Shen, M.; Huang, Y.; Peng, C.; Hou, W.; Zhu, M.; Zhang, G.; Shi, X. Multifunctional Dendrimer-Entrapped Gold Nanoparticles for Dual Mode CT/MR Imaging Applications. Biomaterials, 2013, 34, 1570-1580. (47) Wang, L.; Xing, H.; Zhang, S.; Ren, Q.; Pan, L.; Zhang, K.; Bu, W.; Zheng, X.; Zhou, L.; Peng, W.; Hua, Y.; Shi, J. A Gd-doped Mg-Al-LDH/Au Nanocomposite for CT/MR Bimodal Imagings and Simultaneous Drug Delivery. Biomaterials, 2013, 34, 3390-3401. (48) Chen, Q.; Li, K.; Wen, S.; Liu, H.; Peng, C.; Cai, H.; Shen, M.; Zhang, G.; Shi, X. Targeted CT/MR Dual Mode Imaging of Tumors Using Multifunctional Dendrimer-Entrapped gold Nanoparticles. Biomaterials, 2013, 34, 5200-5209. (49) Rieter, W. J.; Kim, J. S.; Taylor, K. M. L.; An, H.; Lin, W.; Tarrant, T.; Lin, W. Hybrid silica nanoparticles for multimodal Imaging. Angew. Chem. Int. Ed., 2007, 46, 3680-3682. (50) Munoz Ubeda, M.; Carniato, F.; Catanzaro, V.; Padovan, S.; Grange, C.; Porta, S.; Carrera, C.; Tei, L.; Digilio, G. Gadolinium-Decorated Silica Microspheres as Redox-Responsive MRI Probes for Applications in Cell Therapy Follow-Up. Chemistry, 2016, 22, 7716-7720. (51) Carniato, F.; Tei, L.; Cossi, M.; Marchese, L.; Botta, M. A Chemical Strategy for the Relaxivity Enhancement of Gd-III Chelates Anchored on Mesoporous Silica Nanoparticles. Chem.- Eur. J., 2010, 16, 10727-10734. (52) Ahren, M.; Selegard, L.; Soderlind, F.; Linares, M.; Kauczor, J.; Norman, P.; Kall, P.-O.; Uvdal, K. A Simple Polyol-Free Synthesis Route to Gd2O3 Nanoparticles for MRI Applications:


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an Experimental and Theoretical Study. J.Nanopart. Res., 2012, 14, 1006. (53) Chen, H.; Wang, G. D.; Tang, W.; Todd, T.; Zhen, Z.; Tsang, C.; Hekmatyar, K.; Cowger, T.; Hubbard, R. B.; Zhang, W.; Stickney, J.; Shen, B.; Xie, J. Gd-Encapsulated Carbonaceous Dots with Efficient Renal Clearance for Magnetic Resonance Imaging. Adv. Mater., 2014, 26, 67616766.


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Scheme 1. Schematic illustration for the synthesis of DOX-GSCCP nanoplatform.


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Figure 1. (A) low- and (B) high-magnified TEM images, (C) the cross-section compositional profile lines, (D) STEM image, and (E) elemental mapping images of GSCCP.


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Figure 2. (A) UV/Vis absorbance spectra of Gd-Si-Ce6, Gd-Si and Ce6; (B) standard curve of Ce6; (C) relationship between the feeding Ce6 concentrations and the loaded amounts of Ce6 on Gd-Si, the loaded Ce6 ratio is mMCe6/mMSi-Gd; (D) the release profiles of Ce6 from Gd-Si in PBS at different pH values, error bars are based on standard deviations (SD) of triplicate samples.


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Figure 3. (A) Time-course generation of singlet oxygen by GSC under 650 nm laser (0.5 W/cm2) and 808 nm laser (0.5 W/cm2); (B) Generation of singlet oxygen by GSC and free Ce6 at different concentrations under 650 nm laser irradiation (0.5 W/cm2) for 10 min. Error bars are based on the SD of triplicated samples; (C) CLSM image of HeLa cells incubated with Gd-Si, Ce6 and GSCCP under 650 nm laser irradiation. All the cells were marked with DCFH-DA. Scale bars for all images are 100 µm. .


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Figure 4. In vitro and in vivo infrared thermal imaging properties. (A) The infrared thermal photograph of a 96-well HeLa cell-culture plate incubated with (a) pure Ce6 molecules, (b) pure carbon dots, (c) DOX-GSCCP, (d) GSCCP, (e) GSC and (f) GSCP under irradiation of 980 nm (0.5 W/cm2) and 650 nm lasers (0.5 W/cm2); (B) the corresponding temperature profiles; (C) the infrared thermal images of the tumor-bearing mice after injection of DOX-GSCCP and normal saline as a function of the 980 nm irradiation time (0.5 W/cm2); (D) the corresponding temperature profiles of the tumor-bearing mice injected with DOX-GSCCP and normal saline versus irradiation time and the power of the irradiation (0.5 W/cm2).


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Figure 5. Drug-release behavior of DOX-GSCCP at pH = 4.0, 5.5 and 7 phosphate buffers at (A) 37 °C, (B) 50 °C, (C) with and without 980 nm NIR light irradiation at room temperature.


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Figure 6. Confocal laser scanning microscopy (CLSM) images of HeLa cells incubated with (A) Gd-Si, Ce6, GSC and (B) DOX-GSCCP for different times. All the scale bars are 75 µm.


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Figure 7. (A) Cell viability for L929 cells incubated with GSCCP with different concentrations for 24 h; (B) Hemolytic assay of GSCCP to human red blood cells; (C) in vitro cell viabilities of HeLa cells incubated for 24 h with GSCCP, GSC, and DOX-GSCCP at varied concentration with and without laser irradiation; (D) CLSM images of HeLa cells incubated with culture, 980 nm and 650 nm laser irradiation, GSCCP, GSC under 650 nm laser irradiation, DOX-GSCCP under 980 nm laser irradiation, DOX-GSCCP under 980 nm and 650 nm laser irradiation. All the cells are marked with calcein AM and PI. Scale bars for all images are 100 µm.


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Figure 8. In vivo anti-cancer properties. (A) The body weight and (B) relative tumor volume of U14 tumor in different groups versus the treatment time (n = 5); (C) representative photographs of the tumor tissues excised from tumor-bearing mice treated with normal saline, pure laser, pure GSCCP, GSC with 650 nm laser irradiation, DOX-GSCCP with 980 nm laser irradiation and DOX-GSCCP with two types of 980 nm and 650 nm laser irradiation on the 14th day.


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Figure 9. (A) CT imaging properties of DOX-GSCCP. (a) In vitro CT images of DOX-GSCCP with different Gd concentrations; (b) CT values of the aqueous solution of DOX-GSCCP versus the molar concentrations of DOX-GSCCP, CT images of a tumor-bearing Kunming mouse; (c, e) pre-injection and (d, f) after injection in situ. (B) MRI properties of DOX-GSCCP. (a) T1weighted MR images of DOX-GSCCP nanocomposite recorded using a 1.2 T MR scanner, (b) the T1 relaxation rates (r1) of DOX-GSCCP with different Gd concentrations; (c, d) MRI images for in vivo mapping using DOX-GSCCP sample. MR images are taken before (c, left) and after (d, right) injection.


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Figure 10. Representative H&E stained histological images of the heart, liver, spleen, lung and kidney slices. All the scale bars are 75 µm.


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Table of Contents (ToC) Graphic SiO2-Gd-Ce6-CDs theranostic platform exhibits simultaneous thermo/pH-responsive drug release property, photothermal therapy, photodynamic therapy upon two lasers irradiation, leading to excellent synergistic therapeutic efficacy superior to any single/dual-therapy. The result has been firmly verified by in vitro and in vivo assay. Considering the multiple imaging properties (CT, MRI and UCL), this multifunctional system should be of high potential in imaging-guided therapy.


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Photodynamic

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