Theranostic Nanoplatform: Triple-Modal Imaging-Guided Synergistic

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A Theranostic Nanoplatform: Triple-Model Imaging Guided Synergistic Cancer Therapy Based on Liposomes Conjugated Mesoporous Silica Nanoparticles Qi Sun, Qing You, Jinping Wang, Li Liu, Yidan Wang, Yilin Song, Yu Cheng, Siyu Wang, Fengping Tan, and Nan Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13651 • Publication Date (Web): 25 Dec 2017 Downloaded from http://pubs.acs.org on December 26, 2017

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ACS Applied Materials & Interfaces

A Theranostic Nanoplatform: Triple-Model Imaging Guided Synergistic Cancer Therapy Based on Liposomes Conjugated Mesoporous Silica Nanoparticles Qi Sun, Qing You, Jinping Wang, Li Liu, Yidan Wang, Yilin Song, Yu Cheng, Siyu Wang, Fengping Tan, Nan Li*

Q. Sun, Q. You, J. Wang, L. Liu, Y. Wang, Y. Song, Y. Cheng, S. Wang, Prof. F. Tan, Dr. N. Li Tianjin Key Laboratory of Drug Delivery & High-Efficiency School of Pharmaceutical Science and Technology Tianjin University Tianjin, 300072, China E-mail: [email protected]

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Abstract: Mesoporous silica nanoparticles (MSNs) have long since been investigated to provide a versatile drug delivery platform due to its multitudinous merits. Presently, gadolinium (Gd), a T1 magnetic resonance imaging (MRI) contrast agent, was doped into MSNs as a newly emerging theranostic nanocomposite, which has received much research attention. However, it is still concerned about the dispersibility and drug leakage of MSNs. Hence, in this project, we constructed an NIR irradiation triggered, triple-modal imaging guided nanoplatform based on doxorubicin (DOX) @ Gd doped-MSNs, conjugating with indocyanine green (ICG) loaded thermosensitive liposomes (designated as DOX@GdMSNs-ICG-TSLs). In this platform, ICG could contribute to both photodynamic therapy (PDT) and photothermal therapy (PTT) effect, meanwhile, it could also give play to near infrared fluorescence imaging (NIRFI) as well as photoacoustic imaging (PAI). Consequently, NIRFI and PAI from ICG combined with the MRI function of Gd, devoted to triplemodel imaging with success. At the same time, folic acid (FA) modified thermosensitive liposomes were explored to coated onto the surface of DOX@GdMSNs, in order to solve the DOX leakage as well as improve cellular uptake. Under the NIR irradiation, ICG could generate heat, thus leading to the rupture of ICG-TSLs and the release of DOX. Accordingly, the multifunctional nanocomposite appeared to be a promising meritorious theranostic nanoplatform to pave a way for treating cancer. Keywords: Triple-model imaging; GdMSNs; Thermosensitive liposomes; Indocyanine green; Doxorubicin

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1. Introduction Nanocomposites with multiple functions which integrate diagnosis and therapy in one platform have shown great potentials to fight cancer 1-3. Some nanocomposites hold many advantages due to their unique physicochemical features such as large surface areas, tunable sizes, facile modification, considerable biocompatibility, magnetic, thermal effect, etc

4, 5

. Among them, MSNs are

investigated to provide a versatile drug delivery platform since its tunable sizes, high internal surface area, considerable biocompatibility, modification flexibility, etc

6-8

. However, the

dispersibility and inherently low biocompatibility of silica-based nanoparticles limit their bioapplications. To improve its hemocompatibility and realize controlled and/or targeting drug release, it is essential to modify bare MSNs appropriately 9. It has been reported that MSNs coated with lipid bilayers show a superior therapeutic delivery property 10-13. Through this way, the composites could overcome a series of problems such as low solubility, limited stability and poor biodistribution

12, 14

. By a series of improvements, MSNs have been used as platforms in the

bioimaging and anticancer drug delivery of nanomedicine field, including magnetic resonance imaging (MRI), near infrared fluorescence imaging (NIRFI) and photoacoustic imaging (PAI)15-17. Recently, Gadolinium (Gd) doped mesoporous silica nanoparticles (MSNs) as newly emerging nanocomposites, have received much research attention

18, 19

. Gd, which is one of the rare earth

element, has been approved as effective T1 magnetic resonance imaging contrast agents because it possesses a large number of unpaired electrons that may alter the relaxation time of the surrounding water protons. Thus, Gd (III) compounds are attractive to enhance the imaging sensitiveness and quality in MRI

20, 21

. Apart from MRI, other molecular imaging technologies such as NIRFI, PAI

have also showed great potentials in basic biomedical research and have been developed in disease diagnosis and prognosis

22, 23

. Currently, multimodality imaging has been broadly concerned in

researches and clinical applications to improve the accuracy of medical diagnosis for precise and reliable information at the disease sites 24,25. 3



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The traditional cancer therapy via using chemotherapeutic agents has many limitations such as poor bioavailability, internal toxicity and drug resistance, resulting in side effects, including myelosuppression, nausea and vomiting. Hence, an alliance of chemo- and photo-therapy has obtained great attention in current clinical therapy investigation

26-29

. It is worth mentioning that,

indocyanine green (ICG), a Food and Drug Administration (FDA)-approved drug, can not only devote to NIRFI and PAI, but also contribute itself to generate reactive oxygen species (ROS), as well as localized hyperthermia by absorbing near-infrared light (NIR) for photothermal therapy (PTT)

30, 31

. Photodynamic therapy (PDT) relies on local generation of ROS from light activated

photosensitizers (PSs) to kill cancer cells. PTT has received constant attention due to its noninvasiveness and high selectivity by transferring light energy into heat to kill cancer cells 32, 33. The combination of phototherapy and chemotherapy in a single nanoplatform could improve the therapeutic efficacy of malignant tumor, meanwhile, reduce side-effects

34, 35

. As a result, it has

been widely studied as an emerging complementary approach for traditional cancer therapies

36, 37

.

For example, Zhao et al. developed a noninvasive NIR-driven, temperature-sensitive system by coencapsulating doxorubicin (DOX) and ICG, manifesting high efficiency to promote cell apoptosis, and completely eradicate tumor without side-effects 38. In this research, we constructed a multifunctional NIR irradiation triggered, triple-modal imaging guided nanoplatform based on DOX @ Gd doped-mesoporous silica nanoparticles, which coated with ICG loaded thermosensitive liposomes (ICG-TSLs) (designated as DOX@GdMSNs-ICGTSLs). The nanoplatform with chemo- and photo-therapy as well as triple-modal imaging, achieving integrate of diagnosis and treatment for cancer (Figure 1. b). After doping Gd (III) compounds into MSNs, chemotherapeutic drug (DOX) was loaded into the mesopores (designated as DOX@GdMSNs). In order to solve the leakage of DOX as well as improve dispersibility and cellular uptake, DOX@GdMSNs were coated with ICG loaded/ folic acid (FA) modified thermosensitive

liposomes.

Via

intravenous

injection,

DOX@GdMSNs-ICG-TSLs

4


could

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accumulate in tumor sites through the active and passive targeting effect of FA and EPR, respectively. Under the NIR irradiation, ICG could generate heat, thus leading to the rupture of ICG-TSLs and the release of DOX. More importantly, multimodal imaging of NIRFI and PAI, which were both contributed by ICG, as well as MRI via Gd (III) compounds, devote to monitoring particle biodistribution and guiding therapy. Under NIR light irradiation, the nanocomposite acted as an excellent function to kill cancer cells, which was ascribed to the combination of chemotherapy and photothermal/photodynamic therapy. Above all, it could be envisioned that the nanocomposite was an attractive nanosystem for developing versatile functionalities to put good use in caner therapy. 2. Materials and Methods 2.1.

Materials

Cetyltrimethyl ammonium bromide (CTAB) (99 % for molecular biology) was purchased from Tianjin Yuanli Chemical Co., Ltd. (China). Ethanol (99.5 %) was obtained from Tianjin Jiangtian Chemical Technology Co., Ltd. (China). Tetraethyl orthosilicate (TEOS, 28 %) was got from Tianjin Damao Chemical Reagent Co., Ltd. (China). Gadolinium (III) oxide was obtained from Yuanye Biological Technology Co., Ltd. (China). Cholesterol (Chol) was bought from Guangfu Fine Chemical Research Institute (China). Indocyanine green (ICG, 90 %) was purchased from J&K Scientific Ltd. (China). 1, 2-Dialmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyeth-ylene

glycol)-2000]

(DSPE-

PEG2000) and 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate (polyethylene glycol)2000] (DSPE-PEG2000-folate) were purchased from Shanghai Advanced Vehicle Technology Pharmaceutical Co., Ltd. (China). 20, 7'-dichloro-fluorescein diacetate (DCFH-DA) were bought from Tianjin Heowns Biochemical Technology Co., Ltd. (China). 4T-1 (one kind of mouse breast cancer) cells were purchased from Procell life science & technology Co., Ltd. (China). 40, 6Diamidino-2-phenylindole (DAPI), propidium iodied (PI), calcein acetoxymethyl ester (Calcein 5



AM),

1,3-diphenyl

diphenyltetrazolium

iso-benzofuran bromide

(MTT)

(DPBF) were

all

and

3-(4,

purchased

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5-dimethylthiazol-2-yl)-2, from

Sigma-Aldrich

5-

(USA).

Dimethylsulfoxide (DMSO, ≧ 99%) was gained from Beijing Biotopped Science & Technology Co., Ltd. (China). Annexin-V/PI Apoptosis Detection kit was purchased from ComWin Biotech Co., Ltd. (China). Dulbecco's Modified Eagle's Medium (DMEM, high glucose, GIBCO) were gained from Invitrogen (USA). All the chemicals were reagent grade at lowest and used as received without further purification. 2.2.

Preparation of DOX@GdMSNs-ICG-TSLs

The detailed synthesis steps were shown in Figure 1. a. Firstly, GdMSNs were synthesized based on the previously reported traditional methods 18. Briefly, Gd2O3 (0.02 g) was added into NaOH (2 M, 0.85 mL) and dealt with ultrasound for 2 h. Meanwhile, CTAB (0.2 g) was mixed with 100 mL ultrapure water and further stirred at 35 °C for 30 min. Then, Gd2O3/NaOH was mixed with the CTAB aqueous solution, at the moment, the temperature was increased to 80 °C. Following, 1 mL TEOS was dropwise added into above-mentioned miscible liquids and stirred for 2 h at 80 °C. The GdMSNs were gained through centrifugation method at 8000 rpm for 10 min and washed with ethanol for three times. Next, the soft template CTAB was removed by reflux condensation for more than 24 h. The product was then centrifuged and frozen into powder by lyophilization method. Secondly, Dox@GdMSNs were prepared by adding DOX (1mg/mL, 1mL) into the GdMSNs aqueous dispersion, then stirred overnight. Following, unloaded drug was removed via centrifugation method. The last step was to synthesize the functional composites DOX@GdMSNsICG-TSLs. Cholesterol (3.75 mg), DPPC (15.98 mg), DSPE-PEG2000 (1.88 mg), DSPE-PEG2000folate (0.9 mg), and ICG (1 mg/mL, 750 µL), were all solved in methanol /chloroform mixture (15 mL) and sonicated for several minutes in a round-bottom flask. Then, vacuum rotary evaporation was utilized to acquire thin film of lipid. Subsequently, Dox@GdMSNs aqueous dispersion was 6


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dumped into the round-bottom flask. After ultrasonic processing for 2 h, DOX@GdMSNs-ICGTSLs were obtained 10. Excess phospholipids were purified by centrifugating. 2.3.

Characterization of DOX@GdMSNs-ICG-TSLs

Various formulations were monitored by UV-vis spectra (UV-vis spectrophotometer, Agilent, Santa Clara, USA) and IR spectra (infrared spectrophotometer, TENSOR 27, Bruker, German). Morphology was visualized by transmission electron microscopy (TEM, HT7700, Hitachi Ltd, Japan). Energy dispersive X-ray analysis (EDX, Tecnai G2 F20, FEI Ltd, Hong Kong) was used to measure elements distribution. The average particle size, size distribution, and zeta potential measurements were all obtained by using a Malvern Master-sizer (Nano ZS, Malvern Instruments, UK) at normal temperature. The BET (Brunauer–Emmett–Teller) surface area and pore size of GdMSNs were measured by BET Tester (Gemini VII2390, Micromeritics, USA) using nitrogen adsorption-desorption curve and Barett-Joyner-Halenda (BJH) methods, respectively. Thermal images were monitored by an SC300 infrared camera (Fluke TiR, USA). The 808 nm laser light source induced by LSR808NL-2W semiconductor power-tunable laser (Laserver, China) was used to trigger phototherapy effect. 2.4.

In vitro photothermal effect

The photothermal conversion performance of various formulations (PBS, GdMSNs, DOX@GdMSNs, ICG-TSLs and DOX@GdMSNs-ICG-TSLs) were measured under 808 nm laser (1.5 W/cm2) irradiation within 5 min using a digital thermometer. Further, in order to evaluate photothermal conversion efficiency of our optimal formulation, temperature-rise period of DOX@GdMSNs-ICG-TSLs with laser for a span and cool-down process were detected. Further, we used an infrared thermal camera (TiS55, Fluke, USA) to record real-time thermal imaging of formulations (PBS, DOX@GdMSNs, ICG-TSLs and DOX@GdMSNs-ICG-TSLs). 2.5.

In vitro photodynamic effect

DPBF was employed to evaluate the in vitro singlet oxygen generation capability of 7



DOX@GdMSNs-ICG-TSLs within 8 min under continuous 808 nm laser (1.5 W/cm2) irradiation. DPBF, free ICG-TSLs + DPBF and ICG + DPBF were also measured as control groups. DOX@GdMSNs-ICG-TSLs (15 mg/mL ICG, 3 mL) were mixed rapidly with fresh DPBF (0.5 mg/mL, 100 µL) and kept in dark place. Subsequently, the mixture was dealt with the irradiation of 808 nm laser (1.5 W/cm2). Following, we measured the absorbance value at 410 nm wavelength via a UV-vis spectrophotometer. 2.6.

In vitro release experiment

In order to evaluate NIR triggered DOX release characteristics, we investigated in vitro release curves of DOX from DOX@GdMSNs-ICG-TSLs in the presence and absence of NIR trigger, respectively. DOX@GdMSNs-ICG-TSLs (15 mg/mL ICG, 3 mL) were dialyzed in PBS with or without NIR irradiation (808 nm, 1.5 W/cm2) at 2 h and 6 h, respectively. The UV absorption values were measured at different time points. 2.7.

Cell culture

4T1 cells were cultured in cell culture flasks with high-glucose DMEM supplemented containing 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin in a standard incubation condition (37 °C, 5 % CO2). 2.8.

Intracellular uptake and ROS detection

4T1 cells (1×105/plate) were seeded in confocal laser scanning microscope (CLSM) dishes and cultured overnight. Cells were treated with PBS and different formulations at an identical ICG concentration (15 µg/mL) of free ICG and DOX@GdMSNs-ICG-TSLs with or without 808 nm irradiation (1.5 W/cm2, 1 min) for 4 h, respectively. After washing twice by PBS, cells were successively incubated with DAPI (5 µg/mL, 1 mL) for 15 min. Subsequently, cells were washed with PBS for three times before observing under a CLSM. And in order to analyze quantitatively, we further used flow cytometry. 4T1 cells were seeded in six-well plates and incubated for 24 h. Then, cells were given different formulations and incubated for 4 h. Following, flow cytometer (BD Biosciences) was applied to detect fluorescence signals for quantitative analysis. 8


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To further observe the generation of ROS generation, DCFH-DA was employed based on aforementioned method. 4T1 cells were seeded at confocal dishes with the cell density of 1×105. Then, dishes were divided to use culture cells with various formulations for 12 h. Following, cells were successively treated with DAPI (5 µg/mL, 1 mL) for 15 min and DCFH-DA (10 µM, 1 mL) for 50 min. Then, after being washed with PBS, cells were irradiated upon 808 nm laser (1.5 W/cm2, 5 min). Intracellular ROS images were detected by CLSM. 2.9.

In vitro cytotoxicity

The cytotoxicity of various formulations to cancer cells under different disposes was assessed using MTT assay, co-stained experiment and flow cytometry to evaluate respectively. The relative cell viabilities compared with untreated groups were measured by the MTT assay. 4T1 cells (5×104/well) were incubated in 96-well plates aforehand for 24 h. Then, cells were treated with PBS, DOX@GdMSNs, ICG-TSLs + NIR (808 nm, 1.5 W/cm2), DOX@GdMSNs-ICG-TSLs + NIR (808 nm, 1.5 W/cm2) with different concentrations in media for 24 h, respectively. In the course of incubation, NIR irradiation was given at 4 h and 6 h for 5 min, respectively. Afterwards, the cells were further incubated with MTT solution (5 mg/mL, 20 µL) for 4 h. At last, the generated formazan crystals were dissolved with DMSO under mildly shaking. Immediately, the absorption of each well was measured by an enzyme-linked immunosorbent assay reader. As for AM/PI co-stained experiment, the density of 5 × 105 per well 4T1 cells were seeded and treated with PBS, DOX@GdMSNs, ICG-TSLs + NIR (808 nm, 1.5 W/cm2), DOX@GdMSNs-ICGTSLs + NIR (808 nm, 1.5 W/cm2), respectively. At 4 h and 6 h, 808 nm laser (1.5 W/cm2) irradiation was employed for 5 min and follow-up incubated until 24 h. AM (10 ng/mL) and PI (10 ng/mL) were used to stain live and dead cells, respectively. Finally, cells were washed with PBS and observed through confocal laser scanning microscope (CLSM).

9



We further used flow cytometry to evaluate the cytotoxicity. 4T1 cells were seeded in six-well plates and incubated for 24 h. Following, the apoptotic fraction was detected via staining with fluorescein isothiocyanate-conjugated annexin-V and propidium iodide using Annexin-V/PI Apoptosis Detection kit, and then by a FACS Canto cell sorting instrument (BD Biosciences). 2.10.

Animals and tumor models

All animal experiments were performed strictly with the protocols stipulated by Tianjin University. Female nude mice around 4 weeks purchased from Beijing Huafukang Biological Technology Co., Ltd (China), were fed at laboratory animal center of the Chinese Academy of Medical Sciences Institute of Radiology in Tianjin. To construct the tumor model mice, they were treated with 4T1 cells. When the volume of each tumor was around 200 mm3, animal studies were started. 2.11.

Near infrared fluorescence, photoacoustic and magnetic resonance imaging

Free ICG and DOX@GdMSNs-ICG-TSLs with an equivalent ICG concentration (0.86 mg ICG/kg), were intravenously injected into the tumor model mice. The NIRF images were captured at 2 h, 6 h, 12 h, 24 h and 48 h by using an imaging system ((IVIS Lumina, Caliper Life Science, USA). Several among them were sacrificed to use in ex vivo imaging of heart, liver, spleen, lungs, kidneys and tumor tissues. Tumor-bearing mice were injected with free ICG and DOX@GdMSNs-ICG-TSLs with an equivalent of ICG. Photoacoustic images were captured at different time points. The PA signals at the tumor sites were monitored via a Vevo LAZR PAI System. As for magnetic resonance imaging, the cross sections of the mice with various concentrations of Gd (III) in DOX@GdMSNs-ICG-TSLs before- and post-injection were monitored. Mice were narcotized using 4% isoflurane/oxygen, following, they were fixed to an acrylic patient bed in the prone position and maintained on 1% isoflurane/oxygen mixture. T1-weighted images were acquired in the axial plane using a multi-slice spin-echo sequence under the optimal condition (7 T, 10


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spin-echo sequence: repitition time TR = 500 ms, echo time TE = 14.92 ms) performed on a 7.0 T small-animal borizontal bore MRI device (Varian, Palo Alto, US). 2.12.

Infrared thermal imaging

The tumor model mice were injected with designed formulations (PBS, DOX@GdMSNs, ICGTSLs and DOX@GdMSNs-ICG-TSLs with 0.86 mg ICG/kg) for 24 h. At the tumor site of each mouse, it was dealt with 808 nm laser (1.5 W/cm2) over 4 min. Simultaneously, an IR camera (TiS55, Fluke, USA) was used to record thermal images at various time points of different groups. 2.13.

In vivo antitumor efficiency

In order to evaluate the antitumor efficiency, PBS, DOX@GdMSNs, ICG-TSLs and DOX@GdMSNs-ICG-TSLs (0.86 mg ICG/kg) were respectively intravenously (i.v.) administered into the mice. The tumor volume of each groups was estimated every two days after being injected with various formulations according to the calculation formula: V = A*B2/2 (A: maximum diameter of the tumor, B: minimum diameter of the tumor). We recorded weight changes of each group at the same time interval within 21 days. Further, the survival percentage of each group was calculated after 4 weeks anti-tumor treatment. At the end of experiment, the mice were sacrificed, then tumor tissues and major organs including heart, liver, spleen, lungs, and kidneys were excised and saved in 10 % formalin for subsequent experiments. Hematoxylin and eosin (H&E) was employed to monitor changes of each organ and tumor tissues after treatment. In addition to estimate ROS generation in tumor tissues ulteriorly, pre-injected DCFH-DA tumors were isolated and frozen at −80 °C immediately. Frozen sections were prepared and observed using CLSM. It was illustrated according to green fluorescent of intratumoral ROS which was stained by DCFH-DA. Cell nuclei were stained blue with DAPI. ICG distribution within the tumor was indicated with red color. 2.14.

Statistical analysis

Data shown in the article was expressed as mean ± SD. Student's t-test was applied to the comparison results in some studies between two different groups. It was defined to be significant 11



when **p < 0.01, *p < 0.05. 3. Results and Discussion 3.1.

Preparation and Characterization of DOX@GdMSNs-ICG-TSLs

DOX@GdMSNs-ICG-TSLs were prepared as the following steps: Gd2O3 was doped into mesoporous silica nanoparticles under the process of forming MSNs. Then, the chemotherapy drug DOX, was loaded into the pores of GdMSNs. Finally, by coating FA linked ICG-TSLs, DOX@GdMSNs-ICG-TSLs were obtained. The drug loading capacity and efficiency of DOX were 5.17% and 81.81%, respectively. And the same index efficiency of ICG were 5.15% and 98.20%, respectively. The transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed the morphology of DOX@GdMSNs and DOX@GdMSNs-ICG-TSLs. The TEM images indicated that after coating ICG-TSLs, the surface of nanoparticles became much smoother (Figure 2. a, b), which was consistent with SEM images observation. These results confirmed that ICGTSLs were successfully coated onto GdMSNs. The energy dispersive X-ray analysis (EDX) was also employed to detect the distribution of different elements in DOX@GdMSNs-ICG-TSLs (Figure 2. c, d). It respectively showed the distribution of oxygen, silicon and gadolinium, importantly revealing that Gd (III) was doped into MSNs with success. Further, some experiments were utilized in the characterization of formulations. The size distribution was measured by a Malvern Master-sizer, showing the average size of DOX@GdMSNs-ICG-TSLs was around 233.8 nm (Figure 3. a). It was noteworthy that the data was larger than the results in TEM images, which might be attributed to PEGylation and the exist of hydration shell. In addition, polydispersity index (PDI) was 0.306 in double-distilled water. For serum-free and serum-containing cell-culture medium, PDI was 0.291 and 0.298, respectively (Table S1), which illustrated there was almost no influence for serum and cell-culture medium on the dispersion stability of the nanoparticles. Whatos more, the zeta potential (-25.2 mV), showed the good stability of the nanoparticle. 12


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UV-vis spectra was used to verify DOX and ICG were loaded into the carriers (Figure 3. b). The characteristic peaks of DOX and ICG in UV absorption were at 480 nm and 780 nm, respectively. Through comparison, DOX@GdMSNs-ICG-TSLs showed the characteristic peaks of DOX and ICG with a little red shift, which might due to the influence of solvent factors. Besides, from the results of IR spectra, it exhibited the extraction of soft template CTAB from carriers. Compare the results between before and after CTAB extraction, the difference of C-H stretching vibrations which at around 2920 cm-1 and 2850 cm-1 could be observed obviously (Figure 3. c). The sharply weaker of these bands after extraction illustrated the template CTAB was removed effectually, thus forming the pores of carriers. As expected, a detailed analysis of X-ray diffraction (XRD) ulteriorly certified Gd2O3 was doped into MSNs (Figure 3. d, e). To further investigate the structure of GdMSNs, we applied nitrogen adsorption-desorption curve and Barette-Joynere-Halenda (BJH) methods to determine the BET (Brunauere-Emmette-Teller) surface area and pore features by a BET Tester. The shape of the curve showed that the nanoparticles were mesoporous materials. BET surface area, absorption average pore width and average volume of pores were 302.6234 m2/g, 88.8136 Å and 0.6719 m2/g, respectively. Photographs of various formulations (GdMSNs, DOX@GdMSNs, ICGTSLs and DOX@GdMSNs-ICG-TSLs) were shown in Figure S1. In addition, we compared the stability of DOX@GdMSNs and DOX@GdMSNs-ICG-TSLs before and after standing for 8 h (Figure S2.), revealing the enhanced stability of lipid bilayer coated formulation. What’s more, the comparasion (at 8 h and one week) of TEM imaging, particle size, temperature changes within 5 min and change curves of the UV absorbance of DPBF within 5 min were shown in Figure S3 and Table S2, respectively. The results revealed that within one week, the nanoparticles were quite stable. 3.2.

In vitro Efficacy Evaluation

To explore the photothermal effect of the nanosystem, temperature changes induced by 808 nm laser (1.5 W/cm2) irradiation within 5 min was recorded (Figure 4. a). It was significant that 13



DOX@GdMSNs-ICG-TSLs exhibited the effective PTT effect. In contrast, PBS, GdMSNs and DOX@GdMSNs showed inferior ability to increase temperature. Meanwhile, different formulations’ real-time thermal images were recorded using an infrared thermal camera (Figure 4. b). As expected, the results were in accord with the temperature changes curve (Figure 4. a), indicating the essential temperature raise effect of ICG as well as the unexceptionable PTT effect of the optimal formulation. What’s more, we further calculate the photothermal conversion efficiency (ŋ) of DOX@GdMSNs-ICG-TSLs on the basis of temperature change curve and linear time data (Figure 4. c) from the cooling period versus negative natural logarithm of driving force temperature. The ŋ of DOX@GdMSNs-ICG-TSLs was then evaluated to be 11.72 % (Supporting information for details). This demonstrated the photothermal conversion efficiency of DOX@GdMSNs-ICG-TSLs was commendable to dedicate to PTT effect. We also detected the phase transition temperature (Tm) of DOX@GdMSNs-ICG-TSLs, which was 42 °C, verifying the thermal-sensitive nature. Besides, the release profile of ICG was shown in Figure S4. There was minor drug leakage (less than 20 %) at physiological temperature (37 °C), however, the release of DOX@GdMSNs-ICG-TSLs was increased gradually and obviously over the Tm, indicating photothermal-response behavior of our nanosystem. Except for the evaluation of PTT effect, PDT effect was also monitored by using a trapping reagent DPBF to detect singlet oxygen (1O2). Under dark condition, formulations mixed with DPBF were given 808 nm laser (1.5 W/cm2) irradiation for 8 min. By observing the UV absorption changes of DPBF at 410 nm, the ability of 1O2 generation could be evaluated. Free ICG + DPBF, the absorption decreased largely and fleetly. As for DOX@GdMSNs-ICG-TSLs + DPBF group, however, the absorption decreased largely, relatively slower. (Figure 4. d). This illustrated the outstanding PDT effect of DOX@GdMSNs-ICG-TSLs which performed by ICG. For investigation into the release behavior, in vitro release profile of DOX from DOX@GdMSNs-ICG-TSLs had been studied under the different conditions (without or with NIR 14


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laser irradiation). As shown in Figure 4. e, the released amount of DOX under 808 nm laser (1.5 W/cm2) nearly reached to 50%, which was much more than that without NIR laser treated group. What’s more, it was noteworthy that after triggered by NIR, there was significant sudden release, demonstrating that NIR-induced hyperthermia could accelerate drug release. 3.3.

Cellular Uptake and Intercellular ROS Detection

4T1 cells incubated with free ICG and DOX@GdMSNs-ICG-TSLs with the equivalent concentration of ICG (15 µg/mL) for 4 h were utilized to explore the intracellular uptake studies. It showed the ICG fluorescence of both free ICG and DOX@GdMSNs-ICG-TSLs were mainly distributed in the cytoplasm (Figure 5. a). We would like to underline that ICG fluorescence intensity of DOX@GdMSNs-ICG-TSLs + NIR group was the strongest compared with free ICG + NIR and DOX@GdMSNs-ICG-TSLs group. Combined with flow cytometry data in Figure S5, it demonstrated that DOX@GdMSNs-ICG-TSLs were easier to achieve cell internalization under the assists of lipid bilayer and NIR irradiation 39-42. ROS-sensitive probe DCFH-DA was used to detect the results of intercellular ROS. Different groups of 4T1 cells incubated with PBS, free ICG + NIR, DOX@GdMSNs-ICG-TSLs and DOX@GdMSNs-ICG-TSLs + NIR were presented in Figure 5. b. The green fluorescence intensity of DCFH-DA was much stronger for DOX@GdMSNs-ICG-TSLs + NIR group compared with free ICG + NIR group, owning to the higher cell internalization. By contrast, PBS and DOX@GdMSNsICG-TSLs groups without NIR irradiation showed no DCFH-DA fluorescence. In addition, the ICG fluorescence became much weaker after NIR irradiation for 5 min, while the fluorescence intensity of DCFH-DA was obviously enhanced, proving the production of 1O2 induced by ICG under NIR irradiation. 3.4.

Cellular Cytotoxicity Studies

15



MTT assay was applied to quantitatively evaluate the cytotoxicity of DOX@GdMSNs-ICGTSLs with different DOX and ICG concentrations (Figure 6. a). Cells were cultured with PBS, DOX@GdMSNs, ICG-TSLs + NIR and DOX@GdMSNs-ICG-TSLs + NIR, respectively. The 4T1 cell viabilities were decreased with the raise concentration of DOX or ICG. DOX@GdMSNs-ICGTSLs + NIR experienced the most optimal efficiency to damage cancer cells compared with other groups. There were significant differences between solo and synergistic therapy groups under higher concentration, revealing the topping effect of combined phototherapy and chemotherapy . To intuitively evaluate the efficiency of DOX@GdMSNs-ICG-TSLs, calcein-AM and PI costained study was performed (Figure 6. b). 4T1 cells were treated with PBS, DOX@GdMSNs, ICG-TSLs + NIR and DOX@GdMSNs-ICG-TSLs + NIR, respectively. Red and green spots presented dead and living cells, respectively. It was remarkably revealed the formulation (DOX@GdMSNs-ICG-TSLs + NIR) with the synergistic therapy effect had the best behavior of anti-tumor cells. In addition, we used flow cytometry experiment to further quantify the extent of cell apoptosis by Annexin-V/PI Apoptosis Detection kit (Figure 6. c). Non-viable apoptotic or necrotic cells were generally definited to be situate in double positive (PI +/Annexin V+) quadrant. Cells treated with DOX@GdMSNs-ICG-TSLs + NIR groups exhibited the most non-viable apoptotic or necrotic cells (74.3%), showing extremely strong effect to kill cancer cells. 3.5.

In vivo NIRF, PA and MR Imaging

An NIRF imaging system was applied to monitor the in vivo biodistribution of DOX@GdMSNsICG-TSLs at various time points (Figure 7. a). As time went by, DOX@GdMSNs-ICG-TSLs gradually accumulated in the tumor sites at 6 h, reaching the maximal degree at 24 h. Nevertheless, the fluorescence of free ICG group at tumor site nearly disappeared after 12 h, indicating the gradual drugs elimination. These results demonstrated that DOX@GdMSNs-ICG-TSLs had 16


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favorable targeting tumor efficiency and tumor aggregation effect. Additionally, the fluorescence biodistribution of excised tumor tissues and organs was detected with the quantitative data (Figure 7. a, b), which also supported the above conclusions. We could observe from the results intuitively that DOX@GdMSNs-ICG-TSLs were mostly accumulated in the tumor position, by contrast, free ICG was eliminated by the liver quickly. PA imaging as an emerging biomedical imaging modality was also utilized to explore the biodistribution of DOX@GdMSNs-ICG-TSLs. PA signals of the optimal formulation with different concentrations were acquired based on the PA effect induced by NIR light absorption and the subsequent thermal expansion

43

. With the increasing of ICG concentration, PA signal was

enhanced gradually (Figure 7. c). The linear relationship curves between concentration of DOX@GdMSNs-ICG-TSLs and PA intensity was shown in Figure S6. a, What’s more, PA images of free ICG and DOX@GdMSNs-ICG-TSLs at different time points (6 h and 24 h) was exhibited in Figure 7. d. The ICG fluorescence intensity at tumor sites of free ICG treated groups was strong at 6 h, however, the signal sharply decreased at 24 h. As for DOX@GdMSNs-ICG-TSLs group, the fluorescence signal was much stronger at 24 h, showing good efficiency of tumor accumulating as well as outstanding function of PA imaging. To shed more light on the biodistribution performance of DOX@GdMSNs-ICG-TSLs, MR imaging was implemented. Figure 7. f showed T1-weighted MR images of various concentrations of DOX@GdMSNs-ICG-TSLs nanoparticles in water. It could be observed that the MR signal intensity for the optimal formulation of different Gd concentrations was not identical. With increasing Gd concentration in water, the MR signal was enhanced significantly. By calculating the relaxivity r1 (r1 = 25.55 mM-1s-1) according to the linear relationship curves between concentration of DOX@GdMSNs-ICG-TSLs and 1/T (s-1) in MR detection, it showed that our optimal formulation performed satisfactory contrast enhancement (Figure S6. b). What’s more, after injecting our optimal formulation for 24 h, the MR signal enhanced apparently, showing 17



outstanding MRI function of DOX@GdMSNs-ICG-TSLs (Figure 7.f). The quantized data in Figure 7. g further clarified the conclusion, that the nanocomposite was feasible as a safe and effective MRI contrast agent. 3.6.

Therapeutic Efficacy Studies

The excellent photothermal property encouraged us to investigate the in vivo PTT effect of the nanocomposite (Figure 8. a). DOX@GdMSNs-ICG-TSLs with other control formulations were intravenously injected into 4T1 tumor-bearing mice, followed by exposure to 808 nm laser (1.5 W/cm2) irradiation on the tumor sites. In vivo thermal images of full-body were captured using an IR camera at different time intervals. By comparing the obtained thermal images, the temperature in the tumor site of DOX@GdMSNs-ICG-TSLs treated group increased rapidly over 50 °C within 4 min (the maximal temperature about 55 °C). Its ability of temperature raise within short time was prominent compared with other groups, which demonstrated the nanocomposite was capable to give its play to PTT effect to kill tumor cells

44

. We further adopted immunofluorescence of tissue

section to study ROS generation results (Figure S7.). DOX@GdMSNs-ICG-TSLs + NIR treated group exhibited distinct green fluorescence which presented ROS generation. Nevertheless, free ICG + NIR showed weaker green fluorescence intensity. Besides, DOX@GdMSNs-ICG-TSLs without NIR irradiation revealed the intratumoral distribution. These inspiring observations confirmed that DOX@GdMSNs-ICG-TSLs with NIR irradiation experienced owned favorable ability to generate ROS. In the therapeutic efficacy and in vivo toxicity study, relative tumor volume change curve (Figure 8. b) gave the results that the combination therapy formulation performed the best behavior to suppress tumor growth. The tumor volume of the group which treated with PBS increased sharply with 20 days. The relative tumor volume of PBS, DOX@GdMSNs, ICG-TSLs + NIR and DOX@GdMSNs-ICG-TSLs + NIR groups after 20 days were 10.67, 5.03, 5.50 and 1.75, respectively. There was significant difference between combined and mono-therapy (**P < 0.01). 18


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Hence, compared with PBS group, monotherapy groups (DOX@GdMSNs and ICG-TSLs + NIR treated groups), DOX@GdMSNs-ICG-TSLs + NIR had better effect to control tumors. It was worth to note that the variation trend of tumor volume in DOX@GdMSNs-ICG-TSLs + NIR treated group showed rather stable and a little declining, indicating the best suppress tumor effect. The visualized photographs of tumor tissues treated with different formulations and conditions were shown in Figure 8. c. We could observe the results were consistent to the relative tumor volume change curve. Beyond that, mice body weight change curves (Figure 8. d) and percent survival curves (Figure 8. e) were also used to explore the therapeutic efficacy and toxicity. Compared with obvious changes and loss of body weight in PBS, DOX@GdMSNs and ICG-TSLs + NIR treated groups, DOX@GdMSNs-ICG-TSLs + NIR treatment expressed the relatively stable and rise tendency of body weight. As seen with percent survival curves, our optimal treatment performed the satisfactory survival rate. As supplementary evidence, photographs of tumor-bearing mice treated with DOX@GdMSNs-ICG-TSLs + NIR and control group was shown in Figure S8. Compared with PBS treated group, mice treated with DOX@GdMSNs-ICG-TSLs + NIR had obvious ablation of tumor, validating the commendable anti-tumor effect. To further evaluate the antitumor efficacy and in vivo toxicity, we used histological examination by hematoxylin-eosin (H&E) staining (Figure 8. f). Compared with control group, DOX@GdMSNs-ICG-TSLs + NIR treatment showed no significant adverse effect to the major organs including heart, liver, spleen, lung and kidneys. Whereas, there were apparent extensive tumor necrosis and intercellular space decrease in tumor tissue section, showing favorable antitumor effect of DOX@GdMSNs-ICG-TSLs. 4. Conclusions In summary, a multifunctional theranostic nanocomposite with multimodal imaging and combined therapy to a collective way has been created. DOX@GdMSNs-ICG-TSLs nanoplatform accomplished NIRF, PA and MR triple-model imaging to diagnosis. Meanwhile, we endowed the 19



nanocomposite phototherapy effect which contained PDT and PTT, as well as chemotherapy to enhance anti-tumor efficacy. By coating FA modified TSLs, it enabled active target delivery to treat tumor cells and enhanced intracellular uptake, as well as being a gate keeper to control drug release. It was worth emphasizing that DOX@GdMSNs-ICG-TSLs nanoplatform loaded with DOX and ICG displayed NIR laser-triggered controllable programmed chemo- and photo-therapy. The in vitro and in vivo experiment results exhibited DOX@GdMSNs-ICG-TSLs nanoplatform of excellent antitumor efficiency and satisfactory imaging effect of NIRF, PA and MRI. The design of multifunctional theranostic nanocomposite could pin our hope on it to be a powerful theranostic candidate for multimodel imaging-guided chemo- and photo-therapy for destruction of tumors. Supporting Information Calculation of the photothermal conversion efficiency, PDI value, photographs of different samples, TEM images, comparasion data of particle size, temperature and UV absorbance changes of DPBF, release curves, flow cytometry histogram, linear relationship curves about PA and MR, ROS production test, and photographs of tumor-bearing mice Acknowledgements We quite acknowledge financial supports from National Natural Science Foundation of China (81503016), Application Foundation and Cutting-edge Technologies Research Project of Tianjin (Young Program) (15JCQNJC13800), National Basic Research Project (973 Program) of China (2014CB932200), and Peiyang Young Talent Fund of Tianjin University (1701).

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Figures

Figure 1. (a) Schematic illustration of the preparation procedure and (b) functions of DOX@GdMSNs-ICGTSLs theranostic nanocomposite for fluorescence/photoacoustic/magnetic resonance imaging-guided chemoand phototherapy.

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Figure 2. TEM imaging of (a) DOX@GdMSNs and (b) DOX@GdMSNs-ICG-TSLs. Scale bars: 200 nm. SEM imaging of (c) DOX@GdMSNs and (d) DOX@GdMSNs-ICG-TSLs. Scale bars: 200 nm. The energy dispersive X-ray analysis (EDX) of DOX@GdMSNs-ICG-TSLs for oxygen, silicon and gadolinium were shown from panels (f-h) for the corresponding bright field TEM image in panel (e). Scale bars: 50 nm.

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Figure 3. Characterization of DOX@GdMSNs-ICG-TSLs and some other relevant formulations. (a) Size distribution of

the optimal formulaton. (b) UV spectra of free ICG, free DOX, GdMSNs and

DOX@GdMSNs-ICG-TSLs with equivalent ICG concentration. (c) IR spectra of GdMSNs before and after removing CTAB. X-ray diffraction (XRD) spectra of (d) GdMSNs and the control groups (e) MSNs and Gd2O3. (f) N2 adsorption-desorption isotherms of GdMSNs.

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Figure 4. (a) Temperature change curves of PBS, GdMSNs, DOX@GdMSNs, ICG-TSLs and DOX@GdMSNs-ICG-TSLs. (b) Infrared thermographic images of various formulations under 808 nm laser (1.5 W/cm2) irradiation. (c) Photothermal effect of the irradiation of the aqueous dispersion of DOX@GdMSNs-ICG-TSLs for 7 min with an NIR laser (808 nm, 1.5 W/cm2) and then the laser was shut off. And linear time data about the relationship between Time (s) and –lnθ from the cooling period versus negative natural logarithm of driving force temperature. (d) Change curves of the UV absorbance of DPBF at 410 nm in different solutions during 480 s under 808 nm laser (1.5 W/cm2). The free ICG, ICG-TSLs and DOX@GdMSNs-ICG-TSLs were in the equivalent ICG concentration (15 µg/mL). (e) Accumulative drug release of DOX. NIR laser (808 nm, 1.5 W/cm2) was given at 2 h and 6 h. 29



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Figure 5. (a) CLSM images of 4T1 cells incubated with free ICG + NIR, DOX@GdMSNs-ICG-TSLs and DOX@GdMSNs-ICG-TSLs + NIR for 12 h. Scale bar: 50 µm. NIR: 808 nm, 1.5 W/cm2 , 1 min. (b) CLSM images of 4T1 cells treated with PBS, free ICG + NIR, DOX@GdMSNs-ICG-TSLs and DOX@GdMSNsICG-TSLs + NIR for ROS detection. NIR: 808 nm , 1.5 W/cm2, 5 min. Scale bar: 50 µm.

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Figure 6. (a) Cell viability studies by MTT assay of 4T1 cells after incubation for 24 h with various formulations at different concentrations under NIR laser irradiation (808 nm laser, 1.5 W/cm2, 5 min). (b) CLSM images of PI and AM co-stained 4T1 cells treated with PBS and different formulations with or without irradiation. The live cells stained green and dead cells stained red. Scale bar: 100 µm. (c) Flow cytometry analysis of 4T1 cells apoptosis induced by various formulations for 12 h with NIR laser irradiation (1.5 W cm−2, 808 nm, 5 min) by using Annexin V-FITC/PI staining. Data are presented as means ± SD (n = 3), **P < 0.01.

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Figure 7. (a) In vivo NIR fluorescence images after intravenous injection of free ICG and DOX@GdMSNsICG-TSLs in tumor-bearing mice at 2 h, 6 h, 12 h, 24 h, 48 h. And the corresponding fluorescence imaging of different tissues after treated with Free ICG and DOX@GdMSNs-ICG-TSLs at 24 h. (b) Relative fluorescence intensity of ICG in major organs induced by 808 nm laser (1.5 W/cm2) irradiation at 24 h after 33



i.v. administration. (c) US and PA images of DOX@GdMSNs-ICG-TSLs at various concentration of ICG. (d) PA images of tumor-bearing mice after 6 h and 24 h intravenous injection via tail of free ICG and DOX@GdMSNs-ICG-TSLs, respectively. (e) PA intensity of tumor sites after treated with Free ICG and DOX@GdMSNs-ICG-TSLs at 6 h and 24 h. (f) T1-weighted MR images (7T, spin-echo sequence: repitition time TR = 500 ms, echo time TE = 14.92 ms) of DOX@GdMSNs-ICG-TSLs nanoparticles at various Gd concentration. And T1-weighted MR images of tumor-beating mice before and after injected with DOX@GdMSNs-ICG-TSLs for 24 h. (g) The relative MR intensity before and after injecting DOX@GdMSNs-ICG-TSLs. Data are presented as means ± SD (n = 5); *P < 0.05, **P < 0.01.

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Figure 8. (a) Thermographic images of mice post injection of various formulations under 808 nm laser (1.5 W/cm2) irradiation during 4 min. (b) Relative tumor volume curves of different groups (n=10) after various treatments. (c) Photographs of tumor tissues peeled from groups treated with different formulations after 4 weeks. (1): PBS; (2): DOX@GdMSNs; (3) ICG-TSLs + NIR; (4) DOX@GdMSNs-ICG-TSLs + NIR. (d) Body weight change of nude mice bearing 4T1 tumors as a function of days post treatment for various groups (n=10). (e) Percent survival for different treatment groups (n=10) during 4 weeks. (f) Histology staining. Hematoxylin and eosin (H&E) staining of major organs and tumors of 4T1 tumor-bearing mice with various formulations indicated with PBS and DOX@GdMSNs-ICG-TSLs (808 nm, 1.5 W/cm2, 5 min). Scale bar: 50 µm.

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Theranostic Nanoplatform: Triple-Modal Imaging-Guided Synergistic

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