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Selenium nanocomposites as multifunctional nanoplatform for imaging guiding synergistic chemo-photothermal therapy
Accepted Manuscript Title: Selenium nanocomposites as multifunctional nanoplatform for imaging guiding synergistic chemo-photothermal therapy Authors: Xijian Liu, Yeying Wang, Qiyang Yu, Guoying Deng, Qian Wang, Xinhui Ma, Qiugen Wang, Jie Lu PII: DOI: Reference:
S0927-7765(18)30162-0 https://doi.org/10.1016/j.colsurfb.2018.03.018 COLSUB 9219
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
22-7-2017 15-2-2018 14-3-2018
Please cite this article as: Xijian Liu, Yeying Wang, Qiyang Yu, Guoying Deng, Qian Wang, Xinhui Ma, Qiugen Wang, Jie Lu, Selenium nanocomposites as multifunctional nanoplatform for imaging guiding synergistic chemo-photothermal therapy, Colloids and Surfaces B: Biointerfaces https://doi.org/10.1016/j.colsurfb.2018.03.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Selenium nanocomposites as multifunctional nanoplatform for imaging guiding synergistic chemo-photothermal therapy
Xijian Liu,a,‡,*,Yeying Wang,a,‡ Qiyang Yu, a Guoying Deng,b Qian Wang,b Xinhui Ma,c Qiugen Wang b and Jie Lu a,* a
Corresponding Authors
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*E-mail: [email protected]; *E-mail: [email protected] Notes ‡
These authors contributed equally to the paper.
The authors declare no competing financial interest.
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Supporting Information
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Additional figures are presented in the supporting information.
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Graphical Abstract
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College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai, 201620, China. Email: [email protected]; E-mail: [email protected] b Trauma Center, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, NO.650 Xin Songjiang Road, Shanghai, 201620, China. c Shanghai Huaxi Chemical Industry Science & Technology Co.,Ltd. No.555, Huangqiao Road,Shanghai, 201315, China.
Highlights ► The nanocomposite exhibited excellent fluorescence imaging performance. ► The nanocomposite as carrier for co-loading ICG and DOX. ► Triple treatment of photothermal therapy and chemotherapy for selenium and DOX. ► The nanocomposite as a nanoplatform for multimodal imaging guiding synergistic treatment.
Abstract: A multifunctional selenium nanocomposite (selenium@silica core-shell nanoshperes
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for loading indocyanine green(ICG)/Doxorubicin(DOX)) was fabricated to reach visible and efficient cancer treatment. The Se@SiO2-ICG nanocomposites could be used not only as
excellent photothermal agents but also as carriers for DOX delivery. In addition, the Se@SiO2-
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ICG/DOX nanocomposites exhibited excellent fluorescence imaging and infrared imaging
performance. Tumor could be effectively inhibited by Se@SiO2-ICG/DOX due to the triple treatment of photothermal effect and chemotherapy of selenium and DOX. Thus, the Se@SiO2ICG/DOX nanocomposites have a great potential in imaging guiding synergistic treatment of
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cancer.
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Keywords: photothermal therapy, selenium nanocomposites, drug delivery, fluorescence imaging,
1 Introduction
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synergistic treatment
Selenium is an essential trace element for the human body which plays a vital role in most
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of the biochemical and physiological processes.[1-3] Selenium deficiency will accompany with the risk of many health problems and multifarious cancers.[2] These studies
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demonstrated selenium compounds could adjust the level of intracellular reactive oxygen species (ROS) to induce apoptosis of cancer cells.[4-6] Among selenium compounds, the selenium nanoparticles have attracted more attention due to chemical stability, biocompatibility, and low toxicity[7-10]. Recently, the combination of selenium
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nanoparticles with anticancer drugs were studied, which exhibited anticancer synergism due to the fact that selenium nanoparticles sensitize cancer cells for drugs[11-16]. In addition, selenium nanoparticles can decrease the damages of anticancer drugs to normal tissues, such as heart, liver and kidney.[17-19] Especially, Se not only reduces the side effects associated with DOX, but also increases its antitumor activity.[16,19,23] However, selenium nanoparticles have many limitations in clinical treatment due to their narrow margin between the beneficial and toxic effects.[20] Our previous studies have 2
preliminarily explored selenium release (porous Se@SiO 2) system to realize safe treatment for cancer.[21] Selenium can inhibit cancer cell at a long time, but it is not so effectively in eliminating solid tumor at short period. So, it has a great need for selenium nanoparticles combining other methods to eliminate tumor directly. Photothermal therapy (PTT), a minimally invasive surgical technology, uses light-absorbing nanomaterials(PTT agents) to convert NIR energy into heat, which can directly ablate the tumor.[22-25] Among the PTT agents, ICG is a FDA approved dye, which is regarded as promising PTT agent due to its low cytotoxicity and excellent photothermal properties[26, 27].
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ICG can also be used as fluorescence imaging agents owing to NIR fluorescence absorption
around 800 nm. Because of poor photo-stability of free ICG, ICG always was encapsulated inside nanocarriers to improve its performance as a PTT agent and molecular imaging probe[28-31]. It
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will has an excellent instant anticancer effect for ICG and long term anticancer effect for selenium nanoparticles when the selenium nanoparticles were also encapsulated in the ICG nanocarriers. Nevertheless, to our best knowledge, up to now, no studies about the combination of
chemotherapy of selenium nanoparticles and photothermal therapy(ICG) had done. Thus, it is very
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essential to study the synergistic treatment about selenium and ICG.
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Here, we fabricated a multifunctional nanocomposites (Se@SiO2-ICG/DOX) which
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could be used for multimodal imaging (fluorescence imaging and infrared imaging) guiding synergistic treatment (photothermal therapy and chemotherapy of selenium and
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DOX). As shown in Scheme 1, first, the Cu2-xSe nanocrystals were synthesized, then they were oxidized to form Se@SiO2 core-shell nanospheres under microemulsion conditions.
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Afterwards, the Se@SiO2 nanospheres were treated with hot water in the presence of PVP, then the porous Se@SiO2 nanospheres were formed. The anticancer drug (DOX) and photothermal agent (ICG) could be encapsulated into the porous Se@SiO 2 nanospheres to
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form Se@SiO2-ICG/DOX nanocomposites. After the nanocomposites were intravenously injected to the tumor-bearing mice, their pharmacokinetics and biodistribution could be
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tracked in vivo. When the nanocomposites were highly enriched in the tumor site, the photothermal therapy were driven by NIR irradiation, which could efficiently ablate tumor. At the same time, DOX and Se released from the nanocomposites for synergetic chemotherapy, which could kill tumor cells and inhibit their metastasis for a long time. Therefore, the tumor cells can be effectively inhibited and killed due to the triple action of
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photothermal therapy as well as chemotherapy of selenium and DOX. Thus, the Se@SiO2ICG/DOX nanocomposites have a great potential in multimodal imaging guiding synergistic treatment of cancer.
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Scheme 1 Schematic of the synthesis and application of Se@SiO 2-ICG/DOX. 2. Experimental Section 2.1. Chemicals and reagents
All reagents were used without further purification. Copper (I) chloride (CuCl), ammonium
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hydroxide (25%-28%), anhydrous ethanol, polyvinyl pyrrolidone (PVP, K30) are
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analytically pure, which were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China), and tetraethylorthosilicate (TEOS, GR) , selenium powders, oleic acid,
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oleylamine (approximate C18 from 80-90%) were obtained from Aladdin. ICG
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(indocyanine green) was got Meclin Biochemical Technology Co., Ltd. (Shanghai, China), and doxorubicin hydrochloride (DOX) was obtained from Huafeng United Technology
2.2. Characterization
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CO., Ltd. (Beijing, China).
Sizes and morphologies of the nanoparticles were measured by a transmission electron
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microscope (TEM) using JEM-2100F microscope. Optical absorbances of colloidal nanoparticles were measured by a Phoenix 1901 UV-visible-NIR Spectrophotometer. Xray diffraction (XRD) was measured by a D/max-2550 PCX-ray diffractometer (Rigaku,
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Japan).
2.3 Synthesis of Se@SiO2 core-shell nanospheres First, Cu2-xSe nanocrystals were synthesized according to the previous paper.[32] Briefly, the mixtures of 39.5 mg selenium powders and 5 mL oleic acid (OA) were heated to 120
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°C in nitrogen atmosphere for 30 min under stirring. Subsequently, the mixtures were heated to 220 ℃ for formation of Se-OA precursor. At the same time, the mixtures of oleylamine (OAM, 5 mL), OA(5 mL) and CuCl(49.5 mg) were heated to 120 °C for removing moisture. Subsequently, the mixtures were heated to 220 °C, and the above SeOA precursor were quickly injected into the mixtures. And the resulting solution was reacted at 220 °C for 5 min. Finally, the Cu2-xSe nanocrystals were obtained by washing with ethanol, and they were dispersed in 10 mL of normal hexane for later use. 4
Se@SiO2 core-shell nanospheres were prepared according to our previous method. [21] The mixtures of n-hexane(30 mL), n-hexanol(3 mL), Triton X-100(3 mL), deionized water(0.9 mL) and above Cu2-xSe n-hexanol solution(3.3 mL) were stirred for 30 min. Then 0.15 mL TEOS and 0.20 mL aqueous ammonia were dropwise added successively with rapid stirring. The Se@SiO2 particles were obtained by centrifuging after stirring for 24 h. Then, the Se@SiO2 core-shell nanospheres were washed with ethanol three times for later use. 2.4 Synthesis of porous Se@SiO2 core-shell nanospheres.
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The above Se@SiO2 core-shell nanospheres were dispersed in 25 mL of PVP (K30)
solution (10g/L) with stirring for 1h. Then the stir was stop and the temperature of the
mixtures was increased to 95°C and maintained for 2.0 h in standstill condition. Finally,
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porous Se@SiO2 core-shell nanospheres were collected by centrifuging and washing with water three times. 2.5 The Loading and Release performance.
The porous Se@SiO2 nanospheres PBS dispersions (2mg/L, pH 7.4 and 5.0, respectively)
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were vibrated in a shaker at 37 °C. Then the dispersions were centrifuged in a certain
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interval, and the supernatant were filtered by 0.22µm filter for measuring the Se by Leeman ICP-AES Prodigy. The bottom porous Se@SiO2 nanospheres were dispersed in
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fresh PBS solutions with ultrasonication for next analysis.
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2.5 mg of porous Se@SiO2 core-shell nanospheres were mixed with 2 mL DOX aqueous solution (200 µg·mL-1) and 2 mL ICG aqueous solution （200 µg·mL-1）. After the mixtures were stirred for 24 h at room temperature in darkness, the DOX and ICG co-
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loaded Se@SiO2 core-shell nanospheres (Se@SiO2-ICG/DOX nanocomposites) were collected by centrifugation and washed with water two times to remove unbound DOX and
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ICG. The supernatant solution was collected and measured by the 1901 UV-visible-NIR spectrophotometer at 490 nm and 780 nm to calculate the amount of DOX and ICG loading in the nanospheres, respectively.
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The release of ICG and DOX from Se@SiO2-ICG/DOX nanocomposites with or without
NIR laser irradiation were also studied. 2.5 mg of Se@SiO2-ICG/DOX nanocomposites were dispersed in 5.0 mL PBS solutions at pH 5.0 and pH 7.4, respectively. The dispersions were stirred under room temperature and centrifuged in a certain time interval.
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The supernatants were collected, and the bottom Se@SiO2-ICG/DOX nanocomposites were re-dispersed in 5.0 mL fresh PBS solutions under the ultrasonication. In the group of laser irradiation, Se@SiO2-ICG/DOX nanocomposites were additionally irradiated by the NIR laser (1.0 W·cm-2, 808 nm) in a certain time interval (NIR irradiation for 5 min per hour from 0 to 4 hour, and NIR irradiation for 5 min per two hours from 4 to 12 hour). The amounts of released ICG and DOX in the PBS solutions (pH 5.0 and 7.4) were measured by UV-vis-NIR spectrophotometer (480 nm). 5
2.6 Photothermal performance measurement. Aqueous solutions of Se@SiO2-ICG nanocomposites at different concentrations of 0, 25, 50, 100, 200, and 400 µg·mL-1 (the ICG loading efficiency was 10%), and free ICG (40 µg·mL-1) were respectively irradiated by an 808 nm NIR laser (Sfolt Co., Ltd., Shanghai, China) for 5 min with a power density of 1.0 W·cm-2. The temperature changes were recorded per second using a thermal imaging camera (FOTRIC 225) with an accuracy of ±1 °C. 2.7 Cell culture and viability measurements.
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For cell viability measurements, human endothelial cells and HeLa cells were seeded in a 96-well plate and cultured in 5% CO2 at 37 oC for 24 h. The culture mediums were
replaced and cells were incubated with complete mediums containing PBS, ICG, DOX,
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porous Se@SiO2, Se@SiO2-ICG and Se@SiO2-ICG/DOX at a series of concentrations for 6 h. For the NIR treatment group, the cells were irradiated by laser for 5 min (808nm, 1.0 W·cm-2). Then all the cells were further incubated for 18 h. Finally, cell viabilities were
measured by Cell Counting Kit-8 system (CCK-8) according to manufacture's instructions.
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2.8 In Vivo fluorescence imaging.
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HeLa tumor bearing mice were injected with 100 μL aqueous solution containing Se@SiO2-ICG/DOX nanocomposites(2 mg·mL-1, ICG efficiency was 10%, DOX loading
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9%) and free ICG (0.2 mg·mL-1) through intravenous injection, respectively. The mice
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were imaged using a real-time in vivo fluorescence imaging system (Fluoptics FB800) at a certain interval.
2.9 In vivo chemo-photothermal Therapy.
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HeLa tumor bearing mice were from Shanghai General Hospital. When tumors reached about 7~9 mm in diameter, the mice were randomly divided into seven groups: (1) control
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group, (2) PBS+NIR group, (3) DOX group, (4) Se@SiO 2-ICG group, (5) Se@SiO2ICG/DOX group, (6)Se@SiO2-ICG + NIR group, (7) Se@SiO2-ICG/DOX + NIR group. Except for the control group, 100 μL PBS (pH=7.4), DOX (180 µg·mL -1), Se@SiO2-ICG
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(2 mg·mL-1, ICG loading 10%), Se@SiO2-ICG/DOX (2 mg·mL-1, ICG loading 10%, DOX loading 9%) were injected intravenously into the tumor-bearing mice. At 8 hours after injection, the tumor regions of NIR irradiation groups were exposed to NIR light radiation (808 nm, 1 W·cm-2) for 10 min and the thermal images were recorded by a FOTRIC 225
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thermal imaging camera. Before and after treatment, tumor size was measured by a vernier caliper every other day. Tumor volume was calculated in accordance with the following formula: tumor volume = length × width2/2. Relative tumor volume was calculated as V/V 0 (V0 was the initiate tumor volume before treatment). Body weights of each mouse were also recorded every other day.
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All animal experiments were carried out according to protocols approved by the institutional committee for animal care and also in accordance with the policy of the National Ministry of Health. 2.10 Statistical analysis. All data were presented as the mean ± standard deviation (SD). The differences among groups were determined using one-way ANOVA analysis: (*) P < 0.05, (**) P < 0.005,
3 Results and Discussion 3.1 Synthesis and characterization of porous Se@SiO2 nanospheres
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(***) P< 0.001.
The silica coated selenium nanospheres (Se@SiO 2 ) were prepared according to our
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previous method.[21] The XRD patterns of the Se@SiO2 samples are presented in Fig. 1a. All the diffraction peaks can readily correspond to standard hexagonal phase of selenium (JCPDS card no: 65-1876). The TEM image (Fig. 1b) indicates that Se@SiO 2 have
spherical structure with an average diameter of ~ 55 nm, and many small Se particles
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interspersed in the nanospheres. The high-magnified TEM further shows selenium particles
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are irregular and the size of most of them are less than 5 nm(Fig. 1c). After Se@SiO2 nanospheres were treated with water in presence of polyvinyl pyrrolidone (PVP), the
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porous Se@SiO2 nanospheres were successfully obtained(Fig. 1d). As shown in Fig.S1,
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Se@SiO2 nanospheres and porous Se@SiO2 nanospheres have dynamic light scattering (DLS) size of ~83 nm and ~92 nm, respectively, and they demonstrate a narrow polydispersity. The isotherm of porous Se@SiO2 nanospheres after hot water etching
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shows a typical type IV feature (Fig.S2). The Brunauer-Emmet-Teller (BET) surface area and total pore volume of porous Se@SiO2 nanospheres are 152.8 m²/g and 0.3945 cm³/g,
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respectively. The pores of the nanospheres show a broad distribution from 2.0 nm to 43.2 nm. Especially, the pore distribution have a peak at 23.8 nm, which is more than four times of Se particles. It is suitable for release of nano selenium particles. The BET results further
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indicate that the as-prepared porous Se@SiO2 nanospheres possess mesoporous channels, in agreement with the TEM observation in Fig. 1d. The Fourier transform infrared spectroscopy (FTIR) spectrum of porous Se@SiO 2 nanospheres exhibit characteristic peaks of PVP, such as 2925 and 2850 cm−1 and 1640 cm−1(Fig.S3),[21] indicating the
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successful coating of PVP on the nanospheres. Due to good biocompatibility and aqueous solubility of PVP, the colloid stability and biocompatibility of porous Se@SiO 2 nanospheres could be improved.
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Fig. 1 (a) XRD pattern of the Se@SiO2 sample, (b) Low-magnified (c) and high-
magnified TEM of Se@SiO2 nanospheres, (d) TEM of porous Se@SiO2 nanospheres.
3.2 Photothermal performance of Se@SiO2-ICG nanocomposites
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The porous Se@SiO2 nanospheres could be used as carrier due to the porous structures.
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After the mixtures of porous Se@SiO2 core-shell nanospheres and ICG aqueous solution were stirred for 24h, Se@SiO2-ICG nanocomposites were collected by centrifugation and
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washed with water two times to remove ICG. The FTIR spectrum showed Se@SiO2-ICG
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nanocomposites exhibited the characteristic peaks of ICG, indicating successful incorporation of ICG. As shown in Fig. S4, Se@SiO2-ICG nanocomposites demonstrate a strong NIR absorption peak around 808 nm, which endows the Se@SiO 2-ICG
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nanocomposites as a good photothermal agent. Moreover, ICG encapsulated in carriers
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would be more stable than free ICG. [33, 34]
Fig. 2 (a)Temperature changes of the Se@SiO2-ICG nanocomposites dispersions(ICG loading efficiency was 10%) at different concentrations (0 to 400µg·mL-1) and free ICG dispersions (40 µg·mL-1) under the irradiation(808nm, 1.0 W/cm2). (b)Temperature elevations of the Se@SiO2-ICG nanocomposite dispersions at various concentrations(ICG loading efficiency was 10%) under the irradiation (808nm, 1.0 W/cm2) for 5 min. 8
Given the strong absorption in the NIR region, we further evaluated the photothermal conversion properties of Se@SiO2-ICG aqueous dispersions. Dispersions of Se@SiO2-ICG nanocomposite at different concentrations (ICG loading efficiency was 10%) and free ICG (40 µg·mL-1) were respectively irradiated by an 808 nm NIR laser for 5 min with a power density of 1.0 W·cm-2 in room conditions(room temperature was 24.8 °C). As shown in Fig. 2a, the control of pure water demonstrated a negligible temperature increase under NIR irradiation. However, all the Se@SiO2-ICG dispersions exhibited remarkable temperature elevation at the same conditions, suggesting that the Se@SiO 2-ICG could
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efficiently convert the NIR energy into local hyperthermia upon irradiation. The Se@SiO 2ICG dispersions showed a dose-dependent photothermal effect(Fig.2b). If the body temperature is 37 °C , the temperature can easily attain the temperature for the
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hyperthermia therapy(40~45 °C),[35] even at the concentration of Se@SiO2-ICG as low as 50 μg·mL-1 (temperature elevation is 8.8 °C). Additionally, the Se@SiO2-ICG
dispersion had a higher temperature rise than free ICG solution under the same laser irradiation and the same ICG concentration of 40 µg·mL -1 due to shift of NIR absorption
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peak and improvement of stability, according to previous studies[33, 34].
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3.3 Loading and release performance
We also evaluate the Se release from the porous Se@SiO2 nanospheres. As shown in Fig.
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S5, ~0.66% and ~0.85% Se were released from the porous Se@SiO2, which could improve
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the immunity and prevent the cancers.[3, 5] When anticancer drug was loaded into porous Se@SiO2, DOX and Se could simultaneously release to synergistically kill cancer cells. Porous Se@SiO2 nanospheres could also be used for drug delivery due to the porous
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structure. DOX and ICG were co-loaded into Se@SiO2 nanospheres by simple mixing with string under dark conditions. After removing unbound DOX and ICG molecules by
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centrifugation, Se@SiO2-ICG/DOX nanocomposites were obtained, which showed DOX and ICG absorption peaks(Fig.3a). The co-loading efficiency of the porous Se@SiO2 nanospheres can reach to as high as (by weight) 12.9% for ICG and 10.2% (by weight) for
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DOX.
The release of DOX from Se@SiO2-ICG/DOX nanocomposites in PBS buffers at pH 7.4
and pH 5.0 were studied(Fig. 3b). In pH 7.4 (normal tissue environment), DOX release rate was slow. While in pH 5.0, DOX release rate was accelerated due to enhanced water
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solubility of DOX in the acidic conditions.[36] Activated release in acidic conditions is beneficial to drug delivery because the extracellular environment of the tumor is acidic.[37] The excellent photothermal efficiency of Se@SiO 2-ICG/DOX nanocomposites could further promote the release of DOX. The cumulative release of DOX increased from 75.6% to 92.1% by NIR irradiation within 12 h. It can be owing to the heat-stimulative dissociation of interactions between hydroxyl of SiO2 and DOX.[37] The pH-sensitive and NIR-triggered release of DOX can greatly improve the chemo-photothermal therapy 9
effects for cancer cells. At the same time, we also tested the release of ICG from Se@SiO2-ICG/DOX nanocomposites. As shown in Fig.S6, there were no obvious differences of the ICG release profile between pH 5.0 and 7.4, but NIR irradiation could promote the release of ICG. After 12 h, the most amounts of ICG were still incorporated in the nanocomposites without NIR irradiation, which was beneficial for photothermal therapy and fluorescent imaging due to higher tumor accumulation for ICG incorporated in
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nanocomposites by EPR effect.
Fig. 3 (a) UV-Vis-NIR spectra of DOX, Se@SiO2-ICG and Se@SiO2-ICG/DOX aqueous
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dispersions. (b) Cumulative DOX release from the Se@SiO2-ICG/DOX nanocomposites in
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PBS buffer at pH 5.0 and 7.4 with or without NIR laser irradiation (1.0 W cm -2, 808 nm), respectively.
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3.4 In vitro synergistic chemo-photothermal therapy
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To investigate the biocompatibility, human endothelial cells (normal cells) incubated with porous Se@SiO2, Se@SiO2-ICG, ICG and DOX at various concentrations, respectively. As shown in Fig.4a, compared the control group, the cell viabilities of porous Se@SiO2,
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Se@SiO2-ICG(below 200 μg/mL) and ICG(below 2 μg/mL) groups had no obvious difference, indicating they had negligible toxicity for normal cells. However, the DOX
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demonstrated obvious toxicity for normal cells, which means it had toxic side effects for normal tissues. Motivated by the excellent photothermal effect, drug delivery performance and biocompatibility of Se@SiO2-ICG, we further explored in vitro therapeutic efficiency
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of DOX, ICG, porous Se@SiO2, Se@SiO2-ICG, Se@SiO2-ICG/DOX with or without irradiation by CCK-8 assay. As shown in Fig.S7 and Fig.4b, ICG demonstrated no appreciable cytotoxicity for HeLa cells. There were few differences of cell viabilities between porous Se@SiO2 and Se@SiO2-ICG. Compared to control group, the porous
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Se@SiO2 and Se@SiO2-ICG had obvious cytotoxicity to HeLa cells due to release of Se. Porous Se@SiO2, Se@SiO2-ICG and Se@SiO2-ICG/DOX exhibited increased cytotoxicity in a dose-dependent manner. The IC50 values of DOX and porous Se@SiO2 for endothelial cells are 35.96 μg/mL and 1041μg/mL, respectively, while the dose of DOX and porous Se@SiO2 for HeLa cells are 4.02 μg/ml and 352.3μg/ml, respectively. The results demonstrated HeLa cells were much more sensitive for DOX and porous Se@SiO2 than normal cells. Moreover, Se@SiO2-ICG/DOX exhibited enhanced cytotoxicity than porous 10
Se@SiO2 and Se@SiO2-ICG at all concentrations. Because the HeLa cells were inhibited by the double inhibition effect for both selenium and DOX to HeLa cells due to the fact that selenium nanoparticles sensitize cancer cells for drugs [16,19,23] To investigate therapeutic efficiency of the photothermal effect, the cells incubated with PBS, porous Se@SiO2, Se@SiO2-ICG and Se@SiO2-ICG/DOX were irradiated by NIR laser for 5 min. As indicated in Fig.4b and Fig.S7, it had no obvious negative effect for cell viability incubated with PBS(Control group) and porous Se@SiO2 by laser irradiation alone, indicating it is safe for HeLa cells under irradiation (808 nm, 1.0 Wcm -2) and porous
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Se@SiO2 had no obvious photothermal effect. While it led to a drastic decreased percent of cell viabilities in the presence of Se@SiO2-ICG and Se@SiO2-ICG/DOX with NIR
irradiation, which was ascribed to that the excellent photothermal effect to induce the cells
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death by Se@SiO2-ICG and Se@SiO2-ICG/DOX. In addition, Se@SiO2-ICG/DOX
exhibited significantly enhanced cell lethality than Se@SiO2-ICG in all the concentration due to the synergistic effect of photothermal therapy of ICG and chemotherapy of DOX and selenium. The synergistic effect of chemotherapy and photothermal therapy probably
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attributed to enhanced cytotoxicity of DOX at higher temperatures, and higher heat
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sensitivity for the cells exposed to the DOX.[38, 39]. Almost all the HeLa cells were killed by Se@SiO2-ICG/DOX under irradiation at concentration of 200 μg/mL. These results
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demonstrated Se@SiO2-ICG/DOX combined triple therapy ways (photothermal therapy of
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ICG and chemotherapy of DOX and selenium) into a nanocomposites, which could
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efficiently induce cancer cells to death.
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Fig.4 (a)Viability of human endothelial cells (normal cells) incubated with porous Se@SiO2 and Se@SiO2-ICG at various concentrations, respectively. (b)HeLa cells incubated with PBS, Se@SiO2-ICG and Se@SiO2-ICG/DOX nanocomposites at various concentrations, respectively, with or without NIR laser irradiation (808 nm, 1.0 W/cm 2, 5 min). 3.5 In Vivo fluorescence imaging 11
Encouraged by the above outstanding performance of Se@SiO 2-ICG/DOX nanocomposites in photothermal test and drug delivery, the imaging guided synergistic therapy was evaluated on HeLa xenograft model. As shown in Fig. 5, NIR fluorescence imaging was employed to track the in vivo pharmacokinetics and biodistribution of free ICG and Se@SiO2-ICG/DOX. In the group of mice injected with free ICG, at 2 h of postinjection, mice body showed scattered fluorescence for no target biodistribution. At 8 h of post-injection, spleen showed bright fluorescence, indicating major portion of ICG was quickly taken up by spleen. After 24 h of post-injection, no distinct fluorescence signals
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were observed in the mice body, which showed the rapid clearance and degradation of free ICG due to poor stability of free ICG.[30,31] In contrast, Se@SiO2-ICG/DOX
demonstrated a different biodistribution behavior. In the group of mice treated with
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Se@SiO2-ICG/DOX, the tumor site began to appeared bright fluorescence signals 2 h after injection, then the fluorescence signals were strengthened from 2 h to 8 h and maintained strong bright intensity until 24 h, which could be attributed to the EPR effect of
nanomaterials.[40] Subsequently, the fluorescence signals of tumor began to recede, and
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weak fluorescence signals could still be seen 72 h after injection. Compared with free
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ICG, Se@SiO2-ICG/DOX had much higher tumor accumulation and slower body clearance due to enhanced stability and EPR effect of encapsulated ICG. The fluorescence
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results suggested that Se@SiO2-ICG/DOX could efficiently accumulate in the tumor site
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and retain a long time in tumor, which can be used to diagnose and guide therapy.
Fig. 5 In vivo fluorescence imaging of HeLa xenograft mice intravenously injected by the dispersions of free ICG and Se@SiO2-ICG/DOX nanocomposites, respectively.
3.6 In vivo synergistic chemo-photothermal Therapy 12
Encouraged by the outstanding in vitro photothermal-chemotherapy performance of Se@SiO2-ICG/DOX nanocomposites, we further investigated their in vivo therapeutic efficiency. The HeLa xenograft mice were randomized into seven groups when the tumor size reached about 7-9 mm, and intravenously injected with PBS, DOX, Se@SiO 2-ICG and Se@SiO2-ICG/DOX. At 8 h after intravenous injection, the HeLa xenograft mice of NIR treatment groups were irradiated by 808 nm laser(1.0 W/cm 2) with 10 min. As shown in Fig.6a,b, the surface temperature of the tumour had no obvious rise in the control group of mice injected with PBS under irradiation for 10 min, indicating that it would have no
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perceptible hurt to normal tissue by the treatment. In contrast, the tumour temperature of
Se@SiO2-ICG + NIR and Se@SiO2-ICG/DOX + NIR group increased rapidly and reached 61.2 oC and 60.4 oC, respectively, which is sufficient for inducing cancer cell to death.[41]
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After received various treatments, the tumor sizes of seven groups were all measured every two days. As shown in Fig.6c and S8, the tumor sizes of control group mice increased very rapidly owing to rapid growth of cancer cells. And the tumor sizes of PBS+ NIR group mice increased like that of control group due to the fact that the heat generated by PBS
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under irradiation was not sufficient for killing cancer cells. Compared to control group, the
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tumor growths of Se@SiO2-ICG and DOX groups were inhibited in a certain degree due to the anticancer effects of selenium and DOX, respectively. The Se@SiO2-ICG/DOX
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showed a higher tumor inhibitory rate than Se@SiO 2-ICG and DOX for the synergistic
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effect of Se and DOX. The tumor growth were significantly inhibited in Se@SiO 2-ICG+ NIR group, attributing to the excellent photothermal effect of ICG and anticancer effect of selenium. Nevertheless, tumor margins or recesses may be not received sufficient
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irradiation, which resulted in failures in completely removing the tumor or tumor recrudescing after treatment. In order to achieve much enhanced efficacy, Se@SiO2-
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ICG/DOX plus NIR irradiation was carried out in tumor treatment. The tumors growth were severely inhibited and the tumors volume began to decrease after the treatment. Moreover, the tumor completely disappeared after 14 days’ post-treatment, which could be
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visually confirmed by the photos of different treatment groups(Fig.S8). Compared to previous similar studies(chemo-photothermal therapy of DOX and ICG),[28, 33, 42] Se@SiO2-ICG/DOX plus NIR showed enhanced therapy effects. The tumor of mice could completely eliminated by treatment of Se@SiO 2-ICG/DOX plus NIR, which were ascribed
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to the synergistic therapy of chemotherapy (DOX and selenium) and photothermal therapy. The photothermal effect could severely destroys tumor cells in a short time, while slow release of DOX and selenium can kill the residual tumor cells and efficiently restrain them to diffuse and regress in a long period. And, more remarkable, there none tumors reappeared and none mice died in Se@SiO2-ICG/DOX +NIR group after 60 days’ treatment. In addition, the body weight of the mice of all groups did not have noticeable
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changes during the treatment (Fig.6d), indicating the treatment had no obvious systematic
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toxicity.
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Fig. 6 (a) In vivo infrared thermal images and (b) tumor temperature changes of at different time points under NIR irradiation after intravenously injected with PBS, Se@SiO2-ICG
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and Se@SiO2-ICG/DOX, respectively. (c) Tumor growth curves and (d) body weight changes of different groups treatments.
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4 Conclusions
The porous Se@SiO2 nanocomposites were successfully prepared, which could be co-
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loaded for ICG and DOX. The Se@SiO2-ICG/DOX nanocomposites had excellent fluorescence imaging and infrared imaging performance. Tumors even could be completely eliminated by Se@SiO2-ICG/DOX due to the triple treatment of photothermal effect and chemotherapy of selenium and DOX. Thus, the Se@SiO 2-ICG/DOX nanocomposites have
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a great potential in multimodal imaging guiding synergistic treatment of cancer.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Nos. 21176102, 21176215, 21476136, 21171035, 51472049, 8152010831), Science and Technology Commission of Shanghai Municipality (No. 15430501200), the Sino-German Center for Research Promotion (No. GZ935), Innovation Program of Shanghai Municipal 14
Education Commission (No. 14ZZ160), Guidance Fund of Shanghai Committee of Science and Technology (No. 15411968800), the Connotation Construction Project of SUES (No.Nhky-2015-05), Shanghai Municipal Education for foreign visiting scholar Program.
References [1] K. Bierla, A. Flis-Borsuk, P. Suchocki, J. Szpunar, R. Lobinski, Speciation of Selenium in
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Selenium-Enriched Sunflower Oil by High-Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry/Electrospray-Orbitrap Tandem Mass Spectrometry, J. Agr. Food Chem., 64 (2016) 4975-4981.
SC R
[2] Y.F. Wang, J.L. Wang, H. Hao, M.L. Cai, S.Y. Wang, J. Ma, Y. Li, C.B. Mao, S.M. Zhang, In Vitro and in Vivo Mechanism of Bone Tumor Inhibition by Selenium-Doped Bone Mineral Nanoparticles, Acs Nano, 10 (2016) 9927-9937.
[3] C.D. Thomson, Assessment of requirements for selenium and adequacy of selenium status: a
U
review, Eur. J.Clin. Nutr., 58 (2004) 391-402.
[4] T. Li, F. Li, W. Xiang, Y. Yi, Y. Chen, L. Cheng, Z. Liu, H. Xu, Selenium-Containing
N
Amphiphiles Reduced and Stabilized Gold Nanoparticles: Kill Cancer Cells via Reactive Oxygen
A
Species, ACS Appl. Mater. Interfaces, 8 (2016) 22106-22112.
[5] Y. Wang, H. Hao, Y. Li, S. Zhang, Selenium-substituted hydroxyapatite nanoparticles and
M
their in vivo antitumor effect on hepatocellular carcinoma, Colloid. Surface. B, 140 (2016) 297306.
ED
[6] T. Nie, H. Wu, K. H. Wong, T. Chen, Facile synthesis of highly uniform selenium nanoparticles using glucose as the reductant and surface decorator to induce cancer cell apoptosis, J. Mater. Chem. B, 4 (2016) 2351-2358.
PT
[7] S. Skalickova, V. Milosavljevic, K. Cihalova, P. Horky, L. Richtera, V. Adam, Selenium nanoparticles as a nutritional supplement, Nutrition, 33 (2017) 83-90. [8] Y. He, S. Chen, Z. Liu, C. Cheng, H. Li, M. Wang, Toxicity of selenium nanoparticles in male
CC E
Sprague-Dawley rats at supranutritional and nonlethal levels, Life Sci., 115 (2014) 44-51. [9] J. Zhang, X. Wang, T. Xu, Elemental selenium at nano size (Nano-Se) as a potential chemopreventive agent with reduced risk of selenium toxicity: Comparison with Semethylselenocysteine in mice, Toxicol. Sci., 101 (2008) 22-31.
A
[10] P.D. Whanger, Selenium and its relationship to cancer: an update, Brit. J. Nutr., 91 (2004) 1128. [11] W. Liu, X. Li, Y.-S. Wong, W. Zheng, Y. Zhang, W. Cao, T. Chen, Selenium Nanoparticles as a Carrier of 5-Fluorouracil to Achieve Anticancer Synergism, Acs Nano, 6 (2012) 6578-6591. [12] F.P. Gao, Q. Yuan, L. Gao, P.J. Cai, H.R. Zhu, R. Liu, Y.L. Wang, Y.T. Wei, G.D. Huang, J. Liang, X.Y. Gao, Cytotoxicity and therapeutic effect of irinotecan combined with selenium nanoparticles, Biomaterials, 35 (2014) 8854-8866. 15
[13] Y. Huang, L. He, W. Liu, C. Fan, W. Zheng, Y.S. Wong, T. Chen, Selective cellular uptake and induction of apoptosis of cancer-targeted selenium nanoparticles, Biomaterials, 34 (2013) 7106-7116. [14] Y. Xia, P. You, F. Xu, J. Liu, F. Xing, Novel Functionalized Selenium Nanoparticles for Enhanced Anti-Hepatocarcinoma Activity In vitro, Nanoscale Res. Lett., 10 (2015). [15] B. Yu, X. Li, W. Zheng, Y. Feng, Y. S. Wong, T. Chen, pH-responsive cancer-targeted selenium nanoparticles: a transformable drug carrier with enhanced theranostic effects, J. Mater. Chem. B, 2 (2014) 5409-5418.
IP T
[16] L. Tan, X.E. Jia, X.F. Jiang, Y.Y. Zhang, H. Tang, S.Z. Yao, Q.J. Xie, In vitro study on the
individual and synergistic cytotoxicity of adriamycin and selenium nanoparticles against Bel7402 cells with a quartz crystal microbalance, Biosens. Bioelectron., 24 (2009) 2268-2272.
SC R
[17] A. Bhattacharjee, A. Basu, J. Biswas, T. Sen, S. Bhattacharya, Chemoprotective and
chemosensitizing properties of selenium nanoparticle (Nano-Se) during adjuvant therapy with cyclophosphamide in tumor-bearing mice, Mol. Cell. Biochem., 424 (2017) 13-33.
[18] K. Zhu, L. Jiang, Y. Chu, Y.S. Zhang, Protective effect of selenium against cisplatin-induced
U
nasopharyngeal cancer in male albino rats, Oncol. Lett., 12 (2016) 5068-5074.
N
[19] E. Taskin, N. Dursun, The Protection of Selenium on Adriamycin-Induced Mitochondrial Damage in Rat, Biol. Trace Elem. Res., 147 (2012) 165-171.
A
[20] H. Estevez, J. Carlos Garcia-Lidon, J.L. Luque-Garcia, C. Camara, Effects of chitosan-
M
stabilized selenium nanoparticles on cell proliferation, apoptosis and cell cycle pattern in HepG2 cells: Comparison with other selenospecies, Colloid. Surface. B, 122 (2014) 184-193. [21] X.J. Liu, G.Y. Deng, Y.Y. Wang, Q. Wang, Z.F. Gao, Y.G. Sun, W.L. Zhang, J. Lu, J.Q. Hu,
ED
A novel and facile synthesis of porous SiO2-coated ultrasmall Se particles as a drug delivery nanoplatform for efficient synergistic treatment of cancer cells, Nanoscale, 8 (2016) 8536-8541.
PT
[22] M. Abbas, Q. Zou, S. Li, X. Yan, Self-Assembled Peptide- and Protein-Based Nanomaterials for Antitumor Photodynamic and Photothermal Therapy, Adv. Mater., 29 (2017) 1605021. [23] Q. Chen, L. Xu, C. Liang, C. Wang, R. Peng, Z. Liu, Photothermal therapy with immune-
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adjuvant nanoparticles together with checkpoint blockade for effective cancer immunotherapy, Nat. Commun., 7 (2016) 13193. [24] D. Ding, W. Guo, C. Guo, J. Sun, N. Zheng, F. Wang, M. Yan, S. Liu, MoO3-x quantum dots for photoacoustic imaging guided photothermal/photodynamic cancer treatment, Nanoscale, 9
A
(2017) 2020-2029.
[25] Z. Zhou, Y. Wang, Y. Yan, Q. Zhang, Y. Cheng, Dendrimer-Templated Ultrasmall and Multifunctional Photothermal Agents for Efficient Tumor Ablation, Acs Nano, 10 (2016) 48634872. [26] Y. Dai, H. Sun, S. Pal, Y. Zhang, S. Park, C.P. Kabb, W.D. Wei, B.S. Sumerlin, Near-IRinduced dissociation of thermally-sensitive star polymers, Chem. Sci., 8 (2017) 1815-1821.
16
[27] Z. Sheng, D. Hu, M. Xue, M. He, P. Gong, L. Cai, Indocyanine Green Nanoparticles for Theranostic Applications, Nano-Micro Lett., 5 (2013) 145-150. [28] Y. Chen, H. Li, Y. Deng, H. Sun, X. Ke, T. Ci, Near-infrared light triggered drug delivery system for higher efficacy of combined chemo-photothermal treatment, Acta Biomater., 51 (2017) 374-392. [29] H. He, X. Zheng, J. Zhang, S. Liu, X. Hu, Z. Xie, Photothermally induced accumulation and retention of polymeric nanoparticles in tumors for long-term fluorescence imaging, J. Mater. Chem. B, 5 (2017) 2491-2499.
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[30] S. Song, S. He, Y. Tao, L. Wang, F. Han, H. Chen, Z. Zhang, Indocyanine Green Loaded
Magnetic Carbon Nanoparticles for Near Infrared Fluorescence/Magnetic Resonance Dual-Modal Imaging and Photothermal Therapy of Tumor, ACS Appl. Mater. Interfaces, 9 (2017) 9484-9495.
SC R
[31] Z.H. Sheng, D.H. Hu, M.B. Zheng, P.F. Zhao, H.L. Liu, D.Y. Gao, P. Gong, G.H. Gao, P.F. Zhang, Y.F. Ma, L.T. Cai, Smart Human Serum Albumin-Indocyanine Green Nanoparticles Generated by Programmed Assembly for Dual-Modal Imaging-Guided Cancer Synergistic Phototherapy, Acs Nano, 8 (2014) 12310-12322.
U
[32] X. Liu, X. Wang, B. Zhou, W.C. Law, A.N. Cartwright, M.T. Swihart, Size-Controlled
N
Synthesis of Cu2-xE (E = S, Se) Nanocrystals with Strong Tunable Near-Infrared Localized Surface Plasmon Resonance and High Conductivity in Thin Films, Adv. Funct. Mater., 23 (2013)
A
1256-1264.
M
[33] C.C. Hung, W. C. Huang, Y. W. Lin, T. W. Yu, H. H. Chen, S.C. Lin, W. H. Chiang, H.C. Chiu, Active Tumor Permeation and Uptake of Surface Charge-Switchable Theranostic
(2016) 302-317.
ED
Nanoparticles for Imaging-Guided Photothermal/Chemo Combinatorial Therapy, Theranostics, 6
[34] G. Ferrauto, F. Carniato, E. Di Gregorio, L. Tei, M. Botta, S. Aime, Large photoacoustic
PT
effect enhancement for ICG confined inside MCM-41 mesoporous silica nanoparticles, Nanoscale, 9 (2017) 99-103.
[35] J. van der Zee , Heating the patient:a promising approach, Ann. Oncol., 13(2002) , 1173-1184.
CC E
[36] X.C. Qin, Z.Y. Guo, Z.M. Liu, W. Zhang, M.M. Wan, B.W. Yang, Folic acid-conjugated graphene oxide for cancer targeted chemo-photothermal therapy, J. Photoch. Photobio. B, 120 (2013) 156-162.
[37] S. Shen, H. Tang, X. Zhang, J. Ren, Z. Pang, D. Wang, H. Gao, Y. Qian, X. Jiang, W. Yang,
A
Targeting mesoporous silica-encapsulated gold nanorods for chemo-photothermal therapy with near-infrared radiation, Biomaterials, 34 (2013) 3150-3158. [38] H. Park, J. Yang, J. Lee, S. Haam, I. H. Choi, K. H. Yoo, Multifunctional Nanoparticles for Combined Doxorubicin and Photothermal Treatments, Acs Nano, 3 (2009) 2919-2926. [39] S. M. Lee, H. Park, K. H. Yoo, Synergistic Cancer Therapeutic Effects of Locally Delivered Drug and Heat Using Multifunctional Nanoparticles, Adv. Mater., 22 (2010) 4049-4053. [40] M. Wang, M. Thanou, Targeting nanoparticles to cancer, Pharmacol. Res., 62 (2010) 90-99. 17
[41] J.A. Pearce, Improving Accuracy in Arrhenius Models of Cell Death: Adding a TemperatureDependent Time Delay, J. Biomech. Eng. Asme, 137 (2015). 121006 [42] F. Li, H. Yang, N. Bie, Q. Xu, T. Yong, Q. Wang, L. Gan, X. Yang, Zwitterionic Temperature/Redox-Sensitive Nanogels for Near-Infrared Light-Triggered Synergistic Thermo-
A
CC E
PT
ED
M
A
N
U
SC R
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Chemotherapy, ACS Appl. Mater. Interfaces, 9 (2017) 23564-23573.
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S0927-7765(18)30162-0 https://doi.org/10.1016/j.colsurfb.2018.03.018 COLSUB 9219
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
22-7-2017 15-2-2018 14-3-2018
Please cite this article as: Xijian Liu, Yeying Wang, Qiyang Yu, Guoying Deng, Qian Wang, Xinhui Ma, Qiugen Wang, Jie Lu, Selenium nanocomposites as multifunctional nanoplatform for imaging guiding synergistic chemo-photothermal therapy, Colloids and Surfaces B: Biointerfaces https://doi.org/10.1016/j.colsurfb.2018.03.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Selenium nanocomposites as multifunctional nanoplatform for imaging guiding synergistic chemo-photothermal therapy
Xijian Liu,a,‡,*,Yeying Wang,a,‡ Qiyang Yu, a Guoying Deng,b Qian Wang,b Xinhui Ma,c Qiugen Wang b and Jie Lu a,* a
Corresponding Authors
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*E-mail: [email protected]; *E-mail: [email protected] Notes ‡
These authors contributed equally to the paper.
The authors declare no competing financial interest.
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Supporting Information
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Additional figures are presented in the supporting information.
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Graphical Abstract
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College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai, 201620, China. Email: [email protected]; E-mail: [email protected] b Trauma Center, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, NO.650 Xin Songjiang Road, Shanghai, 201620, China. c Shanghai Huaxi Chemical Industry Science & Technology Co.,Ltd. No.555, Huangqiao Road,Shanghai, 201315, China.
Highlights ► The nanocomposite exhibited excellent fluorescence imaging performance. ► The nanocomposite as carrier for co-loading ICG and DOX. ► Triple treatment of photothermal therapy and chemotherapy for selenium and DOX. ► The nanocomposite as a nanoplatform for multimodal imaging guiding synergistic treatment.
Abstract: A multifunctional selenium nanocomposite (selenium@silica core-shell nanoshperes
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for loading indocyanine green(ICG)/Doxorubicin(DOX)) was fabricated to reach visible and efficient cancer treatment. The Se@SiO2-ICG nanocomposites could be used not only as
excellent photothermal agents but also as carriers for DOX delivery. In addition, the Se@SiO2-
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ICG/DOX nanocomposites exhibited excellent fluorescence imaging and infrared imaging
performance. Tumor could be effectively inhibited by Se@SiO2-ICG/DOX due to the triple treatment of photothermal effect and chemotherapy of selenium and DOX. Thus, the Se@SiO2ICG/DOX nanocomposites have a great potential in imaging guiding synergistic treatment of
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cancer.
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Keywords: photothermal therapy, selenium nanocomposites, drug delivery, fluorescence imaging,
1 Introduction
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synergistic treatment
Selenium is an essential trace element for the human body which plays a vital role in most
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of the biochemical and physiological processes.[1-3] Selenium deficiency will accompany with the risk of many health problems and multifarious cancers.[2] These studies
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demonstrated selenium compounds could adjust the level of intracellular reactive oxygen species (ROS) to induce apoptosis of cancer cells.[4-6] Among selenium compounds, the selenium nanoparticles have attracted more attention due to chemical stability, biocompatibility, and low toxicity[7-10]. Recently, the combination of selenium
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nanoparticles with anticancer drugs were studied, which exhibited anticancer synergism due to the fact that selenium nanoparticles sensitize cancer cells for drugs[11-16]. In addition, selenium nanoparticles can decrease the damages of anticancer drugs to normal tissues, such as heart, liver and kidney.[17-19] Especially, Se not only reduces the side effects associated with DOX, but also increases its antitumor activity.[16,19,23] However, selenium nanoparticles have many limitations in clinical treatment due to their narrow margin between the beneficial and toxic effects.[20] Our previous studies have 2
preliminarily explored selenium release (porous Se@SiO 2) system to realize safe treatment for cancer.[21] Selenium can inhibit cancer cell at a long time, but it is not so effectively in eliminating solid tumor at short period. So, it has a great need for selenium nanoparticles combining other methods to eliminate tumor directly. Photothermal therapy (PTT), a minimally invasive surgical technology, uses light-absorbing nanomaterials(PTT agents) to convert NIR energy into heat, which can directly ablate the tumor.[22-25] Among the PTT agents, ICG is a FDA approved dye, which is regarded as promising PTT agent due to its low cytotoxicity and excellent photothermal properties[26, 27].
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ICG can also be used as fluorescence imaging agents owing to NIR fluorescence absorption
around 800 nm. Because of poor photo-stability of free ICG, ICG always was encapsulated inside nanocarriers to improve its performance as a PTT agent and molecular imaging probe[28-31]. It
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will has an excellent instant anticancer effect for ICG and long term anticancer effect for selenium nanoparticles when the selenium nanoparticles were also encapsulated in the ICG nanocarriers. Nevertheless, to our best knowledge, up to now, no studies about the combination of
chemotherapy of selenium nanoparticles and photothermal therapy(ICG) had done. Thus, it is very
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essential to study the synergistic treatment about selenium and ICG.
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Here, we fabricated a multifunctional nanocomposites (Se@SiO2-ICG/DOX) which
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could be used for multimodal imaging (fluorescence imaging and infrared imaging) guiding synergistic treatment (photothermal therapy and chemotherapy of selenium and
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DOX). As shown in Scheme 1, first, the Cu2-xSe nanocrystals were synthesized, then they were oxidized to form Se@SiO2 core-shell nanospheres under microemulsion conditions.
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Afterwards, the Se@SiO2 nanospheres were treated with hot water in the presence of PVP, then the porous Se@SiO2 nanospheres were formed. The anticancer drug (DOX) and photothermal agent (ICG) could be encapsulated into the porous Se@SiO 2 nanospheres to
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form Se@SiO2-ICG/DOX nanocomposites. After the nanocomposites were intravenously injected to the tumor-bearing mice, their pharmacokinetics and biodistribution could be
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tracked in vivo. When the nanocomposites were highly enriched in the tumor site, the photothermal therapy were driven by NIR irradiation, which could efficiently ablate tumor. At the same time, DOX and Se released from the nanocomposites for synergetic chemotherapy, which could kill tumor cells and inhibit their metastasis for a long time. Therefore, the tumor cells can be effectively inhibited and killed due to the triple action of
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photothermal therapy as well as chemotherapy of selenium and DOX. Thus, the Se@SiO2ICG/DOX nanocomposites have a great potential in multimodal imaging guiding synergistic treatment of cancer.
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Scheme 1 Schematic of the synthesis and application of Se@SiO 2-ICG/DOX. 2. Experimental Section 2.1. Chemicals and reagents
All reagents were used without further purification. Copper (I) chloride (CuCl), ammonium
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hydroxide (25%-28%), anhydrous ethanol, polyvinyl pyrrolidone (PVP, K30) are
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analytically pure, which were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China), and tetraethylorthosilicate (TEOS, GR) , selenium powders, oleic acid,
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oleylamine (approximate C18 from 80-90%) were obtained from Aladdin. ICG
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(indocyanine green) was got Meclin Biochemical Technology Co., Ltd. (Shanghai, China), and doxorubicin hydrochloride (DOX) was obtained from Huafeng United Technology
2.2. Characterization
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CO., Ltd. (Beijing, China).
Sizes and morphologies of the nanoparticles were measured by a transmission electron
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microscope (TEM) using JEM-2100F microscope. Optical absorbances of colloidal nanoparticles were measured by a Phoenix 1901 UV-visible-NIR Spectrophotometer. Xray diffraction (XRD) was measured by a D/max-2550 PCX-ray diffractometer (Rigaku,
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Japan).
2.3 Synthesis of Se@SiO2 core-shell nanospheres First, Cu2-xSe nanocrystals were synthesized according to the previous paper.[32] Briefly, the mixtures of 39.5 mg selenium powders and 5 mL oleic acid (OA) were heated to 120
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°C in nitrogen atmosphere for 30 min under stirring. Subsequently, the mixtures were heated to 220 ℃ for formation of Se-OA precursor. At the same time, the mixtures of oleylamine (OAM, 5 mL), OA(5 mL) and CuCl(49.5 mg) were heated to 120 °C for removing moisture. Subsequently, the mixtures were heated to 220 °C, and the above SeOA precursor were quickly injected into the mixtures. And the resulting solution was reacted at 220 °C for 5 min. Finally, the Cu2-xSe nanocrystals were obtained by washing with ethanol, and they were dispersed in 10 mL of normal hexane for later use. 4
Se@SiO2 core-shell nanospheres were prepared according to our previous method. [21] The mixtures of n-hexane(30 mL), n-hexanol(3 mL), Triton X-100(3 mL), deionized water(0.9 mL) and above Cu2-xSe n-hexanol solution(3.3 mL) were stirred for 30 min. Then 0.15 mL TEOS and 0.20 mL aqueous ammonia were dropwise added successively with rapid stirring. The Se@SiO2 particles were obtained by centrifuging after stirring for 24 h. Then, the Se@SiO2 core-shell nanospheres were washed with ethanol three times for later use. 2.4 Synthesis of porous Se@SiO2 core-shell nanospheres.
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The above Se@SiO2 core-shell nanospheres were dispersed in 25 mL of PVP (K30)
solution (10g/L) with stirring for 1h. Then the stir was stop and the temperature of the
mixtures was increased to 95°C and maintained for 2.0 h in standstill condition. Finally,
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porous Se@SiO2 core-shell nanospheres were collected by centrifuging and washing with water three times. 2.5 The Loading and Release performance.
The porous Se@SiO2 nanospheres PBS dispersions (2mg/L, pH 7.4 and 5.0, respectively)
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were vibrated in a shaker at 37 °C. Then the dispersions were centrifuged in a certain
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interval, and the supernatant were filtered by 0.22µm filter for measuring the Se by Leeman ICP-AES Prodigy. The bottom porous Se@SiO2 nanospheres were dispersed in
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fresh PBS solutions with ultrasonication for next analysis.
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2.5 mg of porous Se@SiO2 core-shell nanospheres were mixed with 2 mL DOX aqueous solution (200 µg·mL-1) and 2 mL ICG aqueous solution （200 µg·mL-1）. After the mixtures were stirred for 24 h at room temperature in darkness, the DOX and ICG co-
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loaded Se@SiO2 core-shell nanospheres (Se@SiO2-ICG/DOX nanocomposites) were collected by centrifugation and washed with water two times to remove unbound DOX and
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ICG. The supernatant solution was collected and measured by the 1901 UV-visible-NIR spectrophotometer at 490 nm and 780 nm to calculate the amount of DOX and ICG loading in the nanospheres, respectively.
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The release of ICG and DOX from Se@SiO2-ICG/DOX nanocomposites with or without
NIR laser irradiation were also studied. 2.5 mg of Se@SiO2-ICG/DOX nanocomposites were dispersed in 5.0 mL PBS solutions at pH 5.0 and pH 7.4, respectively. The dispersions were stirred under room temperature and centrifuged in a certain time interval.
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The supernatants were collected, and the bottom Se@SiO2-ICG/DOX nanocomposites were re-dispersed in 5.0 mL fresh PBS solutions under the ultrasonication. In the group of laser irradiation, Se@SiO2-ICG/DOX nanocomposites were additionally irradiated by the NIR laser (1.0 W·cm-2, 808 nm) in a certain time interval (NIR irradiation for 5 min per hour from 0 to 4 hour, and NIR irradiation for 5 min per two hours from 4 to 12 hour). The amounts of released ICG and DOX in the PBS solutions (pH 5.0 and 7.4) were measured by UV-vis-NIR spectrophotometer (480 nm). 5
2.6 Photothermal performance measurement. Aqueous solutions of Se@SiO2-ICG nanocomposites at different concentrations of 0, 25, 50, 100, 200, and 400 µg·mL-1 (the ICG loading efficiency was 10%), and free ICG (40 µg·mL-1) were respectively irradiated by an 808 nm NIR laser (Sfolt Co., Ltd., Shanghai, China) for 5 min with a power density of 1.0 W·cm-2. The temperature changes were recorded per second using a thermal imaging camera (FOTRIC 225) with an accuracy of ±1 °C. 2.7 Cell culture and viability measurements.
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For cell viability measurements, human endothelial cells and HeLa cells were seeded in a 96-well plate and cultured in 5% CO2 at 37 oC for 24 h. The culture mediums were
replaced and cells were incubated with complete mediums containing PBS, ICG, DOX,
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porous Se@SiO2, Se@SiO2-ICG and Se@SiO2-ICG/DOX at a series of concentrations for 6 h. For the NIR treatment group, the cells were irradiated by laser for 5 min (808nm, 1.0 W·cm-2). Then all the cells were further incubated for 18 h. Finally, cell viabilities were
measured by Cell Counting Kit-8 system (CCK-8) according to manufacture's instructions.
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2.8 In Vivo fluorescence imaging.
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HeLa tumor bearing mice were injected with 100 μL aqueous solution containing Se@SiO2-ICG/DOX nanocomposites(2 mg·mL-1, ICG efficiency was 10%, DOX loading
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9%) and free ICG (0.2 mg·mL-1) through intravenous injection, respectively. The mice
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were imaged using a real-time in vivo fluorescence imaging system (Fluoptics FB800) at a certain interval.
2.9 In vivo chemo-photothermal Therapy.
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HeLa tumor bearing mice were from Shanghai General Hospital. When tumors reached about 7~9 mm in diameter, the mice were randomly divided into seven groups: (1) control
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group, (2) PBS+NIR group, (3) DOX group, (4) Se@SiO 2-ICG group, (5) Se@SiO2ICG/DOX group, (6)Se@SiO2-ICG + NIR group, (7) Se@SiO2-ICG/DOX + NIR group. Except for the control group, 100 μL PBS (pH=7.4), DOX (180 µg·mL -1), Se@SiO2-ICG
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(2 mg·mL-1, ICG loading 10%), Se@SiO2-ICG/DOX (2 mg·mL-1, ICG loading 10%, DOX loading 9%) were injected intravenously into the tumor-bearing mice. At 8 hours after injection, the tumor regions of NIR irradiation groups were exposed to NIR light radiation (808 nm, 1 W·cm-2) for 10 min and the thermal images were recorded by a FOTRIC 225
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thermal imaging camera. Before and after treatment, tumor size was measured by a vernier caliper every other day. Tumor volume was calculated in accordance with the following formula: tumor volume = length × width2/2. Relative tumor volume was calculated as V/V 0 (V0 was the initiate tumor volume before treatment). Body weights of each mouse were also recorded every other day.
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All animal experiments were carried out according to protocols approved by the institutional committee for animal care and also in accordance with the policy of the National Ministry of Health. 2.10 Statistical analysis. All data were presented as the mean ± standard deviation (SD). The differences among groups were determined using one-way ANOVA analysis: (*) P < 0.05, (**) P < 0.005,
3 Results and Discussion 3.1 Synthesis and characterization of porous Se@SiO2 nanospheres
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(***) P< 0.001.
The silica coated selenium nanospheres (Se@SiO 2 ) were prepared according to our
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previous method.[21] The XRD patterns of the Se@SiO2 samples are presented in Fig. 1a. All the diffraction peaks can readily correspond to standard hexagonal phase of selenium (JCPDS card no: 65-1876). The TEM image (Fig. 1b) indicates that Se@SiO 2 have
spherical structure with an average diameter of ~ 55 nm, and many small Se particles
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interspersed in the nanospheres. The high-magnified TEM further shows selenium particles
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are irregular and the size of most of them are less than 5 nm(Fig. 1c). After Se@SiO2 nanospheres were treated with water in presence of polyvinyl pyrrolidone (PVP), the
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porous Se@SiO2 nanospheres were successfully obtained(Fig. 1d). As shown in Fig.S1,
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Se@SiO2 nanospheres and porous Se@SiO2 nanospheres have dynamic light scattering (DLS) size of ~83 nm and ~92 nm, respectively, and they demonstrate a narrow polydispersity. The isotherm of porous Se@SiO2 nanospheres after hot water etching
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shows a typical type IV feature (Fig.S2). The Brunauer-Emmet-Teller (BET) surface area and total pore volume of porous Se@SiO2 nanospheres are 152.8 m²/g and 0.3945 cm³/g,
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respectively. The pores of the nanospheres show a broad distribution from 2.0 nm to 43.2 nm. Especially, the pore distribution have a peak at 23.8 nm, which is more than four times of Se particles. It is suitable for release of nano selenium particles. The BET results further
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indicate that the as-prepared porous Se@SiO2 nanospheres possess mesoporous channels, in agreement with the TEM observation in Fig. 1d. The Fourier transform infrared spectroscopy (FTIR) spectrum of porous Se@SiO 2 nanospheres exhibit characteristic peaks of PVP, such as 2925 and 2850 cm−1 and 1640 cm−1(Fig.S3),[21] indicating the
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successful coating of PVP on the nanospheres. Due to good biocompatibility and aqueous solubility of PVP, the colloid stability and biocompatibility of porous Se@SiO 2 nanospheres could be improved.
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Fig. 1 (a) XRD pattern of the Se@SiO2 sample, (b) Low-magnified (c) and high-
magnified TEM of Se@SiO2 nanospheres, (d) TEM of porous Se@SiO2 nanospheres.
3.2 Photothermal performance of Se@SiO2-ICG nanocomposites
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The porous Se@SiO2 nanospheres could be used as carrier due to the porous structures.
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After the mixtures of porous Se@SiO2 core-shell nanospheres and ICG aqueous solution were stirred for 24h, Se@SiO2-ICG nanocomposites were collected by centrifugation and
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washed with water two times to remove ICG. The FTIR spectrum showed Se@SiO2-ICG
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nanocomposites exhibited the characteristic peaks of ICG, indicating successful incorporation of ICG. As shown in Fig. S4, Se@SiO2-ICG nanocomposites demonstrate a strong NIR absorption peak around 808 nm, which endows the Se@SiO 2-ICG
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nanocomposites as a good photothermal agent. Moreover, ICG encapsulated in carriers
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would be more stable than free ICG. [33, 34]
Fig. 2 (a)Temperature changes of the Se@SiO2-ICG nanocomposites dispersions(ICG loading efficiency was 10%) at different concentrations (0 to 400µg·mL-1) and free ICG dispersions (40 µg·mL-1) under the irradiation(808nm, 1.0 W/cm2). (b)Temperature elevations of the Se@SiO2-ICG nanocomposite dispersions at various concentrations(ICG loading efficiency was 10%) under the irradiation (808nm, 1.0 W/cm2) for 5 min. 8
Given the strong absorption in the NIR region, we further evaluated the photothermal conversion properties of Se@SiO2-ICG aqueous dispersions. Dispersions of Se@SiO2-ICG nanocomposite at different concentrations (ICG loading efficiency was 10%) and free ICG (40 µg·mL-1) were respectively irradiated by an 808 nm NIR laser for 5 min with a power density of 1.0 W·cm-2 in room conditions(room temperature was 24.8 °C). As shown in Fig. 2a, the control of pure water demonstrated a negligible temperature increase under NIR irradiation. However, all the Se@SiO2-ICG dispersions exhibited remarkable temperature elevation at the same conditions, suggesting that the Se@SiO 2-ICG could
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efficiently convert the NIR energy into local hyperthermia upon irradiation. The Se@SiO 2ICG dispersions showed a dose-dependent photothermal effect(Fig.2b). If the body temperature is 37 °C , the temperature can easily attain the temperature for the
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hyperthermia therapy(40~45 °C),[35] even at the concentration of Se@SiO2-ICG as low as 50 μg·mL-1 (temperature elevation is 8.8 °C). Additionally, the Se@SiO2-ICG
dispersion had a higher temperature rise than free ICG solution under the same laser irradiation and the same ICG concentration of 40 µg·mL -1 due to shift of NIR absorption
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peak and improvement of stability, according to previous studies[33, 34].
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3.3 Loading and release performance
We also evaluate the Se release from the porous Se@SiO2 nanospheres. As shown in Fig.
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S5, ~0.66% and ~0.85% Se were released from the porous Se@SiO2, which could improve
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the immunity and prevent the cancers.[3, 5] When anticancer drug was loaded into porous Se@SiO2, DOX and Se could simultaneously release to synergistically kill cancer cells. Porous Se@SiO2 nanospheres could also be used for drug delivery due to the porous
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structure. DOX and ICG were co-loaded into Se@SiO2 nanospheres by simple mixing with string under dark conditions. After removing unbound DOX and ICG molecules by
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centrifugation, Se@SiO2-ICG/DOX nanocomposites were obtained, which showed DOX and ICG absorption peaks(Fig.3a). The co-loading efficiency of the porous Se@SiO2 nanospheres can reach to as high as (by weight) 12.9% for ICG and 10.2% (by weight) for
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DOX.
The release of DOX from Se@SiO2-ICG/DOX nanocomposites in PBS buffers at pH 7.4
and pH 5.0 were studied(Fig. 3b). In pH 7.4 (normal tissue environment), DOX release rate was slow. While in pH 5.0, DOX release rate was accelerated due to enhanced water
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solubility of DOX in the acidic conditions.[36] Activated release in acidic conditions is beneficial to drug delivery because the extracellular environment of the tumor is acidic.[37] The excellent photothermal efficiency of Se@SiO 2-ICG/DOX nanocomposites could further promote the release of DOX. The cumulative release of DOX increased from 75.6% to 92.1% by NIR irradiation within 12 h. It can be owing to the heat-stimulative dissociation of interactions between hydroxyl of SiO2 and DOX.[37] The pH-sensitive and NIR-triggered release of DOX can greatly improve the chemo-photothermal therapy 9
effects for cancer cells. At the same time, we also tested the release of ICG from Se@SiO2-ICG/DOX nanocomposites. As shown in Fig.S6, there were no obvious differences of the ICG release profile between pH 5.0 and 7.4, but NIR irradiation could promote the release of ICG. After 12 h, the most amounts of ICG were still incorporated in the nanocomposites without NIR irradiation, which was beneficial for photothermal therapy and fluorescent imaging due to higher tumor accumulation for ICG incorporated in
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nanocomposites by EPR effect.
Fig. 3 (a) UV-Vis-NIR spectra of DOX, Se@SiO2-ICG and Se@SiO2-ICG/DOX aqueous
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dispersions. (b) Cumulative DOX release from the Se@SiO2-ICG/DOX nanocomposites in
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PBS buffer at pH 5.0 and 7.4 with or without NIR laser irradiation (1.0 W cm -2, 808 nm), respectively.
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3.4 In vitro synergistic chemo-photothermal therapy
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To investigate the biocompatibility, human endothelial cells (normal cells) incubated with porous Se@SiO2, Se@SiO2-ICG, ICG and DOX at various concentrations, respectively. As shown in Fig.4a, compared the control group, the cell viabilities of porous Se@SiO2,
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Se@SiO2-ICG(below 200 μg/mL) and ICG(below 2 μg/mL) groups had no obvious difference, indicating they had negligible toxicity for normal cells. However, the DOX
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demonstrated obvious toxicity for normal cells, which means it had toxic side effects for normal tissues. Motivated by the excellent photothermal effect, drug delivery performance and biocompatibility of Se@SiO2-ICG, we further explored in vitro therapeutic efficiency
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of DOX, ICG, porous Se@SiO2, Se@SiO2-ICG, Se@SiO2-ICG/DOX with or without irradiation by CCK-8 assay. As shown in Fig.S7 and Fig.4b, ICG demonstrated no appreciable cytotoxicity for HeLa cells. There were few differences of cell viabilities between porous Se@SiO2 and Se@SiO2-ICG. Compared to control group, the porous
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Se@SiO2 and Se@SiO2-ICG had obvious cytotoxicity to HeLa cells due to release of Se. Porous Se@SiO2, Se@SiO2-ICG and Se@SiO2-ICG/DOX exhibited increased cytotoxicity in a dose-dependent manner. The IC50 values of DOX and porous Se@SiO2 for endothelial cells are 35.96 μg/mL and 1041μg/mL, respectively, while the dose of DOX and porous Se@SiO2 for HeLa cells are 4.02 μg/ml and 352.3μg/ml, respectively. The results demonstrated HeLa cells were much more sensitive for DOX and porous Se@SiO2 than normal cells. Moreover, Se@SiO2-ICG/DOX exhibited enhanced cytotoxicity than porous 10
Se@SiO2 and Se@SiO2-ICG at all concentrations. Because the HeLa cells were inhibited by the double inhibition effect for both selenium and DOX to HeLa cells due to the fact that selenium nanoparticles sensitize cancer cells for drugs [16,19,23] To investigate therapeutic efficiency of the photothermal effect, the cells incubated with PBS, porous Se@SiO2, Se@SiO2-ICG and Se@SiO2-ICG/DOX were irradiated by NIR laser for 5 min. As indicated in Fig.4b and Fig.S7, it had no obvious negative effect for cell viability incubated with PBS(Control group) and porous Se@SiO2 by laser irradiation alone, indicating it is safe for HeLa cells under irradiation (808 nm, 1.0 Wcm -2) and porous
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Se@SiO2 had no obvious photothermal effect. While it led to a drastic decreased percent of cell viabilities in the presence of Se@SiO2-ICG and Se@SiO2-ICG/DOX with NIR
irradiation, which was ascribed to that the excellent photothermal effect to induce the cells
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death by Se@SiO2-ICG and Se@SiO2-ICG/DOX. In addition, Se@SiO2-ICG/DOX
exhibited significantly enhanced cell lethality than Se@SiO2-ICG in all the concentration due to the synergistic effect of photothermal therapy of ICG and chemotherapy of DOX and selenium. The synergistic effect of chemotherapy and photothermal therapy probably
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attributed to enhanced cytotoxicity of DOX at higher temperatures, and higher heat
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sensitivity for the cells exposed to the DOX.[38, 39]. Almost all the HeLa cells were killed by Se@SiO2-ICG/DOX under irradiation at concentration of 200 μg/mL. These results
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demonstrated Se@SiO2-ICG/DOX combined triple therapy ways (photothermal therapy of
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ICG and chemotherapy of DOX and selenium) into a nanocomposites, which could
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efficiently induce cancer cells to death.
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Fig.4 (a)Viability of human endothelial cells (normal cells) incubated with porous Se@SiO2 and Se@SiO2-ICG at various concentrations, respectively. (b)HeLa cells incubated with PBS, Se@SiO2-ICG and Se@SiO2-ICG/DOX nanocomposites at various concentrations, respectively, with or without NIR laser irradiation (808 nm, 1.0 W/cm 2, 5 min). 3.5 In Vivo fluorescence imaging 11
Encouraged by the above outstanding performance of Se@SiO 2-ICG/DOX nanocomposites in photothermal test and drug delivery, the imaging guided synergistic therapy was evaluated on HeLa xenograft model. As shown in Fig. 5, NIR fluorescence imaging was employed to track the in vivo pharmacokinetics and biodistribution of free ICG and Se@SiO2-ICG/DOX. In the group of mice injected with free ICG, at 2 h of postinjection, mice body showed scattered fluorescence for no target biodistribution. At 8 h of post-injection, spleen showed bright fluorescence, indicating major portion of ICG was quickly taken up by spleen. After 24 h of post-injection, no distinct fluorescence signals
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were observed in the mice body, which showed the rapid clearance and degradation of free ICG due to poor stability of free ICG.[30,31] In contrast, Se@SiO2-ICG/DOX
demonstrated a different biodistribution behavior. In the group of mice treated with
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Se@SiO2-ICG/DOX, the tumor site began to appeared bright fluorescence signals 2 h after injection, then the fluorescence signals were strengthened from 2 h to 8 h and maintained strong bright intensity until 24 h, which could be attributed to the EPR effect of
nanomaterials.[40] Subsequently, the fluorescence signals of tumor began to recede, and
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weak fluorescence signals could still be seen 72 h after injection. Compared with free
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ICG, Se@SiO2-ICG/DOX had much higher tumor accumulation and slower body clearance due to enhanced stability and EPR effect of encapsulated ICG. The fluorescence
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results suggested that Se@SiO2-ICG/DOX could efficiently accumulate in the tumor site
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and retain a long time in tumor, which can be used to diagnose and guide therapy.
Fig. 5 In vivo fluorescence imaging of HeLa xenograft mice intravenously injected by the dispersions of free ICG and Se@SiO2-ICG/DOX nanocomposites, respectively.
3.6 In vivo synergistic chemo-photothermal Therapy 12
Encouraged by the outstanding in vitro photothermal-chemotherapy performance of Se@SiO2-ICG/DOX nanocomposites, we further investigated their in vivo therapeutic efficiency. The HeLa xenograft mice were randomized into seven groups when the tumor size reached about 7-9 mm, and intravenously injected with PBS, DOX, Se@SiO 2-ICG and Se@SiO2-ICG/DOX. At 8 h after intravenous injection, the HeLa xenograft mice of NIR treatment groups were irradiated by 808 nm laser(1.0 W/cm 2) with 10 min. As shown in Fig.6a,b, the surface temperature of the tumour had no obvious rise in the control group of mice injected with PBS under irradiation for 10 min, indicating that it would have no
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perceptible hurt to normal tissue by the treatment. In contrast, the tumour temperature of
Se@SiO2-ICG + NIR and Se@SiO2-ICG/DOX + NIR group increased rapidly and reached 61.2 oC and 60.4 oC, respectively, which is sufficient for inducing cancer cell to death.[41]
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After received various treatments, the tumor sizes of seven groups were all measured every two days. As shown in Fig.6c and S8, the tumor sizes of control group mice increased very rapidly owing to rapid growth of cancer cells. And the tumor sizes of PBS+ NIR group mice increased like that of control group due to the fact that the heat generated by PBS
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under irradiation was not sufficient for killing cancer cells. Compared to control group, the
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tumor growths of Se@SiO2-ICG and DOX groups were inhibited in a certain degree due to the anticancer effects of selenium and DOX, respectively. The Se@SiO2-ICG/DOX
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showed a higher tumor inhibitory rate than Se@SiO 2-ICG and DOX for the synergistic
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effect of Se and DOX. The tumor growth were significantly inhibited in Se@SiO 2-ICG+ NIR group, attributing to the excellent photothermal effect of ICG and anticancer effect of selenium. Nevertheless, tumor margins or recesses may be not received sufficient
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irradiation, which resulted in failures in completely removing the tumor or tumor recrudescing after treatment. In order to achieve much enhanced efficacy, Se@SiO2-
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ICG/DOX plus NIR irradiation was carried out in tumor treatment. The tumors growth were severely inhibited and the tumors volume began to decrease after the treatment. Moreover, the tumor completely disappeared after 14 days’ post-treatment, which could be
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visually confirmed by the photos of different treatment groups(Fig.S8). Compared to previous similar studies(chemo-photothermal therapy of DOX and ICG),[28, 33, 42] Se@SiO2-ICG/DOX plus NIR showed enhanced therapy effects. The tumor of mice could completely eliminated by treatment of Se@SiO 2-ICG/DOX plus NIR, which were ascribed
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to the synergistic therapy of chemotherapy (DOX and selenium) and photothermal therapy. The photothermal effect could severely destroys tumor cells in a short time, while slow release of DOX and selenium can kill the residual tumor cells and efficiently restrain them to diffuse and regress in a long period. And, more remarkable, there none tumors reappeared and none mice died in Se@SiO2-ICG/DOX +NIR group after 60 days’ treatment. In addition, the body weight of the mice of all groups did not have noticeable
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changes during the treatment (Fig.6d), indicating the treatment had no obvious systematic
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toxicity.
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Fig. 6 (a) In vivo infrared thermal images and (b) tumor temperature changes of at different time points under NIR irradiation after intravenously injected with PBS, Se@SiO2-ICG
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and Se@SiO2-ICG/DOX, respectively. (c) Tumor growth curves and (d) body weight changes of different groups treatments.
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4 Conclusions
The porous Se@SiO2 nanocomposites were successfully prepared, which could be co-
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loaded for ICG and DOX. The Se@SiO2-ICG/DOX nanocomposites had excellent fluorescence imaging and infrared imaging performance. Tumors even could be completely eliminated by Se@SiO2-ICG/DOX due to the triple treatment of photothermal effect and chemotherapy of selenium and DOX. Thus, the Se@SiO 2-ICG/DOX nanocomposites have
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a great potential in multimodal imaging guiding synergistic treatment of cancer.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Nos. 21176102, 21176215, 21476136, 21171035, 51472049, 8152010831), Science and Technology Commission of Shanghai Municipality (No. 15430501200), the Sino-German Center for Research Promotion (No. GZ935), Innovation Program of Shanghai Municipal 14
Education Commission (No. 14ZZ160), Guidance Fund of Shanghai Committee of Science and Technology (No. 15411968800), the Connotation Construction Project of SUES (No.Nhky-2015-05), Shanghai Municipal Education for foreign visiting scholar Program.
References [1] K. Bierla, A. Flis-Borsuk, P. Suchocki, J. Szpunar, R. Lobinski, Speciation of Selenium in
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Selenium-Enriched Sunflower Oil by High-Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry/Electrospray-Orbitrap Tandem Mass Spectrometry, J. Agr. Food Chem., 64 (2016) 4975-4981.
SC R
[2] Y.F. Wang, J.L. Wang, H. Hao, M.L. Cai, S.Y. Wang, J. Ma, Y. Li, C.B. Mao, S.M. Zhang, In Vitro and in Vivo Mechanism of Bone Tumor Inhibition by Selenium-Doped Bone Mineral Nanoparticles, Acs Nano, 10 (2016) 9927-9937.
[3] C.D. Thomson, Assessment of requirements for selenium and adequacy of selenium status: a
U
review, Eur. J.Clin. Nutr., 58 (2004) 391-402.
[4] T. Li, F. Li, W. Xiang, Y. Yi, Y. Chen, L. Cheng, Z. Liu, H. Xu, Selenium-Containing
N
Amphiphiles Reduced and Stabilized Gold Nanoparticles: Kill Cancer Cells via Reactive Oxygen
A
Species, ACS Appl. Mater. Interfaces, 8 (2016) 22106-22112.
[5] Y. Wang, H. Hao, Y. Li, S. Zhang, Selenium-substituted hydroxyapatite nanoparticles and
M
their in vivo antitumor effect on hepatocellular carcinoma, Colloid. Surface. B, 140 (2016) 297306.
ED
[6] T. Nie, H. Wu, K. H. Wong, T. Chen, Facile synthesis of highly uniform selenium nanoparticles using glucose as the reductant and surface decorator to induce cancer cell apoptosis, J. Mater. Chem. B, 4 (2016) 2351-2358.
PT
[7] S. Skalickova, V. Milosavljevic, K. Cihalova, P. Horky, L. Richtera, V. Adam, Selenium nanoparticles as a nutritional supplement, Nutrition, 33 (2017) 83-90. [8] Y. He, S. Chen, Z. Liu, C. Cheng, H. Li, M. Wang, Toxicity of selenium nanoparticles in male
CC E
Sprague-Dawley rats at supranutritional and nonlethal levels, Life Sci., 115 (2014) 44-51. [9] J. Zhang, X. Wang, T. Xu, Elemental selenium at nano size (Nano-Se) as a potential chemopreventive agent with reduced risk of selenium toxicity: Comparison with Semethylselenocysteine in mice, Toxicol. Sci., 101 (2008) 22-31.
A
[10] P.D. Whanger, Selenium and its relationship to cancer: an update, Brit. J. Nutr., 91 (2004) 1128. [11] W. Liu, X. Li, Y.-S. Wong, W. Zheng, Y. Zhang, W. Cao, T. Chen, Selenium Nanoparticles as a Carrier of 5-Fluorouracil to Achieve Anticancer Synergism, Acs Nano, 6 (2012) 6578-6591. [12] F.P. Gao, Q. Yuan, L. Gao, P.J. Cai, H.R. Zhu, R. Liu, Y.L. Wang, Y.T. Wei, G.D. Huang, J. Liang, X.Y. Gao, Cytotoxicity and therapeutic effect of irinotecan combined with selenium nanoparticles, Biomaterials, 35 (2014) 8854-8866. 15
[13] Y. Huang, L. He, W. Liu, C. Fan, W. Zheng, Y.S. Wong, T. Chen, Selective cellular uptake and induction of apoptosis of cancer-targeted selenium nanoparticles, Biomaterials, 34 (2013) 7106-7116. [14] Y. Xia, P. You, F. Xu, J. Liu, F. Xing, Novel Functionalized Selenium Nanoparticles for Enhanced Anti-Hepatocarcinoma Activity In vitro, Nanoscale Res. Lett., 10 (2015). [15] B. Yu, X. Li, W. Zheng, Y. Feng, Y. S. Wong, T. Chen, pH-responsive cancer-targeted selenium nanoparticles: a transformable drug carrier with enhanced theranostic effects, J. Mater. Chem. B, 2 (2014) 5409-5418.
IP T
[16] L. Tan, X.E. Jia, X.F. Jiang, Y.Y. Zhang, H. Tang, S.Z. Yao, Q.J. Xie, In vitro study on the
individual and synergistic cytotoxicity of adriamycin and selenium nanoparticles against Bel7402 cells with a quartz crystal microbalance, Biosens. Bioelectron., 24 (2009) 2268-2272.
SC R
[17] A. Bhattacharjee, A. Basu, J. Biswas, T. Sen, S. Bhattacharya, Chemoprotective and
chemosensitizing properties of selenium nanoparticle (Nano-Se) during adjuvant therapy with cyclophosphamide in tumor-bearing mice, Mol. Cell. Biochem., 424 (2017) 13-33.
[18] K. Zhu, L. Jiang, Y. Chu, Y.S. Zhang, Protective effect of selenium against cisplatin-induced
U
nasopharyngeal cancer in male albino rats, Oncol. Lett., 12 (2016) 5068-5074.
N
[19] E. Taskin, N. Dursun, The Protection of Selenium on Adriamycin-Induced Mitochondrial Damage in Rat, Biol. Trace Elem. Res., 147 (2012) 165-171.
A
[20] H. Estevez, J. Carlos Garcia-Lidon, J.L. Luque-Garcia, C. Camara, Effects of chitosan-
M
stabilized selenium nanoparticles on cell proliferation, apoptosis and cell cycle pattern in HepG2 cells: Comparison with other selenospecies, Colloid. Surface. B, 122 (2014) 184-193. [21] X.J. Liu, G.Y. Deng, Y.Y. Wang, Q. Wang, Z.F. Gao, Y.G. Sun, W.L. Zhang, J. Lu, J.Q. Hu,
ED
A novel and facile synthesis of porous SiO2-coated ultrasmall Se particles as a drug delivery nanoplatform for efficient synergistic treatment of cancer cells, Nanoscale, 8 (2016) 8536-8541.
PT
[22] M. Abbas, Q. Zou, S. Li, X. Yan, Self-Assembled Peptide- and Protein-Based Nanomaterials for Antitumor Photodynamic and Photothermal Therapy, Adv. Mater., 29 (2017) 1605021. [23] Q. Chen, L. Xu, C. Liang, C. Wang, R. Peng, Z. Liu, Photothermal therapy with immune-
CC E
adjuvant nanoparticles together with checkpoint blockade for effective cancer immunotherapy, Nat. Commun., 7 (2016) 13193. [24] D. Ding, W. Guo, C. Guo, J. Sun, N. Zheng, F. Wang, M. Yan, S. Liu, MoO3-x quantum dots for photoacoustic imaging guided photothermal/photodynamic cancer treatment, Nanoscale, 9
A
(2017) 2020-2029.
[25] Z. Zhou, Y. Wang, Y. Yan, Q. Zhang, Y. Cheng, Dendrimer-Templated Ultrasmall and Multifunctional Photothermal Agents for Efficient Tumor Ablation, Acs Nano, 10 (2016) 48634872. [26] Y. Dai, H. Sun, S. Pal, Y. Zhang, S. Park, C.P. Kabb, W.D. Wei, B.S. Sumerlin, Near-IRinduced dissociation of thermally-sensitive star polymers, Chem. Sci., 8 (2017) 1815-1821.
16
[27] Z. Sheng, D. Hu, M. Xue, M. He, P. Gong, L. Cai, Indocyanine Green Nanoparticles for Theranostic Applications, Nano-Micro Lett., 5 (2013) 145-150. [28] Y. Chen, H. Li, Y. Deng, H. Sun, X. Ke, T. Ci, Near-infrared light triggered drug delivery system for higher efficacy of combined chemo-photothermal treatment, Acta Biomater., 51 (2017) 374-392. [29] H. He, X. Zheng, J. Zhang, S. Liu, X. Hu, Z. Xie, Photothermally induced accumulation and retention of polymeric nanoparticles in tumors for long-term fluorescence imaging, J. Mater. Chem. B, 5 (2017) 2491-2499.
IP T
[30] S. Song, S. He, Y. Tao, L. Wang, F. Han, H. Chen, Z. Zhang, Indocyanine Green Loaded
Magnetic Carbon Nanoparticles for Near Infrared Fluorescence/Magnetic Resonance Dual-Modal Imaging and Photothermal Therapy of Tumor, ACS Appl. Mater. Interfaces, 9 (2017) 9484-9495.
SC R
[31] Z.H. Sheng, D.H. Hu, M.B. Zheng, P.F. Zhao, H.L. Liu, D.Y. Gao, P. Gong, G.H. Gao, P.F. Zhang, Y.F. Ma, L.T. Cai, Smart Human Serum Albumin-Indocyanine Green Nanoparticles Generated by Programmed Assembly for Dual-Modal Imaging-Guided Cancer Synergistic Phototherapy, Acs Nano, 8 (2014) 12310-12322.
U
[32] X. Liu, X. Wang, B. Zhou, W.C. Law, A.N. Cartwright, M.T. Swihart, Size-Controlled
N
Synthesis of Cu2-xE (E = S, Se) Nanocrystals with Strong Tunable Near-Infrared Localized Surface Plasmon Resonance and High Conductivity in Thin Films, Adv. Funct. Mater., 23 (2013)
A
1256-1264.
M
[33] C.C. Hung, W. C. Huang, Y. W. Lin, T. W. Yu, H. H. Chen, S.C. Lin, W. H. Chiang, H.C. Chiu, Active Tumor Permeation and Uptake of Surface Charge-Switchable Theranostic
(2016) 302-317.
ED
Nanoparticles for Imaging-Guided Photothermal/Chemo Combinatorial Therapy, Theranostics, 6
[34] G. Ferrauto, F. Carniato, E. Di Gregorio, L. Tei, M. Botta, S. Aime, Large photoacoustic
PT
effect enhancement for ICG confined inside MCM-41 mesoporous silica nanoparticles, Nanoscale, 9 (2017) 99-103.
[35] J. van der Zee , Heating the patient:a promising approach, Ann. Oncol., 13(2002) , 1173-1184.
CC E
[36] X.C. Qin, Z.Y. Guo, Z.M. Liu, W. Zhang, M.M. Wan, B.W. Yang, Folic acid-conjugated graphene oxide for cancer targeted chemo-photothermal therapy, J. Photoch. Photobio. B, 120 (2013) 156-162.
[37] S. Shen, H. Tang, X. Zhang, J. Ren, Z. Pang, D. Wang, H. Gao, Y. Qian, X. Jiang, W. Yang,
A
Targeting mesoporous silica-encapsulated gold nanorods for chemo-photothermal therapy with near-infrared radiation, Biomaterials, 34 (2013) 3150-3158. [38] H. Park, J. Yang, J. Lee, S. Haam, I. H. Choi, K. H. Yoo, Multifunctional Nanoparticles for Combined Doxorubicin and Photothermal Treatments, Acs Nano, 3 (2009) 2919-2926. [39] S. M. Lee, H. Park, K. H. Yoo, Synergistic Cancer Therapeutic Effects of Locally Delivered Drug and Heat Using Multifunctional Nanoparticles, Adv. Mater., 22 (2010) 4049-4053. [40] M. Wang, M. Thanou, Targeting nanoparticles to cancer, Pharmacol. Res., 62 (2010) 90-99. 17
[41] J.A. Pearce, Improving Accuracy in Arrhenius Models of Cell Death: Adding a TemperatureDependent Time Delay, J. Biomech. Eng. Asme, 137 (2015). 121006 [42] F. Li, H. Yang, N. Bie, Q. Xu, T. Yong, Q. Wang, L. Gan, X. Yang, Zwitterionic Temperature/Redox-Sensitive Nanogels for Near-Infrared Light-Triggered Synergistic Thermo-
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Chemotherapy, ACS Appl. Mater. Interfaces, 9 (2017) 23564-23573.
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