Silica Nanoplatforms for Light-Activated Liver Cancer

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Janus Silver/Silica Nanoplatforms for LightActivated Liver Cancer Chemo/Photothermal Therapy Zheng Wang, Zhimin Chang, Mengmeng Lu, Dan Shao, Juan Yue, Dian Yang, Mingqiang Li, and Wen-Fei Dong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06446 • Publication Date (Web): 24 Aug 2017 Downloaded from http://pubs.acs.org on August 24, 2017

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ACS Applied Materials & Interfaces

Janus Silver/Silica Nanoplatforms for Light-Activated Liver Cancer Chemo/Photothermal Therapy

Zheng Wang†,‡, Zhimin Chang†, Mengmeng Lu$,⊥, Dan Shao*,†,⊥, Juan Yue†,‡, Dian Yang†,‡, Mingqiang Li⊥, Wen-fei Dong*,†

†

CAS Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical

Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, China. ‡

University of Chinese Academy of Sciences, Beijing 100049, China

$

Department of Oral Implantology, Affiliated Hospital of Stomatology, Jiangsu Key

Laboratory of Oral Disease, Nanjing Medical University, Nanjing, 210029, China ⊥

Department of Biomedical Engineering, Columbia University, New York, NY 10027,

USA

Keywords:

Janus,

silver

mesoporous

silica,

liver

chem/photo-thermal therapy, on-demand release

ACS Paragon Plus Environment

cancer,

synergistic


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Abstract Stimuli-triggered nanoplatform have become attractive candidates for combined strategies for advanced liver cancer treatment. In this study, we designed a light-responsive nanoplatform with folic acid (FA)-targeted properties to surmount the poor aqueous stability and photostability of indocyanine green (ICG). In this Janus nanostructure, ICG was released on-demand from mesoporous silica compartments in response to NIR irradiation, exhibiting predominant properties to convert light to heat in the cytoplasm to kill liver cancer cells. Importantly, the silver ions released from the silver compartment that were triggered by light could induce efficient chemotherapy to supplement photothermal therapy. Under NIR irradiation, ICG-loaded Janus nanoplatforms exhibited synergistic therapeutic capabilities both in vitro and in vivo compared with free ICG and ICG-loaded mesoporous silica nanoparticles (MSNs) themselves. Hence, our Janus nanoplatform could integrate ICG-based photothermal therapy and silver ion-based chemotherapy in a cascade manner, which might provide an efficient and safe strategy for combined liver cancer therapy.


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1. INTRODUCTION Patients with advanced liver cancer are diagnosed when they are not eligible for potential resections in 60% of the cases.1,2 In this setting, conventional treatments, including chemotherapy and thermal ablation, remain unsatisfactory due to their ineffectiveness or limitations because of the single modality and the damage they cause to healthy bystander tissues.3,4 Hence, it is urgent to seek combined therapies to enhance therapeutic efficacy and biosafety for advanced liver cancer. Recently, photothermal therapy (PTT) has been recognized as a promising strategy, in which photothermal agents are used to strongly absorb near-infrared (NIR) wavelength irradiation from a laser with superior tissue penetration ability to generate cytotoxic heat to destroy tumor cells.5,6 PTT has attracted increasing interest owing to its controllable, facile and favorable biosafety characteristics.7,8 Various types of photothermal agents, including carbon nanomaterials, gold nanoparticles, copper sulfide nanomaterials and organic NIR dyes, have been widely used for PTT treatments for liver cancer.7-10 Among them, indocyanine green (ICG), a tricarbocyanine dye, has gained great attention as a probe in cancer theranostics owing to its NIR imaging capability and effective photothermal response.11,12 Nevertheless, the clinical application of ICG has been stalled its facile degradation and clearance in vivo and the lack of specific targeting.13 Since folate receptors (FR) were demonstrated to be overexpressed in liver cancer, folic acid (FA) has been considered as a targeting candidate for liver cancer theranostics due to its high



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binding efficacy.14 In this regard, the development of high-performance FA-targeted nanocarriers is of great importance for practical applications using ICG-based PTT for liver cancer. Silver nanomaterials have gained substantial attention because of their broad bio-applications

including

antimicrobial

functions,

bio-imaging

and

wound

dressings.15,16 Several recent studies have demonstrated the anti-tumor activity of silver nanoparticles in vitro and in vivo.17,18 Silver nanoparticles are capable of selectively killing cancer cells because the intracellular release of silver ions generates reactive oxygen species (ROS) and leads to mitochondria-dependent apoptosis.19-21 More importantly, the efficiency of silver nanoparticles in cancer therapy can be significantly enhanced by hyperthermia because the resultant heat could trigger the release of silver ions.22-24 Therefore, the combination of PTT and silver-based chemotherapy might exhibit efficient and safe performance for liver cancer treatment. However, there are several challenges to overcome before realizing the aforementioned objectives: I) ICG as a photothermal agent and silver nanoparticle as a chemotherapeutic agent should be delivered into the tumor sites simultaneously with sufficient doses to achieve maximum synergistic efficacy with minimal adverse effects. II) On-demand release of ICG and silver ions should be precisely controlled in response to external stimuli. III) Targeting moieties should be coupled for improving long-term stability and tumor accumulation, thus providing localized liver cancer therapy.


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Janus mesoporous silica nanoparticles (MSNs) have attracted tremendous attention in biomedicine due to their excellent loading capacity and easy surface modification, especially for noninterfering properties on anisotropic surfaces.25-33 Our group has previously constructed several metal-mesoporous silica Janus nanoparticles as good candidates for cancer theranostics.27-33 Herein, we described Janus-type silver-mesoporous

silica

nanoparticles

(JNPs)

to

realize

combined

chemo/photothermal therapy of liver cancer. These anisotropic nanostructures exhibited superb SPR and enhanced loading capacities due to distinctly architectural preferences. As a result, liver cancer cell-speciﬁc targeting and chemo/photothermal effects were demonstrated using the FA-targeted, ICG-loaded JNPs (FA-JNPs@ICG) both in vitro and in vivo. Hence, this Janus silver/silica nanoparticle may provide an attractive approach for an efficient and safe chemo/photothermal therapy for liver cancer.

2. EXPERIMENTAL SECTION 2.1. Preparation of FA-JNPs and FA-MSNs. To prepare JNPs, the sphere-like silver nanoparticles (Ag NPs) were first synthesized by reducing AgNO3 with glucose in the presence of polyvinylpyrrolidone (PVP), according to our previously reports.33 Then, amino-functionalized JNPs were synthesized using a modified sol−gel method. Briefly, 1 mL of silver nanospheres (8.0 mg mL-1) was mixed with 10 mL of cetyltrimethylammonium bromide (CTAB) (2.5 mg mL-1) in a three-neck bottle under ultrasonic treatment. Then, 15 µL of tetraethyl orthosilicate (TEOS), 5 µL of



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3-aminopropyltrimethoxysilane (APTES), and 500 µL of ammonium hydroxide (14.8 mol/L NH4OH) were sequentially added into the mixture while stirring at 40 °C. After reacting for 30 min, the JNPs-NH2 were collected and washed with ethanol and water three times. To prepare rod-like amino-functionalized mesoporous silica nanoparticles (MSNs) as a control, 25 µL of TEOS, 5 µL of APTES, and 500 µL of NH4OH were added into 20 mL of CTAB (2.5 mg mL-1) with stirring at 40 °C. After 2 h reaction and the MSNs-NH2 were collected and washed with ethanol and water three times. To remove CTAB from the NPs, 20 mg of the as-synthesized products were dispersed in an ethanol solution with NH4NO3 (40 mL, 10 mg mL-1), refluxed overnight and washed with ethanol three times. To synthesis the FA-targeted NPs, 2 mg of FA-PEG-NHS was mixed with 10 mL of the amino-functionalized NPs (JNPs or MSNs) solution (1 mg mL-1) under ultrasonic treatment. The reaction was stirring overnight at room temperature. The FA-JNPs or FA-MSNs were collected and washed with water three times. 2.2. Characterization. The morphology of the NPs was measured using scanning electron microscopy (SEM, FEI quanta 200F) and transmission electron microscopy (JEOL JEM-2100). The size distribution and zeta potential of the NPs were characterized using a Nano-ZS 90 Nanosizer (Malvern Instruments Ltd., Worcestershire, UK). The mesoporous properties were measured using the Brunauer-Emmett-Teller Barrett-Joyner-Halenda

(BET) (BJH)

method

method.

and

Fourier

calculated transform


using

infrared

the (FTIR)

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spectroscopy measurements of the NPs and corresponding materials were measured using

a

Bruker model

VECTOR22

Fourier transform

spectrometer.

The

UV-visible-NIR absorption spectra of the NPs and corresponding materials were measured using a U-3310 spectrophotometer (Hitachi, Japan). The fluorescent spectra of the NPs were measured using a Shimadzu RF-5301 PC spectrophotometer. The temperature was measured with a thermocouple microprobe submerged in the solution. The silver content of the FA-JNPs (30.3±1.5, w/w%) was measured using an inductively coupled plasma mass spectrometer (ICP-MS) (Xseries II, Thermo Scientific, USA). 2.3. Cell Culture, Cytotoxicity and Endocytosis. The FR-positive human liver cancer cell line (SMMC-7721) and FR-negative human normal liver cell line (HL-7702) were purchased from ATCC. The cells were cultured in RPMI-1640 medium supplemented with 10% FBS, penicillin, and streptomycin. To measure the cytotoxicity of the NPs, the cells were seeded in 96-well plates at a density of 5000 cells per well and incubated with different concentrations (400, 200, 100, 50, 25, 12.5, and 6.25 µg mL-1) of MSNs, FA-MSNs, JNPs, FA-JNPs or (120, 60, 30, 15, 7.5 and 3.75 µg mL-1) of Ag NPs. After 24 h, the cell viabilities were assessed using a sulforhodamine B (SRB) assay, as described in our previous work.27-30 To determine the cellular uptake of the NPs, cells were seeded in 24-well plates at a density of 50000 cells per well and incubated with FITC-labeled NPs (12.5 µg mL-1) for 3 h. For the FA competition assay, 1 mM FA was co-incubated with the NPs. Then, the cells were washed and incubated with Lyso Traker Red DND-99 and stained with



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DAPI. The fluorescence of the cells was observed using confocal laser scanning microscopy (CLSM, Olympus FV1000 Japan). To measure the targeting efficacy, the cells incubated with FITC-labeled NPs were washed, trypsinized and resuspended subsequently for flow cytometry measurements (FACS, BD Biosciences, Drive Franklin Lakes, U.S.). 2.4. Loading and release of ICG. To evaluate the ICG loading capacity, 20 mg of the FA-functionalized NPs or amino-functionalized NPs was suspended in ICG solution (10 mL, 0.5 mg mL-1) and stirred at 37 °C for 24 h. The drug-loaded NPs were collected and washed with water twice. The ICG amount in the supernatant before and after loading was measured using UV-vis spectroscopy at 780 nm. The loading efficiency and drug-loading content were calculated according to our previous reports.27 To investigate the NIR irradiation-responsive release behavior, 1 mg of ICG-loaded NPs suspended in 1640 medium supplemented with 10% of FBS was exposed to NIR irradiation (808 nm, 1 W cm-2, 5 min) for 1 h. Then, the releasing process was performed on a shaking table at 37 °C. The amounts of released ICG and silver ions in the supernatant were measured using UV-vis spectrophotometry and ICP-OES at timed intervals, respectively. To measure the intracellular ICG release, the ICG-loaded NPs were incubated with cells for 3 h, and the cells were irradiated under the same conditions. After another 3-h incubation, the ICG distribution and quantitative fluorescent intensity were investigated using the described CLSM and FACS protocols in Experimental Section 2.3, respectively.


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2.5. In Vitro Chemo/Photothermal Effect. To determine the photothermal effect, the ICG-loaded NPs or ICG were incubated with cells for 3 h and then irradiated (808 nm, 1 W cm-2, 5 min). After another 3-h incubation, the cells were washed and stained with calcein-AM and PI according to the manufacturer’s protocol and observed with a fluorescence microscope. To determine the chemo/photothermal effect, ICG-loaded NPs or free ICG at equivalent concentrations (20, 10, 5, 2.5, 1.25 µg mL-1 for ICG) were incubated with cells for 3 h; the cells in the NIR-treated groups were irradiated with an 808-nm laser for 5 min at a power density of 1 W cm-2. All groups of cells were further cultured for 24 h, and the cell viabilities were evaluated using an SRB assay. To further investigate the synergistic effect of the chemo/photothermal therapy, the IC50 values of the FA-JNPs, ICG+NIR and FA-JNPs@ICG+NIR groups were calculated according to their cell viabilities. The combination index (CI50) was determined according to an equation reported previously: CI501.0 represented antagonism.34 2.6. In Vivo Anti-tumor Efficacy and Safety Evaluation. Male nude mice weighing approximately 20 g were injected with 5×106 SMMC-7721 cells in the right shoulder. When the xenografts grew to approximately 80-100 mm3, the animals were randomly divided into six groups (n=3/group), and each mouse in the treated group was intravenously injected with ICG, FA-MSNs@ICG, or FA-JNPs@ICG (equivalent to 2 mg kg−1 free ICG). For the NIR-treated groups, the xenografts were irradiated with an 808-nm laser for 5 min at a power density of 1 W cm-2 at 24 h post injection



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and 48 h post injection. Mice in each group were measured with a digital caliper every two days, and the tumor volumes were calculated using the following formula: Volume=length×width2×0.52. At day 16, all animals were sacrificed, and the tumors were measured, weighed and then embedded in paraffin before being subjected to the fluorescence TUNEL assay to measure the intratumoral late apoptosis. The blood was collected and serum was separated for assessing biochemical parameters, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CRE) according to the manufacturer’s protocol. The heart, liver, spleen, lung, and kidney were fixed in 10% formalin, embedded into paraffin and stained with hematoxylin-eosin (H&E). 2.7. Statistical Analysis. Data were expressed as the mean ±SD and analyzed using Student's test, and P < 0.05 was considered to represent a statistically significant difference.

3. RESULTS AND DISCUSSION 3.1. Preparation and characterization of the FA-JNPs. In our Janus system (Scheme 1), FA-linked PEG was bonded on the surface of JNPs to form FA-JNPs that were stable in biological environments and had liver cancer targeting capabilities. Then the NIR irradiation-triggered release behavior of ICG could improve their stability and avoid rapid elimination, leading to enhanced efficacy for PTT. Importantly, we hypothesized that silver ions could be released from the FA-JNPs in response to photothermal effects, subsequently inducing liver cancer cell death as a


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chemotherapeutic agent. To construct this system, the JNPs were fabricated using a modified sol–gel method using Ag NPs as a substrate, CTAB as a template, and TEOS as a silica source33. Specifically, FA-PEG-NHS was conjugated to the surface of the JNPs. The TEM and SEM images in Fig. 1a and b provide an overview of the as-prepared Janus nanostructures. The FA-JNPs had uniform ball-stick structures with good monodispersity, which consisted of silver spheres with diameters of approximately 80 nm and silica rods with lengths of approximately 200-300 nm. To explore the functions of the Ag NPs in this therapeutic system, rod-like mesoporous silica nanoparticles (MSNs) without Ag NPs were synthesized for contrast. These FA-targeted MSNs (FA-MSNs) had similar dimensions to the silica parts of the FA-JNPs (Fig. S1). The particle sizes of the FA-JNPs and FA-MSNs in the pure water and cell medium and remained constant for 5 days, while both the JNPs and MSNs aggregated in cell culture medium (Fig. S2). These results suggested that the PEGylation process could improve the long-term stability of the nanoparticles.35,36 The UV-visible spectra in Fig. 1c showed that the maximum absorption peak of the AgNPs was located at approximately 410 nm and the absorption peak of the FA-JNPs had red shifted to 420 nm, implying that the Janus structure preserved the optical properties of the silver part. The characteristic absorption peak of FA was observed at approximately 280 nm, indicating the conjugation between FA-PEG-NHS and JNPs or MSNs (Fig. 1c and Fig. S3). The FT-IR spectra are displayed in Fig. S4, new adsorption peaks of the amide carbonyl groups at 1692 and 1550 cm-1 were obviously observed in the FA-JNPs group, demonstrating that FA was linked to the



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surface of the NPs. Besides, the conjugation of FA-PEG-NHS was verified by the appearance of the peak at 1356 and 1093 cm-1 in the spectrum of the FA-JNPs. The zeta potential of NPs before and after conjugation of FA-PEG-NHS was listed in Fig. S5. For FA-JNPs, the zeta potential slightly decreased from +23.1 mV to +18.5 mV, which also confirmed the surface modification and FA-conjugation. Moreover, the N2 adsorption–desorption isotherms showed type IV curves (Fig. 1d) that were indicative of the presence of mesopores. The BET surface area and total pore volume of the FA-JNPs were 512.5 m2g-1 and 0.82 cm3g-1, respectively, which were lower than those of the FA-MSNs (897.3 m2g-1 and 1.08 cm3g-1). The BET equation is used for determining the surface area of MSNs from the nitrogen adsorption isotherm at liquid nitrogen temperatures, so the large specific surface areas of FA-targeted NPs indicate their high drug loading content. The average mesopore diameter was approximately 2 nm for both the FA-JNPs and FA-MSNs (Fig. S6), indicating they might have had a similar drug loading behavior. 3.2. Targeted Endocytosis and Toxicity of the FA-JNPs. To investigate the FA-targeted capacity on liver cancer cells, the cellular uptake of the FITC-labeled FA-JNPs was tested in the SMMC-7721 human liver cancer cell line and HL-7702 human hepatic embryo cell line. As shown in Fig. 2a and b, FR-positive SMMC-7721 cells presented a remarkably higher fluorescence intensity than the bare JNPs after 3-h FA-JNPs’ incubation. The overlay images exhibited the co-localization of the FITC-labeled NPs with lysosomes, indicating that the NPs entered the cells and were mainly internalized in the lysosomes. Due to the competitive-blocking of the FR, the


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fluorescence intensity of the FA-JNP-treated cells was significantly reduced when the free FA was pre-incubated. The fluorescence signals of the FA-JNP and JNP groups were similarly lower in the FR-negative HL-7702 cells (Fig. S7). Consistently, the flow cytometry results also confirmed that the existence of FA could enhance the cellular uptake of FA-JNPs in liver cancer cells compared to it of normal liver cells due to the FA-targeted endocytic effect (Fig. 2b).37,38 Taken together, FA-targeted endocytosis was demonstrated in FR-overexpressed liver cancer cells, which implied the targeted internalization of ICG for photothermal therapy. Next, we studied the biocompatibility of the FA-JNPs on the SMMC-7721 and HL-7702 cells with SRB assay. As shown in Fig. 2c and d, the FA-JNPs exhibited dose-dependent toxicity in the SMMC-7721 cells. The cell viability remained more than 80% even when the FA-JNP concentration reached 50 µg mL-1, while JNPs was less cytotoxic than FA-JNPs. The IC50 of FA-INPs in SMMC-7721 cells and HL-7702 cells were 315.6 and 639.3 µg mL-1, respectively. HL-7702 cells were also less sensitive to the FA-JNP exposure than the SMMC-7721 cells, indicating that the FA-INPs could selectively kill the cancer cells rather than normal cells. This phenomenon was attributed to the FA-enhanced endocytosis as well as the cancer cells was more vulnerable to the released silver ions and suffered greater ROS and DNA damage than the normal cells.39,40 Additionally, both FA-MSNs and MSNs did not exhibit cytotoxic effects in both the cancer and normal cells (Fig. S8). Notably, Ag NPs induced the similar toxic manner to the JNPs (Fig. S9), indicating the therapeutic effect of JNPs were mainly based on the silver part but not the silica part. Collectively,



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these findings demonstrated that the FA-JNPs could selectively kill the FR-positive liver cancer cells and had a minimal effect on the normal liver cells. 3.3. Photothermal Effect and Release of the ICG and Silver Ions. We functionalized the framework of the mesoporous silica body with positively charged amino groups to load negatively charged ICG on the surface and in the nanochannels due

to

strong

electrostatic

attractions.41,42

As

determined

using

UV-vis

spectrophotometry, the loading content of the ICG in the FA-JNPs and FA-MSNs was approximately 4.9% and 5.8%, respectively. Compared with the free ICG, the absorption peaks of the FA-JNPs and FA-MSNs exhibited obvious redshifts (20 and 30 nm, respectively), indicating the different local environments of the NP surfaces after loading the ICG (Fig. 3a). Given the poor aqueous stability and concentration-dependent aggregation properties of ICG, we investigated its photostability in the FA-JNPs@ICG. After 5 min of NIR irradiation, the relative absorbance of the free ICG rapidly decreased below 20%, while the loaded ICG in both the FA-JNPs@ICG and FA-MSNs@ICG maintained 80% of their relative absorbance (Fig. 3b). These results demonstrated that the FA-JNPs endowed ICG had outstanding

photostability.

Moreover,

both

of

the

FA-JNPs@ICG

and

FA-MSNs@ICG exhibited good stability in cell culture medium for 5 days (Fig. S10). The photothermal effects of the free ICG and ICG-loaded NPs were evaluated by tracking the temperature variations at different irradiation intensities. As shown in Fig. 3c, the temperatures of the PBS, FA-MSNs, FA-JNPs, free ICG, FA-MSNs@ICG, and FA-JNPs@ICG reached 26.2, 28.3, 35.5, 43.1, 45.7 and 51.6 °C, respectively, after 5


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min of NIR irradiation. The FA-JNPs@ICG and FA-MSNs@ICG exhibited better PTT effects in contrast to the free ICG, indicating the NPs could enhance the PTT effect of ICG. In addition, the PTT effect of the FA-JNPs was stronger than that of the FA-MSNs, which was attributed to the sufficient absorption of the silver NPs in the NIR region. We further evaluated the ICG release from the FA-JNPs@ICG triggered by the NIR irradiation (Fig. 3d). Without NIR irradiation, less than 20% of the ICG was released during 24 h. In marked contrast, the ICG release reached almost 38.5% for the same duration after irradiation with an 808-nm laser at 1 W cm-2 for 5 min. Consistently, the NIR irradiation also enhanced the ICG release from the FA-MSNs@ICG (Fig. S11). These results also indicated that the silver part of the Janus particles did not affect the release behavior of the FA-JNPs@ICG. Many reports have demonstrated that heat, including photothermal and magnetically induced heating, can trigger the release of silver ions from AgNPs.22-24 To investigate the effect of NIR irradiation on the silver ion release behavior, we quantitatively determined the silver ion concentrations at each time point using ICP-MS. As shown in Fig. 3e, the FA-JNPs@ICG showed the sustained-release behavior of the silver ions over the course of 24 h with and without the NIR irradiation. More importantly, more than 2 times the amount of silver ions was released in response to the NIR irradiation, which was consistent with the photothermal-triggered release behavior reported by other groups.43 In addition, the rough surface of silver nanoparticles were observed from TEM images (Fig. S12), which further confirmed the release of silver ions. Collectively, NIR irradiation could



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be employed as an external stimulus to effectively and precisely control the ICG and silver ion release simultaneously from our FA-JNPs@ICG at the desired time and location. After demonstrating the NIR-triggered release of ICG ex vitro, we further investigated the intracellular release behaviors of the FA-JNPs@ICG in SMMC-7721 cells. As shown in Fig. 3f, the flow cytometry data exhibited the much higher fluorescence intensity of ICG in the FA-JNP@ICG group as compared to the JNP@ICG and free ICG groups. When the cells were irradiated with an 808-nm NIR laser at 1 W cm-2 for 5 min, they also exhibited a significant enhancement in fluorescence intensity. Such results were further characterized using the CLSM images in Fig. S13. These results all consistently revealed that the FA-JNPs significantly increased the internalization of the ICG in liver cancer cells through FA-targeting and NIR irradiation-triggered release properties. Additionally, the same trend was observed for the FA-MSNs@ICG in the SMMC-7721 cells (Fig. S14), which reinforced the liver cancer cell selectivity and NIR irradiation-triggered release behavior of the FA-targeted, ICG-loaded NPs. 3.4.

In Vitro

Synergistic

Chemo/Photothermal Therapy

Using

the

FA-JNPs@ICG. To intuitively evaluate the photothermal effect of the FA-JNPs@ICG, calcein-AM and PI were used to distinguish the live/dead cells after NIR irradiation. As shown in Fig. 4a, NIR irradiation alone showed no obvious effect on the cell viability, suggesting that the laser illumination at a power density of 1 W cm-2 for 5 min was safe for the SMMC-7721 cells. However, cells incubated with FA-JNPs@ICG, FA-MSNs@ICG and free ICG induced more dead cells under NIR


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irradiation. There were no obvious difference in the dead cell count between the FA-JNPs@ICG and FA-MSNs@ICG, possibly due to the slow release content of the silver ions after 3-h incubation. Therefore, the FA-JNPs@ICG and FA-MSNs@ICG could significantly improve the efficiency of photothermal therapy using ICG, which was in line with the photothermal effect results in Fig. 3c. To investigate the chemo/photothermal effect of the FA-JNPs@ICG, we thoroughly detected the cell viability of the SMMC-7721 and HL-7702 cells after treatment with various NPs at the same ICG concentration with and without NIR irradiation. As shown in Fig. 4b and c, the free ICG and FA-MSNs@ICG exhibited good biocompatibility in the SMMC-7721 and HL-7702 cells, and the cell viabilities were higher than 80%, even at a high concentration of 100 µg mL-1 (containing 5 µg mL-1 ICG). Moreover, the FA-JNPs@ICG were toxic toward the SMMC-7721 cells rather than the HL-7702 cells, which coincided with the toxicity results in Fig. 2 c and d. Both of the cells incubated

with

FA-JNPs@ICG,

FA-MSNs@ICG

and

free

ICG

possessed

dose-dependent toxicity when under NIR irradiation. In this situation, the FA-MSNs@ICG were much more toxic to the SMMC-7721 cells than the free ICG due to the FA-enhanced endocytosis of ICG. More importantly, the FA-JNPs@ICG exhibited the highest cell-killing effect of the group because of the combination of the ICG-mediated photothermal therapy and silver ion-induced chemotherapy. In contrast, the cell viabilities of the FA-JNPs@ICG, FA-MSNs@ICG and free ICG in the HL-7702 cells were higher than those in the SMMC-7721 cells under the same conditions. The cell-killing efficiency of the free ICG was higher than that of the



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FA-JNPs@ICG and FA-MSNs@ICG in the HL-7702 cells under NIR irradiation, indicating that the reduced FA-MSNs@ICG could adversely affect the ICG. Additionally, we determined the IC50 values of the FA-JNPs-based chemotherapy, ICG-based photothermal therapy, and FA-JNPs@ICG-based chemo/photothermal therapy, and then calculated the combination index (CI) to investigate the effects of the combined therapy. For the SMMC-7721 cells, the IC50 values of the FA-JNPs and ICG+NIR groups were 315.6 µg mL-1 and 17.8 µg mL-1, respectively, which were much higher than those in the FA-JNPs@ICG+NIR group (53 µg mL-1 for the FA-MSNs and 2.65 µg mL-1 for the ICG). The CI of the FA-JNPs@ICG-based chemo/photothermal therapy was calculated as 0.317, which further confirmed the synergistic effects between the silver-induced chemotherapy and ICG-mediated photothermal therapy. On the basis of these results, the FA-JNPs@ICG could be integrated into a synergistic chemo/photothermal therapy with efficient and safe effects for liver cancer, which further reinforced the targeting effect of the FA. 3.5. Combined Anti-tumor and Safe Effects in vivo. Encouraged by the chemo/photothermal therapeutic effect in vitro, we further investigated the ability of the FA-JNPs@ICG combined with NIR irradiation to inhibit tumor growth in the xenografts of nude mice bearing orthotopic SMMC-7721 cells. The ICG, FA-MSNs@ICG, and FA-JNPs@ICG were administered through intravenous injection, and the tumor regions were irradiated by an 808 nm laser at 1 W cm-2 for 5 min; three mice groups (PBS, PBS+NIR, FA-JNPs) were set as controls. As shown in Fig. 5a and b, the SMMC-7721 tumors treated with PBS plus NIR irradiation or


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solely PBS grew rapidly, indicating that the NIR irradiation had no effect on the tumor growth. The slight anti-tumor efficacy of the FA-JNPs was due to the slow and sustained release of silver ions at the tumor site. The free ICG plus NIR irradiation had a slight inhibitory effect on the tumor growth, which was possibly due to the rapid clearance of free ICG and reduced tumor site accumulation.44 In contrast, the FA-MSNs@ICG and FA-JNPs@ICG plus NIR irradiation remarkably inhibited the tumor growth and tumor weight. More importantly, the FA-JNPs@ICG plus NIR irradiation

displayed

higher

tumor

inhibition

rates

(88.9%)

than

the

FA-MSNs@ICG(23.6%), ICG+NIR (33.6%) and FA-MSNs@ICG+NIR (54.7%) (Fig. 5c). This excellent anti-tumor efficacy was attributed to the FA active targeting, synergistic effect of the silver ion-induced cytotoxicity and ICG-mediated photothermal ablation. To further exploit the mechanism of the combined anti-tumor effect, TUNEL staining of the excised tumors was conducted, and the apoptotic rates were calculated. As shown in Fig. 5d and e, the degree of apoptosis was consistent with that of the tumor inhibition in Fig. 5c. Minimal apoptotic fluorescence signals were observed in the saline and saline+NIR groups. The FA-JNPs and ICG+NIR groups exhibited apoptosis, while the FA-MSNs+NIR group exhibited significantly more apoptosis. As expected, the FA-JNPs+NIR treatment resulted in the highest apoptotic rate in the tumors compared to the other groups, which indicated that the efficient inhibition of the tumor growth resulted from the photothermal- and silver ion-induced apoptosis of the liver cancer cells.



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To further evaluate the systemic toxicity of the combined chemo/photothermal therapy, the body and organ weights and blood chemistry indexes (liver function indexes: alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and kidney function indexes: creatinine (CRE) and blood urea nitrogen (BUN) were examined at the end of the treatment as well as the histological examination of the major organs. As shown in Fig. 6a, compared to the PBS group, only the FA-JNPs and FA-JNPs@ICG+NIR groups showed slightly decreased body weights, while there were no observable weight losses in the other groups. Silver-induced weight loss and organ reduction have been consistently reported by other researchers.45-47 However, the silver-containing groups did not have significant organ weight index changes compared to the PBS group (Fig. 6b). As shown in Fig. 6c-e, the levels of ALT, AST, CRE and BUN were not remarkably changed in all groups, indicating good liver and kidney functions during the combined treatment. Additionally, H&E staining demonstrated that non-evident pathological change were observed in the slices of heart, liver, spleen, lung and kidney of all groups (Fig. 7). Taken together, consistent with our in vitro findings, FA-JNPs@ICG provided an efficient chemo/photothermal therapeutic

agent

in

vivo

with

less

systemic

toxicity.

Although

Janus

silver-mesoporous silica nanoparticles showed some toxicity manner on normal liver cells only at higher concentrations (more than 100 µg mL-1), there were less side effects observed in vivo. Actually, this silver-containing nanoplatform did not exhibited the change of organ weight index, blood chemistry index and pathological conditions when compared to the control group. The toxicity of Janus nanoplatforms


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were reduced because of the targeted delivery and NIR irradiation-induced silver ions release. In this study, we employed NIR irradiation-induced heat to control the release of silver ions from FA-INPs@ICG, resulting in the selective silver-based chemotherapy to supplement ICG-mediated photothermal therapy with minimal side effects.

4. CONCLUSIONS In summary, an FA-targeted Janus silver MSNs-based drug delivery nanoplatform was fabricated for the combined chemo/photothermal therapy of liver cancer. To surmount the poor aqueous stability and photostability of ICG, we obtained a promising NIR-triggered nanoplatform that was could efficiently internalize into the liver cancer cells via FR-mediated endocytosis. Importantly, an ICG-based photothermal effect was able to trigger the simultaneous release of both ICG and silver ions without mutual interference, the former exerted a predominant photothermal ability in the cytoplasm to kill cancer cells; the latter could be employed as an anti-tumor agent for efficient chemotherapy to supplement the photothermal therapy. The Janus nanoplatforms further demonstrated synergistic anti-tumor efficacy with less side effects in vitro and in vivo. Looking forward, our Janus nanoplatform that employs the effector for photothermal therapy as the initiator to activate the chemotherapy, may offer a sequential strategy for combined liver cancer therapy.



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ASSOCIATED CONTENT Supporting Information. Materials, characterization, cytotoxicity, endocytosis, intracellular ICG release. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mails: W.F. D., [email protected], D. S., [email protected]. Suzhou Institute of Biomedical Engineering and Technology (SIBET), Chinese Academy of Sciences (CAS), 88 Keling RD, Suzhou, Jiangsu, China , 215163 Tel: +86-512-6958-8307 Fax: +86-512-6958-8088

Conflict of Interest The authors declare no competing financial interest.

ACKNOWLEDGMENT Prof. Li Chen, Prof. Jing Li, Xiao Zheng and Xin Zhang are acknowledged for their help in preparing the paper. This work was supported by The National Key Research and Development Program of China (Grand No.2017YFF0108600, 2017YFC0211900, and 2016YFF0103800), the National Natural Science Foundation of China (Grand No.81771982, 61535010, 81601609 and 8160071152), Key Research Program of the Chinese Academy of Sciences (No. KFZD-SW-21), the Natural Science Foundation


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of Jiangsu Province (No. BE2015601) and the Science and Technology Department of Suzhou City (No. SS201539 and ZXY201434).

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Figure Captions

Scheme 1. Schematic illustration of the FA-targeted Janus nanoplatform with the NIR irradiation-triggered release behavior of ICG and silver ions for synergistic liver cancer chemo/photothermal therapy.


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Figure 1. Characterization of the FA-JNPs: (a) TEM images; (b) SEM images; (c) UV-vis absorption spectra, and (d) N2 adsorption/desorption isotherms of the FA-JNPs.



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Figure 2. Endocytosis and toxicity of the FA-JNPs: (a) CLSM images of the FA-JNPs, JNPs or free FA+ FA-JNPs treated in SMMC-7721 cells for 3 h; the scale bars represent 10 µm. (b) Quantitative analysis of the internalization of the FITC-labeled NPs in the SMMC-7721 cells and HL-7702 cells after 3 h of exposure using FACS. These data represent three separate experiments and are presented as the mean values ± SD, and *P < 0.05 versus the JNP group, #P

Silica Nanoplatforms for Light-Activated Liver Cancer

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