Janus Gold Nanoplatform for Synergetic Chemoradiotherapy and

Janus Gold Nanoplatform for Synergetic Chemoradiotherapy and Computed Tomography Imaging of Hepatocellular Carcinoma ACS Nano 2017.11:12732-12741. Downloaded from pubs.acs.org by UNIV OF TOLEDO on 06/29/18. For personal use only.

Zheng Wang,†,‡ Dan Shao,*,†,⊥ Zhimin Chang,† Mengmeng Lu,§,⊥ Yingshuai Wang,† Juan Yue,†,‡ Dian Yang,†,‡ Mingqiang Li,⊥ Qiaobing Xu,# and 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, New York 10027, United States # Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, Massachusetts 02115, United States S Supporting Information *

ABSTRACT: There is a pressing need to develop nanoplatforms that integrate multimodal therapeutics to improve treatment responses and prolong the survival of patients with unresectable hepatocellular carcinoma (HCC). Mesoporous silica-coated gold nanomaterials have emerged as a novel multifunctional platform combining tunable surface plasmon resonance and mesoporous properties that exhibit multimodality properties in cancer theranostics. However, their reduced radiation-absorption efficiency and limited surface area hinder their further radiochemotherapeutic applications. To address these issues, we designed Janus-structured gold-mesoporous silica nanoparticles using a modified sol−gel method. This multifunctional theranostic nanoplatform was subsequently modified via the conjugation of folic acid for enhanced HCC targeting and internalization. The loaded anticancer agent doxorubicin can be released from the mesopores in a pH-responsive manner, facilitating selective and safe chemotherapy. Additionally, the combination of chemotherapy and radiotherapy induced synergistic anticancer effects in vitro and exhibited remarkable inhibition of tumor growth in vivo along with significantly reduced systematic toxicity. Additionally, the Janus NPs acted as targeted computed tomography (CT)-imaging agents for HCC diagnosis. Given their better performance in chemoradiotherapy and CT imaging as compared with that of their core−shell counterparts, this new nanoplatform designed with dual functionalities provides a promising strategy for unresectable HCC theranostics. KEYWORDS: Janus, gold mesoporous silica, hepatocellular carcinoma, synergetic chemoradiotherapy, CT imaging survival of unresectable HCC patients.7 Recently, continuing efforts led to the thriving combination of chemotherapy and radiotherapy, ultimately achieving optimal and synergetic treatment of unresectable HCC.8,9 However, chemoresistance and nonselective cytotoxicity result in low therapeutic efficacy and severe side effects, while radioresistance and the limitations

H

epatocellular carcinoma (HCC) has emerged as one of the most common malignancies worldwide and is characterized by poor prognosis and high mortality.1,2 Although surgical resection is considered the best choice for long-term control of this disease, most advanced HCC patients do not meet the criteria for surgical resection.3 Moreover, other single-modality treatments, such as chemotherapy, external radiotherapy, and radio frequency ablation, show limited efficacy.4−6 Thus, there is a pressing need for multimodal therapies to improve therapeutic responses and prolong the © 2017 American Chemical Society

Received: October 23, 2017 Accepted: November 15, 2017 Published: November 15, 2017 12732

DOI: 10.1021/acsnano.7b07486 ACS Nano 2017, 11, 12732−12741

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ACS Nano of the irradiative dosages lead to failure to deracinate the hypoxic HCC foci.10,11 Moreover, such a synergetic treatment remains unsatisfactory, because radiotherapy and anticancer drugs do not simultaneously affect the same HCC regions. Therefore, it is highly desired to employ an efficient radiosensitizer to enhance the response to radiotherapy, thereby providing a targeted strategy for exerting maximal synergistic chemoradiotherapeutic effect with minimal side effects. In light of the integration of nanotechnology in biomedicine, the emergence of nanoplatforms has allowed new approaches for preferentially delivering theranostic agents to the tumor mass while reducing systemic toxicity.12−16 Among various inorganic nanocarriers, gold nanoparticles (Au NPs) have attracted prominent research interest due to their outstanding properties and potential applications in biomedicine.17,18 In addition to having tunable surface plasmon resonance (SPR) properties for photothermal therapy, Au NPs have also emerged as efficient radiosensitizers due to their high radiation absorption.19,20 Intensive preclinical studies have enriched the current understanding of Au NP radiosensitization to a greater degree than their physical dose-enhancement effects.21 Additional chemical and biological contributions, such as tumor accumulation and reactive oxygen species generation, can occur, thereby minimizing healthy tissue damage and improving radiation efficacy.22,23 Mesoporous silica possesses superior properties, including a high surface area, an easily modified surface, and good biocompatibility.24 Consequently, several synthesis strategies have been developed to encapsulate Au cores into mesoporous silica shells to provide Au NPs with synergistic properties.25 However, there are limited studies investigating their combined chemoradiotherapeutic behavior.26 Additionally, these symmetrical structures generally have insufficient surface area available for anticancer-drug loading and reduced radiation-absorption efficiency, which might weaken their synergistic effects for chemoradiotherapy.17 In this regard, it is critical to develop multisurface-based Aumesoporous silica NPs with the optimal intrinsic properties of Au and silica for HCC theranostics. Janus nanocomposites have received substantial attention due to their multifunctional properties on anisotropic surfaces, which allow these particles to house several functional parts for cancer theranostics.27−33 In the present study, Au-mesoporous silica Janus NPs (GSJNs) were fabricated using a modified sol− gel method. Such multifunctional theranostic nanoplatforms are designed to simultaneously integrate chemotherapy, radiotherapy, and in vivo computed tomography (CT) imaging into a single system (Scheme 1). In this system, the folic acid (FA) on the surface of mesoporous silica was modified for HCC targeting, and the subsequent controlled release of the anticancer agent doxorubicin (DOX) was dependent upon acidic stimuli from the tumor microenvironment. The targeted delivery, selective cellular internalization, and pH-controlled drug release of FA-GSJNs-DOX NPs were assessed using hepatoma cell lines and normal liver cell lines with different FAexpression levels. In vitro and in vivo experiments were performed to investigate the chemoradiotherapeutic effects and safety of the FA-GSJNs-DOX. Additionally, the CTimaging ability of this Janus nanoplatform was demonstrated in vivo. Based on the better performance of Janus NPs in targeted chemoradiotherapy and CT imaging as compared with that of their traditional core−shell counterparts, the multifunctional

Scheme 1. Schematic Illustration of the Synthetic Procedure for the DOX-Loaded Au-Mesoporous Silica Janus NPs and Application for Synergetic Chemoradiotherapy and CT Imaging in HCC Theranostics

Janus nanosystem might be a promising platform for achieving efficient and safe HCC theranostics.

RESULTS AND DISCUSSION GSJNs were prepared using a modified sol−gel method reported in previous studies.31−33 The transmission electron microscopy (TEM) image revealed that the length and width of the GSJNs were ∼200 nm to ∼250 nm and ∼100 nm to ∼120 nm, respectively (Figure 1A). The surface of the GSJNs was functionalized with 3-aminopropyltriethoxysilane to obtain GSJNs-NH2, which was then reacted with succinic anhydride to produce GSJNs-COOH. Successful conjugation of the FApolyethylene glycol (PEG) amine and the carboxylate group on the GSJN surface was subsequently confirmed by FTIR analysis (Figure 1B). An increase in intensity was observed in the typical adsorption peaks of the amide carbonyl groups at 1641 and 1539 cm−1, whereas the characteristic adsorption peaks of FA-PEG-NH2 at 1335 and 1100 cm−1 also appeared in the spectrum of the FA-GSJNs. Notably, core−shell-type mesoporous silica nanoparticles (FA-GSCNs) were synthesized in parallel as a contrast to demonstrate the advantages of FAGSJNs in cancer theranostics (Figure S1). The UV−vis spectrum shown in Figure 1C further demonstrated successful FA conjugation, as indicated by the characteristic absorption peak at 275 nm in the FA-GSJNs. The PEG content in FAGSJNs was 8.9 wt %, and three FA molecules were estimated to bind to one NP. As shown in the same figure, the FA-GSJNs exhibited similar SPR properties with a slight red shift in the longitudinal SPR wavelength as compared with that of Au rods, implying that the Janus NPs preserved the optical properties of Au. Although the FA-GSCNs possessed similar SPR properties as Au rods and FA-GSJNs, the red-shift wavelength of FAGSCNs was nearly 100 nm (Figure S2), which was much higher than that of its Janus counterparts. The N2 adsorption− desorption isotherm curve (Figures 1D and S3) indicated that the resulting FA-GSJNs possessed a high BET surface area (726.2 m2/g), a large pore volume (0.47 cm3/g), and a uniform pore size (2.2 nm) for drug loading. The surface area, pore volume, and pore size of FA-GSJNs were slightly lower than those of GSJNs (758.5 m2/g, 0.51 cm3/g, and 2.4 nm, respectively) due to FA conjugation. Notably, the FA-GSJNs exhibited a higher surface area and pore volume than FA12733


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Figure 1. Characterization of FA-GSJNs. TEM images (A), FTIR spectra (B), UV−vis extinction spectra (C), and N2 adsorption−desorption isotherms (D) of FA-GSJNs.

Figure 2. Targeted cell uptake of FA-GSJNs. CLSM images of SMMC-7721 cells (A) and HL-7702 cells (B) incubated with the targeted FAGSJNs, nontargeted GSJNs, or FA-GSJNs+FA for 3 h. Scale bars, 10 μm. Quantitative FACS analysis of internalization of 12.5 μg/mL FITClabeled Janus NPs in SMMC-7721 cells (C) and HL-7702 cells (D) after 3 h of exposure. Data represent three separate experiments and are presented as the mean ± SD *P < 0.05 vs the GSJNs group; #P < 0.05 vs the FA-GSJNs group. 12734


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Figure 3. Selective drug-release behavior of FA-GSJNs-DOX. (A) pH-dependent drug-release profiles of FA-GSJNs-DOX. (B) Quantitative analysis of the fluorescence intensity of DOX in free DOX-, GSJNs-DOX-, or FA-GSJNs-DOX-treated SMMC-7721 or HL-7702 cells for 3 h. Data represent three separate experiments and are presented as the mean ± SD *P < 0.05 vs the DOX group; #P < 0.05 vs the GSJNs-DOX group. (C) CLSM images of SMMC-7721 or HL-7702 cells treated with free DOX or FA-GSJNs-DOX for 3 h. Scale bars, 10 μm.

GSCNs (536.5 m2/g and 0.36 cm3/g, respectively) along with a similar pore size (2.3 nm) (Figures S3 and S4), indicating a higher drug-loading potential for the Janus NPs. Additionally, FA-GSJNs exhibited a Z-average size of 223 nm with a polydispersity index (PDI) of 0.108 (Figure S5A). During a 1 week storage period in water, these PEGylated NPs showed no significant change in size and PDI (Figure S5), suggesting their superior colloidal stability though PEG modification. Because FA is considered a targeting ligand for HCC theranostics,39 we employed the SMMC-7721 human liver cancer cell line with overexpressed folate receptors (FRs) and the HL-7702 human hepatic embryo cell line with low FR expression to investigate the feasibility of effective tumor targeting. As shown in Figure 2A,B, the two cell lines were able to uptake the FA-GSJNs and GSJNs after 3 h of incubation. A large amount of FA-GSJNs and GSJNs was observed to accumulate and colocalize with LysoTracker in the two cell lines, indicating that the FA-GSJNs could be taken up by liver cells and internalized in acidic endolysosomes for efficient drug release. More importantly, the FA-GSJNs showed better cellular uptake than nontargeted GSJNs in SMMC-7721 cells, whereas both FA-GSJNs and GSJNs exhibited similar celluptake behavior in the FR-enriched HL-7702 cells. The strong fluorescence of the FA-GSJNs was significantly attenuated when 1 mM free FA was applied to the SMMC-7721 cells, further indicating FA-mediated cellular uptake. Moreover, FACS data revealed consistent results (Figure 2C,D). These results demonstrated that the FA-modified GSJNs enhanced HCC targeting. To investigate the potential for small-molecule drug delivery, the carboxylate-functionalized NPs were loaded with DOX, a model drug commonly used in the treatment of HCC (Figure S6). The drug-loading content and loading efficiency of DOX in the FA-GSJNs were approximately 21.8% and 61.7%, respectively. Notably, the FA-GSJNs exhibited higher drugloading content than FA-GSCNs (17.2% and 49.5%) and other

core−shell Au-silica NPs due to the large surface area of the Janus structure.40,41 Next, we employed DOX-loaded FAGSJNs as a model system for future studies. As shown in Figure 3A, >40% of the DOX was released at pH 5.5 in contrast to the 5% DOX release at pH 7.4 within 24 h, demonstrating that DOX could be released through the protonation and dissociation of their amine groups in acidic environments. Similarly, >35% of the DOX was released at pH 5.5 in contrast to the 5% DOX release at pH 7.4 within 24 h from DOXloaded FA-GSCNs (Figure S7), further confirming the pHresponsive release behavior. Because this pH-sensitive DOXrelease phenomenon can occur in acid endolysosomes or in the local environment of HCC, thereby enhancing the therapeutic capacity at tumor tissue with fewer side effects in normal tissue, we further investigated DOX internalization within liver cells for 3 h using CLSM and flow cytometry. Quantitative analysis showed that the mean fluorescence intensity of intracellular DOX from the FA-GSJNs was higher than that of the free DOX in SMMC-7721 cells. By contrast, the HL-7702 cells showed more internalization of free DOX than that of DOX from the FA-GSJNs (Figure 3B). Similar DOX accumulation is also shown in Figure 3C, where the majority of DOX was released from FA-GSJNs and transported to the nuclei of HCC cells rather than normal cells due to the above-mentioned pHsensitive DOX-release behavior. We also observed that the quantitative results obtained by DOX internalization were consistent with CLSM imaging and FACS data, further confirming the unique advantage of the present pH-sensitive drug-delivery system in the tumor intracellular environment. Importantly, DOX-loaded Janus NPs exhibited more DOX internalization than DOX-loaded core−shell NPs, which might primarily be due to its higher drug-loading capacity (Figure S8). A consensus has been reached that DOX localization in nuclei can induce irreversible cell death.29,30 Therefore, we further evaluated the cytotoxicity of the FA-GSJNs and FAGSJNs-DOX via SRB assays. After a 24 h incubation, the dose12735


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Figure 4. Cytotoxic profile of FA-GSJNs-DOX. The cytotoxicity of GSJNs, FA-GSJNs, free DOX, GSJNs-DOX, and FA-GSJNs-DOX against SMMC-7721 cells (A, C) and HL-7702 cells (B, D) at different concentrations for 24 h. Data represent three separate experiments and are presented as the mean ± SD *P < 0.05 vs the control group or groups without RT; #P < 0.05 vs the GSJNs-DOX group; &P < 0.05 vs the DOX group. (E) SMMC-7721 cells treated with FA-GSJNs-DOX, RT, or FA-GSJNs-DOX+RT for 6 h and stained with both 4′,6-diamidino-2phenylindole and anti-γ-H2AX (F) followed by quantification of the images. Data represent three separate experiments and are presented as the mean ± SD. Scale bars, 20 μm. *P < 0.05 vs the control group or groups without RT; #P < 0.05 vs the FA-GSJNs-DOX group; &P < 0.05 vs the RT group.

maintained high cell viability. By contrast, the viability of cells treated with Janus NPs with or without DOX (FA-GSJNs-DOX and GSJNs-DOX) and exposed to radiation significantly decreased. Notably, the FA-GSJNs-DOX+RT group showed a more substantial decrease in cell viability than the FA-GSJNsDOX and RT groups, demonstrating the combined effect of the Janus NPs. However, the chemoradiotherapeutic cytotoxicity in HL-7702 cells was less than that observed in SMMC-7721 cells (Figure 4D). These phenomena were consistent with previously reported chemoradiotherapy applications with other mesoporous silica NP-based nanoplatforms.42−44 Additionally, we determined the IC50 of radiotherapy, FA-GSJNsbased chemotherapy, and FA-GSJNs-based radiochemotherapy and calculated the combination index (CI) to investigate the synergistic effects of radiochemotherapy. As shown in Figure S9, the IC50 values of the FA-GSJNs-DOX and RT groups were 9.38 μg/mL and 6.47 Gy, respectively, which were higher than

dependent cytotoxicities of the Janus NPs, free DOX, and DOX-loaded Janus NPs were evaluated and are shown in Figure 4A,B. In the absence of DOX, both FA-GSJNs and GSJNs were reasonably safe up to 12.5 μg/mL. The GSJNsDOX exhibited similar toxicities in SMMC-7721 cells as compared with that of free DOX, and the FA-GSJNs significantly improved the anticancer effect of DOX. However, the HL-7702 cells were more likely to be killed than the SMMC-7721 cells, whereas DOX-loaded Janus NPs displayed reduced cytotoxicity. These findings demonstrated that the distinct anticancer effect of the FA-GSJNs originated from selective endocytosis, as well as pH-responsive drug release, in the HCC cells rather than in normal cells. Encouraged by these findings, we further investigated the antitumor effects of the combination treatment of DOX-loaded Janus NPs and X-ray radiation (RT). As shown in Figure 4C, the SMMC-7721 cells exposed to radiation without any treatment with NPs or DOX 12736


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Figure 5. In vivo CT imaging and biodistribution. (A) CT images of distilled water and FA-GSJNs-DOX as a function of concentration. (B) CT attenuation (HU) of FA-GSJNs-DOX at various concentrations (0.5−10 mg/mL). (C) In vivo CT images of SMMC-7721 tumor-bearing nude mice at 24 h post-injection with FA-GSJNs-DOX or GSJNs-DOX. (D) Quantitative ICP-OES analysis of FA-GSJNs-DOX or GSJNs-DOX in organs and tumors at 24 h post-injection. Data represent the mean ± SD (n = 6). *P < 0.05 vs the GSJNs-DOX group.

that of the FA-GSJNs-DOX+RT group (3.83 μg/mL and 1.53 Gy). The CI of FA-GSJNs-DOX-based radiochemotherapy was calculated at 0.63, which was higher that for FA-GSCNs-DOXbased radiochemotherapy (CI = 0.81; Figure S10). These results further confirmed the synergistic effects between NPbased chemotherapy and X-ray radiation, as well as the improved performance of Janus NP-based chemoradiotherapy, and demonstrated that FA-GSJNs-DOX was able to selectively kill HCC cells through targeted and synergistic chemoradiotherapy. Combined chemotherapy and radiotherapy have been widely used in clinical cancer treatment, showing promise for enhancing therapeutic outcomes.45 On the one hand, X-ray radiation can cause fatal damage to cancer cells by causing breaks in DNA chains; however, cells under a hypoxic tumor microenvironment can also exhibit decreased repair of freeradical-mediated DNA damage.46 On the other hand, DOX not only kills cancer cells by inducing DNA damage but also inhibits the repair of X-ray radiation-induced DNA damage by altering the abilities of DNA-repair enzymes.47 Hence, we observed double-stranded DNA damage via γ-H2AX staining to confirm the synergistic DNA damage induced by NP-mediated DOX and X-ray radiation. As shown in Figure 4E,F, higher levels of γ-H2AX foci in response to DNA damage were observed in the FA-GSJNs-DOX+RT group than in the RT or FA-GSJNs-DOX groups, indicating synergistic double-stranded DNA damage from chemoradiotherapy. In this system, Janus Au-mesoporous silica NPs not only achieved targeted delivery of DOX into liver cancer cells but also acted as radiosensitizers to enhance the efficacy of radiotherapy. By exploiting these synergies, it might be possible to further reduce the effective dose of drugs via targeted delivery of radiosensitizers and anticancer drugs to subsequently achieve efficient and safe HCC treatment. Au NPs have emerged as X-ray contrast agents due to their strong X-ray attenuation properties.48−50 To demonstrate the

CT-imaging ability of the Janus NPs, the Hounsfield unit (HU) of FA-GSJNs-DOX was evaluated using a clinical CT scanner. Figure 5A,B shows the CT images of FA-GSJNs-DOX in the range of 0.5−5 mg/mL. Brighter images were observed as the intensity of the CT signal was enhanced along with increasing Janus NP concentration. We also quantified the relationship between the HU and Janus NP concentration, revealing a linear relationship (R2 = 0.997). To further examine targeted CT imaging in vivo, SMMC-7721 tumor-bearing nude mice were treated with FA-GSJNs-DOX or GSJNs-DOX (5 mg/mL), and after 24 h, CT imaging was employed to track the HCC targeting of the Janus NPs. As shown in Figure 5C, the corresponding tumor site of the mice exhibited a clearly distinguished CT signal in both the FA-GSJNs-DOX and GSJNs-DOX groups based on transverse section images. The CT values at the tumor sites of the mice treated with FAGSJNs-DOX were much higher than those from mice treated with GSJNs-DOX. This result demonstrated that the FAGSJNs-DOX could selectively accumulate at the tumor site, indicating that the Janus NPs might be ideal candidates for use as efficient CT-imaging contrast agents. Additionally, the biodistribution of the Janus NPs demonstrated their predominant accumulation in the liver, spleen, and kidneys of the reticuloendothelial system (Figure 5D).51−54 Notably, FAGSCNs-DOX also exhibited similar and weaker CT-imaging and tumor-targeting properties than those of its Janus counterparts (Figure S11). Furthermore, FA-GSJNs-DOXtreated mice consistently exhibited blood retention similar to that of FA-GSCNs-DOX-treated mice (Figure S12). Collectively, these results indicated that FA-GSJNs-DOX exhibited a higher tumor-accumulation efficiency than both GSJNs-DOX and FA-GSCNs-DOX. SMMC-7721 xenografts were replicated in 24 nude mice to evaluate the synergistic efficacy of the chemoradiotherapy. The mice in eight groups, the control, FA-GSJNs, RT, DOX, GSJNs-DOX, FA-GSJNs-DOX, GSJNs-DOX+RT, and FA12737


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Figure 6. In vivo chemoradiotherapy effect. Tumor photographs (A), weight (B), volume (C), body weight (D), CK levels (E), and weight indexes of the liver, spleen, and kidneys (F) from mice in each group over 23 days. Data represent the mean ± SD (n = 6). *P < 0.05 vs the control group; #P < 0.05 vs the FA-GSJNs+RT group &P < 0.05 vs the GSJNs-DOX group; $P < 0.05 vs the GSJNs-DOX+RT group.

GSJNs-DOX+RT groups, were intravenously injected with drugs or/and received irradiation at 5 Gy every 3 days for a total of six administrations, and the experiment was terminated on day 23. As shown in Figure 6A−C, mice in the FA-GSJNs group displayed tumor growth similar to the control group, indicating that the FA-GSJNs alone had little antitumor effects, whereas the tumors in the other six groups displayed timedependent decreases in volume and reduced tumor weight. Among these groups, the FA-GSJNs-DOX+RT group exhibited the highest level of tumor-growth delay as compared to that of the other groups. In particular, both the FA-GSJNs-DOX and GSJNs-DOX groups displayed faster tumor growth than that of the DOX group, which might be attributed to their unsatisfactory accumulation in the subcutaneous xenografts. Unfortunately, in the DOX group, the body and organ-weight indices of the spleen were greatly reduced (Figure 6D,E). Cardiotoxicity is a known side effect of clinical DOX treatment,55,56 and increased CK levels were observed only in the DOX group (Figure 6F). Other than the DOX-induced side effects, there was no observable weight loss, organ-weight index change, or cardiotoxicity in mice from the other groups. Furthermore, histological assessment confirmed the absence of pathological damage in the major organs during the treatment (Figures 7 and S13), suggesting the excellent biocompatibility of FA-GSJNs-DOX and a decrease in side effects associated with HCC chemoradiotherapy. Importantly, we compared the antitumor effect and safety of FA-GSJNs-DOX- and FA-GSJNsDOX-based chemoradiotherapies. As shown in Figure S14, the FA-GSJNs-DOX+RT group exhibited higher tumor-growth inhibition relative to that observed in the FA-GSCNs-DOX +RT group. Additionally, FA-GSCNs-DOX also showed good biocompatibility (Figure S15). Consequently, these results demonstrated that FA-GSJNs-DOX would be an effective and safe treatment modality for HCC-targeted chemoradiotherapy in vivo.

Figure 7. Effect of radiochemotherapy on the histopathology of organs from SMMC-7721 tumor-bearing mice from each group. Images were obtained at 40× magnification with standard HE staining. Scale bars, 100 μm.

controlled, pH-responsive drug-release behavior after internalization by HCC cells. Importantly, the combination of chemotherapy and radiotherapy mediated synergistic anticancer effects in vitro and remarkable tumor regression accompanied by significantly reduced systematic toxicity in cells treated with FA-GSJNs-DOX rather than FA-GSCNs-DOX. Additionally, we demonstrated the feasibility of using FA-GSJNs-DOX as a targeted CT-imaging agent for HCC diagnosis. Compared with their core−shell counterparts, the Au-mesoporous silica Janus NPs could be developed as efficient and safe nanoagents for synergistic chemoradiotherapy. This work highlighted the potential of the Janus nanoplatform with dual functionalities for multidimensional HCC theranostics.

METHODS Synthesize of FA-GSJNs and GSJNs. GSJNs were synthesized according to our previously published procedure with some minor modifications.31−33 To synthesize FITC-labeled GSJNs, APS was covalently coupled to FITC. The as-synthesized GSJNs or FITClabeled GSJNs were further refluxed in NH4NO3-ethanol solution (60 mL, 10 mg/mL) for 12 h to remove CTAB. To achieve pH-responsive drug release and HCC targeting, we further modified the carboxylate group and FA-PEG-NH2 on the surface of the GSJNs. According to our previous methods, the GSJNs were functionalized with APS

CONCLUSIONS In summary, we developed GSJNs that possess superior SPR and mesoporous properties. The surface conjugation of FAtargeting ligands on the mesoporous silica body effectively enhanced HCC targeting and internalization. Loading of DOX via a pH-sensitive linker onto the silica surface enabled 12738


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synergism, CIX = 1.0 indicates an additive effect, and CIX > 1.0 indicates antagonism. For the immunofluorescence experiment, cells were treated with FA-GSJNs-DOX, X-ray radiation, and FA-GSJNs-DOX plus X-ray radiation for 6 h and fixed in 4% (v/v) paraformaldehyde. The cells were then washed with PBS containing 0.02% (v/v) Triton X-100, followed by incubation with anti-γ-H2AX antibody for 1 h. The cells were incubated with Alexa Fluor (488)-conjugated secondary antibody, and images were investigated using the predescribed CLSM protocols. CT Imaging and Biodistribution Measurement. CT scanning of the NP solution and mice was performed using a Discovery CT590 RT scanner (GE Healthcare, Milwaukee, WI, USA). Hounsfield units (HUs) of the targeted regions were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA). To demonstrate the targeted accumulation of FA-GSJNs-DOX for in vivo CT imaging, SMMC-7721 tumor-bearing nude mice were intravenously injected with FA-GSJNs-DOX or GSJNs-DOX at a dose of 50 mg/kg and scanned 24 h after administration. To quantify the biodistribution of the FA-GSJNs-DOX, mice were sacrificed, and the tumors, heart, lungs, liver, spleen, and kidneys were removed and weighed. The total amount of Au in the solutions was measured using inductively coupled plasma mass spectrometry (ICP-OES; Xseries II; Thermo Fisher Scientific, Waltham, MA, USA), and the contents in each organ were calculated and normalized to the contents of the control group. Furthermore, to assess the in vivo pharmacokinetics and biodistribution of NPs, SMMC-7721 tumor-bearing nude mice were administered FA-GSJNs-DOX (50 mg/kg) via tail-vein injection. At different time points (0.5, 1, 2, 4, 8, 12, 24, and 48 h), blood samples were collected through the orbital sinus, and the total amount of Au in the solutions was measured by ICP-OES. In Vivo Anticancer Effect and Systemic Safety Evaluation. All animal experimental protocols were approved by the Ethics Committee for the Use of Experimental Animals of Jilin University. The SMMC-7721 xenograft-bearing nude mouse model was replicated according to a previously described protocol. The mice were randomized into eight groups (n = 6) and then administered saline, FA-GSJNs, radiotherapy (RT) alone, DOX (1 mg/kg), GSJNs-DOX (5 mg/kg), FA-GSJNs-DOX (5 mg/kg), GSJNs-DOX (5 mg/kg)+RT, and FA-GSJNs-DOX (5 mg/kg)+RT intravenously through the tail vein every 3 days when tumor volume reached a range of 60−100 mm3. In the RT, GSJNs-DOX (5 mg/kg)+RT, and FA-GSJNs-DOX (5 mg/kg)+RT groups, the mice received 5 Gy of X-ray radiation according to group requirements on a clinical linear accelerator (TrueBeam; Varian Medical System) using a 1.5 cm × 1.5 cm radiation field to cover the entire tumor. The mice in each group were measured with a digital caliper every other day, and the formula length × width2 × 0.52 was used for calculations. During the next 23 days, all mice were sacrificed. Subsequently, serum was collected, and the level of phosphocreatine kinase (CK) was assayed. The heart, lungs, liver, spleen, and kidneys from three mice in each group were removed and fixed for hematoxylin and eosin (HE) staining. Statistical Analysis. The data are expressed as the means ± standard deviation (SD). Statistical significance (P < 0.05) was evaluated using Student’s t test when only two groups were compared. If more than two groups were compared, the significance was evaluated using one-way analysis of variance, followed by Bonferroni’s post hoc test.

through the postgrafting method and were then reacted with succinic anhydride to form GSJNs-COOH.34,35 Finally, the modified EDC/ NHS reaction was employed for covalent conjugation between FAPEG-NH2 or PGE-NH2 and GSJNs-COOH according to our previously work.36,37 Briefly, an EDC/NHS aqueous solution was added to the GSJNs-COOH suspension and sonicated for another 30 min. After that, FA-PEG-NH2 or PEG-NH2 was added to the mixed suspension to react another 12 h. Excess reagents were removed by centrifugation at 10,000 rpm and washed with water three times. We named final PEG-conjugated product as FA-GSJNs or GSJNs. HCC-Targeting Evaluation. Human hepatoma cells (SMMC7721) and human hepatic embryo cells (HL-7702) were purchased from ATCC (Manassas, VA, USA). All cells were cultured in RPMI1640 medium with 10% fetal bovine serum, penicillin, and streptomycin. To measure the cell-uptake behavior of the FAGSJNs, fluorescein isothiocyanate (FITC)-labeled FA-GSJNs or GSJNs (12.5 μg/mL) were incubated with the cells for 3 h. In the FA-competition assay, 1 mM FA was co-incubated with 12.5 μg/mL FA-GSJNs for 3 h, followed by washing of the cells three times and staining with Hoechst 33258 for 5 min. Cells were observed by confocal laser scanning microscopy (CLSM; Olympus FV1000; Olympus, Tokyo, Japan). To further quantitate HCC-targeting efficacy, cells were treated with 12.5 μg/mL FA-GSJNs, GSJNs, or FA-GSJNs+FA for 3 h, followed by washing, trypsinization, and subsequent resuspension for fluorescence-activated cell sorting (FACS) by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). DOX Loading and Release. To evaluate DOX-loading capacity, 10 mg FA-GSJNs was dispersed in DOX solution (10 mL; 0.5 mg/ mL), and the DOX-loaded FA-GSJNs and supernatant were collected after stirring overnight. The drug-loading content and loading efficiency were calculated as described in a previous study.29,30 To investigate DOX-release behavior, FA-GSJNs-DOX solution was loaded into a dialysis bag and placed into phosphate-buffered saline (PBS) solutions (pH 7.4 and 5.5). At each time interval, the amount of released DOX was determined by UV−vis spectrophotometry at a wavelength of 480 nm. Additionally, the intracellular drug-release behavior of FA-GSJNs-DOX (12.5 μg/mL) in SMMC-7721 or HL7702 cells for 3 h was investigated through the CLSM and FACS protocols described in section 2.2. To quantitatively determine the internalized DOX, cells in each well were washed three times with PBS, collected by scraping, and lysed in 20 μL of lysis buffer. DOX in the lysates of each group was completely extracted by incubating each cell-lysate sample and detected using high-performance liquid chromatography. The results were normalized to the total protein content through a bovine serum albumin assay in a parallel well. Cytotoxicity Assessment and Immunofluorescence. SMMC7721 or HL-7702 cells were exposed to GSJNs, FA-GSJNs, free DOX, GSJNs-DOX, or FA-GSJNs-DOX at various concentrations (25, 12.5, 6.25, 3.125, and 1.5625 μg/mL for NPs, which is equivalent to DOX concentrations of 5, 2.5, 1.25, 0.625, and 0.3125 μg/mL, respectively) with or without X-ray irradiation (5 Gy for 5 min) for 24 h. The radiation was performed using standard and flattening filter-free 6 MV irradiation beams (TrueBeam; Varian Medical System, Palo Alto, CA, USA), with a mean dose rate of 1 Gy/min. Chemotherapy, radiotherapy, and synergetic chemoradiotherapy effects were assessed using traditional sulforhodamine B (SRB) assays as previously described.32,33 The cell-viability ratio of the control was calculated using the following formula: A/B × 100%, where A is the optical density (OD) value from the experimental cells, and B is the OD value from the control cells. To further investigate the synergistic effect of FA-GSJNs-DOX and X-ray radiation, the 50% inhibitory concentration (IC50) values of FA-GSJNs-DOX, X-ray radiation, and FA-GSJNsDOX plus X-ray radiation were calculated. The combination index (CIX) was determined according to a previously reported equation: CIX = IC50A/IC50A′ + IC50B/IC50B′,38 where IC50A and IC50B represent the IC50 values of drug A alone and drug B alone, respectively, and IC50A′ and IC50B′ represent the concentrations of drug A and drug B in the combination system at the IC50 value. CIX < 1.0 indicates

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b07486. Materials, characterization, analysis of internalized DOX, synergestic anticancer effect, HE staining image of FAGSJNs and FA-GSCNs (PDF) 12739


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AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected], *E-mail: [email protected]. ORCID

Dan Shao: 0000-0002-5243-042X Mingqiang Li: 0000-0002-5178-4138 Wen-fei Dong: 0000-0003-1319-3166 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Key Research and Development Program of China (grant nos. 2017YFF0108600 and 2016YFF0103800), the National Natural Science Foundation of China (grant nos. 61535010, 81371681, 8160071152, 81771982, and 81601609), Key Research Program of the Chinese Academy of Sciences (no. KFZD-SW-21), the Natural Science Foundation of Jiangsu Province (no. BE2015601), and the Science and Technology Department of Suzhou City (nos. SS201539 and ZXY201434). REFERENCES (1) Maluccio, M.; Covey, A. Recent Progress in Understanding, Diagnosing, and Treating Hepatocellular Carcinoma. Ca-Cancer J. Clin. 2012, 62, 394−399. (2) Bruix, J.; Gores, G. J.; Mazzaferro, V. Hepatocellular Carcinoma: Clinical Frontiers and Perspectives. Gut 2014, 63, 844−855. (3) Yang, J. D.; Roberts, L. R. Hepatocellular Carcinoma: A Global View. Nat. Rev. Gastroenterol. Hepatol. 2010, 7, 448−458. (4) Llovet, J. M.; Bruix, J. Systematic Review of Randomized Trials for Unresectable Hepatocellular Carcinoma: Chemoembolization Improves Survival. Hepatology 2003, 37, 429−442. (5) Park, W.; Lim, D. H.; Paik, S. W.; Koh, K. C.; Choi, M. S.; Park, C. K.; Yoo, B. C.; Lee, J. E.; Kang, M. K.; Park, Y. J.; et al. Local Radiotherapy for Patients with Unresectable Hepatocellular Carcinoma. Int. J. Radiat. Oncol., Biol., Phys. 2005, 61, 1143−1150. (6) Livraghi, T.; Goldberg, S. N.; Lazzaroni, S.; Meloni, F.; Ierace, T.; Solbiati, L.; Gazelle, G. S. Hepatocellular Carcinoma: Radio-Frequency Ablation of Medium and Large Lesions 1. Radiology 2000, 214, 761− 768. (7) Graf, D.; Vallböhmer, D.; Knoefel, W. T.; Kröpil, P.; Antoch, G.; Sagir, A.; Häussinger, D. Multimodal Treatment of Hepatocellular Carcinoma. Eur. J. Intern. Med. 2014, 25, 430−437. (8) Cheng, J. C.-H.; Chuang, V. P.; Cheng, S. H.; Huang, A. T.; Lin, Y.-M.; Cheng, T.-I.; Yang, P.-S.; You, D.-L.; Jian, J. J.-M.; Tsai, S. Y.; et al. Local Radiotherapy with or without Transcatheter Arterial Chemoembolization for Patients with Unresectable Hepatocellular Carcinoma. Int. J. Radiat. Oncol., Biol., Phys. 2000, 47, 435−442. (9) Chen, S.-W.; Lin, L.-C.; Kuo, Y.-C.; Liang, J.-A.; Kuo, C.-C.; Chiou, J.-F. Phase 2 Study of Combined Sorafenib and Radiation Therapy in Patients with Advanced Hepatocellular Carcinoma. Int. J. Radiat. Oncol., Biol., Phys. 2014, 88, 1041−1047. (10) Zhai, B.; Sun, X.-Y. Mechanisms of Resistance to Sorafenib and the Corresponding Strategies in Hepatocellular Carcinoma. World J. Hepatol. 2013, 5, 345−52. (11) Wu, J.; Li, Y.; Dang, Y.-Z.; Gao, H.-X.; Jiang, J.-L.; Chen, Z.-N. HAB18G/CD147 Promotes Radioresistance in Hepatocellular Carcinoma Cells: A Potential Role for Integrin Β1 Signaling. Mol. Cancer Ther. 2015, 14, 553−563. (12) Zhang, L.; Chen, Y.; Li, Z.; Li, L.; Saint-Cricq, P.; Li, C.; Lin, J.; Wang, C.; Su, Z.; Zink, J.-I. Tailored Synthesis of Octopus-type Janus Nanoparticles for Synergistic Actively-Targeted and Chemo-Photothermal Therapy. Angew. Chem., Int. Ed. 2016, 55, 2118−2121. 12740


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