Tumor-Microenvironment-Induced All-in-One Nanoplatform for

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Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 25043−25053

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Tumor-Microenvironment-Induced All-in-One Nanoplatform for Multimodal Imaging-Guided Chemical and Photothermal Therapy of Cancer Xiaodong Lin,† Yuan Fang,† Zhanhui Tao,† Xia Gao,† Tianlin Wang,† Minyang Zhao,† Shuo Wang,*,‡ and Yaqing Liu*,†,§

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†

State Key Laboratory of Food Nutrition and Safety, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, P. R. China ‡ Tianjin Key Laboratory of Food Science and Health, School of Medicine, Nankai University, Tianjin 300071, P. R. China § Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University, Beijing 100037, P. R. China S Supporting Information *

ABSTRACT: Precisely locating tumor site based on tumor-microenvironmentinduced (TMI) multimodal imaging is especially interesting for accurate and efficient cancer therapy. In the present investigation, a novel TMI all-in-one nanoplatform, CuSNC@DOX@MnO2‑NS, has been successfully fabricated for chemical and photothermal (Chem-PTT) therapy guided by multimodal imaging on tumor site. Here, the CuS nanocages with mesoporous and hollow structure (CuSNC) acting as nanocarriers provide high capacity for loading the anticancer drug, doxorubicin (DOX). The outer layer of the MnO2 nanoshell (MnO2‑NS) acts as “gatekeeper” to control the DOX release until the nanoplatform arrives at the tumor site, where abundant glutathione and H+ decompose MnO2‑NS into paramagnetic Mn2+. The magnetic resonance imaging and fluorescent imaging were then triggered to locate the tumor, which was further improved by photothermal imaging on account of the intrinsic property of CuSNC. Guided by the multimode imaging, the combination of chemical therapy upon DOX and photothermal therapy upon CuSNC exhibits eminent efficiency on tumor ablation. The nanoplatform exhibits biocompatibility to avoid unwanted harm to normal tissues during trans-shipment in the body. The investigation thus develops a cost-effective TMI nanoplatform with facile preparations and easy integration of Chem-PTT treatment capabilities guided by multimodal imaging for potential application in precise therapy. KEYWORDS: all-in-one nanoplatform, multimodal imaging, precise therapy, photothermal therapy, chemotherapy

1. INTRODUCTION

Passive tumor-targeted drug delivery and releasing responsive system have been considered as a promising strategy to selectively accumulate antineoplastic agent through the enhanced permeability and retention (EPR) effect at the tumor site.21−23 The development of nanotechnology and nano-biomaterials provides infinite possibilities for effective tumor diagnosis and therapy.24,25 In recent years, nanomaterials such as semiconductor nanomaterials, carbon-based nanomaterials, black phosphorus, and noble-metal nanomaterials act as both PTT agent and drug nanocarrier to integrate chemotherapy with photothermal therapy to enhance the cytotoxicity on tumor.26−32 Although promising achievements have been reached, these nanosystems suffer from low capacity of drug loading, since the loaded drug mainly anchors on the external surfaces of the nanomaterials. Mesoporous nanoma-

Up to date, cancer treatment is still a significant challenge for human beings. The conventional cancer treatment strategies such as surgery, chemotherapy, and radiation therapy suffer from their inherent limitations of low therapy efficiency, high recurrence rate, and severe side effects.1−5 As an emerging localized therapeutic modality, photothermal therapy (PTT) specially near-infrared (NIR) light-induced PTT has attracted ever increasing attention for cancer treatment due to the advantages of deeper penetration, less adverse side effects, and less pain.6−12 The PTT assay can also outstandingly improve the chemotherapy efficiency.13−16 Under the synergistic function of chemotherapy and PTT (Chemo-PTT), the therapeutic efficacy is greatly enhanced compared to standalone PTT or chemotherapy treatment.17,18 To further reduce severe the side effect and improve therapy efficiency, it is critical to construct TMI systems for precise diagnosis and therapy.19,20 © 2019 American Chemical Society

Received: May 1, 2019 Accepted: June 20, 2019 Published: June 20, 2019 25043

DOI: 10.1021/acsami.9b07643 ACS Appl. Mater. Interfaces 2019, 11, 25043−25053

Research Article

ACS Applied Materials & Interfaces

imaging and a chemotherapy agent for tumor ablation in vivo. Meanwhile, CuSNC performs functions of tumor-site photothermal imaging and therapy under NIR irradiation. In brief, the as-prepared CuSNC@DOX@MnO2‑NS can serve as an “allin-one” nanoplatform for TMI multimode imaging and chemophotothermal therapy under control of the “gatekeeper”, MnO2‑NS, which presents high safety for human bodies in that Mn is a necessary element in physiological metabolism.46 It is anticipated that the as-prepared multifunctional nanoplatform holds great promise in biomedical engineering and clinical application.

terials with a hollow structure provide an effective way of loading high quantities of therapeutic agents.33,34 Considering the mesoporous structure, the nanocarriers are encapsulated with TMI ensembles to avoid the premature leakage of drugs in biological circulation and also reduce severe side effects. So far, drug-delivery nanosystems coated with molecular or macromolecular ensembles that are widely applied usually require sophisticated synthetic process that are time consuming and have high costs.35 Herein, we aim to develop a new intelligent nanoplatform with a simple and cost-effective way for multimode imagingguided chemo-PTT synergistic treatment triggered by the tumor microenvironment. Copper sulfide (CuS) nanomaterials are regarded as excellent candidates due to their inherent properties of excellent NIR photothermal efficiency, low cost, good biodegradability, and less biological toxicity than other nanomaterials such as semiconductor quantum dots and gold nanomaterials.36−39 Different from the optical absorption of gold nanomaterials based on surface plasmon resonance, the NIR light absorption of CuS nanomaterial originates from the d−d band transition of copper ions. Thus, the absorption wavelength of CuS nanomaterials is not influenced by solvent and surrounding environment,40 presenting advantages over gold nanomaterials.41,42 In our investigation, mesoporous CuS nanocage with a hollow structure (CuSNC) is synthesized as “all-in-one” nanocarrier for the co-delivery of drugs and heat (Scheme 1). Doxorubicin (DOX), one of the most commonly

2. MATERIALS AND METHODS 2.1. Synthesis of CuS with Mesoporous and Hollow Structure (CuSNC). CuSNC was synthesized according to the previously reported method with minor modification.36 Briefly, Cu(NO3)2 solution (50 μL, 0.5 M) was added to deionized water (12.5 mL) containing PVP-K30 (0.12 g) under magnetic stirring. NaOH solution (12.5 mL, pH 9.0) was then added. After 10 min, 4.0 μL of N2H4·H2O (50% in water) was added to generate a brightyellow Cu2O solution. Then, Na2S solution (100 μL, 320 mg mL-1) was added into the above solution, followed by heating at 60 °C for 2 h. After centrifuging at 11 000 rpm for 15 min at room temperature, the CuSNC solution was finally obtained by washing the above centrifugation three times with deionized water. The obtained CuSNC were dispersed in 0.5 mL of deionized water for further experiments. 2.2. Preparation of CuSNC@DOX@MnO2‑NS. DOX with different concentrations and CuSNC (100 μL) were added into PBS (pH 7.4, 10 mM, 400 μL). The mixture was then stirred at room temperature for 12 h. The suspension was centrifuged via 11 000 rpm for 15 min and washed with PBS for three times, producing CuSNC@DOX. The CuSNC@DOX (500 μL) and KMnO4 solution (0.1 M, 5.0 μL) was dispersed in the MES buffer solution (pH 6.0, 10 mM) under ultrasonication for 30 min. After that, PEG was added into the reaction solution for 6 h. The mixing solution was centrifuged at 10 000 rpm for 10 min to produce CuSNC@DOX@MnO2‑NS nanomaterials, which were dispersed into PBS solution. 2.3. Calculating Drug-Loading Efficiency of CuSNC and Stimuli-Responsive Drug Release Efficiency. DOX (0.1 mg mL−1) was added into the CuSNC nanoparticles solution. After stirring for 12 h in dark at room temperature, the CuSNC@DOX nanomaterials were collected via centrifugation at 11 000 rpm for 15 min, which was further used to prepare CuSNC@DOX@MnO2‑NS nanoplatform. The UV−vis spectra of the free DOX from suspension after incubating the nanoplatform in PBS were recorded to determine the loading efficiency in CuSNC according to the calibration curve of the DOX concentration. To assess the stimuli-responsive drug release from CuSNC@DOX@MnO2‑NS, CuSNC@DOX@MnO2‑NS was dispersed into diverse solutions including PBS solution, PBS solution with different pH values, and PBS solution containing GSH in different concentrations. The releasing efficiency of DOX was determined according to the calibration curve of DOX. 2.4. In Vitro Stability of CuSNC@DOX@MnO2‑NS. The stability of CuSNC@DOX@MnO2‑NS was evaluated by monitoring its DOX release efficiency. CuSNC@DOX@MnO2‑NS and CuSNC@DOX were, respectively, incubated in PBS solution (pH 7.4, 10 mM) and fetal bovine serum (FBS) for different times and then centrifuged for 10 min (11 000 rpm). The supernatants were analyzed by a UV−vis spectrometer. 2.5. Cell Culture. HepG2 cells and L-02 cells were cultured in DMEM medium (10% fetal bovine serum, 100 μg mL−1 antibiotics penicillin and 100 μg mL−1 streptomycin) maintained at 37 °C containing 5% CO2 incubator. 2.6. Intracellular Localization Profile of CuSNC@DOX@ MnO2‑NS. HepG2 cells were first cultured in a confocal dish for 12 h. Then, CuSNC@DOX@MnO2‑NS (100 μg mL−1) in the DMEM culture medium were added into the confocal dish. Four hours later, the Hoechst 33258 was added to stain the nucleus for 15 min. Finally,

Scheme 1. Schematic Illustration of Multimodal ImagingGuided Chemical and Photothermal Therapy Mechanism Based on the Tumor-Site-Induced All-in-One Nanoplatform of CuSNC@DOX@MnO2‑NS

used chemotherapeutic drugs, is used as modal chemotherapy agent to be encapsulated in CuSNC (CuSNC@DOX), which is further coated with MnO2 nanoshell (MnO2‑NS) as the “gatekeeper” to achieve the nanoplatform of CuSNC@DOX@ MnO2‑NS in a simple process. It has been demonstrated that the tumor microenvironment is featured with a higher quantity of GSH and H+ than those in normal tissues,43−45 which can decompose MnO2‑NS into Mn2+, endowing the possibility of T1-weighted magnetic resonance imaging (MRI). Accompanied by the decomposition of MnO2‑NS, DOX is then released from CuSNC and acts as both a fluorescent probe for tumor 25044

DOI: 10.1021/acsami.9b07643 ACS Appl. Mater. Interfaces 2019, 11, 25043−25053

Research Article

ACS Applied Materials & Interfaces

Figure 1. TEM images of CuSNC (A), CuSNC@DOX (B), and CuSNC@DOX@MnO2‑NS (C). TEM image of CuSNC@DOX@MnO2‑NS (D) and EDS elemental mapping (E−G) (E: Cu element (red color), F: S element (green color); G: Mn element (purple color)), the spectrum of CuSNC@ DOX@MnO2‑NS (H). XPS of Cu2p (I), S2p (J), and Mn2p (K) raised from CuSNC@DOX@MnO2‑NS. the products were observed by capturing the fluorescence confocal images. 2.7. In Vitro Cytotoxicity Assessment of Nanocomposites. HepG2 cells in DMEM medium were seeded in a 96-well plate with a density of 10 000 cells per well at 37 °C in 5% CO2 atmosphere for 12 h. Then, the cells in the media were treated with PBS, DOX, CuSNC@ DOX@MnO2‑NS, CuSNC, laser, CuSNC + laser (808 nm, 0.5 W cm−2), and CuSNC@DOX@MnO2‑NS + laser (808 nm, 0.5 W cm−2) for 12 h, respectively, where laser irradiation was given for the first 4 h for 5 min. The cell viability was detected by the MTT assay. The absorption of each well was measured by a microplate reader. As for calcein-AM/PI co-stained experiment, calcein-AM and PI were used to stain live and dead cells, respectively. Finally, the cells were washed three times with PBS and monitored through the confocal fluorescence image system. 2.8. Photothermal Conversion Efficiency (η) of CuSNC@ DOX@MnO2‑NS. To examine the photothermal effect of CuSNC@ DOX@MnO2‑NS, the photothermal conversion efficiency was calculated according to the following equation η=

monitored by a Vernier caliper, which was calculated as V = L × W2/2 (L and W are the length and width of the tumor, respectively). 2.11. In Vivo Multimodal Imaging. HepG2 tumor-bearing mice were intravenously injected with CuSNC@DOX@MnO2‑NS (100 μg mL−1, 200 μL). MR imaging was performed under a Siemens Prisma 3.0 T MR scanner (Erlangen, Germany) with the gradient strength up to 80 mT/m. T1 images of HepG2 tumor-bearing nude mice (TR = 800 ms, TE = 12 ms) were obtained by the Fast spin-echo method. PT imaging was recorded by the infrared thermal camera. Fluorescence image was observed through the in vivo imaging system. 2.12. Biodistribution of CuSNC@DOX@MnO2‑NS in Mice. HepG2 tumor-bearing nude mice were intravenously injected with CuSNC@DOX@MnO2‑NS (100 μg mL−1, 200 μL). Major organs (heart, liver, spleen, kidney, and lung) and tumors were obtained at various time points (0, 4, 8, 12, and 24 h) and imaged by the in vivo imaging system. 2.13. In Vivo Fluorescence Image. The in vivo fluorescence image of the tumor-bearing mice intravenously injected with CuSNC@ DOX@MnO2‑NS were monitored through the Berthold NightOWL LB983 in vivo imaging system. (The excitation wavelength was fixed at 500 nm, while the emission wavelength was set to 600 nm via filter. The exposure time of 100 ms was used for each image frame.) 2.14. Hemolysis Assay. Fresh blood sample (0.1 mL) was stabilized by ethylenediaminetetraacetic acid (EDTA) and then mixed with PBS (2.0 mL, 4 °C). After centrifugation (1000 rpm, 10 min), the obtained red blood cells (RBCs) were further washed three times and then dispersed in PBS. To study the hemolysis of CuSNC@ DOX@MnO2‑NS, the RBC dispersions (0.2 mL) were incubated with CuSNC@DOX@MnO2‑NS under various concentrations at 37 °C for 4 h. After centrifugation (12 000 rpm, 10 min), the supernatant was carefully collected for the UV−vis absorbance measurement. Hemolysis percentage (%) = (Asample − APBS)/(ADI water − APBS) × 100%, where Asample, APBS, and ADI water are the absorbance values of the sample group, the PBS group, and the DI water group at 577 nm, respectively. 2.15. H&E Staining. After 14 days of treatment, the major organs (liver, heart, spleen, kidney, and lung) and tumors were obtained from the sacrificed mice in different experimental groups, which were fixed in 10% neutral buffered formalin. After that, these tissues of major organs and tumors were embedded in paraffin sectioned (4 μm thick) and stained with hematoxylin and eosin (H&E). Finally, an optical microscope was used to observe the histological sections. 2.16. Statistical Analysis. Statistical analysis was performed with the Statistical Program for Social Sciences software. All of the data

hs(Tmax − Tsurr) − Q dis I(1 − 10−A808)

Tmax is the equilibrium temperature of CuSNC@DOX@MnO2‑NS. Tsurr is the ambient temperature of the surroundings. Qdis is the heat loss from the light absorbed by the container, I is the laser power (0.5 W cm−2), and A808 is the absorbance value of CuSNC@DOX@MnO2‑NS at 808 nm. 2.9. Animal Experiments. Balb/c-nu mice were purchased from the SPF (Beijing) Biotechnology Co., Ltd. All of the mice were handled under the protocol approved by the Institutional Animal Care and Use Committee of Tianjin. All of the animal experiments were conducted in accordance with the guidelines of the Tianjin Committee of Use and Care of Laboratory Animals. 2.10. Animal Tumor Model. Female nude mice (6−8 weeks old) were subcutaneously inoculated with HepG2 cells (5 × 107) in the right flank to generate HepG2 tumors. The in vivo studies were performed when the tumor volume of mice reached about 100−150 mm3. All of the animal procedures guidelines complied with the requirements of the Chinese Animal Use and Care Committee and the Tianjin Committee of Use and Care of Laboratory Animals. After different treatments, the tumor sizes and body weights were continuously monitored for 14 days. The tumor volume (V) was 25045

DOI: 10.1021/acsami.9b07643 ACS Appl. Mater. Interfaces 2019, 11, 25043−25053

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ACS Applied Materials & Interfaces

Figure 2. (A) Loading efficiency of DOX in CuSNC@DOX@MnO2‑NS. (B) Releasing efficiency of DOX from CuSNC@DOX@MnO2‑NS at different pH values (5.5 and 7.4) in the presence and absence of GSH (5.0 mM), respectively. (C) NIR thermal imagings of PBS in the absence and presence of CuSNC@DOX@MnO2‑NS at different concentrations. (D) Photothermal conversion curves of a suspension of CuSNC@DOX@ MnO2‑NS in various concentrations under NIR laser irradiation (0.5 W cm−2, 808 nm) for 7 min. (E) Photothermal conversion of a suspension of CuSNC@DOX@MnO2‑NS for four alternate cycles of NIR laser on/off (808 nm nm, 0.5 W cm−2). were reported as mean ± standard deviation (SD). Statistical significance was recorded with a two-tailed Student’s t-test. p values: **p < 0.01 and ***p < 0.001.

(Figure 1I−K). The binding energy peaks at 934.8 and 943.6 eV are attributed to Cu 2p3/2 and those at 954.8 and 963.1 eV to Cu 2p1/2.36,47,48 Besides, the peak at 166.1 eV corresponds to S 2p,49 and the binding energy peaks at 649.2 and 639.8 eV are assigned to Mn 2p1/2 and Mn 2p3/2, respectively.50,51 The results are consistent with each other and validate the successful preparation of the CuSNC@DOX@MnO2‑NS nanoplatform. 3.2. Drug Loading, Release, and Photothermal Efficiency. The cavity of CuSNC provides capacity for DOX loading, achieving the maximal loading efficiency of 66% (Figure 2A). The MnO2‑NS stays stable in PBS (pH 7.4) free of GSH and prevents the release of DOX from CuSNC (Figure 2B). Compared with normal tissues, tumor microenvironment is more acidic with a higher amount of GSH.43−45 To validate the TMI drug release, CuSNC@DOX@MnO2‑NS were dissolved in diverse PBS solutions by changing the two factors of GSH amount and pH value. If the pH is changed to 5.5, the MnO2‑NS is reduced to hydrosoluble Mn2+, exposing the mesoporous CuSNC outside. Thirty-eight percent of DOX is then released within 4 h. In the presence of GSH (PBS, pH 7.4), 78.9% of DOX is released from CuSNC owing to the decomposition of the MnO2‑NS. Meanwhile, GSH is oxidized into glutathione disulfide (GSSG),52 which could reduce the negative risk of GSH for human health. The acidic PBS (pH 5.5) containing GSH is used to simulate tumor microenvironment. In this case, the MnO2‑NS is decomposed as follows: MnO2 + 2GSH + 2H+ → Mn2+ + GSSG + 2H2O. About 79% of DOX is rapidly released from CuSNC within 1 h, indicating that the degradation of the MnO2‑NS can be significantly accelerated by the synergistic function of H+ and GSH. In the as-prepared CuSNC@DOX@MnO2‑NS nanoplatform, CuSNC plays functions in not only drug loading but also photothermal imaging and therapy, thanks to its excellent photothermal conversion efficiency in the NIR region. Here, a 808 nm laser is used for the photothermal imaging and therapy in the following experiments due to its minimal absorption coefficient and deep penetration in biological tissues.53 Under NIR irradiation, the photothermal conversion of the CuSNC@

3. RESULTS AND DISCUSSION 3.1. Characterization of CuSNC and CuSNC@DOX@ MnO2‑NS. The structure of CuS nanocage (CuSNC) was characterized by TEM (Figure 1). It shows a uniform hollow and mesoporous morphology of CuSNC with an average diameter of about 68 nm (Figure 1A and Figure S1 in the Supporting Information (SI)). The characteristic absorption located within the near-infrared region from 700 to 1100 nm is observed from the UV−vis−NIR absorption spectrum of CuSNC (Figure S2 in the SI), making it possible to perform NIR photothermal therapy. After loading DOX, named as CuSNC@DOX, the hollow structure of CuSNC still can be observed though not as clearly as before DOX loading, which might be ascribed to the aggregated DOX within CuSNC (Figure 1B). The feature absorption peak of DOX is found at 480 nm, further validating the successful loading of DOX in CuSNC (Figure S2 in the SI). Due to the mesoporous structure of CuSNC, more than thirty percent of DOX would be divulged from CuSNC into PBS solution with increasing incubation time to 4 h (Figure S3A−C in the SI). To avoid drug release, MnO2‑NS, acting as the “gatekeeper”, is coated on the outside of CuSNC@DOX to control the release of DOX, which presents excellent stability in fetal bovine serum (FBS) (Figure S3D in the SI). While the outlayer of MnO2‑NS can be decomposed in GSH-rich tumor microenviroment with endogenous low pH. The coating of the MnO2‑NS enlarges the average diameter of CuSNC@DOX@MnO2‑NS to about 89 nm (Figure 1C and Figure S1 in the Supporting Information). The elemental mapping results (Figure 1D−G) validate the cladding of MnO2‑NS on the outside of CuSNC@DOX, which is further confirmed by the presence of Cu, S, and Mn elements in the energy-dispersive spectroscopy (EDS) (Figure 1H). The absorption spectra still locates within the near-infrared region from 700 to 1100 nm, while the main absorption peak shifts from 890 to 880 nm (Figure S2 in the SI). X-ray photoelectron spectroscopy (XPS) is further measured for elemental analysis 25046

DOI: 10.1021/acsami.9b07643 ACS Appl. Mater. Interfaces 2019, 11, 25043−25053

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Figure 3. (A) Fluorescence imaging of L-02 normal cells and HepG2 cancer cells after being incubated with CuSNC@DOX@MnO2‑NS for different times (from left to right: 0, 1, 4, and 6 h). (B) Fluorescence images of HepG2 cancer cells after being incubated with CuSNC@DOX@MnO2‑NS (a) and Hochest 33258 (b) and the merged image (c).

Figure 4. (A) In real-time fluorescence imaging and quantitative data of major organs and tumors collected from HepG2 tumor-bearing mice after intravenous injection of CuSNC@DOX@MnO2‑NS (100 μg mL−1, 200 μL) for 0, 4, 8, 12, and 24 h. Infrared thermal images (B) and the corresponding mean tumor temperature curves (C) of tumor-bearing mice after being injected with PBS (left) and CuSNC@DOX@MnO2‑NS (right) under NIR laser irradiation (808 nm, 0.5 W cm−2) for 0, 1, 2, and 3 min. **p < 0.01.

upon NIR irradiation for 7 min (808 nm, 0.5 W cm−2, CuSNC@DOX@MnO2‑NS 200 μg mL−1). Meanwhile, the temperature is augmented with increasing the laser power density, and the corresponding photothermal conversion efficiency is calculated as 28% (Figure S4 in the SI), which is much higher than that of the widely used photothermal materials such as the IR-780-loaded liposome (21.9%), the

DOX@MnO2‑NS nanoplatform is investigated by taking thermal photos using an infrared thermal camera. In control experiments, no obvious temperature change is detected in PBS free of the nanoplatform. In contrast, the temperature of the nanoplatform suspensions increases with irradiation time and also the nanoplatform concentration (Figure 2C,D). A rapid temperature elevation from 24.3 to 69.8 °C is realized 25047

DOI: 10.1021/acsami.9b07643 ACS Appl. Mater. Interfaces 2019, 11, 25043−25053

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ACS Applied Materials & Interfaces

Figure 5. (A) T1-weighted in vitro MR images and relaxation rate 1/T1 versus different GSH concentrations (from left to right: 50, 100, 200, 500, and 1000 μM) with CuSNC@DOX@MnO2‑NS in PBS (10 mM, pH 5.5). T1-weighted in vivo MR images (B) and T1-weighted MR signals of the region of interest (C) after being treated with CuSNC@DOX@MnO2‑NS for different times (a: pre, b: 10 min, c: 30 min, d: 1 h, e: 2 h, f: 4 h, g: 12 h, h: 24 h. i: 48 h. Circles indicate the tumor tissue.).

3.4. In Vivo Fluorescence and Photothermal Tumor Imaging. To evaluate and identify the optimal therapeutic time and window of CuSNC@DOX@MnO2‑NS, the fluorescence images and quantitative data of the nanoplatform accumulation in different organs and tumors in vivo were monitored and longitudinally recorded after the intravenous injection of CuSNC@DOX@MnO2‑NS into HepG2-bearing nude mice (Figure 4A). The released DOX could be excreted by the liver and kidney of mice within 24 h, reducing the side effect of chemotherapy for normal organs or tissues. No obvious drug release is found from the heart and lung for the whole monitoring period. At the tumor site, a weak fluorescence signal is observed in the first 4 h after injection. The fluorescence intensity of the tumor region reaches the maximum 12 h after injection, which indicates the prominent EPR effect of CuSNC@DOX@MnO2‑NS and a highly efficient tumor accumulation of DOX in response to the tumor microenvironment. The drug can detained at the tumor sites within 24 h after injection of CuSNC@DOX@MnO2‑NS. Therefore, 12 h is used for the following investigation on the tumor therapy. To further target tumor site in vivo, the photothermal tumor imaging was investigated with tumor-bearing mice as a model as supported by the excellent photothermal conversion capability of CuSNC@DOX@MnO2‑NS. Under the NIR irradiation, the photothermal imaging and the corresponding temperature in tumor sites were recorded after every minute (Figure 4B,C). In the control group, the tumor-bearing mice are not injected with the photothermal nanoplatform of CuSNC@DOX@MnO2‑NS. In this case, the NIR irradiation cannot cause an obvious temperature change, and thus it is hard to distinguish the tumor site from normal tissues. Once CuSNC@DOX@MnO2‑NS is injected into the tumor, the tumor site could be directly visualized from the photothermal responses. The tumor temperature reaches 50 °C, while the temperature of the surrounding normal tissue remains at a safe level of 37 °C. As is known that cancer cells can be killed by heating at 42 °C for at least 10 min or at temperatures above 50 °C for only 5 min.60 Meanwhile, the in vivo fluorescent images indicate that the fluorescence of DOX is monitored at the tumor site, and its intensity is enhanced with incubation time due to the DOX release (Figure S9 in the SI). The results demonstrate that CuSNC@DOX@MnO2‑NS can be used as an ideal nanoreagent for high-precision photothermal imaging and therapy of the tumor site with minimimal adverse effects on normal tissue.

MnO x /TiO2-GR-PVP nanocomposites (18.4%), or the Cu2−xSe crystal (22%).54−56 The as-prepared nanoplatform exhibits outstanding photothermal stability and still can reach about 69.8 °C after four cycles of heating and cooling (Figure 2E). After being irradiated under 808 nm laser for 15 min, negligible change is observed from the absorption spectrum and the hollow structure of CuSNC (Figure S5). This presents advantage over other NIR nanomaterials such as gold nanorods or hollow gold nanospheres, which fuse into solid gold nanoparticles upon NIR laser irradiation, reducing the thermal therapy efficiency.57,58 Meanwhile, it is found that the laser treatment slightly enhances the drug release within the first 1 h of incubation time and then has no obvious influence with further increase in incubation time (Figure S6 in the SI). 3.3. In Vitro Fluorescent Imaging of CuSNC@DOX@ MnO2‑NS. Multimodal imaging can improve the accuracy of tumor detection by integrating the advantages of different imaging modalities.59 Except for the photothermal imaging, here, DOX can act as a fluorophore probe for the fluorescent location of the tumor site. The fluorescence of DOX is quenched after being sealed in the nanoplatform of CuSNC@ DOX@MnO2‑NS, which is then restored after incubating the nanoplatform with GSH and H+ (Figure S7 in the SI). Therefore, it is expected that the fluorescent signal would be triggered once CuSNC@DOX@MnO2‑NS arrives at the tumor site, providing a striking contrast between target and background fluorescence signals. The as-prepared nanoplatform as TMI fluorescent imaging is confirmed in vitro by culturing noncancer cells of L-02 and cancer cells of HepG2 with CuSNC@DOX@MnO2‑NS (Figure 3A). Negligible fluorescence signal can be observed from normal cells after incubating with the nanoplatform for 6 h. Observing from the methyl thiazolyltetrazolium (MTT) results, over 85% noncancer cells still retain high viabilities (Figure S8 in the SI), revealing excellent biocompatibility of the as-prepared nanoplatform on noncancer cells, which is critical for target diagnostics and therapy. In contrast, tumor cells are lightened up within one hour. Such a distinct difference is attributed to the release of DOX on account of degradation of MnO2‑NS in acidic and GSH-rich tumor microenvironment. The intracellular location of CuSNC@DOX@MnO2‑NS (Figure 3B(a)) is distinguished via co-staining cells with nuclear fluorescent marker, Hochest 33258 (Figure 3B(b)). The blue fluorescent signal originating from Hochest 33258 indicates the location of cell nucleus. By merging with the fluorescence images, the CuSNC@DOX@MnO2‑NS nanoplatform is mainly observed in the cytoplasm (Figure 3B(c)). 25048

DOI: 10.1021/acsami.9b07643 ACS Appl. Mater. Interfaces 2019, 11, 25043−25053

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ACS Applied Materials & Interfaces

Figure 6. In vitro cytotoxicity study. The fluorescence imaging of live/dead dye cells (A−G) and MTT assays (H) of HepG2 cells under different treatments: (A) without any treatment, treated by laser (B), CuSNC (C), CuSNC + laser (D), DOX (E), CuSNC@DOX@MnO2‑NS (F), and CuSNC@DOX@MnO2‑NS + laser (G). The laser irradiation lasted for 5 min. **p < 0.01.

Figure 7. In vivo antitumor study: the body-weight changes (A) and tumor volume growth curves (B) of HepG2 tumor-bearing mice treated in different ways. (C) Representative photos of HepG2 tumor-bearing mice and tumor dissection photographs through different treatment (PBS, DOX, CuSNC + laser, CuSNC@DOX@MnO2‑NS, and CuSNC@DOX@MnO2‑NS + laser (from left to right)). (D) H&E staining images of heart, liver, spleen, lung, kidney, and tumor with treatment of CuSNC@DOX@MnO2‑NS + laser and PBS as control. (E) Hemolysis test of red blood cells dispersed in deionized water (a), PBS (b), and CuSNC@DOX@MnO2‑NS with different concentrations (c−g: 10, 20, 50, 100, and 200 μg mL−1). **p < 0.01, ***p < 0.001.

3.5. In Vitro and in Vivo MR Imaging of CuSNC@DOX@ MnO2‑NS. As is well known that Mn2+ could augment T1magnetic resonance (MR) signal, endowing the capability of the as-prepared CuSNC@DOX@MnO2‑NS as the T1-weighted MR contrast agent for tumor-specific detection. In control experiment, CuSNC@DOX@MnO2‑NS was incubated in simulated normal physiological condition (PBS without

GSH, pH 7.4). Mn element is coordinated in MnO2‑NS and shielded from the aqueous solution.61,62 No T1-weighted MR signal is monitored as a result of Mn2+ lacking (Figure 5A). In simulated tumorous condition (PBS with GSH, pH 5.5), T1weighted MR is enhanced with GSH concentration due to the decomposition of MnO2‑NS into Mn2+ under the synergistic function of GSH and H+. A tumor model was further 25049

DOI: 10.1021/acsami.9b07643 ACS Appl. Mater. Interfaces 2019, 11, 25043−25053

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synergistic function of PTT and chemotherapy in one nanoplatform could significantly improve the antitumor efficiency.65,66 The corresponding photographs of tumors on mice further confirm that the treat strategy of CuSNC@DOX@ MnO2‑NS under assistance of laser has the most antitumor efficiency and can induce maximum tumor ablation (Figure 7C). At the end of therapy, the above mice were sacrificed and major organs (heart, liver, spleen, lung, and kidney) and tumors taken our for biodistribution investigation and pathological analysis. The results of hematoxylin and eosin (H&E) stained histological examinations validate that CuSNC@DOX@MnO2‑NS would not cause organ morphology abnormality after 808 nm laser treatment, and no obvious pathological changes are found in all of the treated mice, while the tumor is greatly destroyed, suggesting the target-diagnosis capability of the developed strategy (Figure 7D). Moreover, the hemocompatibility of CuSNC@DOX@MnO2‑NS is evaluated by incubating human red blood cells (RBCs) with the nanoplatform for 4 h. Low hemolysis percentage of the nanoplatform is monitored, which is independent of the tested concentration of the nanocarrier. The hemolysis assay suggests that CuSNC@DOX@MnO2‑NS has no adverse influence on RBCs (Figure 7E). As a result of the above systemic administrations, the as-prepared CuSNC@DOX@MnO2‑NS could be an efficient antitumor nanoreagent for chemophotothermal therapy guided by multimodal imagings and present great potential application in cancer diagnosis and therapy.

developed by implanting cancer cells in the armpit of mice to evaluate the possibility of CuSNC@DOX@MnO2‑NS on in vivo MR imaging tumor. After intravenous injection of CuSNC@ DOX@MnO2‑NS into the mice, the tumor site exhibits a striking contrast enhancement compared with that before the intravenous injection (Figure 5B). The quantification of T1weighted MR signal against time (Figure 5C) clearly shows that the MR signal in the tumors is significantly enhanced within a short time and reaches the maximum 1 h after injection. The MR signal intensity slightly decreased within 12 h, further confirming the high accumulation of the nanoplatform at the tumor site via the EPR effect.6 3.6. In Vitro PTT and Chemotherapy Synergistic Efficacy. By combining two or more different therapeutic assays, the anticancer efficacy could be greatly enhanced.63,64 The above-presented results validate the capability of the asprepared CuSNC@DOX@MnO2‑NS for multiple tumor-imaging guided chemotherapy and the possible PTT, which can improve the accuracy of diagnosis and target therapy. The in vitro therapeutic effects of CuSNC@DOX@MnO2‑NS toward HepG2 cells were subsequently evaluated using the live/dead cell staining and the MTT assay (Figure 6). Fluorescence costaining with propidium iodide (PI) and Calcein-AM was carried out to distinguish live cells (green) from dead cells (red). HepG2 cells keep high cell viability before any treatment. After culturing with CuSNC, cell apoptosis is rarely observed due to the biocompatibility of CuSNC. Exposing HepG2 cells under NIR laser irradiation alone results in ignored cell death in that the NIR irradiation itself has no obvious negative influence. The combination of NIR irradiation and CuSNC significantly elevates the local temperature and thus causes a large amount of cell death. If incubating cancer cells with DOX alone, a lower anticancer efficiency is detected, which might be attributed to the poor water solubility and low uptake efficiency of cell on DOX. A similar result is observed after culturing the cancer cells with CuSNC@DOX@MnO2‑NS caused by the released DOX in the tumor microenvironment. Then, one can understand that tumor is hardly completely eliminated by stand-alone chemotherapy due to the limited therapeutic efficiency.19,20 The synergistic chemotherapy and PTT of CuSNC@DOX@ MnO2‑NS exhibit the most powerful anticancer efficacy, which are in agreement with the result of the MTT assay (Figure 6H). 3.7. In Vivo PTT and Chemotherapy Synergistic Efficacy. Encouraged by the promising in vitro therapeutic efficacy, the tumoricidal efficacy in vivo was further assessed in HepG2 tumor-bearing mice. The tumor-bearing mice were randomly divided into five groups, including the control group (no treatment) and groups treated with DOX, CuSNC + laser, CuSNC@DOX@MnO2‑NS, or CuSNC@DOX@MnO2‑NS + laser. During a two-week therapy, neither death nor appreciable weight fluctuation happens in all of the tested groups, revealing negligible side effects of the treatments (Figure 7A). The relative tumor volume against the initial volume in every group is monitored to assess the antitumor efficacy (Figure 7B). In the control group, tumor volumes greatly increase due to the lack of treatment. The chemotherapy from DOX or CuSNC@DOX@MnO2‑NS alone inhibits the tumor growth slightly. The NIR photothermal therapy arising from CuSNC and laser exhibits better antitumor efficacy. A maximum tumor shrinkage is realized in the group treated with CuSNC@DOX@MnO2‑NS and laser, indicating that the

4. CONCLUSIONS In summary, we have successfully integrated multifunctions of CuSNC and MnO2‑NS for the first time to prepare a TMI all-inone nanoplatform of CuSNC@DOX@MnO2‑NS for precise tumor therapy guided by multimodal tumor-targeting imaging. Here, the MnO2‑NS coating acts as the gatekeeper to guarantee the safe transmission of the nanoplatform in normal tissues and controlling the drug release at tumor sites. The in vitro and in vivo experimental results demonstrate that the nanoplatform can precisely locate the tumor via fluorescent, photothermal and MR imagings by integration advantages of DOX, CuSNC, and MnO2‑NS. Guided by the multimodal images, high efficiency in tumor ablation is realized under the synergistic function of chemotherapy and photothermal therapy. More importantly, the developed multifunctional nanoplatform is cost-effective and simple to synthesis. The investigation validates that the developed multifunctional nanoplatform are particularly attractive in nanomedicine engineering for potential clinical application.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b07643. The used materials and instruments; the DLS of CuSNC, CuSNC@DOX, and CuSNC@DOX@MnO2‑NS; UV−vis− NIR absorption spectra and releasing efficiency of DOX; Photothermal conversion efficiency of CuSNC@DOX@ MnO2‑NS; UV−vis−NIR absorption spectra and TEM of CuSNC; MTT assay evaluating the cytotoxicity of CuSNC and CuSNC@DOX@MnO2‑NS; the in vivo fluorescence image of CuSNC@DOX@MnO2‑NS (PDF) 25050

DOI: 10.1021/acsami.9b07643 ACS Appl. Mater. Interfaces 2019, 11, 25043−25053

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S.W.). *E-mail: [email protected] (Y.L.). ORCID

Yaqing Liu: 0000-0002-2885-1670 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (Nos. 21775108 and 21575138) and the Tianjin Science and Technology Project (Project No. 18PTSYJC00130).

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