Multisynergistic Platform for Tumor Therapy by Mild Microwave

Multisynergistic Platform for Tumor Therapy by Mild Microwave Irradiation-Activated Chemotherapy and Enhanced Ablation Dan Long,†,‡ Tianlong Liu,† Longfei Tan,† Haitang Shi,† Ping Liang,§ Shunsong Tang,† Qiong Wu,†,‡ Jie Yu,§ Jianping Dou,§ and Xianwei Meng*,† †

Laboratory of Controllable Preparation and Application of Nanomaterials, Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China § Chinese PLA General Hospital, 28 Fuxing Road, Beijing, 100853, People’s Republic of China S Supporting Information *

ABSTRACT: Microwave (MW) therapy, as a promising type of thermal therapy, has been attracting more and more attention from scientists. The combination of thermal and chemotherapy is of great significance in the latest studies of synergistic tumor therapy. However, the research on the MW therapy mechanism, especially the nonthermal effect applied in the combined cancer therapy, is not thorough enough. Pleasantly, we have discovered that nonthermal MW irradiation can promote the cellular uptake of nanoparticles and anticancer drugs via experiments in vitro and in vivo. Therefore, multifunctional nanoplatforms have been designed for enhanced tumor inhibition by loading ionic liquids (ILs), doxorubicin hydrochloride (DOX), and phase change materials (PCMs) into ZrO2 hollow nanoparticles. PCMs act as MW switches. The asmade IL-DOX-PCM@ZrO2 nanoplatforms were injected into H22-tumor-bearing mice via the tail vein. Mild microwave irradiation (0.9 W, 450 MHz) was then applied. The thermal effect of MW could cause the temperature of the tumor site to rise (58 °C). On the other hand, it will trigger the MW switch to open and release DOX when the temperature is high enough. At the same time as drug release, a MW nonthermal effect could improve the cellular uptake of nanomaterials and anticancer drugs. The multisynergistic effect can promote the survival rate of the IL-DOX-PCM@ZrO2+MW group to 100%. The results of the tumor therapy experiment in vivo demonstrated that as-made multifunctional IL-DOX-PCM@ ZrO2 nanoplatforms could enhance the therapeutic outcome of combined thermal and chemotherapy under mild MW irradiation. KEYWORDS: multifunctional, nanoplatforms, microwave, enhanced, ablation, nonthermal effect, tumor therapy via changing the concentration of NaCl.12 A DOX- and indocyanine green (ICG)-laden eukaryotic cell-like hybrid (EukaCel) nanoparticle was developed as a multifunctional drug delivery platform4,10,13 for photothermal therapy14,15 and chemotherapy16−19 combined tumor therapy.20 However, satisfactory synergistic therapeutic outcomes are limited by a number of biological obstacles, such as fibroblasts and dense extracellular matrix, which effectively hamper the deep interstitial penetration of the multifunctional nanoplatforms inside the tumor, thus reducing their therapeutic efficacy.13 Therefore, scientists are proposing strategyies to increase the

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ultifunctional nanoplatforms for synergistic cancer therapy hold considerable appeal in the next generation of tumor treatment.1−4 Great effort has been devoted to combine multiple therapeutic modalities in a single formulation with the advantages of enhanced therapeutic efficacy, elicited biological functions, and minimal side effects.5−9 For instant, Fe5C2 nanoparticles coated with bovine serum albumin have been shown to be promising drug carriers that could be modulated by heat and pH stimuli.10 An intelligent biodegradable ZnO-Gd-doxorubicin hydrochloride (ZnO-Gd-DOX) nanoplatform was designed for better chemotherapy, which could release DOX under an acid environment and could serve as a bifunctional probe for both magnetic resonance and fluorescent imaging.11 A kind of bioinspired rodshaped polymer micellar nanoplatform was developed to improve the efficiency of drug delivery and enhance cancer © 2016 American Chemical Society

Received: July 16, 2016 Accepted: September 30, 2016 Published: September 30, 2016 9516

DOI: 10.1021/acsnano.6b04749 ACS Nano 2016, 10, 9516−9528

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Scheme 1. Schematic illustration of the thermal/chemo combined cancer therapy under mild MW irradiation of IL-DOXPCM@ZrO2 drug delivery nanoplatforms. The MW nonthermal effect could promote the cellular uptake of nanomaterials and anticancer drugs. The thermal effect, on the one hand, could cause the temperature of the tumor site to rise and, on the other hand, trigger drug release when the temperature was high enough. In the process of drug release, PCM was used as a MW switch. Hence, the as-made multifunctional nanoplatforms can realize combined therapy that promotes cellular uptake under a nonthermal MW effect and eliminates the tumor cells under thermal and chemotherapy when irradiated by mild MW.

of ZrO2 hollow nanoparticles was used as an ideal carrier for anticancer drugs such as DOX, and the mesoporous structure was employed as a drug release channel. 1-Butyl-3-methylimidazolium hexafluorophosphate, a kind of ionic liquid (IL) with good effect of MW sensitivity, was loaded with DOX into ZrO2 hollow nanoparticles through the physical forced penetration method; then the nanoparticles were further sealed by tetradecanol (phase change materials, PCMs, melting point: 39−40 °C) for higher drug loading and better controlled drug release stimulated by the MW thermal effect (Scheme 1). In this way, the multifunctional drug-loaded IL-DOX-PCM@ZrO2 nanoplatform was finally achieved, with the PCM as a kind of MW switch. The as-made IL-DOX-PCM@ZrO2 nanoparticles were injected into mice through the tail vein and reached the tumor site via the EPR effect. Then mild MW irradiation was applied at the tumor site, and the MW energy was converted into heat to kill tumor cells due to the thermal effect. PCM melted as soon as the temperature exceeded 40 °C, so that the MW switch was opened to release DOX.31−33 At the same time, the tumor cellular uptake of nanoparticles and DOX increased, which led to enhancement of the effect of chemotherapy due to the nonthermal effect of MW. Therefore, the multifunctional IL-DOX-PCM@ZrO2 nanoplatforms could eliminate the tumor cells under the combined action of nonthermal and thermal effects of MW. Our results first utilized the nonthermal effect of MW to promote the cellular uptake of nanoparticles and anticancer drugs, to achieve enhanced thermal/chemo cancer therapy under mild MW irradiation.

permeability of the biological barriers for the delivery of anticancer synergistic agents. Due to the increasing interest in the biological effects of microwave (MW) exposure, MW irradiation is one of the most promising technologies for the next generation of combination therapy.21−23 The thermal effects are the predominant biological response of MW irradiation, whose wavelength lies between 1 m (300 MHz) and 1 mm (300 GHz). Due to the advantages over other hyperthermia methods, such as broader heating zone, faster heat generation, and less susceptibility to the heat-sink effect, MW ablation is prevalently used for clinical tumor treatment with favorable therapeutic efficacy in Far East countries.24−26 Discovered just one year ago, MW-susceptible agents are micro/nanomaterials with unusual MW−thermal transformation efficiency. The enhanced MW hyperthermia with improved survival and pain control is achieved by the agents through targeted and localized heating of tumors. Compared to the thermal effects, the nonthermal effects of MW irradiation are hardly involved in tumor therapy.27−29 It has been demonstrated that rearrangement of structural protein and enhancement of transmembrane movement occur in the cell under MW exposure, which is one key step forward in overcoming the biological barriers for nanoagent passage.30 Nonetheless, to the best of our knowledge, there are no reports on the preparation of multifunctional nanoplatforms for MWinduced combination therapy. Herein, a multisynergistic nanoplatform activated by mild MW irradiation (0.9 W, 450 MHz) was designed for enhanced tumor combination therapy of chemotherapy and ablation. We have pleasantly discovered that nonthermal MW irradiation could promote the absorption of nanoparticles and anticancer drugs via cellular uptake experiments in vitro. The large cavity

RESULTS AND DISCUSSION Synthesis and Characterization of IL-DOX-PCM@ZrO2 Nanoparticles. The template method is one of the important 9517


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Figure 1. Synthesis of hollow ZrO2 nanoparticles with different particle sizes by using different sizes of SiO2 as nanotemplates. TEM images and distribution of particle size histograms of SiO2 nanotemplates and hollow ZrO2 nanoparticles: (a1) 100 nm SiO2 nanoparticles and the corresponding histogram distribution of diameters (a2) 100 nm ZrO2; (a3) synthesis using 100 nm SiO2 nanoparticles as template; (a4) the particle size distribution histogram; (b3 and c3) 200 and 300 nm ZrO2 nanoparticles prefabricated from (b1) 200 nm and (c1) 300 nm SiO2 nanotemplates, respectively. All of the results of particle size histograms were obtained from statistical analysis by measuring about 120 particles.

Figure 2. (a) TEM image, (b) SEM images, and (c) EDS of the as-made IL-DOX-PCM@ZrO2 nanoplatforms. (d) FT-IR spectra of IL, DOX, PCM, ZrO2, and IL-DOX-PCM@ZrO2. (The scale bar in part b inset is 100 nm.)

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Figure 3. In vitro MW heating properties and controllable release of DOX at 37 and 55 °C. (a) In vitro heating curves of the as-made IL-DOXPCM@ZrO2 with different concentrations (1, 2, 10 mg mL−1, saline solution was the control) under MW irradiation at a power of 1.8 W for 5 min. (b) FLIR images based on (a) were recorded once per minute. (c) Temperature change value based on (a) and (b). (d) DOX release behavior of the as-prepared IL-DOX-PCM@ZrO2 nanoparticles, which were dispersed into PBS solution and shaken at 37 °C. After 1 h, the temperature of one group was increased to 55 °C (the red curve), while another group (the dark curve) was maintained at 37 °C. (e) Controlled DOX release behavior of the as-prepared IL-DOX-PCM@ZrO2 nanoparticles under microwave irradiation.

into mesoporous SiO2 nanoparticles by the method. The microwave heating effect of IL@SiO2 and IL@ZrO2 was tested under the same concentration of 20 mg mL−1 (dispersed into saline solution; the saline was set as a control). As shown in Figure S1g, the temperature in the IL@ZrO2 group was 10.6 °C higher than the control group at 3 min, while no significant temperature change was observed in the IL@SiO2 group. The results revealed that the ZrO2 nanoparticles were suitable for loading of IL. In order to realize the effect of enhanced MW hyperthermia and chemotherapy on the induction of MW, the DOX and PCM were packaged into the ZrO2 nanoparticles. Compared with the smooth ZrO2 nanoparticles in Figure 1a3, the surface of the as-made IL-DOX-PCM@ZrO2 became rough (Figure 2a), indicating the drug was successfully loaded into the ZrO2 nanoparticles. Figure 2b shows the scanning electron microscopy (SEM) image of IL-DOX-PCM@ZrO2 nanoplatforms; the SEM inset reveals the hollow structure of the ZrO2 nanoparticles. Energy dispersive spectroscopy (EDS) was used to measure the elements contained in the as-made nanoplatforms. As shown in Figure 2c, the elements Cl, N, P, F, and Zr were detected in the sample, which confirmed the presence of DOX (C27H29NO11·HCl), IL (C8H15N2F6P), and ZrO2. In order to further determine the composition of the as-made nanoplatforms, FT-IR was applied to examine the functional groups (Figure 3d). The stretching vibration absorption of unsaturated H appeared at 3449 cm−1 as the characteristic peak of the aromatic compounds, and the peak aromatic ring of 1620, 1581, 1497, and 1438 cm−1 confirmed the presence of DOX. The sharp absorption peak at 3630 cm−1 belonged to the free O−H stretching vibration; the 3298 cm−1 was the intermolecular hydrogen bond O−H stretching vibration absorption; the 1065 cm−1 was the C−O characteristic peak;

strategies widely used in the preparation of hollow structure materials. The diameter of hollow particles can be controlled by adjusting the size of templates, which is of great significance to biological applications. In this paper, we systematically synthesized ZrO2 hollow nanoparticles with dimensional parameters by using SiO2 nanoparticles of different sizes as templates. Figure 1 shows the TEM images and histogram distribution of SiO2 and ZrO2 nanoparticles of different diameters. Figure 1 a1−a4 show TEM images of 161 ± 6 nm hollow ZrO2 prepared from 123 ± 2 nm SiO2; 210 ± 5 nm SiO2 was used to synthesis 242 ± 4 nm ZrO2 (b1−b4); c1−c4 revealed the 341 ± 4 nm ZrO2 nanoparticles were successfully obtained from SiO2 nanoparticles with a size of 280 ± 4 nm. A controllable method was employed to prepare ZrO2 nanoparticles with uniform and adjustable size using templates of different particle sizes. On the basis of the optimal size for ideal passive targeting effect and suitable for tumor treatment in vivo, the 161 nm ZrO2 hollow nanoparticles were optimized in further experiments. Preliminary confirmation of the successfully synthesis of ZrO2 hollow nanoparticles was simply monitored by measuring the hydrodynamic diameter and zeta potential during the experiment time (Figure S1a−d). When ions or molecules, for instance, inorganic salts or IL, were encapsulated in the micro/nanoparticles, such as urea formaldehyde resin microcapsules,34 gelatin microcapsules,35 sodium alginate microcapsules,22 MoS2 nanoflowers,37 and ZrO2 nanoparticles, the temperature of the particles was significantly increased (>45 °C) under MW irradiation because of the confinement effect, so as to achieve the purpose of tumor ablation. As a kind of ideal MW-sensitive material, IL can be loaded into the hollow ZrO2 nanoparticles by a simple physical forced penetration method. However, it is difficult to load IL 9519


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Figure 4. As-made IL-DOX-PCM@ZrO2 nanoparticles’ cytotoxicity under the nonthermal effect of MW in vitro. (a) Relative viability of HepG-2 cells after being incubated with IL-DOX-PCM@ZrO2 nanoparticles (50, 25, 12.5, 6.25, 5, 2, and 1 μg mL−1, PBS was set as the control group). (b) Relative viability of HepG-2 cells irradiated by different powers of nonthermal MW (2 and 3 W) for different durations. (c) Relative viability of HepG-2 cells after being incubated with IL-DOX-PCM@ZrO2 nanoparticles and treated with nonthermal MW irradiation (2 and 3 W) for different time durations.

and the 683 cm−1 was the O−H plane bending peak. The above characteristic peaks confirmed the PCM in the as-made sample. The characteristic peaks of IL were mainly at the following wavelengths: the characteristic absorption peak of P−F appeared at 838 cm−1; 1169 cm−1 was the imidazole ring stretching vibration; and 1571 and 1468 cm−1 were from the vibration of the imidazole skeleton. The results indicated that IL, PCM, and DOX were successfully loaded into the ZrO2 nanoparticles, implying the IL-DOX-PCM@ZrO2 drug-loaded nanoplatforms were obtained. Due to the sensitivity of IL to MW in a closed space, namely, the confinement proposed by Shi et al.,23 IL was chosen to load into the core of the ZrO2 nanoparticles for MW hyperthermia. To evaluate the heating effect of the as-made nanoparticles in vitro, 1.8 W and 450 MHz MW irradiation was used to irradiate 1 mL of the as-made materials at different concentrations (1, 2, 10 mg mL−1), which were dispersed in saline solution for 5 min, and the saline solution was set as a control. Figure 3a shows that the different concentrations of the as-made nanoparticles revealed different heat effects: with the increase of the concentration of IL-DOX-PCM@ZrO2, the MW heating effect also increased. After being irradiated by MW for 5 min, the temperature of the saline was raised from 29.9 to 45 °C; that is, the temperature changed 15.1 °C. Under the same conditions, the temperature change at the concentrations of 1, 2, and 10 mg mL−1 was 19.6, 21.5, and 26.3 °C, respectively. In other words, the temperature was respectively 4.5, 3.4, and 8.2 °C higher than the control group. The temperature change values of the as-prepared nanoparticles based on Figure 3a are showed in Figure 3c. The corresponding process of temperature change was recorded by a forward-looking infrared (FLIR) imaging instrument (Figure 3b), and a picture was taken every minute. The results revealed the as-prepared ILloaded ZrO2 has a favorable MW heating effect in simulated body fluid. The mesoporous and hollow structure of ZrO2 give the materials the ability to load drug through the physical forced penetration method under subatmospheric pressure. DOX, as a common antitumor drug that is widely used in clinical treatment, was selected to load into the ZrO2 nanoparticles under the cooperative action of the phase change material (PCM). The UV−vis spectrophotometer was employed to examine the concentration of DOX at 483 nm in the supernatant via centrifuging. The amount of DOX release over time was confirmed through a standard calibration curve.

The loading capacity of DOX was 25.21 ± 2.03% (w/w). Thermogravimetric analysis (TGA) was used to determine the content of PCM and IL in IL-DOX-PCM@ZrO2 (Figure S1e), and the drug loading capacities of IL and PCM were 1.67% and 12.83%, respectively. As the nanoparticles were irradiated by the MW, the temperature of the nanoparticles obviously increased, which resulted in the PCM melting. Thus, the DOX in the cavity of the hollow ZrO2 nanoparticles was released due to the opening of the MW switch that was constructed by PCM. As shown in Figure S1f, the DOX IL-DOX@ZrO2 nanoparticles were slowly released in the first 5 h. At 37 °C, the DOX release was only 11.61%, and even if the temperature was 55 °C, the release of DOX was only 13.04% (dispersed into phosphate-buffered saline, PBS, 5 mg mL−1, 48 h). In the preliminary experiments, a temperature over 55 °C at the tumor site during the process of microwave thermal therapy was observed. In order to examine the microwave-controlled release properties of the as-made nanoparticles, 5 mg of the ILDOX-PCM@ZrO2 nanoparticles was dispersed into 1 mL of PBS (0.1 M, pH 7.2) solution, and then the released amount of DOX under the conditions of constant shaking for 1 h at 37 °C was tested. Then the microwave-stimuli release group was placed under 55 °C after 2 h. At 37 °C, the release of DOX was only 3.48%, and when the temperature increased to 55 °C, the release amount reached 37.64% (at the fifth hour). Figure 3d revealed that after 48 h at 37 °C the final release of DOX reached only 14.89%, while when the temperature was 55 °C, the release of DOX could reach 40.62%, which was approximately 3 times as much as at 37 °C. In order to further examine the microwave-controlled release properties of the asmade nanoparticles, 5 mg of the IL-DOX-PCM@ZrO2 nanoparticles was dispersed into 1 mL of PBS (0.1 M, pH 7.2) solution, and the mixed solution was placed into a water bath of 37 °C and was shaken continuously. Every once in a while, the mixture solution was treated by microwave for 5 min. The supernatant of the mixed solution was collected before and after microwave irradiation to measure the released amount of DOX. The release of DOX was only 3.94% at 37 °C in the first hour, and then microwave (1.8 W, 450 MHz) irradiation was applied for 5 min, which resulted in a released amount reaching 9.24%. Then the process was repeated several times. As shown in Figure 3e, the DOX release amount could reach 35.90% after 5 h. Then the release was less and less, and the final released amount was about 39.39% after 24 h. The results revealed that 9520


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Figure 5. Cellular uptake behaviors of nanoparticles and DOX under nonthermal MW irradiation. Fluorescence microscopy images of HepG-2 cells incubated with (a) FITC-SiO2 nanoparticles and (c) DOX for 24 h and irradiated by different powers of MW (2 and 4 W) for different durations. Intracellular fluorescence intensity of (b) FITC-SiO2 and (d) DOX. (e) Fluorescence microscopy images of HepG-2 cells incubated with DOX@ZrO2 nanoparticles. (All of the scale bars are 100 μm.)

increase in both MW power and the prolongation of time, the viability of cells decreased gradually. After being irradiated by 2 W MW for 1, 2, and 3 min, the cell viability decreased to 75%, 72%, and 65%, respectively, and the viability of cells decreased more obviously after 3 W MW irradiation. It reduced to 71%, 62%, and 51% after being irradiated for 1, 2, and 3 min. Nonthermal MW irradiation can promote the uptake of FITCmodified nanoparticles and free DOX by HepG-2 cells, as shown in the cellular uptake studies. It was found that IL-DOXPCM@ZrO2 nanoparticles in combination with nonthermal MW irradiation caused higher cytotoxicity. Therefore, the combination of nonthermal and thermal effects of MW to induce enhanced thermal and chemotherapy for tumor therapy was worth further exploration. Cellular Uptake of Nanoparticles and DOX in Vitro. The nonthermal effects following MW exposure should be looked upon as a quite possible promising mechanism for the development of tumor combination therapy. Different from the increased cell membrane permeability at moderately high temperature (40−43 °C) by the effect of weak photothermal effects to enhance cellular uptake,36 biomembranes could act as oscillating electric dipoles under microwave irradiation.29 The wave passing through the membrane will cause strong polarization and lead to electronic vibrations. The electronic

DOX could be controlled to release from the as-prepared ILDOX-PCM@ZrO2 under microwave stimuli. In Vitro Cytotoxicity. To investigate the cytotoxicity of ILDOX-PCM@ZrO2 on HepG-2 cells, the MTT assay was used. In our previous work, IL@ZrO2 had been demonstrated to have ideal biocompatibility that was suitable for further experiments in vivo.23 After being loaded with DOX, the asmade IL-DOX-PCM@ZrO2 nanoparticles displayed a higher cytotoxicity than IL@ZrO2. As shown in Figure 4a, the ILDOX-PCM@ZrO2 nanoparticles showed a dose-dependent cytotoxicity on HepG-2 cells. The cell viability in groups with a concentration below 6.25 μg mL−1 was above 80%. To test whether the as-made IL-DOX-PCM@ZrO2 will affect cell activity under MW irradiation, HepG-2 cells were incubated with 100 μL of IL-DOX-PCM@ZrO2 (12.5 μg mL−1) and irradiated by MW, and then the MTT assay was used to evaluate the cell viability. Figure 4b showed that the cell viability in the PBS treatment group had no obvious decline under 2 or 3 W MW irradiation (at a frequency of 2450 ± 50 MHz, 2−4 W MW will not cause an obvious thermal effect) for different MW irradiation durations, which indicated the MW irradiation at the experimental powers could not damage tumor cells. While a significant change occurred in the cells that were incubated with the as-made IL-DOX-PCM@ZrO2, with an 9521


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Figure 6. In vivo safety injection-dose evaluation of the as-made IL-DOX-PCM@ZrO2 nanoparticles at different concentrations (20, 40, 100 mg kg−1; PBS-treated group was set as the control). (a) Body weight change curve for 16 days and (b) organ index histogram of each group. (c) Blood routine including ALT, AST, UREA, and CREA; (d) blood biochemistry including RBC, WBC, MCH, MCHC, MCV, HCT, MPV, PLT, and HGB of each group. (e, f) TEM images of ZrO2 nanoparticles in the stool mass of mice 4 h postinjection. (g) Content of Zr4+ (mg kg−1) in the excrement of mice at different time intervals.

vibrations will change the water ordering around the phosphocholine head groups, resulting in the change of membrane water partitioning.27,28 It is reasonable to assume that the dynamic behavior around the interface of biomem-

branes will lead to the migration and transformation of the materials nearby, thereby increasing the cellular uptake of nanoparticles or anticancer drugs. We have performed a number of experiments to test the validity of enhanced 9522


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verify the toxicity of the as-made materials to mice, the biochemistry, including red blood cells (RBC), white blood cells (WBC), mean corpuscular hemoglobin concentration (MCHC), mean capacity hemoglobin (MCH), mean corpuscular volume (MCV), hematocrit (HCT), mean platelet volume (MPV), platelet (PLT), and hemoglobin (HGB), and the blood routine, including alanine aminotransferase (ALT), aspertate aminotransferase (AST), urea (UREA), and creatinine (CREA) (ALT and AST were used to determine liver damage, and UREA and CREA were used to examine renal impairment), were evaluated. Compared with the control group, even the 100 mg kg−1 group (2.5 times the therapeutic dose) did not show obvious abnormalacies in the biochemical and routine parameters. As an important index to estimate the biosafety of nanoparticles, the elimination by excretion has attracted great attention. It has been reported that the inorganic nanoparticles, such as SiO 2 , were found to undergo sequestration by the liver. They are opsonized by serum proteins and thus amenable to hepatobiliary excretion into the gastrointestinal tract.39 To further study if the ZrO2 nanoparticles are eliminated by the process, the excretions of mice under different time intervals after injection via tail vein were collected (the injection dose was 40 mg kg−1). As shown in Figure 6e,f, the ZrO2 nanoparticles were found in the excretions 24 h postinjection. The content of Zr4+ or ZrO2 was further measured by ICP-MS (Figure 6g). The results revealed that the ZrO2 nanoparticles could be eliminated through the gastrointestinal tract. The H&E (hematoxylin and eosin)-stained images (Figure S3) of the main organs (liver, spleen, lung, kidney, and heart) collected from the 20, 40, and 100 mg kg−1 and control group indicated no obvious tissue denaturation or organ damage in administrated mice. Although the most common side effect of DOX was cardiac toxicity, there was no significant change in heart slices in the four groups. The results demonstrated that the materials had no recognizable toxicity on the mice even at a high dose of 100 mg kg−1. Combined with the effective dose for tumor inhibition and the potential toxicity to mice, 40 mg kg−1 was suitable to act as the therapeutic dose.38,39 Antitumor Efficacy Evaluation of the As-Made ILDOX-PCM@ZrO2 in Vivo under Mild MW Irradiation. Taking into account that high-power MW irradiation may cause damage to the skin and superficial organs, we presented the idea of applying a mild MW power therapy to suppress the growth of tumors. However, the effect of hyperthermia induced by IL@ZrO2 could not completely kill the tumor cells under mild MW irradiation (0.9 W, 450 MHz). Fortunately, we recently found that the nonthermal effect of MW can increase cellular uptake of nanoparticles and anticancer drugs, so the loading of DOX can improve the chemotherapy effect under MW irradiation. To find a better way of solving the problem, after attempting to load DOX into the as-made nanoparticles, while combining the temperature sensitivity property of PCM, we finally achieved a kind of MW-stimuli-controlled release of IL-DOX-PCM@ZrO2 nanoparticles that integrates thermal effects and chemotherapy. To determine the targeting ability of the as-made IL-DOXPCM@ZrO2 nanoparticles on the tumor site, H22-tumorbearing mice were injected with the ZrO2 nanoparticles via the tail vein. Then ICP-MS was utilized to measure the content of ZrO2 in organs and tumors in each group. As shown in Figure S4, the as-made IL-DOX-PCM@ZrO2 nanoparticles mainly gathered in the reticuloendothelial system organs (liver and

nanoagent and chemical uptake by tumor cells induced by MW irradiation. To avoid the effects of thermal activation, the use of nonthermal doses of MW for various studies at 2−4 W irradiation (2450 ± 50 MHz) resulted in a negligible temperature increase and cell viability change. As a kind of representative nanomaterials model, SiO2 nanoparticles are widely used as drug carriers. Therefore, FITC-modified SiO2 nanoparticles were adopted as an experimental model to examine whether MW irradiation could effectively improve the cellular uptake of nanomaterials. FITC-modified SiO2 nanoparticles (100 μg mL−1) were added into HepG-2 cells and then irradiated at different powers (2 and 4 W) of MW for 1 min. The group without MW treatment was set as a control. As shown in Figure 5a, the tumor cells were stained with Hoechst33342 after 24 h. Obvious green spots from FITC-modified SiO2 nanoparticles were observed in each group, and much higher FITC fluorescence was found in the groups that received MW irradiation for 1 min. To further verify the results, the green fluorescence intensity in each group was quantified. As shown in Figure 5b, when the cells were incubated with FITCmodified SiO2 for 24 h in the absence of MW irradiation, the fluorescence intensity was only 22, while it reached to 55 and 102 in the groups that were irradiated by MW, which indicated that uptake of nanoparticles was increased by 2.5- and 4.6-fold under 2 and 4 W MW irradiation, respectively. To determine the cellular uptake behavior of DOX@ZrO2 nanoparticles, HepG-2 cells were incubated with DOX@ZrO2 nanoparticles (12.5 μg mL−1) and then irradiated by 2 W MW for 1 min (Figure 5e). Compared with the control group, the red fluorescence in the MW-treated group was significantly enhanced. From the ICP-MS test result as shown in Figure S2b, the content of ZrO2 in HepG-2 cells increased with an increase in incubation time after MW irradiation. The results implied that the increase in cellular uptake of nanoparticles is due to nonthermal effects of MW irradiation. Besides nanoparticles, the cellular uptake of chemotherapeutic agents is another important concern for tumor therapy. It is reasonable to further speculate whether the nonthermal effects of MW irradiation can lead to an increase in cellular uptake of chemotherapeutic agents. Thus, a cellular uptake assay in vitro was performed to investigate the effect of MW irradiation on the tumor cellular uptake of DOX. DOX at different concentrations was added into the cell wells. The results of DOX uptake triggered by MW irradiation are shown in Figure 5c. After exposure to 3 W MW irradiation for 2 min, the red fluorescence of DOX was found to diffuse over the cytosol. The quantitative fluorescence intensity of DOX (Figure 5d) also illustrated the same results. An 11 μg mL −1 concentration of DOX was incubated with HepG-2 cells for different time durations. With the prolonging of the incubation time, most of the red fluorescence gradually covered the blue fluorescence, demonstrating that DOX entered into the cytoplasm and cell nucleus (Figure S2a). Because DOX exerts an antitumor effect in the cell nucleus, the increase in the tumor cellular uptake of DOX suggested a potentially higher chemotherapy efficacy in vivo. The data thus obtained throw light on the cancer treatment of nonthermal effects of MW irradiation by inducing the cellular uptake of therapeutic moieties. Safety Injection-Dose Evaluation in Vivo. Compared with the control group, there was no significance difference in body weight and organ index in each group. The body weight of mice in each group showed a slow upward trend. To further 9523


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Figure 7. In vivo MW irradiation therapy. (a) Distribution of ZrO2 in tumor under multiple mild MW irradiation, with the tumor-bearing mice without MW irradiation set as the control. (b) Temperature change curve at the tumor site for the 0.9 W, 450 MHz MW irradiation-treated groups. The temperature was recorded by an FLIR instrument at 30 s intervals. (c) FLIR images of the tumor site in each group of mice receiving MW irradiation corresponding to (b). (d) Body weight change curve and (e) tumor volume growth curve of MW, IL@ZrO2+MW, IL-DOX-PCM@ZrO2+MW, IL-DOX-PCM@ZrO2, DOX, and control groups for 20 days. The injection dose was 40 mg kg−1, except for 10 mg kg−1 in the DOX group. The therapeutic agents used in the experiment were dispersed in PBS. (f) Survival rate curve of mice in each group.

spleen). To further verify the cellular uptake of nanoparticles in vivo, ZrO2 nanoparticles were injected into the tumor-bearing mice, and then half of the mice were irradiated by fairly mild MW (0.2 W, 30 s) every 2 h at the tumor site. The irradiation power and durations of MW used in the experiment could not cause a thermal effect. Twelve hours postinjection, the tumors of mice were collected for ICP-MS examination. As shown in Figure 7a, the content of Zr4+ in mice undergoing MW irradiation was 2.53 times that without MW treatment. In vivo cell absorption experiments as above demonstrated that the nonthermal effect of MW can effectively improve the targeting

of nanoparticles in the tumor site. To evaluate the inhibition effect of the as-made IL-DOX-PCM@ZrO2 nanoparticles on tumor growth, an H22 liver cancer tumor model was established with ICR mice. Healthy female mice were randomly assigned into six groups and injected with PBS, free DOX, IL@ ZrO2, and IL-DOX-PCM@ZrO2 via tail vein injection, respectively. Mice in the IL@ZrO2 group and half of the mice in the PBS and IL-DOX-PCM@ZrO2 groups were treated with MW irradiation in the tumor site at a mild power of 0.9 W, 450 MHz for 5 min 8 h postinjection, while the rest underwent no further treatment. The temperature increment of the tumor 9524


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ZrO2 nanoplatforms could significantly improve the survival rate of H22-tumor-bearing mice.

site under MW irradiation was time-dependent at the beginning, at 1 to 2 min. Subsequently the temperature was kept at a slow growth level (Figure 7b). Although a higher temperature (>60 °C) could be realized by increasing the power of MW irradiation, empyrosis in the ambient skin and tissue would occur subsequently.40,41 During the irradiation process, the temperature of the tumor region of IL-DOXPCM@ZrO2 group increased to 58 and 56 °C in the IL@ZrO2 group, while the control group reached only 52 °C. The differences in temperature between the IL-DOX-PCM@ZrO2 and IL@ZrO2 groups may be attributed to the presence of HCl (Cl−) in DOX. The thermal effect of MW led to a temperature elevation; the PCM melted and DOX was released at the tumor site under MW stimuli. At the same time, the tumor cellular uptake of nanoparticles and DOX increased due to the nonthermal effect of MW. Through the above process, the cells of the tumor were killed under mild MW and enhanced chemotherapy stimulated by a nonthermal MW effect. The corresponding infrared thermal images (Figure 7c) at the tumor region visually displayed the process of temperature increment under mild MW irradiation. As shown in Figure 7d, only the body weight of the DOX group showed a decline in the first few days and then slowly increased, which was caused by the toxic effect of the antitumor drug. The DOX-loaded group (IL-DOX-PCM@ZrO2 with and without MW irradiation groups) did not indicate an obvious loss in body weight, which further validated the stability of DOX in the IL-DOX-PCM@ZrO2 system without thermal stimulation. After 20 days’ observation, among mice treated with MW irradiation, the IL-DOX-PCM@ZrO2 group expressed a more significant suppression effect in tumor size than the other two groups. From Figure 7e, the mean growth volume of tumor was 585 and 167 mm3 in the PBS+MW and IL@ZrO2+MW groups, respectively, while it was reduced by 92 mm3 in the IL-DOX-PCM@ZrO2+MW group. In mice without MW irradiation, the average growth value was 1962 mm3 in the free DOX group and 2485 mm3 in the IL-DOX-PCM@ZrO2 group, while the value reached 3040 mm3 in the PBS group, which indicated IL-DOX-PCM@ZrO2 alone did not engender an obvious inhibitive effect on tumor growth. Moreover, it was also confirmed that the DOX was relatively stable in the nanoparticles without the heat trigger. The tumor volume reached 167 mm3 in the IL@ZrO2 group versus 585 mm3 in the MW alone group, which illustrated that IL@ZrO2 could not produce enough energy to damage tumor cells completely at a mild MW power and further proved that the MW irradiation we used could not induce tumor cell death. The representative photos of mice as shown in Figure S5 that were taken at different dates before and after various treatments were consistent with the results of tumor volume growth curves. The deaths of mice in the DOX-treatment group were first observed on the 14th day after treatment, and the deaths in the control and IL-DOX-PCM@ZrO2+MW group appeared at 17th and 18th day, respectively (Figure 7f). All of the final survival rates in groups without MW irradiation were 0%, while the other groups were 20% (MW), 60% (IL@ZrO2), and 100% (IL-DOX-PCM@ZrO2+MW), respectively. There were no deaths among the IL-DOX-PCM@ZrO2+MW group during the experimental time. Combined with the H&E-stained images (Figures S6 and S7), IL-DOX-PCM@ZrO2 showed better biocompatibility and therapeutic efficacy than free DOX. The outcomes demonstrated that combined chemo- and MW (nonthermal and thermal) therapy based on IL-DOX-PCM@

CONCLUSIONS In conclusion, we have prepared a kind of promising multifunctional IL-DOX-PCM@ZrO2 nanoplatform for enhanced combined thermal and chemotherapy. Empyrosis in the ambient skin and tissue occurs under high power MW irradiation. However, the complete elimination of tumor cells could not be achieved under lower power. We found that the nonthermal effect of MW can promote cellular uptake of nanoparticles and DOX by measuring the fluorescence intensity change progress. Inspired by the application of the combination of microwave nonthermal and thermal effects in the enhanced synergistic therapy of tumors, the microwave sensitive IL and anticancer drug DOX were loaded into ZrO2 nanoparticles. In the nanoplatforms we have developed, PCM played the role of MW switch. As the temperature increased, PCM melted and DOX was released. The release experiment in vitro showed the release of DOX at 55 °C was about 3 times that at 37 °C, confirming that the microwave heating process will trigger the opening of the MW switch. The MTT test and safety evaluation study in vivo revealed that the IL-DOX-PCM@ ZrO2 nanoparticles were less toxic than free DOX, which demonstrated that the as-made nanoplatforms could effectively reduce the side effects of DOX. Even under mild MW irradiation (0.9 W, 450 MHz), the generated heat due to the thermal effect can kill most of the tumor cells. In a cellular uptake experiment in vitro, compared with the group without MW, the uptake of nanoparticles was increased by 2.5- and 4.6fold under 2 and 4 W MW irradiation, respectively. The results demonstrated the absorption of the anticancer drug DOX in the cell increased by the nonthermal effects, further increasing the number of the residual tumor cells killed. The final survival rate in the groups without MW was 0%, while that of the MW, IL@ZrO2+MW, and IL-DOX-PCM@ZrO2+MW groups was 20%, 60%, and 100%, respectively. The outcomes revealed that the as-made IL-DOX-PCM@ZrO2 nanoplatform showed an ideal treatment effect compared with the other groups, and the survival rate was also significantly increased. EXPERIMENTAL SECTION Materials. Zirconium(IV) propoxide was obtained from Tokyo Chemical Industry Co., Ltd. 1-Butyl-3-methylimidazolium hexafluorophosphate ([Bmim]PF6, IL) was provided by Shanghai Chengjie Chemical Co., Ltd. Tetradecanol (PCM) was obtained from Sinopharm Chemical Reagent Co., Ltd. Doxorubicin hydrochloride was purchased from Beijing Huafeng Chemical Reagent Co., Ltd. Sodium hydroxide and 1,4-dioxane were purchased from the Beijing Chemical Reagents Company. Ethanol, acetonitrile, and aqueous ammonia were commercially available products. The reagents used in this work were of analytical grade without any further purification. Synthesis of ZrO2 Hollow Nanoparticles. SiO2 nanoparticles with a size of 100, 200, and 300 nm acted as templates during the process of preparing ZrO2 hollow nanoparticles (100, 200, and 300 nm). A 2 mL SiO2 ethanol solution (about 180 mg), 150 mL of alcohol and 50 mL of acetonitrile (alcohol:acetonitrile = 3:1), and 1.2 mL of ammonia (25−28%, NH3·H2O) were added into a 250 mL Erlenmeyer flask. Then 0.5 mL of zirconium(IV) propoxide was quickly added into the mixed solution. A layer of ZrO2 will cover evenly and tightly onto the surface of SiO2 nanoparticles to form a SiO2@ZrO2 core−shell structure after over 6 h of reaction under magnetic stirring. In order to remove the SiO2 core layer, the reaction products of SiO2@ZrO2 were added into 100 mL of aqueous solution containing 3 mL of NaOH (1 M) under 80 °C for 4 h. The products 9525


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intervals indicates the accumulated density of nanoparticles or DOX in cancer cells. Then DOX@ZrO2 nanoparticles were used to further detect the cellular uptake behavior (12.5 μg mL−1, 2 W MW irradiation for 0 and 1 min). To detect the intracellular concentration of ZrO2 nanoparticles, HepG-2 cells were added into 35 mm cell culture dishes and cultured for 24 h. A 100 μg mL−1 concentration of ZrO2 nanoparticles was added into the HepG-2 cells and then irradiated by MW (3 W, 2450 ± 50 MHz) for 1 min. Then the cells were cultured for different durations (0, 5, 24, and 48 h). Afterward, the cells were washed with PBS to remove the free ZrO2 in the supernatant. The amount of Zr4+ ion was tested by ICP-MS. In Vitro Cytotoxicity. To estimate the biocompatibility of the asmade IL-DOX-PCM@ZrO2 nanoparticles, the standard MTT assay was implemented on the HepG-2 cells. The cell viability and morphology reflect the cytotoxicity of the IL-DOX-PCM@ZrO2 before and after MW irradiation. Tumor cells were plated into 96well plates (1 × 104 per well) and cultured in a suitable environment for 24 h. Then 100 μL of PBS and IL-DOX-PCM@ZrO2 with or without MW irradiation were added into the plates. MW of different powers was utilized to irradiate the cell well for different durations. Then the cells were incubated for 24 h, and the cell viability was represented as absorbance of formazan at 490 nm. The control (PBS treatment) cells were considered as 100% viable. Safety Injection-Dose Evaluation in Vivo. To investigate the safety injection-dose of IL-DOX-PCM@ZrO2, 12 healthy female mice were randomly and equally divided into four groups. The mice were injected with IL-DOX-PCM@ZrO2 at different doses of 20 to 100 mg kg−1 (20, 40, and 100 mg kg−1, dispersed in PBS) via the tail vein and sacrificed after 15 days; PBS-treated mice were sacrificed as the control group. The blood parameters were collected for further blood analysis (blood routine and blood chemistry examination). Major organs including the heart, spleen, liver, lung, and kidney were harvested for further histological examination. The excretions of mice were collected to evaluate the elimination of ZrO2 nanoparticles. Targeting Ability of IL-DOX-PCM@ZrO2 in H22-TumorBearing Mice. To estimate the targeting ability of IL-DOX-PCM@ ZrO2, female H22-tumor-bearing mice were separated into four groups (three per group). A 0.2 mL amount of IL-DOX-PCM@ZrO2 (40 mg kg−1, dispersed in PBS; PBS was injected into the control group) was injected into the mice via the tail vein. Then the mice were sacrificed after 6 and 24 h postinjection (mice of control group were sacrificed 24 h after injection). The green weight of the body, main organs (heart, liver, spleen, lung, and kidney), and tumor in each group were weighed carefully. To further verify whether the MW nonthermal effect can promote the cellular uptake at the tumor site in vivo, six female H22-tumor-bearing mice were evenly separated into two groups (ZrO 2 and ZrO 2 +MW). A 0.2 mL amount of ZrO 2 nanoparticles (40 mg kg−1, dispersed in PBS) was injected into the mice. The mice of the ZrO2+MW group were irradiated by fairly mild MW (0.3 W, 450 MHz) for 30 s every 2 h; the power and time of MW did not cause a thermal effect. Then the above-mentioned organs and tumor were dried at 40 °C for 48 h, and the dried parts were recorded accurately. Then inductively coupled plasma mass spectrometry (GGL-ICP-MS, NexION 300X from Perkin Elmer, USA) was utilized to measure the content of Zr4+ ion in various organs and tumor of each group, respectively. Antitumor Efficacy Evaluation of the As-Made IL-DOXPCM@ZrO2 in Vivo under Mild MW Irradiation. H22-tumorbearing female mice were randomly divided into the following six groups: PBS, PBS+MW, DOX, IL-DOX-PCM@ZrO2, IL-DOXPCM@ZrO2+MW, and IL@ZrO2+MW. The injection dose was 40 mg kg−1 except for the DOX group (10 mg kg−1), and the abovementioned materials were injected into the mice via tail vein. Mild MW irradiation (0.9 W, 450 MHz) was applied to the tumor site of the mice in the MW groups (PBS+MW, IL-DOX-PCM@ZrO2+MW, and IL@ZrO2+MW) for 5 min at 8 h postinjection. The MW heating effect in vivo was recorded by an FLIR imaging instrument. Then the body weight and tumor were carefully observed for 2 or 3 days during the experiment time. When the size of the tumor in any one direction was more than 20 mm, the mice were sacrificed and marked as state of

were obtained by centrifugation and washing with deionized water three times. Preparation of IL-DOX-PCM@ZrO2 Drug-Loaded Nanoplatforms. 1-Butyl-3-methylimidazolium hexafluorophosphate (IL) acted as the MW-sensitive agent in the drug delivery system. A 1 mL amount of IL, 2 mL of 1,4-dioxane, 10 mL of alcohol, and 80 mg of ZrO2 hollow nanoparticles were added into a 50 mL conical flask. After ultrasonic dispersion for 30 min, the mixture solution was kept under the condition of vacuum pumping until the solvent was drained. The IL@ZrO2 nanoparticles were obtained by centrifuging at 9000 rpm for 10 min and washing with deionized water three times. DOX was used as anticancer drug, and PCM acted as the trigger of drug release at a certain temperature (above 40 °C). A 40 mg amount of DOX, 20 mg of PCM, 10 mL of deionized water, 5 mL of alcohol, and the as-prepared IL@ZrO2 nanoparticles were added into a reactor in a 50 °C water bath. The remaining steps were the same as the process of IL diffusing into the ZrO2 shell. Finally, the IL-DOXPCM@ZrO2 was collected and washed via centrifugation at 4 °C (7000 rpm). The rest of the supernatant was collected for the DOX loading test. Characterization. The morphology and size of SiO2, ZrO2, and IL-DOX-PCM@ZrO2 nanoparticles were measured by a JEM-2100 transmission scanning electron microscope (TEM, JEOL, Japan) and a model 4300 scanning electron microscope (Hitachi). A Zeta-sizer (Malvern Instruments Zetasizer Nano ZS90, U.K.) was used to measure the hydrodynamic zeta-potential and size of the SiO2 and ZrO2 nanoparticles at a temperature of 25 °C. The absorption spectra of the DOX were acquired via a UV−vis spectrophotometer (Jasco V570 UV/vis/NIR spectrophotometer, Shanghai, China). An Excalibur 3100 Fourier transform infrared spectrometer (FT-IR, Varian, USA) was employed to confirm the molecular structure and properties. The N, Cl, P, F, and Zr elements in the IL-DOX-PCM@ZrO 2 nanoplatform were characterized using SEM X-ray (EDS). The progress of MW heating in vivo and in vitro was recorded by an FLIR. Thermogravimetric analysis (Diamond TG/DTA, USA) was used to determine the content of IL and PCM in the IL-DOX-PCM@ZrO2 nanoparticles. An Olympus X71 optical microscope (Japan) was used to observe the paraffin sections and fluorescence. The MW heating effect in vitro of the IL-DOX-PCM@ZrO2 nanoplatforms was evaluated by measuring the temperature at different MW irradiation times. A 1 mL amount of IL-DOX-PCM@ZrO2 nanoplatform at different concentrations (0, 1, 2, 10 mg mL−1) was added into a reaction vessel and irradiated by 1.8 W and 450 MHz MW for 5 min. The saline solution (1 mL) was set as the control group. The temperature was monitored by an optical fiber probe. The corresponding thermal progress imaging was recorded by an FLIR. The controlled release properties of DOX from the IL-DOXPCM@ZrO2 nanoplatform were investigated. IL-DOX-PCM@ZrO2 and IL-DOX@ZrO2 nanoparticles were dispersed in 0.1 M PBS (5 mg mL−1) into a rotary platform at 37 and 55 °C for 48 h (MW-controlled release group was placed under 37 °C at the beginning, and the temperature was increased to 55 °C after 1 h). The solution was continuously shaken by a shaking table so that the nanoparticles were mixed into the PBS solution evenly. Then a UV−vis spectrophotometer was employed to examine the concentration of DOX at 483 nm in the supernatant via centrifuging. The amount of DOX release over time was confirmed through a standard calibration curve. The linear equation was y = 0.05531 + 0.02045x, and the correlation coefficient was 0.99821, where y represents the spectral intensity of DOX at 483 nm and x represents the corresponding concentration of DOX (μg mL−1). Cellular Uptake of IL-DOX-PCM@ZrO2 in Vitro. To examine whether the MW irradiation could enhance the uptake of nanoparticles and DOX in cancer cells, a cellular uptake assay in vitro was carried out. The HepG-2 cells were incubated in Dulbecco’s minimum essential medium (DMEM) added with FITC-modified SiO2 (green fluorescence) and free DOX (red fluorescence, concentration of DOX: 5 and 11 μg mL−1), and the status of cellular uptake before and after MW irradiation (2450 ± 50 MHz) was estimated by fluorescence microscopy. The green or red fluorescence intensity at different time 9526


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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/acsnano.6b04749. Additional information (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (X. Meng). Notes

The authors declare no competing financial interest.

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