High Biocompatible ZIF-8 Coated by ZrO2 for Chemo-microwave

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Biological and Medical Applications of Materials and Interfaces

High Biocompatible ZIF-8 Coated by ZrO2 for Chemomicrowave Thermal Tumor Synergistic Therapy Liuhui Su, Qiong Wu, Longfei Tan, Zhongbing Huang, Changhui Fu, Xiangling Ren, Na Xia, Zengzhen Chen, Xiaoyan Ma, Xudong Lan, Qiang Zhang, and Xianwei Meng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b22177 • Publication Date (Web): 25 Feb 2019 Downloaded from http://pubs.acs.org on March 3, 2019

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ACS Applied Materials & Interfaces

High Biocompatible ZIF-8 Coated by ZrO2 for Chemo-microwave Thermal Tumor Synergistic Therapy Liuhui Su1,2, Qiong Wu2, Longfei Tan2, Zhongbing Huang*,1, Changhui Fu2, Xiangling Ren2, Na Xia1, Zengzhen Chen2, Xiaoyan Ma2, Xudong Lan2, Qiang Zhang*,3, Xianwei Meng*,2 1College

of Materials Science & Engineering, Sichuan University, Chengdu 610065, China.

2Laboratory

of Controllable Preparation and Application of Nanomaterials, CAS Key Laboratory

of Cryogenics, Technical Institute of Physics and Chemistry Chinese Academy of Sciences, Beijing 100190, China. 3Department

of Orthopaedics, General Hospital of Chinese People's Liberation Army, Beijing,

100853, PR China. KEYWORDS nanoscale metal organic frameworks, microwave thermal therapy, ZIF-8, drug delivery, chemotherapy

ABSTRACT Zeolitic imidazolate framework (ZIF-8) is specifically promising for drug carrier due to its excellent intrinsic properties. However, the high toxicity of ZIF-8 nanoparticles severely limits its further research and clinical application. In the work, the biocompatibility of ZIF-8 nanoparticles are greatly improved by coating ZrO2 onto the surface. The survival rate of cells and

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mice in the ZIF-8@ZrO2 nanocomposites group is significantly increased compared with the undecorated ZIF-8 nanoparticles group. Doxorubicin (DOX) as a chemotherapeutic drug is deposited during the ZIF-8 growth by a facile one-pot method. Ionic liquid (IL) is loaded into the pore of the ZIF-8/DOX@ZrO2 nanocomposites for enhanced microwave thermal therapy. The tumor inhibition rate of ZIF-8/DOX@ZrO2@IL nanocomposites with synergistic microwave thermal therapy and chemotherapy is obviously higher than other groups. In addition, the ZIF8/DOX@ZrO2@IL nanocomposites are used for real-time monitoring of the therapeutic outcomes due to the excellent CT contrast agent of ZrO2. Therefore, such ZrO2 coating strategy shows great promise for overcoming high toxicity of ZIF-8 nanoparticles, which offers a new platform for tumor synergistic microwave thermal therapy and chemotherapy using ZIF-8/DOX@ZrO2@IL nanocomposite as a theranostic nanocarrier.

1. INTRODUCTION Chemotherapy is a major clinical tumor treatment, whose efficacy is usually critical limited by side effects, drug degradation and inadequate dosing problems1, 2. In order to reduce side effects and improve therapeutic efficacy, a variety of nanoparticles are prepared for delivering anticancer drugs to tumor region, including liposomes, micelles, nanocapsules, nanoemulsions, mesoporous silica, and hybrid porous solids3-9. In addition, the nanocomposite composed of anticancer drugs and other functional molecules through self-assembly was also a research hotspot. Chuang Gao et al designed a liposome-like capsule which could encapsulate two drugs by self-assembly. The nanocomposite had several characteristics: high drug loading rate, preventing premature release, fluorescence imaging in vivo, and excellent photothermal effect10. Xiangfei Han et al synthesized a PTX-s-s-PTX conjugated nanocomposite by self-assembly. The disulfide bonds of the


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nanocomposites would be break and release PTX to tumor sites. The nanocomposite as a potent nanosystem could combined hyperthermia and chemotherapy for tumor treatment11. Nanoscale metal organic frameworks (NMOFs) have attracted considerable interest for drug delivery system in recent years by reason of large specific surface area, the easy adjustment of the pore size, and high drug-loading capacity12-16. In the field of NMOFs, ZIF-8 nanoparticle is one of the most promising options due to its excellent intrinsic properties, such as high capacity for drug loadings, simple synthesis method, and favourable pH-sensitivity17-19. Zheng et al. synthesized a ZIF-8/DOX nanoparticle with drug-loaded rate up to 20% through stirring the mixture of metal ions, anticancer drugs and ligands in sequential order for 15 minutes at room temperature20. The simple preparation method is gentle without compromising the activity of the drugs. The coordination bonds composed of 2-methylimidazole (2MI) and zinc ions will be dissociated when the pH value is lower than 6.0 because of the protonation effect18, 20, 21. Therefore, ZIF-8 nanoparticles exhibit fantastic properties as a pH-sensitive drug delivery system, which degrade in acid tumor microenvironment while stabilize normal physiological conditions18,

20, 21.

Despite these

encouraging properties, the product of Zinc ions from rapid degradation of ZIF-8 shows markedly toxicity, which severely limits its further research and clinical application. Our group has reported that reducing the toxicity by polydopamine surface coating to regulate the degradation process22. ZrO2 has favorable biocompatibility with an extra property of enhanced CT imaging efficiency23. Surface coating of ZIF-8 with ZrO2 will take advantage of not only the low toxicity and biocompatibility from regulating the degradation process but also efficient CT imaging. However, the design and preparation of such a MOF complex are still nascent area. In our work, the biocompatibility of ZIF-8 nanoparticles was greatly improved by ZrO2 surface coating. The survival rate of mice was 80% when the dose of ZIF-8@ZrO2 nanocomposites


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was high to 100 mg/kg (the content of ZIF-8 nanoparticles was 86.7 mg/kg), while all mice died when the dose of ZIF-8 nanoparticles was 40 mg/kg. The above results manifested that the biocompatibility of ZIF-8 nanoparticles was greatly improved by ZrO2 surface coating. As a widely used anticancer drug in the clinic, DOX as a chemotherapeutic drug was deposited during the synthesis of ZIF-8 nanoparticles. IL was loaded into the pore of the ZIF-8/DOX@ZrO2 nanocomposites

for

microwave

response.

The

as-prepared

ZIF-8/DOX@ZrO2@IL

nanocomposites showed an excellent thermal effect under microwave irradiation in vivo and in vitro. In addition, ZrO2 could be used as a favorable CT contrast agents because of the relatively atomic number of zirconium atoms23, 24. ZIF-8/DOX@ZrO2@IL nanocomposites were also used for real-time surveillance of treatment effects. The anti-tumor effect of synergistic microwave thermal therapy and chemotherapy in vivo was evaluated with H22 tumor mice. The tumorinhibition rate of ZIF-8/DOX@ZrO2@IL nanocomposites group was obviously higher than other treated groups. We solved the toxicity problem of ZIF-8 nanoparticles successfully and eliminated barrier of further research and even clinical application. The ZIF-8/DOX@ZrO2@IL theranostic nanocarrier is promising for chemo-microwave thermal tumor synergistic therapy. 2. EXPERIMENTAL SECTION

2.1. Materials Zirconium (IV) propoxide was supplied by Tokyo Chemical Industry Co., Ltd. 2Methylimidazolate (2MI) was supplied by Shanghai Mackin Biochemical Co., Ltd. Zinc nitrate (Zn (NO3)2·6H2O) was purchased from Tianjin Chemical Reagent 3 Plant. 1-Butyl-3methylimidazolium hexafluorophosphate, a kind of ionic liquid (IL), was supplied from Shanghai Chengjie Chemical Co., Ltd. Ethanol, NaCl and 1, 4-dioxane were obtained from Beijing Chemical


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Works. Doxorubicin (DOX) was supplied by Beijing Huafeng Chemical Reagent Co., Ltd. Ammonia (NH3·H2O) and methanol were provided by Beijing Tongguang Fine Chemicals Co., Ltd. All of the materials used in this work were used as original state and have not been further processed. 2.2. Preparation of ZIF-8/DOX nanoparticles ZIF-8/DOX nanoparticles were prepared by a one-pot method which was reported by Zheng et al16. In short, 200 mg of Zn (NO3)2·6H2O, 30 mg of DOX and 400 mg of 2MI were dispersed into 1 mL, 5 mL and 1 mL of methanol, respectively. Then, Zn (NO3)2·6H2O solution was mixed with DOX solution. The mixture was stirred for 10 min, 2MI solution was dropwise added into above mixture. The reaction mixture was stirred for 6 h. The ZIF-8/DOX nanoparticles were obtained by centrifugation, then washing 3 times with ethanol. The supernatant was collected to quantify the drug loading amount by ultraviolet-visible spectrophotometer (UV-Vis) (V-570, JASCO, Japan). By this means, the ZIF-8/DOX nanoparticles were obtained successfully. 2.3. Preparation of ZIF-8/DOX@ZrO2 nanocomposites 60 mg of ZIF-8/DOX nanoparticles were dispersed into 80 mL of mixed solvent, which composed of acetonitrile and ethanol (v:v = 1:3). 500 μL of NH3·H2O was then added for adjusting the solution to weakly alkaline conditions. 200 μL of zirconium (IV) protoxide solution was added to 20 mL of solvent. Afterwards, zirconium protoxide solution was quickly dumped in the ZIF8/DOX nanoparticles solution. The reaction was sustained magnetic stirring at room temperature for more than 6 h ensuring ZrO2 shell was successfully coated on the surface of ZIF-8/DOX nanoparticles. The amaranthine product was obtained by centrifugal separation, then washing 3 times with ethanol. After that, the ZIF-8/DOX@ZrO2 nanocomposites were obtained.


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2.4. Preparation of ZIF-8/DOX@ZrO2@IL nanocomposites IL was loaded into pore of ZIF-8/DOX@ZrO2 nanocomposites through a physical process. Firstly, 40 mg of ZIF-8/DOX@ZrO2 nanocomposites were dispersed in 6 mL disodium hydrogen phosphate solution. Then, 1 ml IL was added into above system, and reaction system was stirred for 8 h at room temperature. In this case, IL was loaded into pore of the ZIF-8/DOX@ZrO2 nanocomposites. Then, the products were purified via centrifugation (8500 rmp, 5 min) and washed with deionized water 3 times. 2.5. Toxicity tests in vitro The cytotoxicity of ZIF-8 nanoparticles and ZIF-8@ZrO2 nanocomposites was evaluated through cell activity by a methyl thiazolyl tetrazolium (MTT) test. ZIF-8 nanoparticles and ZIF8@ZrO2 nanocomposites with sequential concentrations (6.25, 12.5, 25, 50, 100 and 150 μg/mL) were cultured with L929 cells for 24 h. Then, 0.5 mg/mL MTT solution was added into cells solution to culture for 4 h. Afterward, the MTT solution was poured out and 150 mL of dimethyl sulfoxide (DMSO) was added to measure the absorbance at 490 nm through a scanning multiwell spectrometer. The cells without any treatment in nutrient solution were set as a control group. Six parallel replicas were executed for per cell experiment. 2.6. Toxicity tests in vivo The toxicities of ZIF-8@ZrO2 nanocomposites and ZIF-8 nanoparticles were also evaluated according to the survival rate of mice by injecting different dosages (0, 10, 20, 25, 30, 40, 50, 75, 100 mg/kg) of material into mice. The mice were divided randomly and 5 mice in each group. ZIF8 nanoparticles and ZIF-8@ZrO2 nanocomposites were dispersed into DMEM and getting a series


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of suitable concentrations. The survival rate of mice was calculated after injecting ZIF-8 nanoparticles and ZIF-8@ZrO2 nanocomposites with different concentrations via tail vein. 2.7. Drug release of ZIF-8/DOX@ZrO2@IL The drug release experiment was performed in PBS with different pH value (pH=7.4, 6.5, 5.5 and 4.5), 3 identical replicates per group. 9 mg ZIF-8/DOX@ZrO2@IL nanocomposites were added into each polythene pipe, then 3 mL PBS was added into per pipe at different pH, respectively. Those pipes were placed in a water bath oscillation box with stationary temperature at 37 °C. The liquid supernatant in per pipe was collected by the centrifugal separation at a regular interval and measured via a ultra-violet and visible spectrophotometer to analyze the content of free DOX in supernatant. Because DOX has an absorption peak around 483 nm, and the intensity of the absorption peak is proportional to the concentration of DOX within a small concentration range. The concentration of DOX was calculated basing on a protocol manual. Then, 3 mL PBS at different pH was added into corresponding pipes and dispersing ZIF-8/DOX@ZrO2@IL nanocomposites again to continue this drug release experiment. The time interval was set from 1 h to 12 h. 2.8. Anti-tumor evaluation in vitro Four groups cell experiment were executed to evaluate the inhibiting effect of tumor cells growth. H22 cells were divided into 6-well plates and 2 mL of cells for per well is precultured for 6 h, then the per well was supplemented with 2 mL fresh medium which involved free DOX, ZIF8@ZrO2@IL nanocomposites, ZIF-8/DOX@ZrO2@IL nanocomposites and blank medium, respectively. And the concentrations of material in the ZIF-8@ZrO2@IL and ZIF8/DOX@ZrO2@IL nanocomposites groups were 25 μg/mL. The content of DOX in the free DOX


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group was equal with the ZIF-8/DOX@ZrO2@IL nanocomposites group. Then they were cultured together for 24 h before irradiated for 0, 3, 5, 7 min by microwave. The power of microwave was 0.9 W. After irradiation, the suspension liquid of cells was cultured at 37 °C for 24 h. The cellular activity was obtained via MTT test, which was used as an evidence to assess anti-tumor effects. 2.9. Tumor inhibition evaluation of ZIF-8/DOX@ZrO2@IL nanocomposites in vivo H22 tumor mice (24 ± 2 g) were used to assess the efficiency of inhibiting tumor growth of ZIF8/DOX@ZrO2@IL nanocomposites. The mice with tumor were divided into 6 groups randomly (3 mice per group and the tumor volume was about 150 ± 20 mm3). Those mice were treated with (1) control, (2) free DOX, (3) MW, (4) ZIF-8/DOX@ZrO2@IL, (5) ZIF-8@ZrO2@IL+MW, (6) ZIF-8/DOX@ZrO2@IL+MW (the tail vein injection dose of DOX, ZIF-8@ZrO2@IL, ZIF8/DOX@ZrO2@IL were 15, 50, 50 mg/kg, respectively). The mice in (3), (5) and (6) groups were then irradiated with microwave in tumor sites only once after 6 h of vein injection, the power and time were 1.2 W and 5 min. While the mice in groups (1), (2) and (4) were without irradiation. The formula of (a2 × b) was used to calculate tumor volume, a and b were represented the short and long diameter for a tumor respectively, which was gauged via a vernier scale. 2.10. Histology study The tissue toxicity of materials with different concentration was evaluated by the major organs kidney, lung, spleen, liver, and heart after 14 days of treatments. The formalin solution (4%) was used to fix the obtained organs. Then, those organs were embedded in paraffin blocks and cut into thin paraffin section (the thickness was approximately 5 μm). Finally, those paraffin sections were stained with hematoxylin and eosin (H&E) and inspected by inverted fluorescence microscope (Olympus, X71, Japan) to determine whether there was any tissue damage.


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2.11. System acute toxicity experiment The acute toxicity experiment was performed using BALB/c mice in vivo. Three groups were divided (50 mg/kg, 25 mg/kg and blank group), and then those mice were injected with ZIF8@ZrO2@IL nanocomposites through tail vein injection. The body weight of mice was weighed for 14 consecutive days. The blood of mice was extracted to test blood biochemical analysis and blood routine analysis. 2.12. CT imaging To evaluate the effect of CT imaging in vivo and in vitro, ZIF-8/DOX@ZrO2@IL nanocomposites were dispersed into DMEM and obtained a series of concentrations (0, 3, 5, 10, 20, and 40 mg/ml). CT imaging was performed by a computerized tomography number (CTN). H22 tumor mice were used to perform CT imaging in vivo, the intravenous injection dose of ZIF8/DOX@ZrO2@IL nanocomposites was 50 mg/kg. The time of CT imaging was selected at 0, 3, 6, 9, and 24 h, respectively after tail vein injection. 3. RESULTS AND DISCUSSION Degradation of ZIF-8 nanoparticles will produce large amounts of zinc ions. Zinc ions have obvious biological toxicity22. The oral lethal dose 50 (LD50) of Zn (Zn from zinc chloride) is only 0.35 g/kg25. The high toxicity of ZIF-8 nanoparticles severely limited its further research and clinical application. So, it has become a burning issue to improve biocompatibility and reduce the toxicity of ZIF-8 nanoparticles. In this work, we solved the toxicity issue of ZIF-8 nanoparticles successfully by ZrO2 coating. Then ZIF-8 nanoparticles is used for a promising drug delivery system for chemo-microwave thermal tumor synergistic therapy.


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The preparation procedures were illustrated in Scheme 1. Firstly, ZIF-8/DOX nanoparticles were synthesized by a simple one-pot method. Then, ZIF-8/DOX nanoparticles were coated by ZrO2 to reduce toxicity and improve biocompatibility with an extra property of enhanced CT imaging efficiency. The ZIF/DOX@ZrO2 nanocomposites were loaded by IL to realize microwave thermal therapy. The as-prepared products were defined as ZIF-8/DOX@ZrO2@IL nanocomposites.

Scheme 1. Schematic illustration of preparation process for ZIF-8/DOX@ZrO2@IL nanocomposites: firstly, ZIF-8/DOX nanoparticles were prepared by a simple one-pot method; then, ZIF-8/DOX nanoparticles were covered by ZrO2; finally, IL was loaded into the pore of the ZIF-8/DOX@ZrO2 nanocomposites. And schematic representation: ZIF-8/DOX@ZrO2@IL nanocomposites as an excellent agents for chemo-microwave thermal tumor synergistic therapy.

3.1 Preparation and characterization


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ZIF-8/DOX nanoparticles and ZIF-8/DOX@ZrO2 nanocomposites were synthesized on the basis of the preparation procedure which was introduced in experimental section. Scanning electron microscope (SEM, Model S-4800, Hitachi), transmission electron microscope (TEM, HT7700, Hitachi) and high-resolution transmission electron microscope (HR-STEM, 2100f, JEOL) were selected to characterize their structure, morphology, and elemental composition. ZIF8/DOX nanoparticles had a three-dimensional dodecahedral structure with distinct edges and corners (Figure 1a-b). ZIF-8/DOX@ZrO2 nanocomposites showed approximately spherical with a distinct core-shell structure. The ZIF-8/DOX nanoparticle core still maintained its original appearance (Figure 1c-d), which indicated that ZIF-8/DOX@ZrO2 nanocomposites were successfully synthesized. Dark field high-resolution image (Figure 1f) and elemental mapping (Figure 1g-j) of ZIF-8/DOX@ZrO2 nanocomposites were executed to verify the structure of coreshell. The element of O was symmetrical distributed throughout whole nanocomposites, while Zr was in shell and Zn and N were in core, respectively. In addition, TEM energy dispersive spectrometer (EDS) results also gave the details about structural and compositional, which indicated ZrO2 has been coated onto the surface of ZIF-8 nanoparticles and constituting the structure of core-shell successfully (Figure 1k). The results of TEM and SEM indicated that diameters of ZIF-8/DOX@ZrO2 nanocomposites and ZIF-8/DOX nanoparticles were around 280 nm and 240 nm, respectively (Figure 2). The size distribution of ZIF-8/DOX nanoparticles, ZIF8/DOX@ZrO2 nanocomposites and ZIF-8/DOX@ZrO2@IL nanocomposites was showed in Figure S1, basing on Dynamic Light Scattering (DLS) (MAN0486-01, Malvern, England). The average hydrodynamic sizes of ZIF-8/DOX nanoparticles, ZIF-8/DOX@ZrO2 nanocomposites and ZIF-8/DOX@ZrO2@IL nanocomposites were 251.9, 292.6, and 306.2 nm, respectively. Figure 3a was the XRD patterns of ZIF-8/DOX@ZrO2 nanocomposites, ZIF-8@ZrO2


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nanocomposites, ZIF-8/DOX nanoparticles, and ZIF-8 nanoparticles. It indicated that all nanocomposites were high crystalline, which demonstrated the formation of ZIF-8 according to the data of crystal structure21, 26. The XRD pattern of ZIF-8/DOX@ZrO2 nanocomposites, ZIF8@ZrO2 nanocomposites, ZIF-8/DOX nanoparticles indicated the integrity of crystal structural for ZIF-8 nanoparticles was unvaried even though coating ZrO2 or loading drugs. On the contrary, the characteristic peak intensity of ZIF-8 nanoparticles in ZIF-8@ZrO2 nanocomposites, and ZIF8/DOX@ZrO2 nanocomposites decreased because of coating ZrO2. The result of XRD further confirms that ZrO2 has been coated onto ZIF-8 nanoparticles successfully. Figure 3b indicated that the content of ZIF-8 nanoparticles in ZIF-8@ZrO2 nanocomposites was 86.7%. In addition, there were not characteristic peak and diffraction spots about ZrO2 in Figure S2 and Figure S3, which indicated pure ZrO2 was an amorphous compound. The porosities and pore-size distributions of ZIF-8 nanoparticles, ZIF/DOX nanoparticles, and ZIF-8/DOX@ZrO2 nanocomposites were investigated by Nitrogen adsorption-desorption measurements. As shown in Figure 3c, ZIF-8/DOX@ZrO2 nanocomposites, ZIF-8/DOX nanoparticles, and ZIF-8 nanoparticles all exhibited a type I isotherm, the N2 uptake was obvious increases when the relative pressure was very low (0.05). There results indicated the biocompatibility of ZIF-8@ZrO2 nanocomposites was excellent enough for in vivo tumor treatment.


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Figure 4. (a) Cytotoxicity of ZIF-8 nanoparticles and ZIF-8@ZrO2 nanocomposites incubating with L929 for 24 h. (b) Survival rate figure of ZIF-8 nanoparticles and ZIF-8@ZrO2 nanocomposites at a series of doses. (c) Weight change figure of mice in acute toxicity test after injecting different doses (0, 25, 50 mg/kg) of ZIF8@ZrO2 nanocomposites. (d) The H&E staining images of ZIF-8@ZrO2 nanocomposites (50 mg/kg) for main organs (spleen, lung, liver, kidney, and heart). The scale bar is 50 μm.


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Figure 5. The H&E staining images of mice for the major organs in acute toxicity experiment. The scale bar is 50 μm.


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Figure 6. The blood routine test result of ZIF-8@ZrO2 nanocomposites (0, 25, 50 mg/ kg) (including HGB, PLT, MPV, HCT, MCV, MCHC, MCH, WBC, and RBC).

Figure 7. The blood biochemical analysis result of ZIF-8@ZrO2 nanocomposites (including CREA, UREA, AST, and ALT).

3.3. Microwave heating properties of ZIF-8/DOX@ZrO2@IL in vitro Due to the sensitivity to microwave of IL in a closed space23, 24, 27, it was loaded into the pores of ZIF-8/DOX@ZrO2 nanocomposites for microwave response. 1 mL of ZIF-8/DOX@ZrO2@IL nanocomposites at different concentrations (3, 6, 9 mg/mL saline solution) was irradiated by a 450 MHz microwave at 1.8 W for 5 min to evaluate the heating effect of ZIF-8/DOX@ZrO2@IL


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nanocomposites in vitro. The control group is saline solution. Figure 8a showed that ZIF8/DOX@ZrO2@IL nanocomposites possessed of different heating effects due to different concentrations of ZIF-8/DOX@ZrO2@IL nanocomposites in per group. With the concentration of ZIF-8/DOX@ZrO2@IL nanocomposites increased, the microwave heating effect enhanced gradually. The temperature of saline raised from 26.5 to 46.5 °C after 5 min microwave irradiation. The value change of temperature was 20.0 °C. The value changes in temperature of ZIF8/DOX@ZrO2@IL nanocomposites at concentrations of 3, 6, and 9 mg/mL were 27.5, 34.4, and 37.2 °C, respectively in the same conditions. The temperature was 7.5, 14.4, and 17.2 °C higher than normal saline solution, respectively. The change values in temperature of ZIF8/DOX@ZrO2@IL nanocomposites were indicated in Figure 8b. Forward-looking infrared (FLIR) imaging instrument was used to record heating process and temperature change was recorded every minute (Figure 8c). The results indicated that ZIF-8/DOX@ZrO2@IL nanocomposites had an excellent microwave heating effect in simulated body fluid, which could be used for microwave thermal tumor therapy in vivo.


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Figure 8. (a) Temperature rising curves of ZIF-8/DOX@ZrO2@IL nanocomposites in vitro at a series of concentrations (0, 3, 6, 9 mg/mL and saline). (b) The change values of temperature at a series of concentrations based on (a). (c) FLIR images of a series of concentrations based on (a). (d) pH sensitive release behaviors of ZIF-8/DOX@ZrO2@IL nanocomposites.

3.4. Drug release in vitro DOX as a widely used chemotherapy drug was loaded into the pores of ZIF-8 nanoparticles by means of a one-pot method. The concentration of DOX was measured by UV-vis spectrophotometer at 483 nm in supernatant after centrifugalization. The cumulative release amount of DOX over time was calculated according to a standard calibration curve. The drugloading rate of DOX was 28.75% (w/w). ZIF-8/DOX@ZrO2@IL nanocomposites showed pH


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responsive release behaviors. The amount of DOX release had a significant difference at different pH (Figure 8d and Figure S5). When the PBS with pH=4.5, 5.5, 6.5 and 7.4, the cumulative release amount of DOX was 29.3%, 22.5%, 19.1% and 16.2%, respectively in 72 h under 37 °C shaking. The results showed ZIF-8/DOX@ZrO2@IL nanocomposites had excellent pH responsive release behaviors. The cumulative release amount of DOX was significantly increased with acidity increase.

3.5. Anti-tumor effects in vitro H22 cells were used to measure the cytotoxicity and the inhibitory effect of ZIF8/DOX@ZrO2@IL

nanocomposites

in

vitro.

The

cytotoxicity

of

ZIF-8@ZrO2@IL

nanocomposites was evaluated by MTT assay (Figure 9a). The cell activity was 74.9% when the concentration of ZIF-8@ZrO2@IL nanocomposites reached 150 μg/mL, which indicated that the relatively low toxicity of ZIF-8@ZrO2@IL nanocomposites. In order to assess the inhibitory effect of ZIF-8/DOX@ZrO2@IL nanocomposites on tumors, H22 cells were treated with control, free DOX,

ZIF-8@ZrO2@IL

nanocomposites,

and

ZIF-8/DOX@ZrO2@IL

nanocomposites,

respectively. As shown in Figure 9b, cellular activity was gradually decreases as microwave time increases. And the cell survival rates of each group was 41.4% (control), 23.7% (free DOX), 28.8% (ZIF-8@ZrO2@IL), and 14.6% (ZIF-8/DOX@ZrO2@IL), respectively after 7 min under microwave irradiation. The results indicated that ZIF-8/DOX@ZrO2@IL nanocomposites could guide drug controlled release and induce localized hyperthermia under microwave irradiation for effective chemo-microwave thermal tumor synergistic therapy.


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Figure 9. (a) Cytotoxicity of ZIF-8@ZrO2@IL nanocomposites incubating with H22 for 24 h. (b) H22 cells were incubated with free DOX, ZIF-8@ZrO2@IL, and ZIF-8/DOX@ZrO2@IL, then microwave irradiation for 0, 3, 5, 7 min. The cellular activity were evaluated by MTT method. ***p

High Biocompatible ZIF-8 Coated by ZrO2 for Chemo-microwave

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