Doxorubicin-Loaded MnO2@Zeolitic Imidazolate Framework-8 Nanoparticles as a Chemophotothermal System for Lung Cancer Therapy.

Doxorubicin-loaded MnO2@zeolitic imidazolate framework-8 (DOX/MnO2@ZIF-8) nanoparticles, a smart multifunctional therapeutic platform, were prepared for the treatment of lung cancer. The morphology, structure, and redox and photothermal properties of MnO2@ZIF-8 were characterized by the corresponding methods. The anticancer drug DOX released from the DOX/MnO2@ZIF-8 nanoparticles was measured. The cell viability of Lewis lung cancer (LLC) cells treated with MnO2@ZIF-8 or DOX/MnO2@ZIF-8 nanoparticles was determined using the cell counting kit-8 (CCK-8) method. The cellular uptake of DOX/MnO2@ZIF-8 nanoparticles into LLC cells was observed using a confocal laser scanning microscope. TUNEL staining was performed to evaluate the in vivo therapeutic efficacy of DOX/MnO2@ZIF-8 nanoparticles. The results showed that the as-prepared MnO2@ZIF-8 nanoparticles had an average particle size of 155.59 ± 13.61 nm and the DOX loading efficiency was 12 wt %. MnO2@ZIF-8 could react with H2O2 to generate O2 and showed a great photothermal conversion effect both in vitro and in vivo. Up to 82% of total DOX could be released from DOX/MnO2@ZIF-8 nanoparticles at pH = 5.0. The CCK-8 assay showed that MnO2@ZIF-8 had low cytotoxicity to LLC cells, while DOX/MnO2@ZIF-8 can significantly reduce the cell viability. DOX/MnO2@ZIF-8 can be accumulated in LLC cells over time. Compared with PBS and DOX/MnO2@ZIF-8 groups, the mice in the DOX/MnO2@ZIF-8 + NIR group had the most apoptotic cells and significantly reduced tumor volume. In conclusion, these findings suggest that the as-prepared MnO2@ZIF-8 nanoparticles with synergetic therapeutic effects by photothermal therapy and improved tumor microenvironment and as a pH-responsive nanocarrier for delivering the nonspecific anticancer drug DOX might be applied in the treatment of lung cancer.

Introduction

Lung cancer is the second most common cancer and is the leading cause of cancer-related deaths in both men and women.[1−3] Though the survival rates for most cancers have increased steadily with progress in therapeutic strategies, the advance is very slow for lung cancer.[1,2] A total of 57% of lung cancer patients were diagnosed at a distant stage with a 5-year survival rate of only 5%, and 22% cases were diagnosed at a regional stage with a 5-year survival rate of about 30%.[1−3] Thus, it is urgent to develop more novel therapies for the treatment of lung cancer.

It is well known that the therapeutic potential of monotherapy, such as chemotherapy, surgery, and radiotherapy, is usually undermined owing to the heterogeneity, diversity, and complexity of cancers.[4] Thereby, the current multimodal synergistic therapy is considered to enhance the therapeutic efficacy. Manganese dioxide (MnO2)-based nanoplatforms like the hMnO2/Ce6-DOX nanoplatform of combined chemo-photodynamic therapy,[5] MnO2/DVDMs of combined photodynamic-photothermal therapy,[6] and mSiO2-DOX@MnO2 nanoagent of combined chemo-chemodynamic therapy[7] have been widely used in the synergistic therapy for cancers.[8]

Hypoxia and a slightly acidic microenvironment of tumor is a typical character of solid cancers such as lung cancer, which contributes to cancer progression and metastasis and diminishes the efficacy of chemotherapies.[9−11] It has been reported that hypoxia manipulates the lung cancer development, metastasis, invasion, and chemoresistance and is associated with poor prognosis.[12−14] Thereby, hypoxia is suggested as a potential target for the treatment of lung cancer.[11,12]

Metal–organic frameworks are widely applied in drug delivery due to their unique properties of easy functionalization, tunable pore size, and flexible composition.[15] Among these metal–organic frameworks, owing to the pH-sensitive dissolution properties of zeolitic imidazolate framework-8 (ZIF-8),[15,16] it was widely used in the pH-controlled drug release systems, such as UCNP@ZIF-8 for anticancer drug 5-fluorouracil delivery[17] and polyacrylic acid@zeolitic imidazolate framework-8 for doxorubicin (DOX) delivery.[18]

The synergetic therapeutic effects for cancers have attracted great research attention. Here, we fabricated intelligent MnO2@ZIF-8 nanoparticles with synergetic therapeutic effects of PDT/PTT as well as a pH-responsive nanocarrier for delivering the nonspecific anticancer drug DOX.

Results and Discussion

Characterization and Performance Analysis of MnO2@ZIF-8 and DOX/MnO2@ZIF-8 Nanoparticles

MnO2@ZIF-8 nanoparticles were successfully fabricated with 34.47% loading of MnO2 (Figure S1) and the black dots in the MnO2@ZIF-8 particle are MnO2 (Figure A). MnO2@ZIF-8 nanoparticles showed a typical I-type isotherm, which suggested their microporous characteristic (Figure B). The infrared absorption spectrogram (400–4000 nm) of MnO2@ZIF-8 is shown in Figure C. MnO2@ZIF-8 nanoparticles had an average particle size of 155.59 ± 13.61 nm (Figure D). After loading the anticancer drug DOX, the resulting DOX/MnO2@ZIF-8 nanoparticles showed an increased particle size to 206.22 ± 21.00 nm with a loading efficiency of 12 wt %.

Figure 1

Characterization of MnO2@ZIF-8 and DOX/MnO2@ZIF-8 nanoparticles. (A) TEM image, (B) N2 adsorption–desorption isotherms, and (C) Fourier transform infrared spectrum of MnO2@ZIF-8. (D) Particle size of MnO2@ZIF-8 and DOX/MnO2@ZIF-8.

It is reported that MnO2 can react with H2O2 to generate O2.[19,20] To test whether MnO2@ZIF-8 nanoparticles could react with H2O2 to produce O2, different concentrations of MnO2@ZIF-8 (10 and 50 μg/mL) nanoparticles were incubated with H2O2 and 3,3′,5,5′-tetramethylbenzidine. The color changes are shown in Figure A. The reaction was observed to be higher for 50 μg/mL MnO2@ZIF-8 nanoparticles compared with that for 10 μg/mL MnO2@ZIF-8. The ultraviolet–visible (UV–vis) spectra revealed that MnO2@ZIF-8 showed high reactivity with H2O2 to generate O2 (Figure B). While injected into the tumor site, they would regulate oxygen levels and the local pH to improve the hypoxia microenvironment in cancers.

Figure 2

Redox performance of MnO2@ZIF-8 nanoparticles. (A) Color change of different concentrations of MnO2@ZIF-8 with 3,3′,5,5′-tetramethylbenzidine and H2O2. (B) UV–vis spectra of MnO2@ZIF-8 with 3,3′,5,5′-tetramethylbenzidine and H2O2.

In Vitro and In Vivo Photothermal Assays of MnO2@ZIF-8 Nanoparticles

MnO2 nanoparticles have been reported to exhibit photothermal conversion capabilities. Here, after 2.5 min of near-infrared (NIR) laser irradiation (1 W/cm2), the temperature of MnO2@ZIF-8 solutions (100 μg/mL) increased to 60.8 °C, (Figure A,C). Moreover, the photothermal conversion effect of MnO2@ZIF-8 was confirmed in vivo (Figure B,D). The local temperature of the tumor site shot up under the NIR laser irradiation. These results indicate the great photothermal conversion effect of MnO2@ZIF-8 nanoparticles.

Figure 3

Photothermal performance of MnO2@ZIF-8 in vitro and in vivo. In vitro (A) and in vivo (B) photothermal images under NIR irradiation. (C) Photothermal heating curve of MnO2@ZIF-8 solutions. (D) Temperature curve in the mouse tissues.

pH-Responsive Release of DOX from DOX/MnO2@ZIF-8 Nanoparticles

ZIF-8 was widely used in the pH-controlled drug release system due to its pH-sensitive dissolution properties.[15,16] Whether DOX was pH-responsively released from DOX/MnO2@ZIF-8 nanoparticles was determined. As shown in Figure , compared with 15% of total DOX released from DOX/MnO2@ZIF-8 nanoparticles at pH = 7.4, up to 53% DOX released from DOX/MnO2@ZIF-8 nanoparticles at pH = 6.0 and 82% released from DOX/MnO2@ZIF-8 nanoparticles at pH = 5.0. It is well known that the pH value in the tumor tissues (pH = 5.5–6.0) is lower than that in the normal tissues (pH = 7.4).[21] Thus, the anticancer DOX drug loaded in the MnO2@ZIF-8 nanoparticles could be pH-responsively released in the areas of tumor tissues.

Figure 4

Cumulative DOX release from DOX/MnO2@ZIF-8 nanoparticles.

Cytotoxicity of DOX/MnO2@ZIF-8 Nanoparticles Against Lung Cancer Cells

The cytotoxicity of the nanoparticles must be first considered while applied in the medicine.[15] To test the cytotoxicity of MnO2@ZIF-8 nanoparticles and the anticancer efficacy of DOX/MnO2@ZIF-8 nanoparticles containing 12% DOX, the cell viabilities of Lewis lung cancer (LLC) cells incubated with different concentrations of MnO2@ZIF-8 or DOX/MnO2@ZIF-8 nanoparticles were measured. Over 80% of LLC cells survived after 100 μg/mL MnO2@ZIF-8 treatment (Figure ), suggesting low cytotoxicity of MnO2@ZIF-8. Notably, the cell viability was significantly reduced while treated with 60 μg/mL DOX/MnO2@ZIF-8. The cells treated with 100 μg/mL DOX/MnO2@ZIF-8 had the lowest cell viability. Therefore, DOX/MnO2@ZIF-8 nanoparticles might be effective for the treatment of lung cancer.

Figure 5

Cell viability of MRC-5 normal cells and LLC cells incubated with different concentrations of nanoparticles. p* <0.05, p <0.001, and p*** <0.0001.

Cellular Uptake of DOX/MnO2@ZIF-8 Nanoparticles

The cellular uptake of DOX/MnO2@ZIF-8 was observed using a confocal laser scanning microscope (CLSM). As shown in Figure , there was remarkably increased red fluorescence of DOX in the LLC cells after incubating with DOX/MnO2@ZIF-8 for 3 h. The intracellular distribution of DOX increased over time. These results indicate that DOX/MnO2@ZIF-8 gradually accumulated in the LLC cells.

Figure 6

Confocal laser scanning microscope images of LLC cells incubated with MnO2@ZIF-8 for different times.

In Vivo Therapeutic Efficacy

To test the therapeutic efficacy of DOX/MnO2@ZIF-8 under NIR laser irradiation, a terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay was performed to show apoptotic cells in the sections from tumors. Compared with those injected with PBS, the mice injected with DOX/MnO2@ZIF-8 presented remarkably more apoptotic cells (Figure A). The mice injected with DOX/MnO2@ZIF-8 plus NIR laser irradiation had the most apoptotic cells. Moreover, the tumor volume of the mice in the DOX/MnO2@ZIF-8 + NIR group significantly reduced compared with PBS and DOX/MnO2@ZIF-8 groups (Figure B). Additionally, no significantly adverse reactions were observed in multiple organs of mice in the DOX/MnO2@ZIF-8 + NIR group (Figure S2). These results indicate the dramatic therapeutic effect of DOX/MnO2@ZIF-8 + NIR, which might be caused by the combination of the redox and photothermal properties of MnO2 and the loading and pH-responsive performance of ZIF-8.

Figure 7

Assessment of therapeutic efficacy in vivo. (A) TUNEL staining of cancer tissues harvested from mice with different treatments. (B) Comparison of tumor growth in mice with different treatments. PBS: the mice were subjected to tail vein injection of PBS; DOX/MnO2@ZIF-8: the mice were subjected to tail vein injection of DOX/MnO2@ZIF-8 nanoparticles; and DOX/MnO2@ZIF-8 + NIR: the mice were subjected to tail vein injection of DOX/MnO2@ZIF-8 nanoparticles and then treated with NIR irradiation for 10 min 6 h after injection. p** <0.001 and p*** <0.0001. Scale bar: 100 μm.

Conclusions

In conclusion, DOX/MnO2@ZIF-8 nanoparticles were prepared and employed as an anticancer system. Because of the presence of MnO2, the system exhibited good photothermal and photodynamic performance and an improved hypoxia cancer microenvironment. Due to the high storage and pH-responsive dissolution properties of ZIF-8, the loading efficiency of DOX was 12 wt % and 80% of the total amount of DOX was released from DOX/MnO2@ZIF-8 at pH 5.0. These findings suggest that the as-prepared MnO2@ZIF-8 nanoparticles with synergetic therapeutic effects of photothermal and photodynamic properties as well as a pH-responsive nanocarrier for delivering the nonspecific anticancer drug DOX might be applied in the treatment of lung cancer.

Material And Methods

Synthesis of MnO2 and MnO2@ZIF-8

The synthesis of MnO2 and MnO2@ZIF-8 nanoparticles was performed in accordance with the reported methods with some modifications.[22,23] MnO2 nanoparticles were synthesized by mixing 9 mL of KMnO4 solution (3.5 mg/mL) with 1 mL of poly(allylamine hydrochloride) (37.4 mg/mL) at room temperature. After full reaction for 15 min, MnO2 nanoparticles were obtained and then washed several times using DDH2O.

To fabricate the MnO2@ZIF-8 nanoparticles, 15 mL of methanol solution of Zn(NO3)2•6H2O (223.05 mg) and MnO2 nanoparticles (32.55 mg) were added into 7.5 mL of methanol solution of 2-methylimidazole (492.6 mg) and polyvinylpyrrolidone (281.4 mg). The mixture was stirred for 1.5 h at room temperature. The prepared MnO2@ZIF-8 precipitate was then washed with methanol solution and stored for further experiments.

DOX/MnO2@ZIF-8 was prepared by adding the above resultant MnO2@ZIF-8 into the DOX solution (40 mg/mL) followed by 24 h of stirring.

Experimental Animals and Cells

The 4–6-week-old BALB/c nude mice (18–22 g) were purchased from the Animal Experiment Center of Chongqing Medical University. All animal experiments were approved by the Animal Ethics Committee of People’s Hospital of Deyang City and carried out in accordance with the corresponding guidelines.

Mouse LLC cells were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). LLC cells were cultured in a Roswell Park Memorial Institute-1640 medium supplemented with 10% fetal bovine serum (Invitrogen) at 37 °C at 5% CO2.

Characterization of MnO2 and MnO2@ZIF-8

The morphology of MnO2@ZIF-8 nanoparticles was characterized by scanning electron microscopy using a Hitachi FESEM S-4800 instrument. Their Brunauer–Emmett–Teller surface area was measured using N2 adsorption–desorption isotherms with a quartz tube (Quantachrome 2200e). The UV–vis spectra were recorded using a spectrometer. The particle size distribution of MnO2@ZIF-8 and DOX/MnO2@ZIF-8 was determined by dynamic light scattering (Malvern Zetasizer Nano ZS). The redox properties of MnO2@ZIF-8 nanoparticles were determined by incubating with H2O2 and 3,3′,5,5′-tetramethylbenzidine.

The photothermal performance of MnO2@ZIF-8 was evaluated in vitro and in vivo. An Eppendorf tube containing 1 mL of MnO2@ZIF-8 solution (100 μg/mL) was irradiated using an 808 nm NIR laser (1 W/cm2). The mice subcutaneously injected with 100 μL of MnO2@ZIF-8 solution (100 μg/mL) were also irradiated with an 808 nm NIR laser (1 W/cm2). The photothermal images were taken using an infrared camera.

Drug Loading and Release Assay

DOX was dissolved in an ethanol solution to prepare the DOX solution. The anticancer drug DOX was loaded into MnO2@ZIF-8 nanoparticles by adding the nanoparticles into the DOX solution and then stirring for 24 h at room temperature. The release of DOX from DOX/MnO2@ZIF-8 was measured by adding DOX/MnO2@ZIF-8 into a phosphate buffer with different pH values (pH = 7.4, 6.0, and 5.0). A Lambda Bio40 UV–vis spectrometer was used to read the fluorescence intensity of the above mixture at intervals.

Cytotoxicity Assay

The cell viability of LLC cells incubated with different concentrations of MnO2@ZIF-8 or DOX/MnO2@ZIF-8 was measured using the cell counting kit-8 (CCK-8; CP002, Signalway Antibody, MD, USA) according to the manufacture’s instruction. First, 100 μL of cell suspension (3 × 104 cells/mL) was seeded into 96-well plates and incubated with different concentrations of DOX/MnO2@ZIF-8 at 37 °C for 48 h. Then, the CCK-8 reagent (10 μL) was added into each well and the resulting mixture was cultured at 37 °C for another 1 h. The absorbance of the mixture at 450 nm was recorded and the cell viability was calculated.

Cellular Uptake Assay

The cellular uptake of DOX/MnO2@ZIF-8 was observed using a CLSM. LLC cells were incubated with DOX/MnO2@ZIF-8 solution (100 μg/mL). After 3, 6, 12, and 24 h of incubation, the cells were rinsed with phosphate buffer and then treated with 4% paraformaldehyde solution followed by 0.01% Triton X-100. Subsequently, the cells were stained with DAPI and FITC-phalloidin for 12 min, respectively. After rinsing with phosphate buffer, the cells were observed under different wavelengths of laser (405 nm for nuclei, 488 nm for DOX, and 562 nm for F-actin) using a CLSM.

In vivo Anticancer Assay

LLC cells (5 × 106 cells per mouse) were subcutaneously injected into the mice. Then, after 14 days of breeding, the mice were randomly divided into three groups (n = 6 per group): PBS, DOX/MnO2@ZIF-8, and DOX/MnO2@ZIF-8 + NIR. The mice of the PBS group were subjected to tail vein injection of PBS, the mice of the DOX/MnO2@ZIF-8 group with DOX/MnO2@ZIF-8 nanoparticles, and the mice of the DOX/MnO2@ZIF-8 + NIR group with DOX/MnO2@ZIF-8 nanoparticles and then treated with 10 min NIR irradiation 6 h after injection. DOX (5 mg/kg) was used in the in vivo experiment. The tumor volume was measured every 2 days. The tumor volume was calculated using the following formula:where “S” and “L” are the short and long axes of the tumor nodule, respectively.

TUNEL Staining

The harvested tumor tissues were first fixed with 4% paraformaldehyde. Then, they were embedded in an optimal cutting temperature compound followed by cutting into 5 μm-thick sections. The sections were treated using Triton X-100 at room temperature for 10 min and then blocked with 5% bovine serum albumin. The apoptotic cells were measured with a TUNEL apoptosis assay kit (C1088, Beyotime Biotechnology, Shanghai, China) in accordance with the manufacturer’s instruction. DAPI (C1006, Beyotime Biotechnology) was selected for nuclear staining.

Statistical Analysis

All experiments were performed three times independently. The data were calculated as mean ± standard deviation. Statistical difference was analyzed using two-way ANOVA. P value < 0.05 was set as a significant difference.