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Dec 29, 2017 - The cellular uptake in HeLa cells of 3-MA encapsulated in ZIF-8 (3-. MA@ZIF-8 ..... drug-loaded ZIF-8 was covered by a little 3-MA via ...
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Research Article Cite This: ACS Appl. Mater. Interfaces 2018, 10, 2328−2337

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MOF Nanoparticles with Encapsulated Autophagy Inhibitor in Controlled Drug Delivery System for Antitumor Xuerui Chen,† Rongliang Tong,‡,§ Zheqi Shi,† Beng Yang,‡,§ Hua Liu,‡,§ Shiping Ding,∥ Xu Wang,⊥ Qunfang Lei,† Jian Wu,*,‡,§ and Wenjun Fang*,† †

Department of Chemistry, Zhejiang University, Hangzhou 310027, China Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, School of Medicine, and §The Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou 310003, China ∥ The National Education Base for Basic Medical Sciences, School of Medicine, Zhejiang University, Hangzhou 310058, China ⊥ Hangzhou Medical College, No. 481 Binwen Road, Hangzhou 310053, China

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ABSTRACT: High porosities, large surface areas, and tunable functionalities made metal−organic frameworks (MOFs) as effective carriers for drug delivery. One of the most promising MOFs is the zeolitic imidazolate framework (ZIF-8) crystal, an advanced functional material for smallmolecule delivery, due to its high loading ability and pH-sensitive degradation. As a novel carrier, ZIF-8 nanoparticles were used in this work to control the release of an autophagy inhibitor, 3-methyladenine (3MA), and prevent it from dissipating in a large quantity before reaching the target. The cellular uptake in HeLa cells of 3-MA encapsulated in ZIF-8 (3MA@ZIF-8 NPs) is facilitated through the nanoparticle internalization with reference to TEM observations and the quantitative analyses of zinc by ICPMS. The autophagy-related proteins and autophagy flux in HeLa cells treated with 3-MA@ZIF-8 NPs show that the autophagosome formation is significantly blocked, which reveals that the pH-sensitive dissociation increases the efficiency of autophagy inhibition at the equivalent concentration of 3-MA. In vivo experiments, when compared to free 3-MA, 3-MA@ZIF-8 NPs show a higher antitumor efficacy and repress the expression of autophagy-related markers, Beclin 1 and LC3. It follows that ZIF-8 is an efficient drug delivery vehicle in antitumor therapy, especially in inhibiting autophagy of cancer cells. KEYWORDS: drug delivery, autophagy, ZIF-8, 3-methyladenine, antitumor, metal−organic frameworks

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low cell membrane permeability.13,14 In many cases, high drug doses certainly increase the potential of side effects while offsetting the poor pharmacokinetics of these drugs.15 Nanomaterial drug delivery can overcome these limits by stabilizing the drug through encapsulation or surface attachment, promoting cellular internalization, targeting to a specific cell population, and controlling the drug to release at the designated target.16 One kind of promising MOF is the zeolitic imidazole framework (ZIF) which has favorable structures with large pore volume, inherent biodegradability, and huge surface area.17 When scaled down to the nanoscale level, ZIF is regarded as an ideal vehicle with loading abundant drugs and controlling to release small molecules, such as gases, fluorescent probes, and therapeutic agents.16,18 Among the ZIF family, ZIF-8 nanoparticles (ZIF-8 NPs) are regarded as the favorable delivery vehicle owing to the good characteristics that the nanoparticles

INTRODUCTION Metal−organic frameworks (MOFs) have well-regulated crystalline networks with unprecedented porosity, and their structures are assembled by coordinated metal ions and organic ligands.1−3 Various functional materials including metal nanoparticles, quantum dots, graphene, carbon nanotubes, and biomolecules have been integrated into MOFs to form the composite/hybrid materials.4−8 Two major synthetic strategies have been explored to inject functional organic molecules into MOFs. One is impregnation procedure that functional molecules were loaded into pores of MOFs via capillary forces, electrostatic interactions, and coordination reactions.9 Another is the in situ encapsulation by the introduction of functional organic molecules in the growth of MOFs to construct the molecules/MOFs composites.1,10 One of the most attractive aspects is to make MOFs as the delivery of drugs, which has potentials in improving the drug effectiveness by controlling drug to be released at the target location.11,12 Direct administration of therapeutic agents to patients suffers from the inherent limitations of the small molecule drugs including weak physiological stability, nonspecific targeting, and © 2017 American Chemical Society

Received: October 30, 2017 Accepted: December 29, 2017 Published: December 29, 2017 2328

DOI: 10.1021/acsami.7b16522 ACS Appl. Mater. Interfaces 2018, 10, 2328−2337

Research Article

ACS Applied Materials & Interfaces remain firm under general neutral conditions and undergo fast degradation in low-pH environments due to the effects of protonation, which release the drug in tumor whose pH is approximately between 5.0 and 6.5.19−21 This pH-sensitive degradation makes ZIF-8 NPs as a hopeful drug carrier, while the effects of ZIF-8 NPs on some special biological behaviors such as autophagy are rather absent.9 There are few investigations on the effectiveness of autophagy drugs loaded in ZIF-8 NPs and on the autophagy behavior in vivo with this novel hybrid material.22 Autophagy may be capable of protecting cancer cells from antitumor therapies by blocking apoptotic pathways, whereas other cancer cells are observed to undergo autophagic cell death following antitumor therapy.23−26 As one of the autophagy inhibitors, 3-methyladenine (3-MA) can significantly inhibit the class III PI3K (Vps34)/Beclin-1 complex and thus can interfere the formation of autophagosomes.27,28 Through inhibiting autophagic process, 3-MA can induce the apoptosis process and kill cancer cells.29,30 As a novel attempt of in vivo study on autophagy study with drug-loaded MOFs, the effects on autophagy inhibition and the antitumor efficiency of 3-MA encapsulated ZIF-8 NPs (3-MA@ ZIF-8 NPs) are investigated in this work. The size, stability, and drug loading capacity of synthesized 3-MA@ZIF-8 NPs are characterized by a transmission electron microscope (TEM), a scanning electron microscope (SEM), powder X-ray diffraction patterns (PXRD), BET, thermogravimetric analysis (TGA), and UV−vis. The morphological and biochemical markers of autophagy in HeLa cells are determined by TEM and confocal laser scanning microscopy (CLSM). Antitumor efficacy and toxicity are demonstrated by in vivo experiments and autophagic analyses of xenograft tumor.

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DLS (ZEN3600, Malvern) was used to detect the particle size. N2 adsorption isotherms at 77 K were applied to measure pore size distribution of ZIF nanoparticles through the nonlocal density functional theory. TEM (JEM-1200EX, JEOL) and SEM (S-4800, Hitachi) were exploited to visualize their morphology and shape. PXRD were recorded on a Bruker D8-focus Bragg−Brentano X-ray powder diffractometer equipped with a Cu sealed tube (λ = 1.541 78 Å) at room temperature. Inductively coupled plasma mass spectrometry (ICP-MS, Elan DRCII, PerkinElmer Sciex) was utilized to determine the zinc contents in vitro and in vivo experiments. FTIR spectra were recorded on a Shimadzuir 460 spectrometer in a KBr matrix. FTIR spectra were used to confirm the presence of 3-MA within the MOF matrix. TGA were carried out from 35 to 700 °C at the heating rate of 10 °C min−1 to check the thermal stability under a nitrogen atmosphere. Drug Loading Efficacy of 3-MA@ZIF-8 NPs. The amount of 3MA loaded was determined from the absorbance at 273 nm in the UV/vis, and the standard curve between absorbance and concentration was analyzed. 10 mg of 3-MA@ZIF-8 NPs was dissolved in 1 M HCl and diluted by ultrapure water. To evaluate the 3-MA loading efficiency, the concentration of 3-MA in the acid solution was determined from absorbance at 273 nm by UV/vis and obtained through standard curve. The 3-MA drug loading efficiency (DLE %) of ZIF-8 was calculated by the equation

DLE % =

weight of drug in NPs × 100% weight of NPs taken

Release of 3-MA from 3-MA@ZIF-8 NPs at Different pH Values. In a normal release system, 10 mg of 3-MA@ZIF-8 NPs was suspended in 20.0 mL of PBS (pH 7.4, 6.5, and 5.0) at 37 °C. The release system was then maintained at 37 °C under shaking (shaking frequency = 100 rpm). The release medium was sampled with 1 mL at each time, and UV/vis spectrophotometry was used to determine the percentage of released 3-MA; the sample was back to the original release system. The amount of 3-MA loaded in ZIF-8 NPs was determined from the UV−vis absorbance at 273 nm. The release percentages of 3-MA were calculated according to the formula

MATERIALS AND METHODS

Reagents. Zn(NO3)2·6H2O (CAS No. 10196-18-6), MeIM (CAS No. 693-98-1), methanol (CAS No. 67-56-1), 3-methyladenine (3MA) (CAS No. 5142-23-4), dimethyl sulfoxide (DMSO), trypsin/ EDTA solution (1.7 × 105 U L−1 of trypsin and 0.2 g L−1 of EDTA), phosphate buffered saline (PBS), fetal bovine serum (FBS), anti-βactin antibody, cacodylate, Dulbecco’s Modified Eagle Medium (DMEM), and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) were purchased from Sigma-Aldrich and used as received. The LC3-II primary antibodies were purchased from Cell Signaling Technologies. The HeLa cell line was provided by the Institute of Immunology, School of Medicine, Zhejiang University. Ultrapure water with a resistivity higher than 1.82 × 106 Ω·m at 25 °C was produced from the Millipore Q3 system. Synthesis and Characterization of 3-MA@ZIF-8 NPs. Stock solutions of 3-MA (2.5 mg mL−1) were prepared in methanol. First, 0.2 g (0.66 mmol) of Zn(NO3)2·6H2O was dissolved in 0.8 mL of methanol. Then, 4 mL of 3-MA stock solution was added into the Zn(NO3)2 solution. After being stirred for 5 min, 10 mL of MeIM (24.36 mmol) solution was added dropwise. They were stirred for 15 min. The product was collected by centrifugal separation and washed at least three times with methanol. The powder products (3-MA@ZIF8 NPs) were dried at 51 °C under vacuum. The loading amount of 3MA was tuned by changing the concentration of the 3-MA stock solution. 0.2 g (0.66 mmol) of Zn(NO3)2·6H2O was dissolved in 0.8 mL of ultrapure water. 10 mL of MeIM (24.36 mmol) solution was added dropwise. The reaction mixture was stirred for 15 min. The product was collected by centrifugal separation and washed at least three times with ultrapure water. The powder products (ZIF-8 NPs, 150 nm) were dried at room temperature under vacuum. The powder products (ZIF8 NPs, 80 nm) were dried at 51 °C under vacuum by changing the ultrapure water to methanol during reaction.

release percentage (%) = M r /Ml Mr is the amount of released 3-MA, while Ml is the total amount of loaded 3-MA. Cell Culture. The cervical cancer cell line (HeLa) was cultured in DMEM, supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO2. MTT Assays in HeLa Cell Line. HeLa cells were seeded onto 96well plates with 1 × 105 cells per well, 12 h prior to the test. 100 μL per well of the cell suspension was added into 96-well plates to preculture for 24 h. The medium was replaced by a fresh medium that contained each sample (3-MA, 3-MA@ZIF-8 NPs, or ZIF-8 NPs+3MA) at various concentrations. 100 μL of MTT solution (0.5 mg mL−1) was then added into per well. After 4 h, 100 μL of DMSO was introduced into each well. The absorbance at 570 nm was detected. Intracellular Reactive Oxygen Species (ROS) Quantification. Dichlorofluorescein diacetate (DCFH-DA) was used as the fluorescent probe to detect the level of intracellular ROS. 1 × 107 HeLa cells were cultured in a six-well plate and were treated with 3-MA or 3-MA@ZIF8 NPs for 24 h. Cells were trypsinized and collected by centrifugal separation and then incubated with 10 mmol/L DCFH-DA for 15 min at 37 °C. Subsequently, cells were washed twice with PBS and analyzed by a flow cytometer. Confocal Laser Scanning Microscope (CLSM) Assay. HeLa cells were transfected with GFP-LC3 using mRFP-GFP-LC3 (adenovirus, Hanheng, China) according to the manufacturer’s protocol. Transfection cells were underwent selection in DMEM medium. Cell colonies exhibiting green fluorescence and red fluorescence were selected by a confocal laser scanning microscope (Zeiss Lsm710nlo). Transmission Electron Microscopy (TEM) Assay. HeLa cells were washed with PBS and centrifuged at 1000 rpm for 5 min after 2329

DOI: 10.1021/acsami.7b16522 ACS Appl. Mater. Interfaces 2018, 10, 2328−2337

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ACS Applied Materials & Interfaces incubation for 24 h with 3-MA@ZIF-8 NPs. The collected cells were fixed with a solution containing 2.5% glutaraldehyde plus 2% paraformaldehyde in 0.1 mol L−1 cacodylate buffer, pH = 7.3, for 1 h. Then, the samples were immersed in 1% OsO4 in 0.1 mol L−1 cacodylate buffer for 30 min. Finally, for the autophagy vesicles, the morphological evidence of autophagy was found when the samples were observed under the TEM (JEM-1200EX, JEOL, Japan). Western Blotting Analysis. Cells protein was extracted by the General Protein Extraction Reagent (Bioteke, Beijing, China) supplemented with 1% protease inhibitor. 25 μg of protein was loaded and separated on 12% SDSPAGE. Then they were transferred by electrophoresis to 0.45 μm poly(vinyl difluoride) membranes. The following antibodies were used: LC3, Atg5, p62, and Beclin 1, which were purchased from Cell Signaling Technology (CST, Danvers, MA). β-Actin antibody was purchased from Zhong Shan Jin Qiao (Beijing, China). All of the antibodies were used at a dilution ratio of 1:1000. The membrane was developed using an Immobilon Western chemiluminescent HRP substrate (Millipore). Animals Experiment. All the animal experiments were performed according to the guidelines of the National Institutes of Health (Guide for the Care and Use of Laboratory Animals, 2011). Eighteen nude mice were divided into three groups (3-MA@ZIF-8 group at a dose of 10 mg/kg; 3-MA group at a dose of 2 mg/kg and control group), and each group has six mice. HeLa cells were resuspended in 100 μL of PBS and injected subcutaneously into the left flank of nude mice (3 × 106 cells per mouse). The free 3-MA or 3-MA@ZIF-8 groups were treated with 3-MA or 3-MA@ZIF-8 NPs via tail-vein injection six times with the same dosage of 3-MA (10 mg per kg body weight; day 8, day 11, day 13, day 15, day 17, and day 19). The tumor sizes were measured. The tumor volume was calculated as below: larger diameter × (smaller diameter)2/2. The xenograft tumors were collected in the 19th day. Before analysis, tissue samples were stored in 4% polyoxymethylene or at −80 °C. Immunohistochemistry Assay. The xenograft tumors were embedded by paraffin after deparaffinization and rehydration and then immersed in a 10 mM citrate buffer solution and treated with 3% H2O2 in PBS for 5 min. Then, 10% normal goat serum have blocked the sections for 10 min and incubated overnight at 4 °C with primary antibody (LC3, Beclin 1, p62). After being treated with the avidin− biotin affinity system for 30 min at room temperature and stained with 3,3′-diaminobenzidine substrate, the sections were examined under an optical microscope (Olympus X71-F22PH, Japan). Double-blind conditions were used to evaluate the positive cells. Image-pro Plus software (Media Cybernetics, United States) was used to calculate the average integrated optical density (IOD) per stained area (μm2) (IOD/area) for positive staining. Pharmacokinetics and Biodistribution Study. 3-MA@ZIF-8 NPs at a single dose of 10 mg kg−1 body weight were injected into five rats. Blood samples (0.5 mL) from the tail vein were collected into Eppendorf tubes (containing heparin) at specific points (0, 0.5, 1, 2, 3, 4, 6, and 24 h after injection) and quantified via ICP-MS. The tissue distribution was evaluated in an ICR mouse model. Briefly, nine mice were randomly divided into three groups: 3-MA@ ZIF-8 NPs (10 mg kg−1), 3-MA (2 mg kg−1), and saline; they were administered in three groups every 3 days for four total times, respectively. Seven days after the last injection, the six major organs (lung, liver spleen, heart, kidney, and brain) were excised, weighed, and quantified via ICP-MS. Biochemical Test. Every blood sample (1 mL) was collected into Eppendorf tubes with heparin and then centrifuged at 4000 rpm for 5 min to obtain serum. Typical biochemical markers of hepatic cytolysis, such as circulating alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), and renal function including creatinine (CR), blood urea nitrogen (BUN), and uric acid (UA) were determined.

with encapsulated 3-MA is presented in Figure 1. Metal ions (Zn2+) and target organic molecules (3-MA) self-assemble to

Figure 1. Schematic representation of 3-MA@ZIF-8 NPs.

form coordination polymers. Organic linkers (2-methylimidazole) are added to disassemble the metal ions from the target organic molecules and subsequently form ZIF-8 NPs by the assembly of the metal ions and linkers. The target molecules are encapsulated during the formation of ZIF-8 NPs, resulting in hierarchical nanoparticles. 3-MA@ZIF-8 NPs display the uniform size of 80 nm through dynamic lights cattering (DLS) analysis. Furthermore, the morphology and crystallography of 3-MA@ZIF-8 NPs were identified by TEM, SEM, and PXRD. Dissociation of the bonding between the zinc and the MeIM was responsible for the loss of the characteristic crystalline nature in the acidic environment. The PXRD patterns of 3-MA@ZIF-8 NPs are similar to that of the standard ZIF-8. Additionally, the intensity of peaks is decreased due to the partial loss of crystallinity resulting from the filling of pores with the drug and the high degree of disorder (Figure 2d). The TGA recorded the decrease of the known mass by heating under a nitrogen gas flow. ZIF-8 samples and 3-MA@ ZIF-8 samples were observed to have three weight loss regions. The less weight loss of 3-MA reveals that 3-MA coordinate with ZIF-8 via capillary forces, electrostatic interactions, and coordination reactions (Figure 2g). N2 adsorption shows that the large surface areas of 3-MA@ZIF-8 NPs are similar to that of ZIF-8 NPs, which demonstrates that 3-MA was encapsulated into ZIF-8 crystals instead of in the pores and channels of ZIF-8 (Figure 2e,f). The representative Fourier transform infrared (FTIR) spectra of ZIF-8 NPs and 3-MA@ZIF-8 NPs are shown in Figure 2h. The peak at 1471 cm−1 corresponding to CN of 3-MA disappeared in the FTIR spectra of 3-MA@ZIF-8, and the peak at 1624 cm−1 due to the CC group of the ZIF-8 framework appeared in that of 3-MA@ZIF-8; these results indicate that the vibration of 3-MA was restrained due to the shielding effect of the ZIF-8 framework. The zeta potentials of 3-MA@ZIF-8 and ZIF-8 were characterized to be 3.2 ± 2.2 and 24.1 ± 5.5 mV, respectively. The zeta potentials of 3-MA@ZIF8 NPs were higher than that of ZIF-8 because the surface of drug-loaded ZIF-8 was covered by a little 3-MA via strong adsorption. In addition, the PXRD peak of 3-MA did not occur in the pattern of 3-MA@ZIF NPs, presumably suggesting that 3-MA may be encapsulated inside of ZIF-8, rather than physically adsorbed on the surface of ZIF-8 (Figure 2d). The size of ZIF-8 NPs was 90 nm, which is a little larger than that of 3-MA@ZIF-8 NPs (Figure 2c and Figure S1). The changes of size in 3-MA@ZIF-8 NPs might result from the coordination bonding between metallic and polymeric parts enhanced by the encapsulation of 3-MA in ZIF-8 NPs. Furthermore, in Figure S2, the red-shift of the UV−vis spectrum and comparison with that of a free 3-MA solution conform that 3-MA molecules are coordinated with Zn2+ ions. At the same time, the absorbance

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RESULTS AND DISCUSSION Synthesis and Characterization of 3-MA Encapsulated in ZIF-8 NPs. The process for one-pot synthesis of ZIF-8 NPs 2330

DOI: 10.1021/acsami.7b16522 ACS Appl. Mater. Interfaces 2018, 10, 2328−2337

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Figure 2. (a) TEM image of 3-MA@ZIF-8 NPs. (b) SEM image of 3-MA@ZIF-8 NPs. (c) Representative particle size distribution of 3-MA@ZIF-8 NPs determined by DLS. (d) PXRD patterns of ZIF-8 NPs and 3-MA@ZIF-8 NPs. (e) N2 adsorption/desorption isotherms of ZIF-8 NPs and 3MA@ZIF-8 NPs. (f) Pore width distribution of 3-MA@ZIF-8 NPs. (g) TGA curves for ZIF-8 NPs and 3-MA@ZIF-8 NPs. (h) FTIR spectra of ZIF8 NPs, 3-MA, and 3-MA@ ZIF-8 NPs.

delivery system for antitumor, the pH-responsive releases of 3MA from 3-MA@ZIF-8NPs were detected in PBS of pH 5.0, 6.0, and 7.4. TEM analysis and PXRD results reveal that the spheres maintained their size, shape, and crystal structure after being immersed in neutral PBS for one month (Figure S4). However, immersed in PBS of pH 5.0 and 6.0, the NPs have round edges, smaller sizes, and more little fragments than those

of 3-MA increases a little after 3-MA@ZIF-8 NPs suspending in neutral PBS for 24 h, which reveals that the ZIF-8 nanoparticles have low solubility in PBS of pH 7.4. With the UV/vis absorbance in 273 nm (Figure S3), the 3-MA loading in NPs was calculated as 19.8%. pH-Responsive Release of 3-MA from 3-MA@ZIF-8 NPs. To study the potential of 3-MA@ZIF-8 NPs as a drug 2331

DOI: 10.1021/acsami.7b16522 ACS Appl. Mater. Interfaces 2018, 10, 2328−2337

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Figure 3. (a) Release behavior of 3-MA in PBS with the pH-dependent changes at 37 °C. (b) Different weight ratios of 3-MA@ZIF-8 release profiles in PBS (pH 7.4) at 37 °C.

Figure 4. (a) Cell viability when incubated with the ZIF-8 scale in 80 and 150 nm. (b) Cell viability of 3-MA@ZIF-8 NPs in theoretical ratio of 3MA. (c) Comparison of cell viability in HeLa cells exposed to 3-MA@ZIF-8 NPs, a mixture of ZIF-8 and 3-MA (ZIF-8 + 3-MA), or free 3-MA.

Figure 5. TEM images of a HeLa cell. The inset is an enlarged image of the area marked by the square showing individual 3-MA@ZIF-8 NPs (red arrows) and autophagosomes (yellow arrows). Control (a, b); 3-MA@ZIF-8 NPs (c, d); 3-MA (e, f); ZIF-8 NPs (g, h).

drug release in acidic environments. In general, the rapid drug release earns more concerns in the future, and it is expected that the feature of control release can further enhance its biological functions.9 Cytotoxicity Tests. The cytotoxicity of 3-MA@ZIF-8 NPs, free 3-MA, and a mixture of free 3-MA and ZIF-8 were evaluated by determining cellular viability through an MTT assay. At relatively high concentrations, the viabilities of ZIF-8 NPs at 80 and 150 nm were still high, 80−90% (Figure 4a). The toxicities of 3-MA@ZIF-8 NPs, a mixture of 3-MA and ZIF-8 NPs (denoted 3-MA + ZIF-8), and free 3-MA on HeLa cells for 24 h were compared. As shown in Figure 4c, 3-MA@ ZIF-8 NPs is toxic to HeLa cells in a dose-dependent manner. After being treated with 3-MA@ZIF-8 NPs at a concentration of 7.5 μg mL−1 (equivalent to a concentration of 3-MA of 1.5 μg mL−1) for 24 h, the mitochondrial function fell from the baseline level to 60% in HeLa cells. The values were even lower

in PBS of pH 7.4 (Figures S5 and S6). As shown in Figure 3b, different weight ratios of 3-MA in 3-MA@ZIF-8 NPs released 3-MA slowly in PBS of pH 7.4, in which system less than 70% 3-MA was released at the end of 24 h. It is conducive to avoiding dissipation before 3-MA getting to the target. However, a rapid release of ≈40% during the initial 4 h was monitored in PBS of pH 6.0 (Figure 3a). With the fact of fast degradation of ZIF-8 in acidic environment, it was proved that drug release was closely correlated with ZIF-8 NPs dissociation. The induction period is associated with the dissolution of the peripheral 3-MA-free shells of ZIF-8, which act as a protective capsule around the 3-MA. The release mechanism of 3-MA@ ZIF-8 system is different from that of other drug delivery systems, such as mesoporous silica. In the cases of mesoporous silica and other MOFs, drugs are released through the pores, and the drug carriers remain intact.2,31,32The disintegration of ZIF-8 NPs driven by the low pH can be exploited for selective 2332

DOI: 10.1021/acsami.7b16522 ACS Appl. Mater. Interfaces 2018, 10, 2328−2337

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investigated. In detail, a total of four autophagy-related proteins of HeLa cells treated with 3-MA@ZIIF-8 NPs were analyzed by Western Blotting. The LC3-II/LC3-I ratio of 3-MA@ZIF-8 group was significantly lower than that of free 3-MA. The increase of p62 in the 3-MA@ZIF-8 group, which is a substrate preferentially degraded by autophagy, together with the decrease of Atg5 and Beclin1, suggests that the autophagic activity was effectively inhibited through the control release of 3-MA in 3-MA@ZIF-8 NPs (Figure 7). It reveals that ZIF-8 NPs enhances the inhibition of 3-MA and thus blocks the autophagosome formation.

(below 10%) after the cells had been treated with 3-MA@ZIF-8 NPs at concentration of 10 μg mL−1 (Figure 4b). A possible reason for the toxicity is perhaps resulted from the released MeIM from the framework of the ZIF-8, leading to an alkaline environment in the cells, which breaks the pH balance in the target cells.33 The toxic effect of 3-MA@ZIF-8 NPs was significantly higher after 24 h than that of 3-MA + ZIF-8 (Figure 4c). At the same time, ROS generation was studied as an important mechanisms of cellular toxicity. It reveals that 3MA@ZIF-8 NPs have serious effects on the elevation of ROS and show strong toxicity than 3-MA+ZIF-8 (Figure S7). The greater efficacy was not caused by the simple additive effect of ZIF-8 NPs and 3-MA, as shown by the experiments using the same assay for the mixture of free 3-MA and ZIF-8 NPs at the same concentrations. This increase in toxicity is not a result of the effects of pure ZIF-8 NPs or the mixture of ZIF-8 NPs and 3-MA but a result of the synergistic effect of 3-MA and ZIF-8 NPs in 3-MA@ZIF-8 NPs. Cellular Uptake of 3-MA@ZIF-8 NPs. The cellular uptake and uptake efficiency of 3-MA@ZIF-8 NPs were evaluated by TEM and ICP-MS. These results confirmed successful uptake of the 3-MA@ZIF-8 NPs into the cells with uniformly dispersed NPs (Figure 5). The ZIF-8 NPs were localized mainly in the cytoplasm and subcellular organelles. They were smaller than the as-prepared ZIF-8 NPs, presumably due to partial dissolution of ZIF-8 NPs in the cells. Additionally, the cells treated with 3-MA@ZIF-8 NPs show a number of nanoparticles inside and more autophagosomes than that of 3MA. Furthermore, the differences in distribution patterns of 3MA in the cells treated with free 3-MA and 3-MA@ZIF-8 NPs show that ZIF-8 alters the 3-MA delivery pathway in the cells. Cells incubated with ZIF-8 NPs without 3-MA have more autophagic vesicas than cells treated with 3-MA@ZIF-8 NPs. To quantify the relation between the concentration of ZIF-8 NPs and cellular uptake efficiency, the concentration of zinc atoms in cell solutions was directly assessed with ICP-MS, which estimates the amount of ZIF-8 NPs uptake by cells. The number of Zn atom in cells through cellular uptake was calculated by Zn concentration and the number of cells in the solution (Figure 6). It indicates that the selective cellular uptake of ZIF-8 NPs is heavily dependent upon endocytosis. Autophagy Efficiency in Vitro. The 3-MA solution shows dose-dependent inhibition of cell proliferation, and it is revealed to have a weaker cytotoxicity than 3-MA@ZIF-8 NPs. The effects of 3-MA@ZIF NPs on autophagy were further

Figure 7. LC3 lipidation, Becline-1, p62, and Atg5 assayed by Western Blotting analysis on HeLa cells treated with 3-MA@ZIF-8 NPs for 24 h.

To confirm that the role of 3-MA in ZIF-8 development is relevant to autophagy process, we examined autophagy flux using HeLa cells expressing tandem mRFP-GFP-LC3 and observed that the ratio of red:yellow puncta in 3-MA-treated cells was higher than that in 3-MA@ZIF-8 group (Figure 8). Red puncta were used to detect lysosomes and autolysosomes,

Figure 6. Cellular uptake of 3-MA@ZIF-8NPs. Zn concentration after HeLa cells incubated with 3-MA@ZIF-8NPs (20 μg mL−1) for 24 h.

Figure 8. Representative cell images showing punctate GFP-LC3 distribution after 3-MA@ZIF-8 NPs treatments (scale bar: 20 μm). 2333

DOI: 10.1021/acsami.7b16522 ACS Appl. Mater. Interfaces 2018, 10, 2328−2337

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Figure 9. Detection of autophagy in xenograft tumor-loaded mice. The relative tumor volume (a) and relative body weight (c) of HeLa cancer bearing mice as a function of time (2 mg 3-MA)/(kg body weight). Photo of the excised tumors (b) on the 19th day and the concentration of Zn2+ in tumor by ICP-MS analysis (d). *p < 0.05 and ***p < 0.001.

and green puncta were used to detect autophagosomes. With the fact that it is located at autophagic membrane, LC3 is proved to be associated with number of autophagosomes. When autophagy is activated, cells could exhibit a high number of fluorescence punctate structures. As shown in Figure 8, 3MA@ZIF-8 NPs inhibited punctate GFP-LC3 in the HeLa cells. Consistently MDC (fluorescence indicators of autophagic vacuole) were loaded into cells treated with 3-MA@ZIF-8 NPs or 3-MA, and the intensity of the fluorescence was measured in Figure S4, normalized for the intensity of autophagy. The relative fluorescence intensities of cells treated with 3-MA@ ZIF-8 NPs were significantly lower than that of cells treated with free 3-MA. Antitumor Effects in Vivo. Currently, in vivo assays of 3MA@ZIF-8 NPs remain largely unexplored. The xenograft tumor of cervical cancer HeLa cell was established to evaluate the antitumor effect of 3-MA@ZIF-8 NPs. The inhibitory effects of 3-MA and 3-MA@ZIF-8 NPs were measured through the changes of tumor volume. As shown in Figure 9a, the tumor volumes are in the order of saline > free 3-MA > 3-MA@ZIF-8 group, indicating that 3-MA@ZIF-8 shows obvious higher antitumor efficacy than free 3-MA on HeLa tumor cell. The photo of the excised tumors is shown in Figure 9b, and the average weights of the mice in the three groups are approximately same (Figure 9c). The results of quantitative ICP-MS measurement estimate ZIF-8 was internalized via tailvein injection, and they carried 3-MA to the tumor (Figure 9d). The autophagic regulation proteins were then estimated by immunohistochemistry. Compared to free 3-MA, 3-MA@ZIF-8 NPs resulted in upregulating of p62 and downregulating of Beclin 1 and LC3 (Figure 10). Pharmacokinetics and Biodistributions. Pharmacokinetic studies was conducted to determine the fate of 3-MA@

Figure 10. Autophagic regulation proteins of xenograft tumor estimated by immunohistochemistry (scale bar: 200 μm).

ZIF NPs in the mice body, which detect zinc concentration in the blood, urine, and feces at special time by ICP-MS. Following dosing, the zinc concentration in blood gradually decreased over time (Figure 11a), with an elimination half-life of 2.33 h and an endogenous zinc level of 4.07 μg mL−1. The excretion analysis of Zn concentration in urine and feces revealed that the accumulated zinc was excreted within 24 h, respectively, which implied that the ZIF-8 NPs is metabolizable and nontoxicity in vivo (Figure 11b,c). Furthermore, the biodistribution of 3-MA@ZIF-8 NPs was quantified by ICP-MS (Figure 12). The Zn accumulations of 3MA@ZIF-8 group in the tissues such as the heart, kidney, and brain were similar to that of the saline and 3-MA groups. However, much more Zn accumulations were observed in the 2334

DOI: 10.1021/acsami.7b16522 ACS Appl. Mater. Interfaces 2018, 10, 2328−2337

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Figure 11. (a) Time−concentration curve of zinc in blood from SD rats after a single dose iv administration of 10 mg kg−1 3-MA@ZIF-8 NPs (n = 5). The solid line shows a three-compartment pharmacokinetic fit to the observed points. (b, c) Excretion of zinc in urine and feces from SD rats after a single dose injection (n = 5). The tested animals were half male and female. **p < 0.01.

organs of reticuloendothelium systems (RES) including the lung, liver, and spleen which is probably due to the particle size and size distribution of the nanoparticles. It reveals that 3MA@ZIF-8 NPs were internalized via tail-vein injection, and 3MA was carried to several tissues through general blood circulation. Biocompatibility in Vivo. The liver and renal function have been regarded as typical biochemical markers, which determine the hepatic and renal cytolysis of mice. The alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in the 3-MA@ZIF-8 NPs group were lower than that in 3-MA group and saline group. However, the activities of alanine aminotransferase (ALT) in 3-MA@ZIF-8 group increased, in comparison with that in the saline group on the seventh day.

Figure 12. Biodistribution in major tissues from ICR mice 7 days after a four times injection of ZIF-NPs at a dose of 10 mg kg−1 (n = 3). *p < 0.05; **p < 0.01.

Figure 13. Activities of typical biochemical markers of the liver (a) and renal (b) function. 2335

DOI: 10.1021/acsami.7b16522 ACS Appl. Mater. Interfaces 2018, 10, 2328−2337

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Technology Department of Zhejiang Province No. 2015C03034. X.W. received funding from the National Natural Science Foundation of China No. 21503190.

Additionally, creatinine (CR), blood urea nitrogen (BUN), and uric acid (UA) were monitored to identify renal function, in which the 3-MA@ZIF-8 group showed negligible changes except UA. It is suggested that 3-MA@ZIF-8 NPs have low hepatic and renal cytolysis.

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CONCLUSION In this contribution, an effective autophagy inhibition and accurate tumor target have been realized by encapsulating 3MA, the autophagy inhibitor, into the ZIF-8 with high loadings (19.798 wt %). Their uniform shapes and sizes, excellent biocompatibilities, and high stabilities at physiological conditions indicate ZIF-8 NPs are the promising and applicable drug delivery vehicles. The 3-MA@ZIF-8 NPs show a higher cytotoxicity and lead to a severely blocked autophagosome formation and autophagy flux in comparison with the equivalent concentration of free 3-MA. Moreover, the encapsulations of 3-MA contribute to a more precise drug control release, a more efficient antitumor and autophagy inhibition effectiveness for xenograft tumor, as well as a relatively moderate toxicity for mice than free 3-MA. The cell uptake results of xenograft tumor indicate that the 3-MA@ZIF8 NPs can increase the drug accumulations in tumors for targeted cancer therapy. This work highlights that ZIF-8 framework is effective in drug control release and holds immense potentials in controlling autophagy by encapsulating autophagy inhibitors. Our constructed pH-sensitive and tumortargeting drug delivery system based on the ZIF-8 provides a hopeful platform in cancer treatment for enhancing therapeutic efficacy and reducing side effects, broadening the applications of ZIF-8 in biomedical field.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b16522. Particle size distribution of ZIF-8 NPs; UV/vis spectra of a 3-MA solution, solution of 3-MA@ZIF-8; loading efficacy for 3-MA@ZIF-8 NPs; TEM image and PXRD analysis of 3-MA@ZIF-8 immersed in physiological fluids (pH = 7.4) for one month; particle size distribution of 3MA@ZIF-8 NPs in the PBS of pH 5.0 and 6.0 for 12 h; ROS generation of HeLa cells incubated with 3-MA@ ZIF-8 and 3-MA; autophagic vesicles in HeLa cells incubated with 3-MA@ZIF-8 from MDC staining assay (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected]; Tel +86 571 88981416; Fax +86571-88981416. *E-mail [email protected]. ORCID

Wenjun Fang: 0000-0002-5610-1623 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS X.C., Z.S., S.D., Q.L., and W.F. received funding from the National Natural Science Foundation of China No. 21473157. R.T., B.Y., H.L., and J.W. received funding from Science 2336

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