MOF Nanoparticles with Encapsulated Autophagy Inhibitor in

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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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16522 • Publication Date (Web): 29 Dec 2017 Downloaded from http://pubs.acs.org on December 30, 2017

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ACS Applied Materials & Interfaces

MOF Nanoparticles with Encapsulated Autophagy Inhibitor in Controlled Drug Delivery System for Antitumor Xuerui Chen,1Rongliang Tong,2,3Zheqi Shi,1Beng Yang,2,3 Hua Liu,2,3Shiping Ding,4Xu Wang,5Qunfang Lei,1Jian Wu*,2,3Wenjun Fang*,1

1. Department of Chemistry, Zhejiang University, Hangzhou 310027, China

2. Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China.

3. The Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou 310003, China.

4. The National Education Base for Basic Medical Sciences, School of Medicine, Zhejiang University, Hangzhou 310058, China

5. Hangzhou Medical College, No. 481 Binwen Road, Hangzhou 310053, China

KEYWORDS: drug delivery, autophagy, ZIF-8, 3-methyladenine, antitumor, metal-organic frameworks

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 1

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advanced functional material for small-molecule 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 (3-MA), 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 (3-MA@ZIF-8 NPs) is facilitated through the nanoparticle internalization with reference to TEM observations and the quantitative analyses of zinc by ICP-MS. 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.

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 2


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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 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 MOFs 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 ZIF family, ZIF-8 3



nanoparticles (ZIF-8 NPs) are regarded as the favorable delivery vehicle owing to the good characteristics that the nanoparticles 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 5.0-6.5.19-21 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 class III PI3K (Vps34)/Beclin-1 complex, 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 transmission electron microscope (TEM), scanning electron microscope (SEM), 4


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Powder X-ray diffraction patterns (PXRD), BET, thermo gravimetric 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.

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 (3-MA) (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-β-actin antibody, cacodylate, dulbecco's modified eagle medium (DMEM), [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. 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 of 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 methanol. Then, 4 mL 3-MA stock solution was added into

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the Zn(NO3)2 solution. After stirred for 5 min, 10 mL 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@ZIF-8 NPs) were dried at 51 oC under vacuum. The loading amount of 3-MA 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 ultrapure water. 10 mL 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 (ZIF-8 NPs 80 nm) were dried at 51 oC under vacuum by changing the ultrapure water to methanol during reaction.

DLS (ZEN3600, Malvern) was used to detect the particle size. N2 adsorption isotherms at 77K was 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 = 1.54178 Å) at room temperature. Inductively coupled plasma mass spectrometry (ICP-MS, Elan DRCII, Perkin-Elmer Sciex) was utilized to determine the zinc contents in vitro and in vivo experiments, respectively. FTIR spectra were recorded on a Shimadzuir 460 spectrometer in a KBr matrix. FTIR 6


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spectra were used to confirm the presence of 3-MA within the MOF matrix. TGA were carried out from 35 to 700 oC at the heating rate of 10 oC⋅min-1 to check the thermal stability under nitrogen atomosphere

Drug Loading efficacy of 3-MA@ZIF-8 NPs The amount of 3-MA 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 3-MA@ZIF-8 NPs were 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 were calculated by the equations given below.

DLE % =

×100 %

Release of 3-MA from 3-MA@ZIF-8 NPs at Different pH values In a normal release system, 10 mg 3-MA@ZIF-8 NPs was suspended in 20.0 mL of PBS (pH 7.4, 6.5 and 5.0, respectively) at 37 °C. The release system was then maintained at 37 °C under shaking (shaking frequency = 100 rpm). The release medium was sampled with 1mL at each time, and UV/vis spectrophotometry was used to determine the percentage of released 3-MA, and 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

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the formula,

Release Percentage (%) = Mr/ Ml

Mr is the amount of released 3-MA, while Ml is the total amount of loaded 3-MA.

Cell culture The breast carcinoma cell line (HeLa) was cultured in DMEM, supplemented with 10 % fetal bovine serum (FBS) at 37 oC in a humidified atmosphere containing 5 % CO2.

MTT Assays in HeLa Cell Line HeLa cells were seeded onto 96-well 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. Medium was replaced by a fresh medium that contained each sample (3-MA, 3-MA@ZIF-8 NPs or ZIF-8 NPs+3-MA) at various concentrations. 100 µL 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 Dichlorofluoresceindiacetate (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@ZIF-8 NPs for 24 h, respectively. Cells were trypsinized and collected by centrifugal separation, 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 8


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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 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 incubated for 24 h with 3-MA@ZIF-8 NPs. The collected cell 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, the autophagy vesicles, the morphological evidence of autophagy were found when the samples were observed under the TEM (JEM-1200EX, JEOL, Japan).

Western Bolting Analysis. Cells protein was extracted by General Protein Extraction Reagent (Bioteke, Beijing, China) supplemented with 1% protease inhibitor. 25 µg protein was loaded and separated on 12% SDSPAGE. Then they were transferred by electrophoresis to 0.45 µm polyvinyl difluoride membranes. The following antibodies were used: LC3, Atg5, p62 and Beclin 1, which were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). β-Actin antibody was purchased from Zhong

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Shan Jin Qiao (Beijing, China). All of the antibodies were used at a dilution ratio of 1:1000.

The

membrane

was

developed

using

ImmobilonTM

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, respectively. HeLa cells were resuspended in 100 µl 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 with3-MA or 3-MA@ZIF-8 NPs via tail-vein injection six times with the same dosage of 3-MA (10 mg per kg bodyweight; Day8, Day11, day13, day15, day17 and day 19). The tumor sizes were measured. 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 -80oC.

Immunohistochemistry Assay. The xenograft tumors were embedded by paraffin after deparaffinisation 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 treated with avidin-biotin affinity system for 30 min at room temperature, and stained with 3-3’ diaminobenzidine substrate, the 10


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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, respectively. Blood samples (0.5 mL) from tail vein were collected into Eppendorf tubes (containing heparin) at specific points (0, 0.5, 1, 2, 3, 4, 6, 24 h after injection) and quantified via ICP-MS.

The tissue distribution was evaluated in an ICR mouse model. Briefly, 9 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 group every three days for four total times, respectively. 7 days after the last injection, the six major organs (lung, liver spleen, heart, kidney, 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.

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Results and Discussions

Synthesis and Characterization of 3-MA Encapsulated in ZIF-8 NPs The process for one-pot synthesis of ZIF-8 NPs with encapsulated 3-MA is presented in Figure 1. Metal ions (Zn2+) and target organic molecules (3-MA) self-assemble to 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 resulted from the filling of pores with the drug and the high degree of disorder (Fig. 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 (Fig.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 12


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pores and channels of ZIF-8 (Fig.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 mV and 24.1±5.5 mV, respectively. The zeta potentials of 3-MA@ZIF-8 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 (Fig.2d). The size of ZIF-8 NPs was 90 nm, which is a little larger than that of 3-MA@ZIF-8 NPs (Fig.2c and S1). The changes of size in 3-MA@ZIF-8 NPs might results from the coordination bonding between metallic and polymeric parts enhanced by the encapsulation of 3-MA in ZIF-8 NPs. Furthermore, in Fig.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 of 3-MA increase 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 (Fig. S3), the 3-MA loading in NPs was calculated as 19.8%. 13



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

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Fig.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 3-MA@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 ZIF-8 NPs, 3-MA and 3-MA@ ZIF-8 NPs.

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 delivery system for antitumor, the pH-responsive releases of 3-MA 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 15



and crystal structure after immersed in neutral PBS for one month (Fig. 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 in PBS of pH7 .4 (Fig. S5 and S6). As shown in Fig.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 hours was monitored in PBS of pH 6.0 (Fig. 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 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

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

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 nm and 150 nm were still high, 80−90 % (Fig. 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 Fig.4c, 3-MA@ZIF-8 NPs is toxic to HeLa cells in a dose-dependent manner. After 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 (below 10 %) after the cells had been treated with 3-MA@ZIF-8 NPs at concentration of 10 µg⋅mL−1 (Fig. 4b). A possible reason for the toxicity is perhaps resulted from the released 2-methylimidazole (MeIM) from the framework of the ZIF-8, leading to an alkaline environment in the cells, which breaks

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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 (Fig. 4c). At the same time, ROS generation was studied as an important mechanisms of cellular toxicity. It reveals that 3-MA@ZIF-8 NPs have serious effects on the elevation of ROS and show strong toxicity than 3-MA+ZIF-8(Fig.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 synergistic effect of 3-MA and ZIF-8 NPs in 3-MA@ZIF-8 NPs.

Fig.4 (a) Cell viability when incubated with the ZIF-8 scale in 80 nm and 150 nm;(b) Cell viability of 3-MA@ZIF-8 NPs in theoretical ratio of 3-MA;(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.

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

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successful uptake of the 3-MA@ZIF-8 NPs into the cells with uniformly dispersed NPs (Fig. 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 3-MA. Furthermore, the differences in distribution patterns of 3-MA 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 (Fig. 6). It indicates that the selective cellular uptake of ZIF-8 NPs is heavily dependent upon endocytosis.

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Fig.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).

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

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 investigated. In detail, a total of four autophagy related protein 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 3-MA@ZIF-8 group, which is a substrate preferentially degraded by autophagy, together with the decrease of Atg5 and Beclin1, suggest that the

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autophagic activity was effectively inhibited through the control release of 3-MA in 3-MA@ZIF-8 NPs (Fig.7). It reveals that ZIF-8 NPs enhances the inhibition of 3-MA, thus blocks the autophagosome formation.

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 (Fig. 8). Red puncta were used to detect lysosomes and autolysosomes, 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, 3-MA@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 Fig.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.

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

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

Antitumor Effects in Vivo Currently, in vivo assays of 3-MA@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 is in an 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 23



shown in Figure 9b, and the average weights of the mice in the three groups are approximately same (Fig. 9c). The results of quantitative ICP-MS measurement estimate ZIF-8 was internalized via tail-vein injection and they carried 3-MA to the tumor (Fig. 9d). The autophagic regulation proteins were then estimated by immunohistochemistry. Compared to free 3-MA, 3-MA@ZIF-8 NPs resulted in up-regulating of p62 and down-regulating of Beclin 1 and LC3 (Fig. 10).

(a) 1500

Saline 3-MA 1000

3-MA@ZIF-8

*

500

19

17

15

13

11

0

8

Mean Tumor Volume (mm3 )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

time(day)

Fig.9 The 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). * indicates p < 0.05 while *** indicates p < 0.001. 24


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Fig.10

Autophagic

regulation

proteins

of

xenograft

tumor

estimated

by

immunohistochemistry (scale bar: 200 µm).

Pharmacokinetics and Biodistributions pharmacokinetic studies was conducted to determine the fate of 3-MA@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 (Fig. 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 non-toxicity in vivo (Fig.11b,c).

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Furthermore, biodistribution of 3-MA@ZIF-8 NPs was quantified by ICP-MS (Fig. 12). The Zn accumulations of 3-MA@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 organs of reticulo-endothelium 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 3-MA@ZIF-8 NPs were internalized via tail-vein injection and 3-MA was carried to several tissues through general blood circulation.

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Fig.11 (a) Time-concentration curve of zinc in blood from SD rats after a single dose i.v. 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. ** indicates p < 0.01.

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

Biocompatibility In Vivo The liver and renal function have been regarded as typical 27



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 7th day. 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 show negligible changes except UA. It is suggested that 3-MA@ZIF-8 NPs have low hepatic and renal cytolysis.

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

Conclusion

In this contribution, an effective autophagy inhibition and accurate tumor target have been realized by encapsulating 3-MA, the autophagy inhibitor, into the ZIF-8 with high loadings (19.798 wt %). Their uniform shapes and sizes, excellent 28


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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@ZIF-8 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 tumor-targeting 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.

ASSOCIATED CONTENT

Supporting Information.

Particle size distribution of ZIF-8 NPs; UV/Vis spectra of a 3-MA solution, solution of 3-MA@ZIF; 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

29



month; particle size distribution of 3-MA@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.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86 571 88981416. Fax: +86-571-88981416.

*E-mail: [email protected].

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT

Authors Xuerui Chen, Zheqi Shi, Shiqing Ding, Qunfang Lei and Wenjun Fang received funding from the National Natural Science Foundation of China NO.21473157. Authors Rongliang Tong, Beng Yang, Hua Liu and Jian Wu received funding from Science Technology Department of Zhejiang Province No. 2015C03034. Author Xu Wang received funding from by the National Natural Science Foundation of China NO.21503190.

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Graphical abstract

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MOF Nanoparticles with Encapsulated Autophagy Inhibitor in

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