Nanoscale Zeolitic Imidazolate Framework-8 as Efficient Vehicles for

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Nanoscale zeolitic imidazolate framework-8 as efficient vehicles for enhanced delivery of CpG oligodeoxynucleotides Huijie Zhang, Wei Chen, Kai Gong, and Jinghua Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09583 • Publication Date (Web): 25 Aug 2017 Downloaded from http://pubs.acs.org on August 27, 2017

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ACS Applied Materials & Interfaces

Nanoscale zeolitic imidazolate framework-8 as efficient vehicles for enhanced delivery of CpG oligodeoxynucleotides

Huijie Zhang1*, Wei Chen2, Kai Gong1, Jinghua Chen1* 1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China 2. School of Basic Medical Sciences, Xi’an Jiaotong University, Xi’an 710061, China

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Corresponding author. Tel: +86-510-85197769; Fax: +86-510-85197769

E-mail: [email protected] (Huijie Zhang); [email protected] (Jinghua Chen)

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Abstract CpG oligodeoxynucleotides (ODNs) activate immune system and induce Th 1 responses by stimulation of Toll-like receptor 9 (TLR9). Thus, CpG ODNs become immunotherapeutics against various diseases including cancers, allergies, and infection. However, applications of CpG ODNs are largely limited because of their easy degradation by DNase as well as inefficient cellular uptake. Development of efficient delivery systems capable of transferring CpG ODNs into immune cells is important to enhance their therapeutic efficacy. Herein, for the first time, we demonstrated the construction of a novel CpG ODNs delivery system by encapsulating CpG ODNs into zeolitic imidazolate framework-8 (ZIF-8) nanoparticles. ZIF-8 possessed high CpG ODNs loading capacity due to its porous structure. ZIF-8/CpG ODNs complexes exhibited good stability in physiological environment but effectively released CpG ODNs in acid conditions corresponding to the TLR 9 localized endolysosomes. ZIF-8/CpG ODNs complexes had no cytotoxicity in contrast to ZIF-8. ZIF-8 significantly increased the intracellular uptake of CpG ODNs in RAW264.7 cells, which further enhanced the secretion of immune cytokines both in vitro and in vivo. Our results suggest that nanoscale metal-organic-frameworks (MOFs) can serve as ideal vehicles for the delivery of CpG ODNs.

Keywords: CpG oligodeoxynucleotides, zeolitic imidazolate framework-8, drug delivery, immunostimulation, nanomedicine

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1. Introduction Bacterial and viral DNA possess immunostimulatory effect because their genomic DNA contain the unmethylated CpG motifs. toll-like receptor 9 (TLR9).

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CpG DNA is recognized by endolysosome localized

Activation of TLR9 by CpG ODNs initiates MyD88-dependent

signaling pathways such as MAPK and NFκB, and further induces the expression of multiple proinflammatory cytokines.1,

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These cytokines and chemokines are important for Th-1

immune responses. Synthetic CpG oligodeoxynucleotides (ODNs) mimic microbe DNA and own similar immunostimulatory activity.1,

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Thus, CpG ODNs become promising

immunotherapeutics against various diseases including cancers, allergies, and infection.4,

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However, cellular uptake of CpG ODNs is low due to the electrostatic repulsion between the negatively charged CpG ODNs and electronegative cell membrane. 10-12 Moreover, CpG ODNs are prone to DNase degradation.

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These shortcomings greatly limit the therapeutic

applications of CpG ODNs. Therefore, construction of novel delivery systems able to efficiently transfer CpG ODNs into immune cells is crucial to enhance their therapeutic efficacy. Recently, various CpG ODNs assemblies or nanomaterials-based delivery systems have emerged for CpG ODNs delivery.11-12, 15-26 Zhu’ group developed mesoporous silica nanoparticles (MSNs)-based CpG ODNs delivery systems, which obviously improved the efficiency of CpG ODNs delivery and TLR9-mediated cytokine induction.27-30 Although these strategies were effective in improving the efficiency of CpG ODNs delivery and expanding their therapeutic applications, a simpler and more efficient CpG ODNs delivery vehicles are still in demand. Metal-organic frameworks (MOFs) are highly crystalline hybrid porous materials constructed by organic linkers and metal ions connectors.31-34 MOFs possess advantageous properties including ultrahigh surface area, tunable shape, uniform pore sizes, good thermal and chemical stability, and versatile functionality. Thus, MOFs hold great promise in gas separation

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and storage, catalysis, and sensing.31, 35-36 Over the past few years, biomedical applications of MOFs has emerged by virtue of their intrinsic biodegradability and low cytotoxicity.37-38 Nanoscale MOFs have become ideal candidates for drug delivery, bioimaging, and other theranostic applications.39-44 Zeolite imidazole frameworks-8 (ZIF-8) is a sub-family of MOFs， which is built from zinc ions and 2-methylimidazole (MIM). ZIF-8 exhibits intriguing structural features of higher porosity and specific area, which is beneficial for drug encapsulation.38 Furthermore, ZIF-8 exhibits exceptional thermal and chemical stability.31 ZIF-8 was recently reported to possess low cytotoxicity and pH-sensitive release properties.38, 45-46 ZIF-8 was stable in neutral conditions but quickly decomposed when suspended in acid solution45. These unique advantages enable ZIF-8 as promising vehicles for drug delivery and controlled release of pharmaceuticals. As we know, CpG ODNs interact with TLR9 in acidic endolysosomal compartments and stimulate immune responses. Delivery of CpG ODNs using ZIF-8 as carrier can guarantee the controlled release of CpG ODNs in endolysosomes. Therefore, ZIF-8 is considered promising as vehicle for CpG ODNs delivery. ZIF-8 as carriers for delivery of various anticancer drug, such as 5-fluorouracil, doxorubicin, and curcumin has been reported, which possessed high drug encapsulation and delivery efficiency.38, 45, 47 However, no studies on ZIF-8 based CpG ODNs delivery system has been reported until now. In this study, nano-sized ZIF-8 was synthesized and utilized as carrier for CpG ODNs delivery (Scheme 1). ZIF-8 possessed high CpG ODNs loading capacity and significantly enhanced their cellular uptake. ZIF-8/CpG ODNs complexes had no cytotoxicity and exhibited good stability in physiological conditions. ZIF-8/CpG ODNs complexes efficiently released CpG ODNs in acid conditions corresponding to the TLR9-localized endolysosomes. Cytokine assay showed that ZIF-8/CpG ODNs complexes exhibited enhanced immunostimulatory activity. The current study highlights the potential of ZIF-8 as theranostic nanoplatforms and will

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facilitate the biomedical application of ZIF-8.

Scheme 1. Schematic showing the construction of ZIF-8 based CpG ODNs delivery system for enhanced immunostimulation.

2. Methods 2.1. Synthesis of ZIF-8 50 mL of Zn (NO3)2 ⋅6 H2O (25 mM in methanol) were mixed with 50 mL of 2-Methylimidazole (50 mM in methanol) at room temperature, followed by ultrasonication for 15 min. Then, the mixture was centrifuged. The product was washed for several times with methanol and dried in a vacuum oven. 2.2. Preparation of ZIF-8/CpG ODNs complexes ZIF-8 (50 µL, 0.5 mg/mL) was added to 50 µL of CpG ODNs solution (40 µM). The mixture was stirred for 1 h at room temperature. Then, ZIF-8/CpG ODNs complexes were

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obtained by centrifugation. The unbound CpG ODNs in the supernatant were analyzed to calculate the loading capacity. 2.3. Cytokine secretion in RAW264.7 cells RAW264.7 cells were cultured in 96-well plate at 5×105 cells/well and incubated for 24 h. Then, the cells were washed with PBS and treated with ZIF-8/CpG ODNs complexes. The final concentration of ZIF-8 was 25 µg/mL. After 8 h or 24 h, the medium was collected for determining the secretion of TNF-α and IL-6, which were measured by ELISA assay using manufacturer's protocols. 2.4. In vivo cytokine assay CpG ODNs or ZIF-8/CpG ODNs complexes (10 µg CpG ODNs/mouse) were injected into mice through tail vein. The blood of the mice was collected 6 h post injection. Thereafter, the serum was prepared by centrifugation. The serum levels of TNF-α and IL-6 were determined by ELISA assay following manufacturer's protocols. 3. Results and discussion 3.1. Synthesis of ZIF-8 ZIF-8 were synthesized in the mixture of Zn (NO3)2, 2-Methylimidazole and methanol. The morphology and size of the prepared ZIF-8 were examined by SEM and TEM. ZIF-8 exhibited a well-defined polyhedral shape and the size was approximately 200 nm (Figure 1a, 1b). Powder XRD were further used to verify the formation of the desired structure of ZIF-8. XRD pattern of the as-synthesized ZIF-8 corresponded well with the simulated ZIF-8 pattern, and no impurity peaks could be found (Figure 1c,). This result suggested that the prepared ZIF-8 was pure-phased ZIF-8 crystals and was well crystallized. Formation of nanoscale ZIF-8 was further evidenced by the FTIR (Figure 1d). In the spectrum of ZIF-8, the peak at 420 cm-1 was ascribed to the Zn–N stretching. The bands at 758, 995, 1307, 1583, 2856, 2927, 2959 and

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3135 cm-1 corresponded to the C–H stretching of 2-methylimidazole.48 The porous structure of ZIF-8 was characterized by N2 sorption measurements. As shown in Figure 1e, ZIF-8 exhibited a type I isotherm, with a rapid increases in N2 adsorption at low relative pressure (

Nanoscale Zeolitic Imidazolate Framework-8 as Efficient Vehicles for

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