Reduction-Responsive Codelivery System Based on a MetalâOrganic

Subscriber access provided by UNIVERSITY OF TOLEDO LIBRARIES

Biological and Medical Applications of Materials and Interfaces

The Reduction–Responsive Co-Delivery System Based on MetalOrganic Framework for Eliciting Potent Cellular Immune Response Yong Yang, Qianqian Chen, Jian-Ping Wu, Thomas Brett Kirk, Jiake Xu, Zonghua Liu, and Wei Xue ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01680 • Publication Date (Web): 29 Mar 2018 Downloaded from http://pubs.acs.org on March 29, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31 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

ACS Applied Materials & Interfaces

The Reduction–Responsive Co-Delivery System Based on MetalOrganic Framework for Eliciting Potent Cellular Immune Response Yong Yang,† Qianqian Chen,† Jian-Ping Wu,‡ Thomas Brett Kirk,‡ Jiake Xu,§ Zonghua Liu,*,† and Wei Xue*,†,ǁ,# †

Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of

Biomedical Engineering, Jinan University, Guangzhou 510632, China ‡

3D Imaging and Bioengineering Laboratory, Department of Mechanical Engineering, Curtin

University, Perth, Australia §

The School of Pathology and Laboratory Medicine, University of Western Australia, Perth,

Australia ǁ

The First Affiliated Hospital of Jinan University, Guangzhou 510632, Guangdong, China

#

Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, Guangdong,

China KEYWORDS: Metal-organic framework, reduction-responsive, antigen delivery, cellular immunity, cytotoxic T lymphocyte ABSTRACT: Utilizing nanoparticles to deliver subunit vaccines can be viewed as a promising strategy for enhancing the immune response, especially with regards to cellular immunity to fight against infectious viruses and malignant cancer. Nevertheless, its applications are still far from practical due to some limitations like the high cost, non-biocompatibility, non-biodegradability, and the inefficient stimulation of cytotoxic T lymphocyte (CTL) response. In this study, we use

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 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

Page 2 of 31

metal-organic framework (MOF) MIL-101-Fe-NH2 nanoparticles as carriers to fabricate an innovative reduction-responsive antigen delivery system for co-transporting the antigen model, ovalbumin (OVA) and an immune adjuvant, unmethylated cytosine-phosphate-guanine oligonucleotide (CpG). In vitro cellular tests show that the MOF nanoparticles can not only greatly improve the uptake of OVA by the antigen presenting cells (APCs), but also smartly deliver both OVA and CpG into the same cell. By feat of the reductively controllable release of OVA and the promoting function of CpG, the delivery system can elicit strong cellular immunity and CTL response in mice. Moreover, the increased frequencies of effector memory T cells inspired by the delivery system indicate that it can induce a potent immune memory response. These results demonstrate that MOF nanoparticles are excellent vehicles for co-delivering antigen and immune adjuvant, and may find wider applications in the biomedical fields. INTRODUCTION The stimulation of cytotoxic T lymphocytes (CTL)-based cellular immunity is a crucial aspect of the adaptive immune response since antigen-specific CTLs can specifically eliminate infectious viruses or malignant tumor cells.1-2 New-generation subunit vaccines that are safe, cost-effective, and easily prepared and transported, compared to traditional attenuated or live pathogen vaccines, have been extensively explored to elicit vigorous antigen-specific humoral and cellular immune responses.3-4 Nevertheless, free proteins or peptides, when used as subunit vaccines, are not easily ingested by the antigen presenting cells (APCs) due to their ultrasmall sizes, and are prone to be decomposed by proteases, which leads to poor cross-presentation by the APCs and weak immunogenicity.5 To overcome these barriers, one valid strategy is utilizing nanoparticles to freight the antigen into cells.6-8 By imitating the size, morphology and surface property of pathogen vaccines, a nanoparticle-based antigen delivery system can efficiently facilitate cellular


2

Page 3 of 31 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


uptake by the APCs and effectively protect the antigen against degradation. Furthermore, nanoparticles can co-deliver an immune adjuvant with the antigen to helpfully induce a much stronger immune response.9-10 Although until now numerous nanoparticles, such as liposomes,1112

polymers13-15 and inorganic nanomaterials,16-17 have been developed as antigen carriers, these

nanomaterials possess disadvantages like poor biocompatibility, non-biodegradation, high cost, inefficient eliciting CTL response, etc.18 Metal-organic frameworks (MOFs)19 comprise a well-known class of crystalline materials that have been widely explored in the biomedical domain.20-21 Considering their low cost, excellent biodegradability, high loading performance, diverse structure and other virtues, MOFs can serve as ideal vehicles for drug delivery,22-24 biosensing,25-26 bioimaging,27-28 and the transporting of subunit vaccines, as has been reported in recent years. Qu’s group used ZIF-8 to co-deliver antigen and immune adjuvant by encapsulating ovalbumin (OVA) into ZIF-8 and attaching CpG on the surface through electrostatic adsorption. The pH responsive delivery system induced systemic immune response and potent immune memory effect.29 Zhang et al adopted an analogous method to fabricate the pH responsive delivery system with a high OVA loading efficiency of 55% (w/w). As a result, the antigen delivery system enhanced the CTL response and showed an antitumor effect on B16-OVA melanoma in vivo.30 Despite these outcomes, a one-pot synthesis for antigen loading cannot be extensively applied since most MOFs need to be prepared in relatively strict conditions, such as high temperature and inadaptable pH, which do harm to the structural stability and bioactivity of antigens. Therefore the post-loading of antigens onto the already synthesized MOFs through conjugation, adsorption or other manners stands for a common way.31 The reduction-sensitive disulfide bond is stable in the ordinary extracellular condition, though


3


Page 4 of 31

it breaks down in the reductive ambience of the cell cytosol due to the high concentration of glutathione (2.5-10 mM). Hence, antigens can be conjugated to the vectors through disulfide bonds, and then taken up by APCs and controllably released in the cytosol to produce strong cross-presentation and CD8+ T cell response.32-35 Hubbell’s group, for example, linked OVA to poly(propylene sulfide) nanoparticles via disulfide bonds. The nanoparticles as prepared could be specifically targeted to dendritic cells (DCs) and induce potent mucosal and systemic CD8+ T cell immunity.36-37 Li et al used dextran nanogels as a carrier to load OVA and the results of in vitro study showed that the release of OVA occurred precisely in the DCs, boosting the MHC-I antigen presentation.38 Furthermore, in an in vivo study, the combination of poly(I:C) and OVAloaded nanogels induced a strong therapeutic effect against melanoma.39 De Geest’s group also designed hydrogel nanoparticles based on disulfide bonds to deliver a peptide antigen. After PEGylation, the hydrogel NPs could efficiently target the draining lymph nodes and improve the priming of T cells.40 Based on the previously mentioned results, in this study we used MIL-101-Fe-NH241 as the antigen carrier due to its biodegradability, biosafety, and more important tailorability of amino groups. As shown in Scheme 1, with undergoing chemical modification, the well explored antigen model OVA was conjugated to the surface of nanoparticles via disulfide bonds. It was expected to smartly discharge into the cytosol of APCs when encountering the presence of glutathione. Furthermore, to take advantage of the stimulatory function of CpG, an immune adjuvant previously proved to be effective in boosting cellular immunity through the Toll-like receptor 9 (TLR9),42-46 the positively charged MOF nanoparticles were co-loaded with the electronegative CpG through the electrostatic adsorption. We expected this co-delivery system for OVA and CpG would probably elicit strong cellular immunity, which was desired to shed


4

Page 5 of 31 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


light on the application of MOF in the vaccine delivery field.

Scheme 1. Schematic representation of the preparation of MOF-based antigen delivery system and its role in eliciting strong cellular immunity response.

EXPERIMENTAL SECTION Materials. Ferric chloride hexahydrate, 2-aminoterephthalic acid, glutathione (reduced) and dithiothreitol were purchased from Energy Chemical (Shanghai, China). N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was purchased from TCI Shanghai (Shanghai, China). DMF and ethanol were bought from Guangzhou Chemical Reagent Factory (Guangzhou, China). Ovalbumin (OVA) and FITC were obtained from SigmaAldrich (MO, USA). CpG (5′-TCC ATG ACG TTC CTG ACG TT-3′) and CpG-Texas red were purchased from Sangon Biotechnology Inc. (Shanghai, China). The Cell Counting Kit-8 was bought from DOJINDO (Japan). The medium for cells culture was complete RPMI 1640 (Gibco, CA, USA) with 0.5% (v/v) penicillin– streptomycin (Gibco, CA, USA) and 10% (v/v) fetal bovine serum (Gibco, CA, USA). All enzyme-linked immunosorbent assay (ELISA) kits for detecting cytokines were purchased from Biolegend (CA, USA). All fluorochrome-conjugated anti-mouse antibodies for flow cytometry were purchased from eBioscience (CA, USA). Characterization. TEM images and elemental mapping were obtained by using a JEOL JEM-2100 transmission electron microscope equipped with an Oxford TSR energy disperse spectroscope at 120 kV. SEM


5


Page 6 of 31

images were recorded by a Zeiss Ultra 55 field emission scanning electron microscope. X-ray measurement was performed with an X-ray diffractometer (Miniflex 600, Rigaku, Japan). The zeta potentials of MOFs nanoparticles and the nanoparticles-OVA complexes were recorded by a Zetasizer Nano ZS (Malvern, UK). FT-IR spectra were obtained by an FT-IR spectrophotometer (VERTEX 70, BRUKER, Germany). The UV-vis absorption spectra were measured by using an UV-3100 spectrophotometer (Mapada, China). The fluorescence emission spectra were recorded by an F-700 fluorescence spectrophotometer (Hitachi, Japan). The flow cytometry was carried out on a Beckman CytoFLEX flow cytometry. The immunohistochemical and histopathological graphs were acquired by using a ZEISS Axio Observer A1 microscope. The absorbance of 96-well plates was measured by a Multiskan MK3 photometric microplate reader. The intracellular location of fluorescently-labeled OVA and CpG was observed with a confocal laser scanning microscope (LSM 700, Zeiss). Synthesis of MIL-101-Fe-NH2 (MOF). MIL-101-Fe-NH2 was synthesized as reported previously by the literature.41 The typical procedure was that 2-aminoterephthalic acid (375 mg, 2.07 mmol) and FeCl3·6H2O (1125 mg, 4.16 mmol) were dissolved in DMF (12.5 mL) respectively, and then these two solutions were put into a stainless steel autoclave with 50 mL total volume. After the mixture was treated for 24 h at 120˚C, the brown powder was collected by centrifugation (8000 rpm, 10 min) and washed with DMF and ethanol three times in sequence. At last, the product was dried at 50˚C for 8 h in vacuum. Introduction of Disulfide Bond to MOF. Theoretical numbers of amine groups from 1 mg of MIL-101-FeNH2 were calculated according to the result of elemental analysis. To explore the modification rate, different molar ratios of amine groups to linkers (1:0.5, 1:1 and 1:2) were evaluated (Table S1). As the optimized reaction condition, MOF (2 mg) was dispersed in deionic water (1.7 mL) by ultrasonic. Then N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP, 1.3 mg) dissolved in DMF (0.3 mL) was added into the suspension. The reaction was kept at room temperature for 4 h under agitation. Unreacted SPDP was removed by centrifugation (8000 rpm, 10 min) and washing of three times with the mixture of DMF and water (1:1, v/v). The product (denoted as MOF-S-S) was dried at 45˚C for 8 h in vacuum. Conjugation efficiency was confirmed through incubation of MOF-S-S (1 mg) in dithiothreitol solution (2 mL, 40 mM) overnight. Due to disulfide-thiol exchange, pyridine-2-thione was released from MOF-S-S and could be detected at 343 nm by using an UV-vis


6

Page 7 of 31 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


spectrophotometer after centrifugation (8000 rpm, 10 min). Modification rate of amine groups was calculated as the following formula: Modification rate= mole of SPDP conjugated/mole of amine groups Conjugation efficiency of SPDP was calculated as the following formula: Conjugation efficiency= mole of SPDP conjugated/mole of SPDP charged Synthesis of OVA-FITC. FITC labeled OVA was prepared according to the literature.29 OVA (20 mg) was dissolved in sodium carbonate buffer (10 mL, pH=9.8, 25 mM) and then mixed with FITC solution (400 µL, 1 mg/mL). The mixture was stirred in the dark at 4˚C for 18 h and then dialyzed for 2 days. The purified solution was lyophilized to bring forth dried OVA-FITC. Conjugation of OVA or OVA-FITC to MOF. Firstly, in order to prepare thiolated OVA or OVA-FITC, OVA or OVA-FITC (5 mg) was dissolved in PBS buffer solution (1 mL, pH=7.4), into which solution Traut’s reagent solution (20 µL, 14 mM) was added. The solution was agitated at room temperature for 1 h. Then the OVA solution was pipetted into MOF suspension made from MOF-S-S (5 mg) dispersed in ultrapure water (2.5 mL) by ultrasonic. After 3 hours’ agitation at room temperature, the product (denoted as MOF-S-S-OVA or MOF-S-S-OVA-FITC) was recovered by centrifugation (10000 rpm, 5 min) and the supernatant was used to determinate the amount of unconjugated OVA or OVA-FITC by bicinchoninic acid (BCA) assay. At last, the product was washed three times with ultrapure water and dried under vacuum. The result of BCA assay showed that 1.4 mg of OVA or OVA-FITC was attached on the surface of MOF nanoparticles through disulfide bonds. Co-Loading of CpG to MOF-S-S-OVA. CpG (84 µg) was dissolved in ultrapure water (1 mL), in which solution MOF-S-S-OVA (1.3 mg) was suspended by ultrasonic. The suspension was under stirring at room temperature for 0.5 h. The product (denoted as MOF-S-S-OVA@CpG) was collected by centrifugation (10000 rpm, 5 min) and the supernatant was analyzed at 260 nm by a UV-vis spectrophotometer. The result indicated that 41 µg of CpG was loaded on the nanoparticles. In Vitro and in Vivo Toxicity Evaluation. The degradability of the MOF material was tested in PBS (pH=7.4). The MOF material (10 mg) was dispersed in 10 mL of PBS under ultrasound. Then the suspension was incubated at 37˚C with stirring. At every time point, the suspension was centrifuged (10000 rpm, 15 min),


7


Page 8 of 31

2 mL of supernatant was taken out, and 2 mL of PBS was replenished. UV-vis spectra was used to quantify the amount of released 2-aminoterephthalic acid at the wavelength of 328 nm and SEM was used to observe the morphological change of MOF nanoparticles. In vitro cell viability was evaluated by the CCK-8 assay. Firstly, DC 2.4 cells were incubated in 96-well plate at a density of 7000 cells per well for 24 h. Then MOF and MOF-S-S-OVA@CpG dispersed in DMEM at various concentrations (62.5, 125, 250, 500 and 1000 µg/mL) were added into wells and the plate was sustained in the cell incubator for 24 h. Finally, the standard CCK-8 assay was used to determine the relative cell viability. In vivo toxicity was explored by histopathological study of tissue organs from mice injected with PBS and other ones immunized with MOF-S-S-OVA@CpG. 100 µL of PBS or suspension of MOF-S-S-OVA@CpG (141.5 µg) in PBS was subcutaneously injected into mice on day 0, 10 and 20 respectively. On day 30, mice were sacrificed to collect tissue organs. After paraffin embedding, slicing and HE staining, the morphostructure of tissue organs was investigated through a fluorescence microscope. In Vitro OVA Release from MOF-S-S-OVA-FITC. Two copies of MOF-S-S-OVA-FITC (8 mg) were dispersed in PBS (4 mL, pH=7.4) and agitated at 37 ˚C. At the seventh hour, one suspension was mixed with glutathione to achieve the concentration of 5 mM, while the other one not. After centrifugation (10000 rpm, 5min), 400 µL of supernatant was taken from these two suspensions at different time points and 400 µL of PBS or glutathione solution (5 mM) was replenished. The supernatant was analyzed by a fluorescence spectrophotometer to quantify the released OVA. In Vitro CpG Release from MOF-S-S-OVA@CpG-Texas red. MOF-S-S-OVA@CpG-Texas red (8 mg) was suspended in PBS (4 mL, pH=7.4) and under stirring at 37 ˚C. At every given time the suspension was centrifuged (10000 rpm, 5min), 400 µL of supernatant was extracted and 400 µL of PBS was replenished. The supernatant was analyzed by a fluorescence spectrophotometer to quantify the released CpG. In Vitro Cellular Uptake Study. The efficiency of nanoparticles’ ingestion by DC2.4 cells was confirmed by flow cytometry. DC2.4 cells (1.5×105 cells/mL) were seeded in a 24-well plate for 24 h. Then the mixture of OVA-FITC with CpG-Texas red or MOF-S-S-OVA-FITC@CpG-Texas red was respectively shifted into wells at the concentration of 10 µg/mL of OVA and 0.15 µg/mL of CpG. After incubation of 4 h, DC2.4 cells


8

Page 9 of 31 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


were trypsinized and washed three times with PBS. At last, cell suspensions were analyzed by flow cytometry in the channel of FITC or Texas red respectively. For imaging of cellular location, DC2.4 cells (2.5×104 cells/mL) were cultured in 4-chamber glass bottom dishes for 24 h. And then cells were incubated with the mixture of OVA-FITC with CpG-Texas red or MOF-SS-OVA-FITC@CpG-Texas red for additional 4 h. After washed three times with PBS, the DC2.4 cells were fixed in 4% paraformaldehyde for 1h. Later on, following three times’ washing again, the imaging of intracellular location was performed with a confocal laser scanning microscope. For observing the release of OVA in the cellular condition, fractional amino groups of MOF were modified with FITC, and then conjugated with OVA-Cy5 via disulfide bonds. DC2.4 cells (1×105 cells/mL) were cultured in 4-chamber glass bottom dishes for 24 h. After washed with PBS several times, DC2.4 cells were treated with MOF-S-S-OVA for 3 h or 6 h. Cells were fixed with 4% paraformaldehyde and stained with DAPI. Images were obtained by a confocal laser scanning microscope. In Vitro Cytokine Assays. RAW264.7 cells were seeded on 6-well plates (5×105 cells per well) and cultured for 24 h. Cells were washed with PBS three times, and then treated with OVA, OVA+CpG, MOF-SS-OVA and MOF-S-S-OVA@CpG for 8 h. After centrifugation (12000 rpm, 10 min), the supernatants were collected and stored at -20˚C. The levels of IFN-γ and TNF-α were measured by ELISA according to the protocols of kits. Animals and Immunization Studies. Female C57BL/6 mice (6-8 weeks old) were purchased from HFK Biological Technology Co., Ltd (Beijing, China). All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, and obtained permission from Experimental Animal Ethics Committee in Jinan University. The mice were randomly divided into different groups (five mice in each group). In the beginning, 100 µL of OVA, OVA+CpG, MOF+OVA, MOF-S-S-OVA and MOF-S-SOVA@CpG with equivalent doses (30 µg of OVA, 4.3 µg of CpG) were subcutaneously injected into the mice three times at 10 days interval. On day 30, mice were sacrificed under anesthesia and splenocytes were harvested for subsequent assays. Meanwhile the serum was collected for IgG titer measurement. Expression of MHC-II and Co-Stimulatory Molecules on Dendritic Cells in Spleen. Splenocytes were stained with APC-anti-CD11c, FITC 450-anti-CD80, PerCP-Cy5.5-anti-CD86 and PE-anti-MHC-II. The


9


Page 10 of 31

expression of MHC-II and co-stimulatory molecules was detected by flow cytometer. Splenocytes Proliferation Assay. To evaluate OVA-specific splenocytes activation, a portion of splenocytes (2.5×106 cells/mL) was stimulated with OVA (60 µg/mL), while another portion not. After splenocytes were incubated in 96-well plates for 72 h, CCK-8 solution (10 µL) was added to each well. The plates were maintained in the incubator for additional 4 h. The ratio of OD for stimulated samples to OD for unstimulated samples, which was measured at 450 nm by a Multiskan MK3 photometric microplate reader, was defined as the proliferation index. Measurement of Memory T Cells Response and Cytokine Levels. Splenocytes (2.5×106 cells/mL) derived from the immunized mice were restimulated with OVA (60 µg/mL) for 60 h in the cell incubator. The supernatant was collected via centrifugation (1000 rpm, 5min) and used to test cytokine levels by ELISA. After washed three times with PBS, the splenocytes were stained with FITC 450-anti-CD4, PerCP-Cy5.5-antiCD8α, APC-anti-CD62L and PE-anti-CD44. Soon afterwards the cells were examined by flow cytometer. Measurement of IgG Titer. The OVA-specific IgG titer was quantitatively measured by ELISA assay. Briefly, 96-well plates were coated with OVA solution (100 µL, 10 µg/mL of concentration, dissolved in 0.05 M carbonate buffer solution, pH=9.6) at 4 ˚C for 12 h. Then the plates were washed three times with PBST (0.01 M PBS mixed with 0.05% Tween 20, pH=7.4) and blocked with bovine serum albumin solution (BSA dissolved in PBST at 20 µg/mL) at 37 ˚C for 1 h. After washing with PBST, appropriate sera dilutions (100 µL) were pipetted into wells and the plates were maintained at 37 ˚C for 1 h with shaking (400 rpm). The plates were then washed and incubated with horseradish peroxidase-conjugated anti-mouse IgG antibody (diluted in PBST at 1:8000) for 1 h. Thereafter, plates were washed three times again and incubated with 3,3′,5,5′-tetrame-thylbenzidine for 15 min at room temperature in the dark. After the reaction was stopped by adding H2SO4 solution (100 µL, 1 M) per well, the absorbance was read at 450 nm by a Multiskan MK3 photometric microplate reader. Evaluation of Immunohistochemical Assay. C57BL/6 mice divided into different groups (four mice in each group) were injected subcutaneously with 100 µL of OVA, OVA+CpG, MOF+OVA, MOF-S-S-OVA and MOF-S-S-OVA@CpG with equivalent doses (30 µg of OVA, 4.3 µg of CpG). On day 2 or 7, the spleens from euthanized mice were collected, fixed in 4% paraformaldehyde, embedded into paraffin and cut into


10

Page 11 of 31 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


slices. The slices were dealt with 3% H2O2 for 30 min after deparaffinage. Then the sections were blocked with anti-ovalbumin antibody at 37˚C for 2 h. Afterwards the slices were incubated with goat anti-rabbit IgG antibody HRP and then stained with hematoxylin. At last the slices were inspected and the immunohistochemical images were documented by a ZEISS Axio Observer A1 microscope. In Vivo CTL Assay. The CTL assay was performed like the literature reported.47 C57BL/6 mice were randomly divided into five groups (five mice in each group). E.G7-OVA cells (50×104) was subcutaneously inoculated into the right backs of C57BL/6 mice. On day 7, the mice were injected with 100 µL of OVA, OVA+CpG, MOF+OVA, MOF-S-S-OVA and MOF-S-S-OVA@CpG (30 µg of OVA, 4.3 µg of CpG). On day 14, splenocytes were harvested and suspended in RPMI-1640 medium (10% FBS, 5% penicillin-streptomycin, 50 µM 2-ME and 20 U/mL recombinant murine IL-2). After incubated with mitomycin C treated E.G7-OVA cells for 3 days, the splenocytes served as effector cells for the CTL assay. When the ratio of effector cells to target cells (E.G7-OVA cells) was 20:1 or 40:1, the lactate dehydrogenase (LDH) cytotoxicity assay was used to detect the CTL activity. Statistical Analysis. All statistical analyses were performed by using Origin8.5 or SPSS16.0 software, and all the data were expressed as means ± SD. Differences between two groups were tested by using independentsamples T test. Significant differences between the groups were expressed as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.

RESULTS AND DISCUSSION Preparation of the Co-Delivery System for OVA and CpG. In this study, the antigen delivery system of MOF-S-S-OVA@CpG was prepared according to the detailed procedure in the experimental section. Scanning electron microscopy (SEM) images (Figure 1A and B) demonstrated that MOF-S-S-OVA@CpG had similar particle sizes of about 300 nm with the pure MOF, while its surface morphology turned to be rough due to the co-loading of OVA and CpG, which could be further confirmed by the Fourier transform infrared (FT-IR) spectra and transmission electron microscopy (TEM) elemental mappings. As shown in Figure 1E, the weak


11


Page 12 of 31

peak at 540 cm-1 could be attributed to the stretching vibration of disulfide bond, which proved that the surface of MOF was smoothly modified with functional disulfide bond (Table S1). The obvious stretching vibrations of hydroxyl at 3450 cm-1 and carbonyl at 1645 cm-1 verified the conjugation of OVA. Meanwhile, the TEM elemental mappings (Figure 1D) illustrated the rational distribution of Fe, C, N, S and P elementals on the same particle, which could be viewed as the proof of co-occurrence of OVA and CpG. In addition, the gradual decrease of nanoparticles’ zeta potential, which can be seen in Figure S2, also indicated the loading of negatively charged OVA and CpG. The nitrogen sorption measurement revealed that after the co-loading OVA and CpG, the BET surface area decreased from 1709 m2 g-1 for MOF to 1033 m2 g-1 for MOF-S-S-OVA@CpG (Figure S3). At last, to quantify the chemically linked OVA and physically adsorbed CpG, the bicinchoninic acid (BCA) assay and UV-vis absorbance measurement were carried out (Figure S4 and S5). The results revealed that 280 µg of OVA had been loaded onto 1 mg of MOF nanoparticles. Even though the higher initial concentration of CpG resulted in a higher loading amount (Table S2), the loading amount of 41 µg was adopted according to the report45.


12

Page 13 of 31 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


Figure 1. A) SEM image of pure MIL-101-Fe-NH2 (MOF). B) SEM image of MOF-S-S-OVA@CpG. C) TEM image of MOF-S-S-OVA@CpG. D) Bright-field TEM image of MOF-S-S-OVA@CpG and relevant elemental mappings of Fe-K edge, C-K edge, N-K edge, S-K edge and P-K edge signals in the STEM mode. E) FT-IR spectra of pure MOF, MOF-S-S, MOF-S-S-OVA and pure OVA. F) Cumulative release of OVA-FITC from MOF-S-S-OVA-FITC in PBS (pH=7.4) or glutathione solution at 37˚C.

In Vitro Release Behaviors of OVA and CpG. The loading of antigen on the nanoparticles via disulfide bonds has been proved to be an effective pathway for purposefully delivering the antigen into the cytosol of APCs. Thereby, to verify the efficiency of OVA release in the nonreductive or reductive media, the release process was studied in PBS (pH=7.4) or glutathione solution of PBS (pH=7.4, 5mM) simulating the cytosol. Before being loaded on the MOF, OVA


13


Page 14 of 31

was labelled with FITC, thus the amount of released OVA in the supernatant could be quantitatively ascertained by a fluorescence spectrophotometer. As shown in Figure 1F, little released OVA could be detected in both suspensions before 6 h until glutathione was added into one of the two suspensions at the 7th hour. During the rapid interchange reaction between thiol and disulfide bond, about 76% of OVA was released within an hour and almost all OVA was released over the following eight hours. While in the suspension without glutathione added all along, OVA was slowly discharged from the delivery system just because of degradation of MOF in PBS.48 Furthermore, as shown in Figure S6, the separation of green and red fluorescence indicated the release of OVA from MOF-S-S-OVA. These results demonstrated that the delivery system in this work would be effective for releasing the antigen in reductive cytosol and expectedly induce a strong immune response. In addition, as far as the release mechanism of CpG, even though there is a relatively high electrostatic attraction between ferric ion and phosphate ion of CpG, CpG still gradually dissociated from nanoparticles into the PBS solution under the dual effects of ion exchange and degradation of MOF (Figure S7). About 90% of CpG was discharged into the solution after 10 hours, which suggested the delivery system could reasonably release CpG at a slow rate. In Vitro and in Vivo Toxicity Assessment. As shown in Figure S9 and S10, most of the MOF particles degraded into small debris after being incubated in PBS (pH=7.4) for 15 days, leading to a release amount of about 73% of the organic linker. This result indicated that the MOF used in this study owned good biodegradability. Prior to the delivery system being applied in animal experiments, in vitro cell viability assay was used to evaluate its cytotoxicity on DC2.4 cells. The result (Figure S11) revealed that both pure MOF and MOF-S-S-OVA@CpG hardly caused harm to the cells until the concentration exceeded 500 µg/mL. Thereafter,


14

Page 15 of 31 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


histopathological study was implemented to test the possibility of the delivery system damaging tissue organs. The images of susceptible organs’ sections (heart, liver, spleen, lung and kidney) from PBS or MOF-S-S-OVA@CpG injected mice are displayed in Figure S12, which showed no recognizable morphologic abnormality. Based on above results, MOF-S-S-OVA@CpG was confirmed to possess good biocompatibility and biosafety. In Vitro Cellular Uptake Study. It is known that nanoparticles can greatly improve the efficiency of antigen uptake by APCs. In addition, the co-delivery of OVA and CpG to the same APCs can facilitate to play their roles and induce a strong cellular immune response, which has been proved by many reports.42-46 Herein, FITC-labelled OVA and Texas red-labelled CpG were utilized to verify the ingesting efficiency of DC2.4 cells and the function of co-localization by using the flow cytometry and confocal laser scanning microscope (CLSM). Resort to comparing the mixture of OVA-FITC and CpG-Texas red with MOF-S-S-OVA-FITC@CpG-Texas red, the large shift of fluorescence curves and enhancement of mean fluorescence intensity in both FITC channel and Texas red channel, which are evident in Figure 2A, suggested that MOF nanoparticles indeed promoted the uptake efficiency. Moreover, as shown in Figure 2B, strong green and red fluorescence signals in the DC2.4 cells incubated with MOF-S-S-OVAFITC@CpG-Texas red were detected and indicated that MOF nanoparticles reliably facilitated OVA and CpG to be efficiently internalized by the same cell.


15


Page 16 of 31

Figure 2. A) Flow cytometry analysis of DC2.4 cells treated with OVA-FITC+CpG-Texas red and MOF-S-SOVA-FITC@CpG-Texas red and corresponding mean fluorescence intensity a) in the channel of FITC and b) in the channel of Texas red. ***p < 0.001. B) Fluorescence images of DC2.4 cells incubated with OVAFITC+CpG-Texas red and MOF-S-S-OVA-FITC@CpG-Texas for 4 h respectively.

Common

Evaluations

of

OVA-Specific

Immune

Response.

Firstly,

the

immunohistochemical study was performed to determine the existence of OVA in spleens. It is can be seen in Figure 3A that, both at the early stage of 2 days and at the late stage of 7 days post-immunization, the abundant OVA could be detected in the spleens of the mice immunized with MOF-S-S-OVA and MOF-S-S-OVA@CpG. This phenomenon could be ascribed to the enhanced ingestion of chemically loaded OVA by APCs, which increased the possibility of OVA being transported into the spleen.


16

Page 17 of 31 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


Afterwards, the maturation of DCs in spleens was explored because mature DCs mainly play the role of antigen presentation and are the initiators of immune response. As shown in Figure S13, MOF-S-S-OVA@CpG could promote the high expression of CD80 and MHC II, which is the mark of mature DCs, so that it meant the delivery system had the potential of activating strong immune response. Next, a splenocytes proliferation assay measured by CCK-8 kit was used to evaluate the efficacy of various OVA complexes activating splenocytes, which could indirectly reflect the extent of immune response. The result in Figure 3B showed that MOF-S-SOVA@CpG more efficiently facilitated splenocytes proliferation than soluble OVA, soluble OVA mixed with CpG and soluble OVA mixed with MOFs nanoparticles. Therefore, it was believed that OVA loaded on MOF nanoparticles via disulfide bonds could induce potent OVAspecific immune response with the assistance of CpG. Finally, to measure antibody responses induced by various OVA complexes, serum was collected from mice subcutaneously immunized three times and then IgG titers were investigated by ELISA. As shown in Figure 3C, there were higher IgG titers when OVA was loaded on nanoparticles by disulfide bonds than the simple mixture of OVA and MOF, which indicated that disulfide bonds indeed promoted controllable release of OVA. Furthermore, MOF-S-SOVA@CpG elicited the strongest antibody response than other OVA complexes owning to the immunoregulatory role of CpG.


17


Page 18 of 31

Figure 3. A) Representative graphs of antigen level in spleens collected from the mice at the second or seventh day after immunized with various OVA formulations. The antigen level was determined by immunohistochemical assay and the yellow areas signified available OVA. The study was performed at 20 times magnification by a ZEISS Axio Observer A1 microscope. B) Proliferative responses of splenocytes restimulated by antigen ex vivo. C) Antigen-specific IgG antibody responses in the mice immunized with various vaccine formulations. *p < 0.05, **p < 0.01.

Measurement of Cytokine Levels. As is known, the secretion of various cytokines is the sign of on-going immune response. Therefore, levels of Th1 cytokines (IFN-γ, TNF-α) and Th2 cytokines (IL-4, IL-10) stemming from restimulated splenocytes by OVA were determined by


18

Page 19 of 31 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


ELISA. IFN-γ and TNF-α are typical markers of cellular immune response, which play critical roles in fighting against viral infection and cancer. As shown in Figure 4A and B, MOF-S-SOVA and MOF-S-S-OVA@CpG produced higher concentrations of IFN-γ and TNF-α cytokines in comparison with soluble OVA, soluble OVA and CpG, or the mixture of OVA and MOFs, which indicated that chemically linked OVA could be more effectually delivered and initiate stronger cellular immunity. Further on, under the promotion of immune adjuvant CpG, MOF-SS-OVA@CpG could trigger the strongest secretion of IFN-γ and TNF-α. These results were in line with those of in vitro cytokine level measurements (Figure S14) based on the same mechanism. Besides, MOF-S-S-OVA@CpG also facilitated the secretion of IL-4 and IL-10 (Figure 4C and D), which suggested that it could efficaciously enhance both cellular and humoral immunity.


19


Page 20 of 31

Figure 4. Cytokines secreted by splenocytes. The levels of A) IFN-γ, B) TNF-α, C) IL-4 and D) IL-10 were determined by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001.

Measurement of Memory T Cells Response. The memory effect of immune response is a crucial aim of vaccination, as it can facilitate the immune system to quickly respond to the second infection and kill invasive viruses or metastatic cancer cells. Memory T cells are divided into two subsets of central memory T cells, which are mainly resident in the secondary lymphoid


20

Page 21 of 31 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


tissues and afford slow and long-term protection, and effector memory T cells, which locate in lymphoid or non-lymphoid tissues and can cause immediate protection against the second infection. Therefore, the splenocytes harvested from the mice immunized three times were stained with FITC 450-anti-CD4, PerCP-Cy5.5-anti-CD8α, APC-anti-CD62L and PE-anti-CD44, and then analyzed by flow cytometry to determine the proportions of memory T cells. Regarding central memory T cells, as a result, there were no significant differences among various groups in the present work. However, as shown in Figure 5A and B, MOF-S-S-OVA@CpG induced higher frequencies of both CD4+ and CD8+ effector memory T cells in comparison with soluble OVA, mixture of OVA with CpG or mixture of OVA with MOFs. Especially with respect to more important CD8+ effector memory T cells, MOF-S-S-OVA@CpG was superior to MOF-SS-OVA because of immune adjuvant’s aid, which implied MOF-S-S-OVA@CpG could assist the immune system to more rapidly defeat the reinvasion of virus or migratory tumor cells.


21


Page 22 of 31

Figure 5. Frequency of effector (CD44hiCD62Llow) memory A) CD4+ T and B) CD8+ T cells in splenocytes harvested from immunized mice. The frequency was ascertained by ELISA. C) Representative graphs of flow cytometry results. *p < 0.05, **p < 0.01.

In Vivo Stimulation of OVA-Specific CTL. The antigen-specific CTL response is essential for decimating infectious virus and cancer cells. To evaluate the magnitude of CTL response, mice were immunized with various formulations of OVA to promote the activation and proliferation of CTLs in spleen, which were set as effector cells. After E.G7-OVA cells serving


22

Page 23 of 31 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


as target cells were treated by effector cells for 4 h, as shown in Figure 6, the percentage of cell lysis caused by MOF-S-S-OVA@CpG was largest, whether the ratio of effector cells to target cells was 20:1 or 40:1. Owing to the increase of CTLs, cell lysis resulting from MOF-S-SOVA@CpG at the ratio of 40:1 was distinctly more powerful than that at the ratio of 20:1, and led to about 67% of E.G7-OVA cells being dead. These results demonstrated MOF nanoparticleloaded OVA and CpG could efficiently arouse the OVA-specific CTL response.

Figure 6. The induction of OVA-specific CTL response from mice immunized with OVA, OVA+CpG, MOF+OVA, MOF-S-S-OVA or MOF-S-S-OVA@CpG. In the LDH assay the ratio of effector cells to target cells was set as 20:1 or 40:1. *p < 0.05, **p < 0.01, ***p < 0.001.

CONCLUSIONS In summary, we developed a novel and efficacious reduction-sensitive antigen delivery system based on MOF that can elicit much strong cellular immunity. After being chemically modified, OVA can be loaded onto the MOF nanoparticles via disulfide bonds. In addition, due to the


23


Page 24 of 31

electropositivity of ferric ions, MOF nanoparticles can co-load negatively charged CpG through electrostatic adsorption. The as-prepared antigen delivery system has several advantages as following. Firstly, appropriately sized-particles can be a benefit for the delivery system when they are taken up by APCs to enhance the antigen immunogenicity. Secondly, the MOF nanoparticles can transport OVA and CpG into the same antigen presenting cell and controllably release OVA into the reductive cytosol, which both contribute to the robust cellular immunity and the CTL response. Thirdly, besides instigating a potent cellular immunity, the delivery system can trigger a noteworthy memory effect in the immune response, allowing the organism to respond rapidly to the second invasion of viruses or migrating tumor cells. Inspired by these results, we conclude that MOF nanoparticles are superior vaccine platforms and may prove to be effective in various applications in the biomedical field. ASSOCIATED CONTENT Supporting Information XRD pattern of MIL-101-Fe-NH2 (MOF), table of modification rate of amine groups of MOF, standard curves of UV-vis spectra and fluorescence spectra, in vitro and in vivo biosafety assessment images, expressions of co-stimulatory molecule CD80 and MHC II on CD11c+ DCs in spleens. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected]. Notes The authors declare no competing financial interest.


24

Page 25 of 31 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


ACKNOWLEDGMENTS This study was financed by the China Postdoctoral Science Foundation (2017M622909). REFERENCES (1) Rappuoli, R.; Mandl, C. W.; Black, S.; De Gregorio, E. Vaccines for the Twenty-First Century Society. Nat Rev Immunol 2011, 11 (12), 865-872. (2) Irvine, D. J.; Swartz, M. A.; Szeto, G. L. Engineering Synthetic Vaccines Using Cues from Natural Immunity. Nat Mater 2013, 12 (11), 978-990. (3) Baxter, D. Active and Passive Immunity, Vaccine Types, Excipients and Licensing. Occup MedOxford 2007, 57 (8), 552-556. (4) Jaberolansar, N.; Toth, I.; Young, P. R.; Skwarczynski, M. Recent Advances in the Development of Subunit-Based RSV Vaccines. Expert Rev Vaccines 2016, 15 (1), 53-68. (5) Moser, M.; Leo, O. Key Concepts in Immunology. Vaccine 2010, 28, C2-C13. (6) Irvine, D. J.; Hanson, M. C.; Rakhra, K.; Tokatlian, T. Synthetic Nanoparticles for Vaccines and Immunotherapy. Chem Rev 2015, 115 (19), 11109-11146. (7) Yang, L.; Li, W.; Kirberger, M.; Liao, W. Z.; Ren, J. Y. Design of Nanomaterial Based Systems for Novel Vaccine Development. Biomater Sci-Uk 2016, 4 (5), 785-802. (8) Zhu, G. Z.; Zhang, F. W.; Ni, Q. Q.; Niu, G.; Chen, X. Y. Efficient Nanovaccine Delivery in Cancer Immunotherapy. Acs Nano 2017, 11 (3), 2387-2392. (9) Tao, Y.; Ju, E. G.; Li, Z. H.; Ren, J. S.; Qu, X. G. Engineered CpG- Antigen Conjugates Protected Gold Nanoclusters as Smart Self- Vaccines for Enhanced Immune Response and Cell Imaging. Adv Funct Mater 2014, 24 (7), 1004-1010. (10) Fischer, N. O.; Rasley, A.; Corzett, M.; Hwang, M. H.; Hoeprich, P. D.; Blanchette, C. D. Colocalized Delivery of Adjuvant and Antigen Using Nanolipoprotein Particles Enhances the Immune Response to Recombinant Antigens. J Am Chem Soc 2013, 135 (6), 2044-2047.


25


Page 26 of 31

(11) Yoshizaki, Y.; Yuba, E.; Sakaguchi, N.; Koiwai, K.; Harada, A.; Kono, K. Potentiation of pHSensitive Polymer-Modified Liposomes with Cationic Lipid Inclusion as Antigen Delivery Carriers for Cancer Immunotherapy. Biomaterials 2014, 35 (28), 8186-8196. (12) Fan, Y. C.; Sahdev, P.; Ochyl, L. J.; Akerberg, J. J.; Moon, J. J. Cationic Liposome-Hyaluronic Acid Hybrid Nanoparticles for Intranasal Vaccination with Subunit Antigens. J Control Release 2015, 208, 121-129. (13) Wilson, J. T.; Keller, S.; Manganiello, M. J.; Cheng, C.; Lee, C. C.; Opara, C.; Convertine, A.; Stayton, P. S. pH-Responsive Nanoparticle Vaccines for Dual-Delivery of Antigens and Immunostimulatory Oligonucleotides. Acs Nano 2013, 7 (5), 3912-3925. (14) Rosalia, R. A.; Cruz, L. J.; van Duikeren, S.; Tromp, A. T.; Silva, A. L.; Jiskoot, W.; de Gruijl, T.; Lowik, C.; Oostendorp, J.; van der Burg, S. H.; Ossendorp, F. CD40-Targeted Dendritic Cell Delivery of PLGA-Nanoparticle Vaccines Induce Potent Anti-Tumor Responses. Biomaterials 2015, 40, 88-97. (15) Acharya, A. P.; Sinha, M.; Ratay, M. L.; Ding, X. C.; Balmert, S. C.; Workman, C. J.; Wang, Y. D.; Vignali, D. A. A.; Little, S. R. Localized Multi-Component Delivery Platform Generates Local and Systemic Anti-Tumor Immunity. Adv Funct Mater 2017, 27 (5), 1-11. (16) de Faria, P. C. B.; dos Santos, L. I.; Coelho, J. P.; Ribeiro, H. B.; Pimenta, M. A.; Ladeira, L. O.; Gomes, D. A.; Furtado, C. A.; Gazzinelli, R. T. Oxidized Multiwalled Carbon Nanotubes as Antigen Delivery System to Promote Superior CD8(+) T Cell Response and Protection against Cancer. Nano Lett 2014, 14 (9), 5458-5470. (17) Almeida, J. P. M.; Lin, A. Y.; Figueroa, E. R.; Foster, A. E.; Drezek, R. A. In Vivo Gold Nanoparticle Delivery of Peptide Vaccine Induces Anti-Tumor Immune Response in Prophylactic and Therapeutic Tumor Models. Small 2015, 11 (12), 1453-1459. (18) Fischer, N. O.; Rasley, A.; Blanchette, C. Nanoparticles and Antigen Delivery: Understanding the Benefits and Drawbacks of Different Delivery Platforms. Nanomedicine-Uk 2014, 9 (4), 373-376.


26

Page 27 of 31 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


(19) Ockwig, N. W.; Delgado-Friedrichs, O.; O'Keeffe, M.; Yaghi, O. M. Reticular Chemistry: Occurrence and Taxonomy of Nets and Grammar for the Design of Frameworks. Accounts Chem Res 2005, 38 (3), 176-182. (20) Della Rocca, J.; Liu, D. M.; Lin, W. B. Nanoscale Metal-Organic Frameworks for Biomedical Imaging and Drug Delivery. Accounts Chem Res 2011, 44 (10), 957-968. (21) Gimenez-Marques, M.; Hidalgo, T.; Serre, C.; Horcajada, P. Nanostructured Metal-Organic Frameworks and Their Bio-related Applications. Coordin Chem Rev 2016, 307, 342-360. (22) Zheng, H. Q.; Zhang, Y. N.; Liu, L. F.; Wan, W.; Guo, P.; Nystrom, A. M.; Zou, X. D. One-pot Synthesis of Metal Organic Frameworks with Encapsulated Target Molecules and Their Applications for Controlled Drug Delivery. J Am Chem Soc 2016, 138 (3), 962-968. (23) Wu, M. X.; Yang, Y. W. Metal-Organic Framework (MOF)-Based Drug/Cargo Delivery and Cancer Therapy. Adv Mater 2017, 29 (23), 1-20. (24) Zhang, F. M.; Dong, H.; Zhang, X.; Sun, X. J.; Liu, M.; Yang, D. D.; Liu, X.; Wei, J. Z. Postsynthetic Modification of ZIF-90 for Potential Targeted Codelivery of Two Anticancer Drugs. Acs Appl Mater Inter 2017, 9 (32), 27332-27337. (25) Yassine, O.; Shekhah, O.; Assen, A. H.; Belmabkhout, Y.; Salama, K. N.; Eddaoudi, M. H2S Sensors: Fumarate-Based fcu-MOF Thin Film Grown on a Capacitive Interdigitated Electrode. Angew Chem Int Edit 2016, 55 (51), 15879-15883. (26) Bhardwaj, N.; Bhardwaj, S. K.; Mehta, J.; Kim, K. H.; Deep, A. MOF-Bacteriophage Biosensor for Highly Sensitive and Specific Detection of Staphylococcus aureus. Acs Appl Mater Inter 2017, 9 (39), 33589-33598. (27) Li, Y. T.; Tang, J. L.; He, L. C.; Liu, Y.; Liu, Y. L.; Chen, C. Y.; Tang, Z. Y. Core-Shell Upconversion Nanoparticle@Metal-Organic Framework Nanoprobes for Luminescent/Magnetic DualMode Targeted Imaging. Adv Mater 2015, 27 (27), 4075-4080.


27


Page 28 of 31

(28) Sava Gallis, D. F.; Rohwer, L. E. S.; Rodriguez, M. A.; Barnhart -Dailey, M. C.; Butler, K. S.; Luk, T. S.; Timlin, J. A.; Chapman, K. W. Multifunctional, Tunable Metal-Organic Framework Materials Platform for Bioimaging Applications. Acs Appl Mater Inter 2017, 9 (27), 22268-22277. (29) Zhang, Y.; Wang, F. M.; Ju, E. G.; Liu, Z.; Chen, Z. W.; Ren, J. S.; Qu, X. G. Metal-OrganicFramework-Based Vaccine Platforms for Enhanced Systemic Immune and Memory Response. Adv Funct Mater 2016, 26 (35), 6454-6461. (30) Duan, F.; Feng, X. C.; Yang, X. J.; Sun, W. T.; Jin, Y.; Liu, H. F.; Ge, K.; Li, Z. H.; Zhang, J. C. A Simple and Powerful Co-Delivery System Based on pH-Responsive Metal-Organic Frameworks for Enhanced Cancer Immunotherapy. Biomaterials 2017, 122, 23-33. (31) Doonan, C.; Ricco, R.; Liang, K.; Bradshaw, D.; Falcaro, P. Metal-Organic Frameworks at the Biointerface: Synthetic Strategies and Applications. Accounts Chem Res 2017, 50 (6), 1423-1432. (32) Brulisauer, L.; Kathriner, N.; Prenrecaj, M.; Gauthier, M. A.; Leroux, J. C. Tracking the Bioreduction of Disulfide-Containing Cationic Dendrimers. Angew Chem Int Edit 2012, 51 (50), 1245412458. (33) Li, P.; Luo, Z. C.; Liu, P.; Gao, N. N.; Zhang, Y. J.; Pan, H.; Liu, L. L.; Wang, C.; Cai, L. T.; Ma, Y. F. Bioreducible Alginate-Poly(ethylenimine) Nanogels as an Antigen-Delivery System Robustly Enhance Vaccine-Elicited Humoral and Cellular Immune Responses. J Control Release 2013, 168 (3), 271-279. (34) Kapadia, C. H.; Tian, S. M.; Perry, J. L.; Luft, J. C.; DeSimone, J. M. Reduction Sensitive PEG Hydrogels for Codelivery of Antigen and Adjuvant to Induce Potent CTLs. Mol Pharmaceut 2016, 13 (10), 3381-3394. (35) Rincon-Restrepo, M.; Mayer, A.; Hauert, S.; Bonner, D. K.; Phelps, E. A.; Hubbell, J. A.; Swartz, M. A.; Hirosue, S. Vaccine Nanocarriers: Coupling Intracellular Pathways and Cellular Biodistribution to Control CD4 vs CD8 T Cell Responses. Biomaterials 2017, 132, 48-58. (36) Stano, A.; van der Vlies, A. J.; Martino, M. M.; Swartz, M. A.; Hubbell, J. A.; Simeoni, E. PPS Nanoparticles as Versatile Delivery System to Induce Systemic and Broad Mucosal Immunity after Intranasal Administration. Vaccine 2011, 29 (4), 804-812.


28

Page 29 of 31 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


(37) Nembrini, C.; Stano, A.; Dane, K. Y.; Ballester, M.; van der Vlies, A. J.; Marsland, B. J.; Swartz, M. A.; Hubbell, J. A. Nanoparticle Conjugation of Antigen Enhances Cytotoxic T-cell Responses in Pulmonary Vaccination. P Natl Acad Sci USA 2011, 108 (44), E989-E997. (38) Li, D. D.; Kordalivand, N.; Fransen, M. F.; Ossendorp, F.; Raemdonck, K.; Vermonden, T.; Hennink, W. E.; van Nostrum, C. F. Reduction-Sensitive Dextran Nanogels Aimed for Intracellular Delivery of Antigens. Adv Funct Mater 2015, 25 (20), 2993-3003. (39) Li, D. D.; Sun, F. L.; Bourajjaj, M.; Chen, Y. N.; Pieters, E. H.; Chen, J.; van den Dikkenberg, J. B.; Lou, B.; Camps, M. G. M.; Ossendorp, F.; Hennink, W. E.; Vermonden, T.; van Nostrum, C. F. Strong in Vivo Antitumor Responses Induced by an Antigen Immobilized in Nanogels via Reducible Bonds. Nanoscale 2016, 8 (47), 19592-19604. (40) De Koker, S.; Cui, J. W.; Vanparijs, N.; Albertazzi, L.; Grooten, J.; Caruso, F.; De Geest, B. G. Engineering Polymer Hydrogel Nanoparticles for Lymph Node-Targeted Delivery. Angew Chem Int Edit 2016, 55 (4), 1334-1339. (41) Bauer, S.; Serre, C.; Devic, T.; Horcajada, P.; Marrot, J.; Ferey, G.; Stock, N. High-Throughput Assisted Rationalization of the Formation of Metal Organic Frameworks in the Iron(III) Aminoterephthalate Solvothermal System. Inorg Chem 2008, 47 (17), 7568-7576. (42) Molino, N. M.; Anderson, A. K. L.; Nelson, E. L.; Wang, S. W. Biomimetic Protein Nanoparticles Facilitate Enhanced Dendritic Cell Activation and Cross-Presentation. Acs Nano 2013, 7 (11), 9743-9752. (43) de Titta, A.; Ballester, M.; Julier, Z.; Nembrini, C.; Jeanbart, L.; van der Vlies, A. J.; Swartz, M. A.; Hubbell, J. A. Nanoparticle Conjugation of CpG Enhances Adjuvancy for Cellular Immunity and Memory Recall at Low Dose. P Natl Acad Sci USA 2013, 110 (49), 19902-19907. (44) Wang, C.; Sun, W. J.; Wright, G.; Wang, A. Z.; Gu, Z. Inflammation-Triggered Cancer Immunotherapy by Programmed Delivery of CpG and Anti-PD1 Antibody. Adv Mater 2016, 28 (40), 8912-8920.


29


Page 30 of 31

(45) Zeng, Q.; Li, H. M.; Jiang, H.; Yu, J.; Wang, Y.; Ke, H.; Gong, T.; Zhang, Z. R.; Sun, X. Tailoring Polymeric Hybrid Micelles with Lymph Node Targeting Ability to Improve the Potency of Cancer Vaccines. Biomaterials 2017, 122, 105-113. (46) Zhang, H. J.; Chen, W.; Gong, K.; Chen, J. H. Nanoscale Zeolitic Imidazolate Framework-8 as Efficient Vehicles for Enhanced Delivery of CpG Oligodeoxynucleotides. Acs Appl Mater Inter 2017, 9 (37), 31519-31525. (47) Yuba, E.; Kanda, Y.; Yoshizaki, Y.; Teranishi, R.; Harada, A.; Sugiura, K.; Izawa, T.; Yamate, J.; Sakaguchi, N.; Koiwai, K.; Mono, K. pH-Sensitive Polymer-Liposome-Based Antigen Delivery Systems Potentiated with Interferon-Gamma Gene Lipoplex for Efficient Cancer Immunotherapy. Biomaterials 2015, 67, 214-224. (48) Zhang, Y.; Liu, C. Q.; Wang, F. M.; Liu, Z.; Ren, J. S.; Qu, X. G. Metal-Organic-FrameworkSupported Immunostimulatory Oligonucleotides for Enhanced Immune Response and Imaging. Chem Commun 2017, 53 (11), 1840-1843.


30

Page 31 of 31 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


Table of Contents Graphic 62x15mm (600 x 600 DPI)


Reduction-Responsive Codelivery System Based on a MetalâOrganic

Recommend Documents

Reduction-Responsive Codelivery System Based on a MetalâOrganic