Simultaneous delivery of DNA vaccine and hydrophobic adjuvant using reducible polyethylenimine-functionalized graphene oxide for activation of dendritic cells
Graphical abstract
Introduction
Dendritic cells (DCs) are regarded as major antigen-presenting cells (APCs), and their maturation and antigen presentation are closely related to the expression of costimulatory signals (e.g. CD80, CD86) and major histocompatibility complex (MHC) class I and II, which are crucial for T-cell activation in cancer immunotherapy [1], [2], [3], [4], [5], [6]. Previous studies have indicated that antigens with a nanocarrier or nanoadjuvant greatly enhance their immunogenicity for treating infectious diseases and cancers mainly by promoting the cellular uptake of antigens in DCs through an endogenous pathway [7], [8], [9], [10], [11], [12], [13]; however, most of them required modification and covalent conjugation of antigens with carriers, which might hinder their release in the intracellular environment or slightly alter their original structure.
Graphene oxide (GO) is a single-layer graphene derivative containing oxygen-functionalized groups, including carboxyl groups and ether groups. Compared with other non-viral carriers, the superior physicochemical properties and potentials of GO nanoparticles and their derivatives have been proven in biomedical fields [14], [15], [16]. Owing to its large and hydrophobic surface, GO can interact with hydrophobic or aromatic drugs [17], and doxorubicin and other aromatic anticancer drugs was loaded onto GO for delivery to tumor [18], [19]. GO derivatized with ether or carboxyl groups can provide flexibility of the chemical modifications on the surface and allow the development of novel multifunctional materials [20], [21], [22], [23], [24]. The modifications of the functional groups on the surface of GO can avoid strong ĻāĻ stacking forces between graphene sheets and increase their colloidal stability under physiological conditions [24]. Recent studies have shown that GO, which is biocompatible and nontoxic, can be degraded in the presence of human peroxidase and cleared from the body, ensuring the safety of GO application [25]. In addition, functionalized GO can activate DCs through a variety of toll-like receptor (TLR) pathways [26], [27], [28].
Since the discovery of GO, GO has been explored as a nanocarrier for the delivery of genes, anticancer drugs, and antigens [29]. It has rarely been adopted for the co-delivery of DNA vaccines and hydrophobic immune adjuvants in immunotherapy, and the synergistic effects of functionalized GO and other immune adjuvants have rarely been investigated. Compared with conventional vaccines, DNA vaccine has been considered a promising approach in immunotherapy for treating infectious diseases and cancers [30], [31], [32]. Owing to the aforementioned advantages of GO, we designed a simple strategy to achieve co-delivery of a DNA vaccine and hydrophobic immune adjuvant (resiquimod, R848) and enhance the ability of immune modulation via the synergistic effect between functionalized GO and R848. As low-molecular weight polyethylenimine (LMW PEI) can be cleared out of the body, we adopted thiol-modified LMW PEI (TPEI1.8) to link 4-aminothiophenol-modified GO (TGO) via the formation of disulfide bonds. TGO with its assembled TPEI1.8 could not only load R848 but also provide positive charges to interact with plasmid DNA encoding model antigen, ovalbumin (OVA). Owing to the reducibility of the disulfide bond [33], [34] and the āproton spongeā effect of PEI in the cellular environment, the DNA vaccine could be rapidly released in the cytosol. This system significantly promoted the expression of the DNA vaccine and successfully stimulated the expression of CD86 of DC for its maturation and antigen presentation (Scheme 1).
Section snippets
Materials
4-Aminothiophenol, LMW PEI (PEI 1.8K), ethylene sulfide, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), methanol, tetrahydrofuran (THF), Resiquimod (R848), phosphate-buffered saline (PBS), ethidium bromide, 6Ć loading dye, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 4ā²,6-diamidino-2-phenylindole (DAPI), an Alexa Fluor 488 DNA Labeling Kit, a 10% formalin solution, 1% penicillin/streptomycin, Ī²-mercaptoethanol, and GM-CSF were purchased from SigmaāAldrich (St.
Characteristics of TGO and TPEI1.8
We used 4-aminothiophenol for the modification of GO because 4-aminothiophenol not only provided thiol groups but also disrupted the ĻāĻ stacking forces between the original GO sheets. In general, the X-ray photoelectron spectra (XPS) exhibited signals corresponding to carbon, oxygen, and sulfur, as shown in Fig. 1A. The existence of a characteristic thiol (sulfur 2p binding energy peak around 164Ā eV) of TGO was observed (Fig. 1B), indicating that 4-aminothiophenol was successfully conjugated to
Conclusions
In this study, we designed a thiolated graphene (TGO)-based co-delivery system for a DNA vaccine and hydrophobic immune modulator, R848, to BMDC. TGO-TPEI1.8/pOVA/R848 can stimulate the maturation of BMDCs and presentation of the OVA antigen on MHC class I molecules, suggesting the synergic effect of the GO and its loaded R848. The interactions of activated DCs and naive T cells in draining lymph nodes may facilitate the generation of antigen-specific T cells for cancer immunotherapy.
Acknowledgements
This research was supported by National Research Foundation of Korea (NRF) grants (2010-0027955, 2017M3A9F5032628, 2018M3A9B5021319) funded by the Ministry of Science and ICT (MSIT), Industrial Strategic Technology Development Program (10077704) funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) (HI18C1174) funded by the Ministry of Health & Welfare.
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The authors contributed equally to this study.