Simultaneous delivery of DNA vaccine and hydrophobic adjuvant using reducible polyethylenimine-functionalized graphene oxide for activation of dendritic cells

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Highlights

  • ā€¢

    GO modified with PEI via a disulfide linkage was used as a carrier for the simultaneous cellular delivery of TLR agonist (R848) and DNA vaccine.

  • ā€¢

    Owing to the reducibility of the disulfide bond in the cellular environment, the DNA vaccine could be readily released to produce the antigen.

  • ā€¢

    The system achieved the enhanced expression of the costimulatory signals and the antigen presentation to DCs for their activation and maturation.

Abstract

The activation and maturation of dendritic cells are critical in immunotherapy, and the potency of DCs is associated with high levels of antigen presentation on major histocompatibility complex (MHC) class I and II and the expression of costimulatory signals. Graphene oxide (GO) and its derivatives have many excellent physicochemical properties, including large and hydrophobic surface available for interacting with hydrophobic or aromatic drugs via Ļ€ā€“Ļ€ stacking forces, flexibility of the chemical modification on the surfaces, and capacity of DC activation. In this study, we designed a simple strategy to achieve the co-delivery of a DNA vaccine and hydrophobic immune adjuvant (R848) and to enhance the adjuvanticity of R848 via the synergistic effect of GO and R848. Thiolated low-molecular-weight polyethylenimine (TPEI1.8) was crosslinked with 4-aminothiophenol-modified GO (TGO) via the formation of disulfide bonds. Thus, TGO with its assembled TPEI1.8 could not only load R848 but also electrostatically interact with the DNA vaccine. Owing to the reducibility of the disulfide bond in the cellular environment, the DNA vaccine could be readily released. This system can significantly enhance the DNA transfection, the expression of the costimulatory signal, and the level of antigen presentation to MHC class I DCs for their activation and maturation.

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 Ļ€ā€“Ļ€ 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 ā€œproton 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, 6Ɨ loading dye, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 4ā€²,6-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 Sigmaā€“Aldrich (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 Ļ€ā€“Ļ€ 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.

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