Carbohydrate-Functionalized rGO as an Effective Cancer Vaccine for

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Carbohydrate-Functionalized rGO as an Effective Cancer Vaccine for Stimulating Antigen-Specific Cytotoxic T Cells and Inhibiting Tumor Growth Arjyabaran Sinha,† Bong Geun Cha,† Youngjin Choi,† Thanh Loc Nguyen,† Pil J. Yoo,† Ji Hoon Jeong,‡ and Jaeyun Kim*,†,§,∥ †

School of Chemical Engineering, ‡School of Pharmacy, §Department of Health Sciences and Technology, Samsung Advanced Institute for Health Science & Technology (SAIHST), ∥Biomedical Institute for Convergence at SKKU (BICS), Sungkyunkwan University (SKKU), Suwon 16419, Republic of Korea S Supporting Information *

ABSTRACT: Efficient delivery of antigens to dendritic cells (DCs), potent antigen-presenting cells, and subsequent antigen presentation to initiate the production of activated cytotoxic T cells are vital parameters that determine the success of cancer immunotherapy. Here, we report dextran-functionalized reduced graphene oxide (rGOdextran) as an antigen delivery carrier for cancer immunotherapy. We synthesized dextran-functionalized rGO, where the dextran component facilitated good colloidal stability by exposing hydroxyl groups on the surface of reduced graphene oxide (rGO) and also enhanced cellular uptake via interaction with carbohydrate receptors present on DCs. High surface area and intrinsic hydrophobic surface of rGO facilitated high loading of the model antigen, ovalbumin (OVA). We found that rGO-dextran efficiently delivered OVA to DCs and enhanced the antigen presentation via major histocompatibility complex class I (MHC-I). In addition, the release of inflammatory cytokines, IL-12 and TNF-α, for DCs incubated with OVA-loaded rGO-dextran was remarkably higher than that for those incubated with soluble OVA. We also demonstrated that OVA-loaded rGO-dextran induced production of antigenspecific cytotoxic T cells in vivo and significantly inhibited tumor growth. Therefore, the proposed rGO-dextran could be a potent candidate for cancer vaccine and other immunotherapy.

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INTRODUCTION Effective production of activated T cells is an essential requirement in cancer immunotherapy.1,2 Activated T cells can kill cancer cells after recognizing antigens expressed by cancer cells. This antigen recognition is mediated by specific binding of T cells via surface receptors to antigenic peptide coupled with major histocompatibility complex (MHC) presented by antigen presenting cells.3−5 Dendritic cells (DCs) are highly effective antigen-presenting cells (APCs) that engulf antigens, present antigenic peptides on their surfaces in the form of MHC complexes, and become mature.6,7 ̈ T Mature DCs migrate to lymph nodes and interact with naive 3−5 cells to invoke antigen-specific activated T cells. Therefore, uptake of antigens and presentation of antigen-MHC complexes on the surfaces of DCs play a key role in DC maturation and the subsequent activation of T cells. However, poor uptake efficiency of small soluble antigens such as proteins and peptides by DCs leads to ineffective production of activated T cells.8,9 Thus, it is necessary to develop a suitable delivery system for cancer immunotherapy that can efficiently deliver antigens to DCs and produce antigen-specific cytotoxic T cells.10,11 © 2017 American Chemical Society

Nanoparticles are now extensively used as delivery carriers in cancer vaccines because of their high loading efficiency, resistance to enzyme degradation, and ability to effectively deliver antigens to antigen-presenting cells.12−14 Liposomes,15−17 porous silica nanoparticles,18,19 carbon-based nanoparticles,20 iron oxide nanoparticles,21 gold nanoparticles,22 and protein cages and polymeric nanoparticles23−26 have already been used for vaccine delivery in cancer immunotherapy. Although these nanoparticles successfully deliver protein, they suffer from low loading efficiency, require a complicated and lengthy synthesis procedure, have poor structural stability, require modification with antigens or other molecules, and are structurally complex because of covalent conjugation to proteins or antigens. Recently, graphene-based nanomaterials have received great attention as promising alternatives to drug and vaccine delivery systems because of their high surface area, hydrophobic surfaces, excellent adsorption capability, and biocompatible Received: May 29, 2017 Revised: July 27, 2017 Published: July 28, 2017 6883

DOI: 10.1021/acs.chemmater.7b02197 Chem. Mater. 2017, 29, 6883−6892

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Scheme 1. Schematic Representation of the Preparation of OVA@rGO-Dextran and Its Application as a Vaccine Delivery Carrier for Cancer Immunotherapy

Figure 1. (a) TEM image of rGO-dextran-40. Digital images of a colloidal solution of OVA@rGO-dextran-40 in water, PBS, and serum containing RPMI cell culture medium at day (b) 1 and (c) 7. (d) Hydrodynamic size of rGO-dextran-6 and rGO-dextran-40 before and after OVA loading. (e) Zeta-potential of rGO, rGO-dextran-6, and rGO-dextran-40 before and after OVA loading.

properties.27−32 However, preparation of stable colloidal graphene solution is difficult because of strong π−π interactions between graphene sheets.33 Hence, several approaches have been developed to prepare stable graphene solutions via surface modification with water-soluble molecules or polymers.31,34−39 For example, graphene oxide (GO) has been modified with polyethylene glycol,31,41 polyethylene amine,31,40,41 or carnosine,30 and then proteins or antigens have been loaded on the functionalized GO surfaces by chemical modification or physical adsorption for use as vaccine delivery carriers. Although the ability of a few GO-based materials with low loading efficiency of antigens to activate the immune system has been investigated, the application of graphene for vaccine

delivery has not yet been explored. Reduced GO (rGO) facilitates adsorption of proteins higher than that of GO because residual polar functional groups present in chemically reduced GO contribute hydrophilic groups to the intrinsically hydrophobic surface of graphene, but modification of the surface of graphene is very challenging because of graphene’s poor water solubility, paucity of functional groups, and high susceptibility toward chemical conjugation.33,42 Thus, to obtain good dispersion in water, high antigen loading, efficient cellular uptake, and inhibit nonspecific binding, surface modification of graphene with suitable biomolecules is necessary. Here, we report a simple method for the synthesis of an rGO-based vaccine delivery carrier and characterize the effects 6884

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Figure 2. (a) FTIR spectra of rGO before and after dextran functionalization and of dextran only. Spectra of rGO displayed the characteristic peak of dextran after functionalization as highlighted by the dotted line. (b) Raman spectra of GO and rGO before and after dextran functionalization. The graph exhibits the D and G bands of graphite-type materials and the calculated of ID/IG ratios of GO and the various functionalized rGO materials. (c) TGA graph of rGO and dextran-functionalized rGO. In the temperature region of 260−350 °C, weight loss of around 35−40% was observed due to dextran functionalization.

formation of reduced GO.28,35,36 The resultant dextranfunctionalized rGO (rGO-dextran) was highly stable in water because the hydroxyl groups of dextran were exposed on the surface of rGO. Finally, OVA was loaded on the surface of rGO-dextran via simple mixing based on hydrogen bonding and hydrophobic interaction between rGO-dextran and the proteins. The resulting OVA@rGO-dextran was retrieved via repetitive centrifugation and washing, dispersed in water, and stored at 2−8 °C for further study. Characterization of Materials. The morphology of rGOdextran was observed by transmission electron microscopy (TEM) (Figure 1a), which clearly showed the sheet-like morphology of graphene. Colloidal stability of a material is an important parameter for its biological application. Generally, graphene aggregates in water due to strong π−π interactions between graphene sheets. To evaluate the colloidal stability of OVA@rGO-dextran, samples were dispersed in water, phosphate buffered saline (PBS), and 10% serum-containing cell culture medium, and aggregation was investigated over time (Figures 1b and c). OVA@rGO-dextran showed no aggregation in any of the tested media, even after aging for up to seven days, suggesting that OVA@rGO-dextran had good colloidal stability. This good colloidal stability of OVA@rGO-dextran is probably attributed to the large amount of hydroxyl groups contributed by dextran on the graphene surface.28,35 Figure 1d shows the hydrodynamic sizes of rGO-dextran and OVA@ rGO-dextran prepared with dextran of different molecular weights obtained from a dynamic light scattering study. The hydrodynamic sizes of rGO-dextran-6 (prepared with dextran with MW of 6 kDa) and rGO-dextran-40 (prepared with dextran with MW of 40 kDa) were 290 and 295 nm,

of this carrier on the maturation of DCs, production of antigenspecific activated T cells, and inhibition of tumor growth. Functionalization of dextran, a carbohydrate molecule, on the surface of rGO afforded good water dispersibility, minimized nonspecific binding similar to polyethylene glycol, and facilitated efficient cellular uptake of particles by interaction with carbohydrate receptors present on the surfaces of DCs. In addition, rGO allowed high loading of ovalbumin, a model antigenic protein, because of its hydrophobic surface. rGOdextran effectively delivered model antigens to DCs and promoted their maturation. Furthermore, an animal study based on subcutaneous vaccination of OVA-loaded rGOdextran revealed that vaccination induced systemic activation of antigen-specific cytotoxic T cells and thus inhibited tumor growth. All of these properties make rGO-dextran a promising vaccine delivery carrier for cancer immunotherapy.

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RESULTS AND DISCUSSION

Synthesis of OVA-Loaded rGO-Dextran. The synthesis strategy to prepare dextran-functionalized, OVA-loaded rGO (OVA@rGO-dextran) and its application as vaccine delivery carrier for cancer immunotherapy is shown in Scheme 1. First, water-soluble colloidal GO prepared via the modified Hummer’s method43 was mixed with dextran followed by the addition of ammonia under stirring condition. In this step, dextran was attached to the surface of GO via hydrogen bonding, van der Waals interactions, and covalent binding between the hydroxyl groups of dextran and epoxide groups of GO. Next, GO was reduced chemically by the addition of hydrazine at 75 °C, and the color of the resulting solution changed from brown to black within a few minutes due to 6885

DOI: 10.1021/acs.chemmater.7b02197 Chem. Mater. 2017, 29, 6883−6892

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Figure 3. (a) Viability of BMDCs after a 24 h incubation with different concentrations of rGO-dextran-6 or rGO-dextran-40, which was estimated using CCK-8 assay. (b) Fluorescent microscopic images of BMDCs incubated with soluble OVA-FITC or OVA-FITC@rGO-dextran-40 followed by staining with lysotracker red and DAPI. Green, OVA-FITC; red, lysotracker red; blue, DAPI (scale bar indicates 50 μm). (c) Flow cytometry quantification results for uptake of OVA-FITC by BMDCs after incubation with soluble OVA-FITC or OVA-FITC@rGO-dextran-40 for 6 h. *p < 0.01.

biomedical fields. We investigated the cytotoxicity of rGOdextran by cell counting kit-8 assay (CCK-8) by adding different doses of materials to BMDCs followed by a 24 h incubation (Figure 3a). Cells maintained more than 80% viability even at the highest concentration of rGO-dextran tested (100 μg/mL), suggesting that the materials were nontoxic in the range of concentrations we evaluated. Efficient uptake of antigen by APCs is a key requirement for antigen presentation and stimulating immune responses. To measure cellular uptake efficiency, OVA labeled with fluorescein isothiocyanate (OVA-FITC) was loaded on the surface of rGO-dextran by simple mixing of OVA-FITC and rGO-dextran followed by the separation of free OVA-FITC by extensive washing with water. Absorbance spectrum of an aqueous dispersion of OVA-FITC@rGO-dextran showed characteristic absorbance peak of FITC, revealing successful loading of OVA-FITC (Supporting Information, Figure S1a). To measure the binding efficiency of other protein on rGOdextran materials, bovine serum albumin (BSA) and glucose oxidase (GOx) were also loaded on rGO-dextran. The results show that 1.4, 1.1, and 1.7 mg of OVA, BSA, and GOx were loaded per mg of rGO-dextran, respectively, suggesting effective binding of protein with rGO-dextran (Supporting Information, Figure S1b). We also determined the release kinetics of OVAFITC from OVA-FITC@rGO-dextran in phosphate buffered saline (PBS). The amount of OVA released was calculated by measuring the fluorescence intensity of the supernatants. Release profile showed no significant release of OVA-FITC from rGO-dextran (Supporting Information, Figure S1c), demonstrating that the interaction between OVA and rGOdextran was strong enough to hold OVA antigen on the surface

respectively. Hydrodynamic size of OVA@rGO-dextran increased to around 350 nm for both samples due to adsorption of OVA on the surface of the rGO-dextran. Zeta-potential measurement showed a highly negative surface charge due to presence of hydroxyl groups on the rGO surface (Figure 1e). Functionalization of dextran on the surface of rGO was further confirmed by Fourier transform infrared spectroscopy (FTIR) (Figure 2a). FTIR spectra of rGO-dextran-6 and -40 showed characteristic bands at 1110, 1155, 1640, and 2921 cm−1 corresponding to C−O/C−C stretching/−O−H bending vibrations, C−O−C stretching/−O−H bending vibrations, H− OH vibrations of hydroxyl groups, and CH2 stretching vibrations, respectively, indicating the successful functionalization of dextran on rGO.35,36 All Raman spectra of GO, rGO, and rGO-dextran samples showed the characteristic D and G bands of graphene-based materials at 1380 and 1590 cm−1, respectively (Figure 2b). The intensity ratio of D and G bands (ID/IG) of GO and rGO used to determine the extent of disorder were 0.92 and 1.1, respectively, indicating that successful reduction did take place after the addition of hydrazine. The ID/IG ratio further increased in rGO-dextran compared to that in rGO, probably due to the increase in edge defects upon functionalization with dextran.42,44 The amount of dextran attached on the surface of rGO was estimated by thermogravimetric analysis (TGA) (Figure 2c). The rGO displayed no significant weight loss in the temperature range of 260−350 °C, while rGO-dextran exhibited around 35 wt % extra weight loss in the same temperature range, indicating that around 35 wt % dextran was present on the rGO surface. Cytotoxicity and Cellular Uptake. Toxicity of nanomaterials has to be evaluated prior to their application in 6886

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Figure 4. Expression of (a) costimulatory molecule CD86 and (b) SIINFEKL-MHC I on CD11c+ cells after incubation of BMDCs with rGOdextran, OVA, and OVA@rGO-dextran-40, which was assessed by flow cytometry. Release of cytokines (c) IL-12 and (d) TNF-α in the cell culture supernatant of BMDCs incubated with different doses of materials, which was measured using ELISA. *p < 0.01.

on the DCs surfaces and the carbohydrate moieties of dextran present on the surface of rGO.46 Enhancement of OVA uptake by rGO-dextran could facilitate presentation of antigen and stimulate adaptive immune responses. In Vitro DC Maturation. Mature DCs express antigenic peptides on their cell surfaces via MHC complexes that interact ̈ T cells.4−6,47 In with T cell receptors (TCRs) on naive addition, mature DCs express high levels of the costimulatory molecules CD80/86 that interact with CD 28 expressed on ̈ T cells. Both interactions are required to successfully naive induce cytotoxic T cells that can recognize target cells and kill them in an antigen-specific manner.4,5 Hence, the induction of mature DCs plays a key role in the production of cytotoxic T cells, which is crucial for an effective immune response. To examine the effect of rGO-based nanomaterials on the maturation of DCs, soluble OVA, a mixture of dextran and soluble OVA, OVA@rGO, rGO-dextran, and OVA@rGOdextran were incubated with immature BMDCs for 16 h; the amounts of OVA and rGO-dextran used were the same as those present in the OVA@rGO-dextran sample. The maturation of DCs was evaluated by flow cytometry by measuring the expression level of CD86 costimulatory molecules and MHC class I molecules complexed with SIINFEKL peptide (an epitope of OVA) by BMDCs. First, surface modification of rGO with dextran of higher molecular weight (40 kDa) resulted in higher expression of the maturation marker (Supporting Information, Figure S2) than surface modification of rGO with dextran of lower molecular weight (6 kDa), demonstrating that rGO-dextran-40 had a positive adjuvant role on the maturation of DCs greater than that of rGO-dextran-6. On the basis of this

of rGO-dextran in media. This is advantageous for the delivery of a large amount of antigens to DCs without significant loss of antigen after injection into the body until reaching to DCs. Next, we investigated intracellular delivery of OVA@rGOdextran to bone marrow-derived dendritic cells (BMDCs). After incubating soluble OVA-FITC and OVA-FITC@rGOdextran with BMDCs for 6 h followed by staining with lysosome tracker (red), BMDCs were analyzed by fluorescent microscopy (Figure 3b). Higher green emission was observed in BMDCs incubated with OVA-FITC@rGO-dextran than that observed cells incubated with soluble OVA-FITC, representing that rGO-dextran facilitated the cellular uptake of OVA presumably via an enhanced endocytosis. The merged images show that most of the green fluorescence overlapped with the red fluorescence of lysosome tracker, indicating that OVA trafficked to lysosome. In contrast, some localization and separation of green dots from red signals were observed in the merged image of BMDCs incubated with OVA-FITC@rGOdextran. This data represents that a certain extent of lysosome escape occurred in the case of OVA-FITC@rGO-dextran, which is important for antigen cross-presentation.45 To further quantify cellular uptake of the model antigen, the cultured BMDCs were analyzed by flow cytometry (Figure 3c). The fluorescence intensity of FITC was much higher in cells incubated with OVA-FITC@rGO-dextran than in cells incubated with the same amount of soluble OVA-FITC. These results suggest that OVA-FITC@rGO-dextran significantly enhanced the cellular uptake of OVA compared to soluble OVA. This enhanced uptake of OVA by BMDCs might arise due to interaction between carbohydrate receptors present 6887

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Figure 5. (a) Schematic representation of experimental procedure. Mice were immunized twice subcutaneously with PBS, OVA, or OVA@rGOdextran-40 with a seven day interval between immunizations. At day 14, mice were sacrificed, and cells were collected from the lymph node and spleen for analysis of T cell populations using flow cytometry. (b−d) Flow cytometry results for CD8+, CD4+, and tetramer+ T cells population, respectively, collected from lymph node. (e−g) Flow cytometry results for CD8+, CD4+, and tetramer+ T cell populations, respectively, collected from the spleen. *p < 0.05.

those incubated with soluble OVA and rGO-dextran for all tested doses of materials, indicating that OVA@rGO-dextran significantly enhanced the immunostimulatory capability of DCs. In Vivo activation of CD8+ and CD4+ T cells. Motivated by the positive in vitro results, we further investigated the ability of OVA@rGO-dextran to activate CD8+ and CD4+ T cells in vivo, as these cells are essential for eliminating tumors, viruses, and pathogen infection. Prior to conducting our in vivo activation experiments, we first evaluated if rGO-dextran can be successfully delivered to draining lymph nodes, where adaptive immune responses are initiated.53 To investigate antigen delivery efficiency to lymph nodes, mice were subcutaneously injected with rGO-dextran loaded with rhodamine 6G (R6G) dye. Twenty-four hours after injection, mice were sacrificed and fluorescently imaged on an optical imaging system. Fluorescent images clearly showed a strong fluorescence signal in the draining lymph node (Supporting Information, Figure S4), indicating effective delivery of rGO-dextran to the lymph nodes after subcutaneous injection, probably through lymphatic flow. To measure in vivo adaptive immune responses, materials were subcutaneously injected in mice twice at an interval of seven days (Figure 5a). Seven days after the final immunization, cells were collected from the spleen and drained lymph node and stained with anti-CD8a, anti-CD4, anti-CD3e, and MHC class I tetramer, and activation of antigen specific CD8+ and CD4+ T cells was evaluated by flow cytometry. Vaccination with OVA@rGO-dextran activated both CD8+ (Figures 5b and e) and CD4+ (Figures 5c and f) T cells in the lymph node (Figures 5b and c) and spleen (Figures 5e and f) more

result, we selected rGO-dextran-40 for further in vitro and in vivo studies. Flow cytometry data showed that enhanced expression of CD86 (Figure 4a, Supporting Information, Figure S3) and MHC I-SIINFEKL (Figure 4b) were induced on BMDCs incubated with OVA@rGO-dextran compared to BMDCs incubated with soluble OVA, a mixture of dextran and soluble OVA, OVA@rGO, and rGO-dextran. Taken together with the results of the intracellular uptake study, OVA@rGO-dextran promoted maturation of DCs via enhanced intracellular delivery of antigens into DCs in vitro. Inflammatory Cytokine Secretion from DCs. Cytokines are small proteins secreted from activated immune cells that stimulate the differentiation and activation of a variety of T cells called natural killer cells.48−50 For example, interleukin 12 (IL12), an inflammatory cytokine produced by activated immune cells, is essential for differentiation and activation of both innate and acquired immunity.51 Tumor necrosis factor alpha (TNFα), another pro-inflammatory cytokine, is also produced by activated immune cells and stimulates the production of reactive nitrogen intermediates and the migration of immune cells to the infection site.52 Hence, the secretion of IL-12 and TNF-α by DCs is necessary for viral infection, clearance of pathogens, and cancer immunotherapy. We investigated the amount of IL-12 and TNF-α released into cell culture supernatant by enzyme-linked immunosorbent assay (ELISA) after incubating immature BMDCs with soluble OVA, rGOdextran, or OVA@rGO-dextran for 16 h (Figures 4b and c). Both IL-12 and TNF-α were released from DCs in a dosedependent manner, and the concentration was remarkably higher for cells incubated with OVA@rGO-dextran than for 6888

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Figure 6. (a) Schematic representation of experimental procedure used for prophylactic vaccination of C57BL/6 mice. Mice were vaccinated twice via subcutaneous injection with PBS, soluble OVA, or OVA@rGO-dextran-40 at seven day intervals. Tumors were generated by inoculation of mice with B16-OVA cells 7 days after the final vaccination, and mice were sacrificed after 18 days. The size of tumors was measured using calipers every 1 or 2 days, and the changes in tumor volume are presented in panel b (n = 6). (c) Digital images showing the size of the final tumors after isolation. (d) Flow cytometry results of CD3+CD4+ and CD3+CD8+ T cell populations collected from the spleen. *p < 0.01.

can adsorb large amounts of antigen. Furthermore, antigenic proteins are strongly adsorbed on the graphene surface and not easily released from the surface of rGO-dextran until uptake by DCs after administration into the body. Last, dextran present on rGO-dextran facilitates high cellular uptake via interaction with carbohydrate receptors on DCs. Small doses of rGOdextran were therefore able to deliver a large amount of OVA to DCs and significantly activate antigen-specific T cells.

efficiently than soluble OVA, although the same amount of OVA was present in the injected materials. In vivo generation of antigen-specific cytotoxic T cells was accessed by tetramer staining (Figures 5d and g). MHC class I tetramer specifically binds to T cell receptors that recognize the antigen epitope (SIINFEKL) expressed on target cells. OVA@rGO-dextran significantly increased the tetramer+CD8+ T cell population in both lymph nodes (Figure 5d) and the spleen (Figure 5g) compared to soluble OVA. All of these results suggest that OVA@rGO-dextran can successfully induce adaptive cellular immunity in vivo. Prophylactic Vaccination Study. To further evaluate the effectiveness of OVA@rGO-dextran as a vaccination platform, we vaccinated mice twice with PBS, soluble OVA (140 μg), and OVA@rGO-dextran loaded with 140 μg OVA with a 7 day interval (Figure 6a). Seven days after the immunization, we challenged the mice with tumor cells by subcutaneous inoculation of OVA-expressing B16 melanoma (B16-OVA) cells and monitored tumor growth over time (Figures 6b and c). Vaccination with OVA@rGO-dextran significantly inhibited tumor growth compared to that with vaccination with soluble OVA and PBS. This indicates that OVA@rGO-dextran can significantly activate antigen-specific cytotoxic T cells and induce strong antitumor immunity. To further confirm activation of T cells, mice were sacrificed at 18 days posttumor inoculation, and the spleen was isolated to analyze CD4+ and CD8+ T cells (Figure 6d). Higher population of CD4+ and CD8+ cells were found in animals vaccinated with OVA@rGO-dextran than in animals vaccinated with soluble OVA or PBS. Taken together, these data strongly indicate that OVA@rGO-dextran can be used as a vaccine platform for cancer immunotherapy because it enhances antigen presentation by DCs in lymph nodes and activates antigen-specific T cells systemically after subcutaneous vaccination. The strong potential of rGO-dextran as a vaccination delivery platform is due to its high dispersibility in serum and its flat surfaces that

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CONCLUSION In summary, we studied the potential application of rGOdextran as a vaccine delivery carrier for cancer immunotherapy. Our rGO-based delivery system had good colloidal stability, was biocompatible, and showed high loading efficiency for a model antigenic protein. In vitro studies revealed that rGOdextran could deliver antigens to DCs efficiently, induce maturation of DCs expressing high levels of CD86 costimulatory molecules and MHC class I-SIINFEKL complexes, and induce the production of high concentrations of pro-inflammatory cytokines. Subcutaneous administration of OVA@rGO-dextran resulted in delivery to the lymph nodes. Furthermore, mature DCs generated by OVA@rGO-dextran were able to effectively activate CD4+ and CD8+ T cells in vivo, which in turn suppressed tumor growth in a prophylactic cancer vaccine experiment. Together, our results indicate that dextran-functionalized rGO is a suitable delivery system for cancer immunotherapy.

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EXPERIMENTAL SECTION

Materials and Reagents. Graphite powder, ovalbumin, bovine serum albumin (BSA), glucose oxidase (GOx), RPMI 1640 (Roswell Park Memorial Institute), heat-inactivated fetal bovine serum (HIFBS), and cell counting kit-8 (cck-8) were purchased from SigmaAldrich. Dextran of molecular weights 6 and 40 kDa was purchased from TCI chemicals. Lysotracker red and 4′,6-diamidino-2-phenylindole (DAPI) were received from Invitrogen. Sulfuric acid (H2SO4), sodium nitrate (NaNO3), sodium chloride (NaCl), potassium 6889

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Chemistry of Materials permanganate (KMnO4), hydrazine, and ammonium solution (28%) were purchased from JUNSEI and used as received. Granulocytemacrophage colony-stimulating factor (GM-CSF) was obtained from Preprotech. Anti-CD11c-APC, anti-CD86-PE, anti-H-2kb/SIINFEKLPE-Vio770, anti-CD8a-APC, anti-CD4-PE-Vio770, anti-CD3e-FITC, and tetramer-SIINFEKL-PE were purchased from Miltenyi Biotec and used as received. Animal Information. Six to nine week old female C57BL/6 mice (DBL, Korea) were used in all animal experiments. All animal experiments were conducted with the approval of the SKKU School of Pharmacy Institutional Animal Care and Use Committee. Synthesis of GO. Water-soluble colloidal GO was prepared via the modified Hummer’s method. In brief, 0.2 g of graphite powder and 0.1 g of NaNO3 were mixed with 5 mL of concentrated H2SO4 under stirring in an ice bath. Next, 0.6 g of KMnO4 was added in a stepwise manner, and the temperature of the solution was maintained at lower than 20 °C. The temperature of the solution was then raised to 36 °C, and the reaction was allowed to proceed for 30 min. After that, the solution was diluted with 14 mL of water, and the temperature was increased to 98 °C for 15 min. The color of the solution changed from black to brown at this stage. Next, 0.5 mL of 3 wt % H2O2 was added to remove the excess permanganate. The resulting particulates were extensively washed with warm water until the pH of the solution becomes neutral and then air-dried. The solid powder was mixed with water and sonicated for 30 min. Finally, the solution was centrifuged at 3000 rpm for 30 min to remove larger particles, and the supernatant was used as GO. Synthesis of rGO-Dextran. To synthesize rGO-dextran, 250 mg of dextran (molecular weight 6 or 40 kDa) was added to 10 mL of 1 mg/mL GO solution followed by addition of 100 μL of NH3 solution under stirring condition. After 30 min, 50 μL of hydrazine was added to this solution, and the resulting solution was heated to 75 °C for 1 h. Next, to remove excess reagent, rGO-dextran was precipitated by the addition of NaCl and centrifuged. Finally, rGO-dextran was extensively washed with water and dispersed in water via sonication for further use. rGO-dextran-6 and rGO-dextran-40 represent rGO functionalized with dextran of molecular weight 6 and 40 kDa, respectively. Loading of Various Proteins (OVA, BSA, and GOx) in rGODextran. To load protein, 2 mL of 20 mg/mL protein (OVA, BSA, and GOx) solution was added to 2 mL of 1.5 mg/mL rGO-dextran solution and stirred for 6−8 h. Next, protein loaded rGO-dextran (OVA/BSA/GOx@rGO-dextran) was separated by centrifugation, and the supernatant was discarded. Protein loaded rGO-dextran was washed another two times with water to remove weakly adsorbed protein and redispersed in 2 mL of water for further use. The amount of protein loaded on rGO-dextran was calculated by the difference in absorbance of the protein solution before and after adsorption on the rGO-dextran surface. The calculated loading amount of OVA, BSA, and GOx were 1.4, 1.1, and 1.7 mg per mg of rGO-dextran, respectively. Cytotoxicity Assay. For cytotoxicity testing, BMDCs were first cultured in a 24-well plate in RPMI-based cell culture medium (10% HI-FBS, 1% penicillin/streptomycin) containing 20 ng/mL GM-CSF. Next, rGO-dextran was added to the cell culture medium, and cells were incubated for 24 h. Then, cells were washed three times with PBS, and 0.5 mL of fresh culture medium was added followed by addition of 10 μL of CCK-8 solution. After incubation for 3 h, the absorbance of solution at 450 nm was read using a microplate reader. Cell viability was expressed as a percentage of untreated cells (100%). Maturation of BMDCs. Bone marrow cells were collected from C57BL/6 mice and cultured in a Petri dish at a concentration 2 × 106 cells in complete RPMI medium containing 20 ng/mL GM-CSF to generate BMDCs. On day 7, BMDCs were seeded into 6-well plates (1 × 106 cells per well) and stabilized for 12 h. Next, different concentrations of rGO-dextran-6 and rGO-dextran-40 were mixed with the medium followed by another 16 h incubation. Then, cells were collected from the plate and washed with PBS and fluorescentactivated cell sorting (FACS) buffer. Collected cells were incubated with anti-CD11c-APC, anti-CD86-PE, and anti-H-2kb/SIINFEKL-PEVio770 for 20 min at 4 °C. Finally, cells were washed with FACS

buffer and analyzed using a FACS instrument (Miltenyi Biotec, MACSQuant VYB). Cellular Uptake by BMDCs. To measure OVA uptake efficiency, BMDCs were cultured in a 24-well plate in RPMI medium (1 × 106 cells per well). Next, cells were incubated with different doses of OVAFITC and OVA-FITC@rGO-dextran-40 containing the same concentration of OVA. After 6 h, cells were washed with PBS to remove excess particles. After that, cells were incubated with lysotracker red and DAPI for staining of lysosome and nucleus, respectively, according to the manufacturer’s instructions. The stained cells were imaged under a fluorescent microscope (Ti-U, Nikon). For quantitative analysis of cellular uptake of OVA, cells were incubated with OVA-FITC and OVA-FITC@rGO-dextran-40 for 6 h, collected from the plate, washed with a buffer, and analyzed by flow cytometry (Miltenyi Biotec, MACSQuant VYB). Effect of OVA, OVA@rGO-Dextran-40, rGO-Dextran-40, OVA@rGO, and Mixture of Dextran-40 and OVA on the Maturation of BMDCs. Overall procedure was similar to the BMDC maturation experiments based on rGO-dextran except concentrations of 10, 20, and 50 μg/mL rGO-dextran-40, OVA@rGO-dextran-40, OVA@rGO, dextran-40/OVA mixture solution, respectively, and soluble OVA at the same concentration present in OVA@rGOdextran were used. Quantification of IL-12 and TNF-α. BMDCs (1 × 106 cells per well) were incubated with different doses of rGO-dextran-40, OVA@ rGO-dextran-40, or soluble OVA for 16 h. Cell culture medium was collected and centrifuged at 2000 rpm for 5 min at 4 °C. Levels of IL12 and TNF-α present in the supernatants were assessed by ELISAs (BD Bioscience) following the protocols recommended by the manufacturer. Effect of Materials on In Vivo Activation of T cells. C57BL/6 mice were used to investigate T cell activation. Mice were subcutaneously injected twice with 100 μL of PBS, soluble OVA (140 μg), or OVA@rGO-dextran (100 μg rGO-dextran loaded with 140 μg OVA) dispersed in 100 μL PBS with a 7 day interval. Seven days after the final immunization, mice were sacrificed, and cells were collected from the spleen and drained lymph node. Retrieved cells were stained with anti-CD8a-APC, anti-CD4-PE-Vio770, anti-CD3eFITC, and tetramer-SIINFEKL-PE following the protocol recommended by the manufacturer. Finally, flow cytometry was used to analyze cells. Prophylactic Vaccination Study against B16-OVA Tumor. For the prophylactic vaccination study, 6−8 week old C57BL/6 mice were vaccinated twice with 100 μL of PBS, soluble OVA (140 μg), or OVA@rGO-dextran (100 μg rGO-dextran loaded with 140 μg OVA) dispersed in 100 μL PBS with a 7 day interval. Fourteen days after the final vaccination, mice were subcutaneously challenged with 0.8 × 106 B16-OVA cells suspended in 0.1 mL PBS. Every two, three, or four days after B16-OVA cell inoculation, tumor size was measured using calipers. Tumor volume was estimated using the equation V = 1 /2(width)2 × length, where length was the longer dimension and width the shorter dimension. Mice were sacrificed 18 days post-tumor inoculation, and the spleen was retrieved for further T cell analysis. Statistical Analysis. All values in this study were presented as mean ± standard deviation (as error bar) with three or more than three replicates. Statistical analysis was performed with F-test and student’s t-test (two-tailed).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b02197. Characterization of OVA-FITC loading, release profile of OVA-FITC from rGO-dextran, effect of rGO-dextran40/rGO-dextran-6 on activation of BMDCs, effect of dextran OVA mixture and OVA@rGO on the maturation of BMDCs, and in vivo imaging of lymph node after 6890

DOI: 10.1021/acs.chemmater.7b02197 Chem. Mater. 2017, 29, 6883−6892

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Chemistry of Materials

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subcutaneous injection of R6g@rGO-dextran in mice (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Tel.: +82-31-290-7252; Fax: +82-31-290-7272. ORCID

Jaeyun Kim: 0000-0002-4687-6732 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants funded by the National Research Foundation (NRF) under the Ministry of Science, ICT & Future Planning, Republic of Korea (Grants 20100027955 and 2015R1A2A2A01005548) and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant HI17C0076).

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