3.1 NaVs Preparation and Characterization
DOPE and CpG ODN were covalently linked using SPDP as the crosslinking agent. Successful synthesis of DOPE-S-S-CpG ODN was confirmed by agarose gel electrophoresis (Figure S1). The resulted DOPE-S-S-CpG ODN and OVA (as model antigen) self-assembled forming into NaVs. The morphology of NaVs was characterized by TEM and the image showed the formulations were uniform nanocapasules (Fig. 1A). The diameter of NaVs determined by dynamic light scattering (DLS) was 192.4 ± 0.66 nm, while the blank nanocapsule without OVA (named as SNCs) was 138.2 ± 0.90 nm (Fig. 1B and Table S1). And the size of NaVs remained stable in PBS solution for 45 days approximately (Fig. 1C). The loading capacity of OVA in NaVs determined by an Enhanced-BCA Protein Assay Kit was (26.4 ± 0.77) %. OVA had a burst release of 30% from NaVs during the first two days under conditions that mimic intracellular reducing environment (4 mM DTT in 7.4 pH buffer) and followed with a controlled release reaching about 65% in 25 days, which was faster than the release behavior of OVA (44.99 ± 1.69) % in the condition without DTT (Fig. 1D), indicating the NaVs did possess the redox-responsive property allowing antigens quick release after uptake into cells, and the nanovaccines could provide strong initial antigens stimuli and long-term sustained antigens exposure to immune cells which was important for eliciting powerful immune responses.
3.2 Immunization Studies in vitro
3.2.1 Cytotoxicity Assessment
The cell viability of BMDCs treated with NaVs at various concentrations were assessed by CCK-8 assay. According to the experimental result, even when the concentration of OVA was up to 60 µg/ml, cell viability remained high (102.1 ± 5.77) %, which demonstrated that these nanovaccines showed excellent safety to BMDCs (Fig. 2A).
3.2.2 Endocytosis Mechanism of NaVs
We next investigated antigen uptake capacity of NaVs. After co-incubated with free FITC-OVA or NaVs for 8 h respectively, images taken with confocal fluorescence microscopy showed that much more significant OVA signal appeared in BMDCs treated with NaVs than free OVA group. Fluorescence analysis of representative image using Image J software indicated that the mean fluorescence intensity (MFI) of OVA in NaVs group far exceeded that in free OVA group, which was enhanced about 6-fold. The results of the flow cytometry also demonstrated that NaVs facilitated uptake of antigens in BMDCs (93.0% vs 17.3%) (Fig. 2B-D).
Although Chad A. Mirkin et al. reported that high density oligonucleotides on the surface of nanoparticles could bind scavenger receptors on the surface of macrophages and promote nanoparticles uptake into macrophages [10], in an attempt to further explore the endocytic pathways and investigate the mechanism of nanoparticles uptake in DCs which is important for initiating immune responses for vaccines, we pretreated BMDCs with methyl-β-cyclodextrin (MBCD), chlorpromazine (CPM) and fucoidan (FCD), respectively. MBCD can remove cholesterol from the cell membrane effectively inhibiting caveolin-dependent endocytosis, CPM can block the formation of clathrin-coated pits, while FCD is a pharmacological inhibitor of class A scavenger receptor (SR-A) [15–18]. As shown in Fig. 2E and 2F, compared to untreated cells, FCD led to a drastic reduction of antigens uptake of NaVs (dropped up to 5.2%), MBCD reduced the uptake of cells to 36.0%, while CPM caused an uptake decrease to 24.9%. All the data suggested that NaVs internalized into BMDCs through multiple receptor-mediated endocytosis pathways, but mainly mediated by SR-A. Taken together, NaVs constructed with the conjugate of adjuvants CpG ODN and biomimetic materials DOPE having a "3D" structure could load antigens in the cavity of nanocapsules as vaccine delivery nanocarriers, and the high density nucleotides on the surface of nanocapsules could bind the various receptors on the surface of BMDCs and promote antigens endocytosis circumventing the drawbacks of free antigens, which in turn facilitated CpG ODN interaction with TLR9 receptor in endosomes and activated antigen presenting cells [19–25].
3.2.3 BMDCs Activation and Antigens Cross-Presentation
In order to further verify whether NaVs could promote BMDCs maturation and activation, which was essential for initiation of antigen-specific immune responses, we co-incubated immature BMDCs with PBS, Free OVA, Free OVA + CpG and NaVs for 6 h, respectively. The expression level of co-stimulatory molecules CD86 and CD40 on BMDCs treated with NaVs was nearly doubled compared to other groups (Fig. 3A and 3B). Moreover, we demonstrated that the SIINFEKL OVA-CD8+ epitope presented by MHC class I H-2Kb molecules on the surface of BMDCs treated with NaVs increased nearly 4 times compared with PBS groups (Fig. 3C), indicating that NaVs could promote antigens cross-presentation via MHC-I pathway which would subsequently initiate CD8+ T cell responses which are critical for cancer treatment, while exogenous antigens are mainly presented via the MHC-II peptide complex [26, 27]. Furthermore, the results of enzyme-linked immunosorbent assay (ELISA) of cytokines showed that BMDCs co-incubated with NaVs had a remarkable enhanced level of TNF-α compared with other groups (Fig. 3D) and IL-6 (Fig. 3E), especially IL-6 was enhanced up to ten-fold compared to PBS group. IL-6 can stimulate the production of chemokines and promote the activation and proliferation of B cells [28, 29], while TNF-α has important roles in stimulating T cell expansion and inducing a potent tumor-specific CTLs [30, 31]. Therefore, these in vitro results showed that NaVs could promote DCs maturation, activation and antigen cross-presentation favoring induction of antigen-specific CTLs-polarizing immune responses.
3.3 Immunization Studies in vivo
3.3.1 Antigen Release Behavior of NaVs in vivo
Encouraged by the light of results in vitro, we next sought to examine antigen depot effect and release studies of NaVs in vivo using the Maestro imaging system. As shown in the Fig. 4A, fluorescence Cy7 intensity signals of free OVA reached zenith about 6 hours after injection and then decreased gradually until it is not detected at 120 h. In contrast, the initial fluorescence intensity of NaVs increased relatively slowly and reached peak value at 12 h and still 25% remained at the sites after 7 days (Fig. 4B). The above results indicated that NaVs could exhibit strong initial immune stimulation and sustained long-term antigen supply in vivo.
3.3.2 Therapeutic Effect of NaVs
We then established E.G7-OVA tumor models and further examined the in vivo therapeutic effect of NaVs. As shown in Fig. 4C and 4E, compared with PBS group, NaVs vaccination could significantly suppress tumor growth, OVA + CpG moderately inhibited tumor growth, while free OVA only offered a slight tumor growth inhibition. In terms of survival prolongation, most the animals immunized with NaVs survived over 40 days, while mice treated with other groups almost died within 41 days (Fig. 4D). So, NaVs vaccination could offer a significant protection and remarkably prolong the life span of immunized mice. Moreover, the results of H&E staining (Fig. 4F) and TUNEL apoptosis of tumor tissues (Figure S2) showed that, compared with other groups, NaVs vaccination could effectively promote necrosis and apoptosis of tumor cells.
3.3.3 OVA-Specific Antibody Production
In order to further verify the type of immune response triggered by the NaVs and confirm the direction of lymphocyte differentiation, we first analyze the type and amount of IgG in the blood of tumor-bearing mice which reflects the subtype of Th cells. It's generally acknowledged that the level of IgG1 antibodies are associated with Th2 immune response, whereas the production of IgG2a antibodies are characterized for Th1 responses [32, 33]. As shown in Figure S3, mice immunized with the NaVs exhibited the highest OVA-specific IgG levels among all the groups, especially characterized with high IgG2a/IgG1 ratio as well, which meant NaVs polarized immune response towards Th1 type bias. In addition, Th1-dominated responses have potential for induction of CTL responses to eliminate antigen-specific tumor cells [34]. Therefore, NaVs not only had the property of significantly inducing the production of IgG antibodies, but also had the trend of eliciting Th1-type cell immune responses.
3.3.4 T Immune Cells Activation
For gaining a more in-depth understanding of the immune responses and relative mechanism of NaVs in vivo, the spleen lymphocytes of the immunized mice were stained with anti-mouse APC-CD3, FITC-CD4 and Cy5.5-CD8 antibodies. As shown in Fig. 5A, compared to other groups, the percentage of CD4+ T cells from mice treated with NaVs greatly enhanced from 8.9–26.5% (Fig. 5B), while the percentage of CD8+ T cells increased from 5.4–13.6% (Fig. 5C and 5D). Taken together, NaVs could significantly promote the expansion of CD4+ T cells and CD8+ cells in the immune system of tumor-bearing mice.
Furthermore, the cytokine levels of supernatant of splenocytes after antigen re-stimulation in vitro were detected. As shown in Fig. 5E, NaVs increased the cytokine level of TNF-α approximately 4 times compared with the PBS group, which is consistent with the in vitro results. NaVs also significantly elevated the level of cytokine IL-10 (Fig. 5F). IL-10 as an anti-inflammatory factor can specifically limit Th17 inflammation to eliminate protumor environment and promote Th1-type anti-tumor immunity to recognize and eliminate tumors cells, and hence inhibit tumor development and metastasis [35, 36]. It can also activate NK cells and CD8+T cells directly or indirectly to enhance anti-tumor effects and contribute to the formation of immune memory [37]. As stated in the previous study, here the cytokines of TNF-α and IL-10 were upregulated mainly mediated by NF-κB and AP-1 activation through mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B inducing kinase -IkB kinase-IkB (NIK-IKK-IkB) pathways which resulted from TLR-9 receptor binding with the dense CpG ODNs after NaVs internalized into endosomes [5, 38]. Altogether, we conclude that NaVs could enhance both humoral and cellular immune responses.
3.3.5 Memory T Cell Immune Response
An important principle of tumor nanovaccine is to stimulate the immune system producing long-lasting antigen-specific immune memory, which prevents tumor formation as well as recurrence. So, we examined the memory T cell immune responses. CD44 is the most reliable marker of mouse memory cells and generally is used to define primary and memory T cells, while CD62L, an important adhesion molecule on central memory T cells, is usually used to distinguish between central memory T cells (CD44HiCD62LHi) and effector memory T cells (CD44HiCD62LLo) [39–41]. After the spleen lymphocytes from mice treated with NaVs were re-stimulated by the antigens, TCM proliferated in a large amount of both CD4+T (PBS 6.5% vs NaVs 15.9%) cells and CD8+T cells (PBS 8.9% vs NaVs 18.2%) (Fig. 5G and 5H). These data suggested that NaVs could effectively induce the production and proliferation of TCM, so that the body's immune system can maintain the memory of tumor antigens and provide long-term immune surveillance.