A Simple Self-Adjuvanting Biomimetic Nanovaccine Self-Assembled with the Conjugate of Phospholipids and Nucleotides Can Induce Strong Cancer Immunotherapeutic Effect

Background: Biomimetic nanoparticles have potential applications in many elds for their favorable properties. Results: Here, we developed a self-adjuvanting biomimetic anti-tumor nanovaccine, which was self-assembled with an amphiphilic conjugate synthetized with phospholipids of 1, 2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and hydrophilic Toll-like receptor (TLR9) agonists CpG ODN. The nanovaccine could not only provide effective initial antigens stimulation and sustained long-term antigen supply with a controlled release, but also induce antigens cross-presentation via MHC-I pathway initiating CD8 + T-cell responses. Moreover, the dense nucleotides shell around the nanovaccine could promote antigens endocytosis via various receptor-mediated pathways into dendritic cells. And CpG ODN interacted with TLR9 triggering the cytokines secretion of TNF-α and IL-10 further boosted the anti-tumor humoral and cellular immune responses, which led to signicant tumor suppressive effect and remarkable survival prolongation. Conclusions: So, this self-assembled with phospholipid-nucleotide serve BMDCs: 4, 6-diamidino-2-phenylindole; LSM: laser FCD: fucoidan; CPM: chlorpromazine; MBCD: methyl-β-cyclodextrin; TNF-α: tumor necrosis factor-α; IL-6: interleukin-6; MFI: mean uorescence intensity; H&E: hematoxylin and eosin; IL-10: interleukin-10; SD: standard deviation; DLS: dynamic light scattering; SR-A: class A scavenger receptor; SNC: Spherical nano capsule; MAPK: mitogen-activated protein kinase; NIK-IKK-IkB: nuclear factor kappa-B inducing kinase -IkB kinase-IkB.


Background
Nanovaccine has been investigated in recent years as an emerging eld in cancer immunotherapy owing to their ability to cross biological barriers, prolong circulation times, and induce enhanced long-lasting protective immune effect [1][2][3]. Generally, nanovaccine carriers are required to encapsulate and codelivery antigens and adjuvants to overcome intrinsic di culties of functional cargos and trigger delightful immune responses [4][5][6]. Recently, it has been reported that nucleic acids arranging in a dense spherical spatial form (SNAs) on a core made of gold or other polymers, can endow them unique properties, such as rapidly uptake through receptor-mediated mechanism and e cient protection against nuclease degradation [7][8][9][10]. However, the preparation of popular polymeric nanocarriers is complicated and the traditional core polymers have potential physiological toxicity [6,[11][12][13][14].
Biomimetic NPs to mimic the chemical and structural features of biological systems possess distinct properties, including favorable biocompatibility, extended circulation and targeted drug delivery. Here, we reported a simple self-adjuvanting biomimetic nanovaccine (NaVs) self-assembled with the conjugate of phospholipids and nucleotides which had a dense nucleotide shell similar to SNAs. In this study, hydrophobic biomimetic phospholipids of DOPE and hydrophilic adjuvanting Toll-like receptor (TLR9) agonists CpG ODN were exploited and covalently linked together. The resultant amphiphilic molecules similar to the phospholipid bilayer of cells membrane self-assembled forming nanocapsules. So, the nanovaccine mimicked both the chemical and structural characteristics of cells displaying good biocompatibility. Furthermore, CpG ODN not only as adjuvants enhanced the immune responses, but also the dense nucleotides shell formed with CpG ODN promoted antigens endocytosis via various receptor-mediated pathways and subsequently boosted the anti-tumor humoral and cellular immune responses. Therefore, the biomimetic nanovaccine offered a simple, safe and effective strategy for cancer treatment.

Preparation of Self-Adjuvanting Biomimetic Nanovaccines
The preparation of self-adjuvanting biomimetic nanovaccines was involved three steps. Brie y, DOPE (Sigma-Aldrich Co., USA) was pyridyldithiol-activated by 3-(2-Pyridyldithio) propionic acid Nhydroxysuccinimide ester (SPDP). Then the activated DOPE was conjugated with thiol terminated CpG ODN (Sangon Biotech Ltd., China) via a disul de bond in the presence of phase transfer catalyst. At last, the resulted amphiphiles of DOPE-S-S-CpG ODN and ovalbumin (OVA, a model antigen, Sigma-Aldrich Co., USA) solutions were mixed and stirred at 25℃ for the formation of self-adjuvanting biomimetic nanovaccines (named as NaVs) with self-assembly. NaVs were washed with distilled water and lyophilized.

Characterization of DOPE-S-S-CpG ODN Amphiphiles and NaVs
The conjugate of DOPE-S-S-CpG ODN was con rmed by agarose gel electrophoresis. The particle sizes and Zeta potential of NaVs were analyzed using Zetasizer Nano ZP (Malvern Instruments Ltd., UK). The redox-responsive property of NaVs was studied through observing the changes of size after reaction with excessive DL-Dithiothreitol (DTT). To investigate the stability of NaVs, the size of NaVs was measured every ve days until the nanoparticles aggregated. The morphology of NaVs was observed using transmission electron microscopy (TEM) (Tecnai-F20, FEI, The Netherlands).
The OVA loading capacity of NaVs was determined using an Enhanced-BCA Protein Assay Kit. To evaluate the release behavior of antigens from NaVs in vitro, 5 mg of lyophilized NaVs powder was dissolved with 1 mL of phosphate buffer saline (PBS, pH7.4) and placed in a dialysis bag (molecular weight: 50000 Da) which was immersed in 10 ml PBS at 37℃, and the OVA concentration in dialysate were measured with BCA kits at regular intervals.

Cytotoxicity Assessment
Bone Marrow-Derived Dendritic Cells (BMDCs) derived from C57BL/6 mouse femur and cultured with IL-4 and GM-CSF (Peprotech, USA) for 6 days before further experiments. BMDCs with a density of 1 × 10 5 cells/mL were co-incubated with NaVs at a series of concentrations for 24 h, the cytotoxicity of NaVs was evaluated using a CCK-8 kits (Dojindo Molecular Technologies Inc, Japan).

Antigen Uptake in BMDCs
To observe whether NaVs could facilitate the antigen uptake by BMDCs, after co-incubation severally with free OVA and NaVs (each concentration of FITC-OVA: 20 µg/mL) for 8 h at 37℃, BMDCs were washed using immunol staining wash buffer to remove the extracellular FITC-OVA or NaVs. Afterwards, BMDCs were labeled with 4, 6-diamidino-2-phenylindole (DAPI) and imaged with a laser scanning confocal microscope (Zeiss LSM 800, Germany).

Endocytic Mechanism of NaVs in BMDCs
For further exploring the endocytic pathways of DCs to NaVs, we used several uptake inhibitors to pretreat cells separately to block the corresponding endocytic pathway. Brie y, BMDCs were co-cultured with fucoidan (FCD, 10 µg/mL), chlorpromazine (CPM, 10 µg/mL) or methyl-β-cyclodextrin (MBCD, 10 µg/mL) for 1 hour, respectively. Afterwards, the pretreated cells were co-incubated with free FITC-OVA or NaVs (containing FITC-OVA) for hours while untreated cells were used as negative control. Then the quantity of FITC-OVA in BMDCs was detected using a ow cytometer (BD Biosciences, CA, USA) and the reduction of antigens uptake of NaVs compared to negative control group was calculated.

Effect of NaVs on BMDCs in vitro
In order to verify whether NaVs could promote BMDCs activation and antigens cross-presentation, immature BMDCs were co-cultured with PBS, free OVA, OVA + CpG and NaVs at 37℃ in 5% CO2 cell incubator for 6 h, respectively. Then BMDCs were subsequently stained with uorescence-labeled antibodies against CD11c, CD86, CD40 and OVA257-264 peptide SIINFEKL (Thermo Fisher Scienti c Inc., USA). Events were collected and plotted using a ow cytometer. Meanwhile, the cytokines of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) released in culture supernatants were quanti ed with ELISA kits (Thermo Fisher Scienti c Inc., USA).

Antigen Depot Effect
Six-week-old female C57BL/6 mice (n = 5) were used to study the release behavior of NaVs in vivo. 100 µL suspension of NaVs and free OVA (each containing 20 µg Cy7-labeled OVA) were intramuscularly injected into mice, respectively. Then Cy7 uorescent signals of the immunization site were detected at predetermined intervals with the Maestro imaging system (CRI, USA) and the mean uorescence intensity (MFI) was calculated at each checkpoint.

Tumor Challenge and Therapeutic Effect
All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of the Chinese Academy of Medical Science and Peking Union Medical College. After getting acclimated to the SPF barrier environment, healthy female C57BL/6 mice were used to establish xenograft tumor models. E.G7-OVA (5 × 10 5 ) cells in logarithmic growth phase were injected subcutaneously into the right back of the mice. Until the tumor reached 5 mm in diameter, immunotherapy was performed on mice. Divided tumor-bearing mice into 4 groups, then respectively injected 50 µL suspension of free OVA, OVA + CpG, NaVs (each formulation containing 20 µg OVA), using PBS as a negative control. The immunization was performed three times at intervals of every other week.
To further evaluate the anti-tumor effect of NaVs, the length and width of mouse tumors were recorded with digital vernier calipers every two days, while observing the mental state of the mice. Tumor volume (mm 3 ) was calculated according to the following formula: (length × width 2 )/2. Two days after the last immunization, mice were sacri ced and their peripheral blood, tumors and spleens were collected immediately for the following immunological experiments. The solid tumor tissues collected from tumorbearing mice were stained with hematoxylin and eosin (H&E) and TUNEL apoptosis kits (Beyotime Biotechnology, China) to observe the in uence of NaVs on the necrosis and apoptosis of tumor cells.

Secretion of OVA-Speci c Antibodies
Before mice were dislocated, the blood was drawn from the postorbital venous plexuses and blood serum were isolated through centrifugation twice at 3000 rpm for 10 min. Then the level of OVA-speci c antibodies including IgG, IgG1, and IgG2a secreted in blood were determined by ELISA kits.

T Cells Immune Responses and Immune Memory
The spleen from the immunized mice were made into a single cell suspension and the lymphocytes were extracted. Part of the splenocytes were co-incubated with anti-mouse antibodies against CD4, CD8 and CD3, and detected by a ow cytometer to analyze the proliferation of T cells. The leftover cells were restimulated with OVA for 3 days and stained CD8, CD4, CD62L and CD44 antibodies labeled with different uorescence to analyze the generation of memory T cells. Meanwhile, the cell supernatant was harvested and the secretion of TNF-α and interleukin-10 (IL-10) were evaluated using ELISA kits to further con rm the type of immune responses.

Statistical Analysis
Data were expressed as mean ± standard deviation (SD). The differences were assessed using ANOVA or Student's t-test and Tukey's post-test. P value no more than 0.05 was considered signi cant.

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 con rmed 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 rst 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.

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).

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 uorescence microscopy showed that much more signi cant 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 uorescence intensity (MFI) of OVA in NaVs group far exceeded that in free OVA group, which was enhanced about 6-fold. The results of the ow 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 caveolindependent endocytosis, CPM can block the formation of clathrin-coated pits, while FCD is a pharmacological inhibitor of class A scavenger receptor (SR-A) [15][16][17][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][20][21][22][23][24][25].

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-speci c 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-speci c CTLs [30,31]. Therefore, these in vitro results showed that NaVs could promote DCs maturation, activation and antigen cross-presentation favoring induction of antigen-speci c CTLs-polarizing immune responses.

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, uorescence 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 uorescence 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 longterm antigen supply in vivo.

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 signi cantly 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 signi cant 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.

OVA-Speci c Antibody Production
In order to further verify the type of immune response triggered by the NaVs and con rm the direction of lymphocyte differentiation, we rst analyze the type and amount of IgG in the blood of tumor-bearing mice which re ects 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-speci c 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-speci c tumor cells [34]. Therefore, NaVs not only had the property of signi cantly inducing the production of IgG antibodies, but also had the trend of eliciting Th1-type cell immune responses.

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 signi cantly 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 signi cantly elevated the level of cytokine IL-10 ( Fig. 5F). IL-10 as an anti-in ammatory factor can speci cally limit Th17 in ammation 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.

Memory T Cell Immune Response
An important principle of tumor nanovaccine is to stimulate the immune system producing long-lasting antigen-speci c 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 de ne 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 (CD44 Hi CD62L Hi ) and effector memory T cells (CD44 Hi CD62L Lo ) [39][40][41]. After the spleen lymphocytes from mice treated with NaVs were re-stimulated by the antigens, T CM 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 T CM , so that the body's immune system can maintain the memory of tumor antigens and provide long-term immune surveillance.

Conclusions
In summary, as shown in Fig. 6, we developed a self-adjuvanting biomimetic nanovaccine self-assembled with amphiphilic conjugate synthetized with hydrophobic phospholipids and hydrophilic TLR9 agonists CpG ODN. The nanovaccine with 3D structure could load antigens in the cavity, provide effective initial immune stimulation and sustained long-term antigen supply, and promote antigens cross-presentation via MHC-I pathway initiating CD8 + T-cell responses. Moreover, the dense nucleotides shell around the nanovaccine could promote antigens endocytosis via various receptor-mediated pathways into dendritic cells. And CpG ODN interaction with TLR9 triggering the cytokines secretion of TNF-α and IL-10 further boosted the anti-tumor humoral and cellular immune responses as well as generated effective immune memory, which led to signi cant tumor suppressive effect and remarkable survival prolongation. So, the self-adjuvanting biomimetic nanovaccine can serve as a great promising immunotherapeutic approach for tumor treatment. Moreover, the method reported here could be applied to produce various types of nanovaccines for treatment of infectious diseases though loading different antigens in the cavity of the capsule, or to prepare siRNA nanocarriers for gene therapy through replacing CpG ODN shell around the nanovaccine with siRNA.