Silica-Based Nanoparticles Targeting Dendritic Cells for Cancer Immunotherapy

Vaccination is a promising anticancer strategy, but the limited delivery routes and short retention of antigens and immunomodulatory agents are problems that need to be solved in vaccine design. Because silicon nanoparticles have a tunable pore size and high loading capacity, they have been used in a variety of drug delivery systems, but their roles in tumor vaccine and tumor immunotherapy need to be examined. CD40 mAb was attached to mesoporous silica nanoparticles (MSNs) through covalent conjunction, and MSN-CD40/OVA/CpG nanoparticles were examined by Fourier transform-infrared spectroscopy, transmission electron microscopy and nanoparticle analyzer. In vitro functions of nanoparticles were detected by cytotoxicity, cellular uptake, DC maturation, cross-presentation and T cell priming. In vivo functions were monitored by tumor elimination, DC maturation, cross-presentation and T cell activity. strongly increased peptide-specic into the cell supernatant, induced dendritic expression of major histocompatibility complex-II, and stimulated lymphocyte CD80 and CD86 expression. The MSN-CD40/OVA/CpG


Introduction
Tumor immunotherapy has provided impressive results in animal models of human diseases, such as prostate cancer, colon cancer, and breast cancer, as well as in human clinical studies. 1,2 The main purpose of therapeutic vaccines is to eliminate the cause of a given disease. Their activity is dependent on the induction of antigen-speci c CD8 + T cells to generate cytotoxic T lymphocytes (CTLs) primed to reject cancerous or infected cells. 3 Dendritic cells (DCs) are speci c antigen-presenting cells in the immune system that capture the antigen and present the peptide to active CD8 + T cells. 4 However, the infectious pathogens or cancerous cells may escape from immune surveillance and induce immune tolerance, 5 and the generation of robust CTL in vivo remains a major challenge. CD40 is a promising target on DCs for cancer immunotherapy. 6 In patients with advanced solid malignancies, CD40 agonists showed antitumor activity and had a controlled toxicity pro le. CD40 is a member of the tumor necrosis factor (TNF) family of cell-surface receptors that is highly expressed on DCs. Binding of CD40 to its receptor on DCs enhances the expression of costimulatory mediators, such as CD80 and CD86, and major histocompatibility complex (MHC) molecules, and induces the release of immune-stimulatory cytokines. 7 It also plays a vital role in the maturation of DCs into fully competent antigen-presenting cells (APCs) and is a key signal for CD4 + T helper-dependent CD8 + T cell priming. 8,9 Immature DCs express pathogen recognition receptors, such as toll-like receptors (TLR), to control the danger signals capable of stimulating the innate immune system. 10 Many types of engineered materials have been developed and their potential immunomodulatory properties have been evaluated in several platforms, including poly(lactide-co-glycolide) nanoparticles, [11][12][13] gold nanoparticles, 13,14 liposomes, [15][16][17] mesoporous silica, [18][19][20] and other materials. [21][22][23][24] Mesoporous silica nanoparticles (MSNs), which have a distinctive mesoporous structure, good biocompatibility, and a large surface area, have attracted much attention for their potential biomedical applications. 25 Moreover, some antibody constructs have been developed that can encourage antigen internalization in early endosomes 26 and promote antigen-speci c T cell responses. 27 Despite the advantages of anti-CD40 antibodies, 28,29 there are many adverse side effects, such as vascular leakage, cytokine release syndrome, 30 and liver damage. 31 In this study, we evaluated the use of MSN-CD40/ovalbumin (OVA)/TLR9 agonist (CpG) nanoparticles (Scheme 1) to facilitate the delivery of an antigen to DCs in vitro and induce DC maturation. Vaccination with the MSN-CD40/OVA/CpG nanoparticles elicited a robust antigen-speci c CD8 + T cell response, controlled tumor growth, and had low toxicity in vivo.

Materials And Methods
Page 4/25

Synthesis of CD40 mAb-engineered MSNs
CD40 mAb was covalently attached to the surface of the MSN-NH 2 particles using 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) coupling chemistry. CD40 mAb-bound EDC (120 μg, nal concentration 2mM) and N-hydroxysuccinimide ( nal concentration 5 mM) were dissolved in 0.1 M MES buffer (pH 6.0) for 15 min at room temperature. Simultaneously, MSN-NH 2 particles (3 mg) were dispersed in phosphate-buffered saline (PBS; 100 mL, pH 8.5). The two solutions were mixed together for 2 h at room temperature. The MSN-CD40 nanoparticles were washed three times with PBS (pH 7.4) at 12,000 rpm. The nanoparticles were collected in KBr pellets, and the morphology and size of the nanoparticles were analyzed by Fourier transform-infrared spectroscopy (FT-IR; Nicolet iS 50; Thermo Fisher Scienti c) and transmission electron microscopy (TEM; JEOL JEM-200CX, Japan) at 200 kV, respectively. The zeta potentials and hydrodynamic sizes of MSN-CD40/OVA/CpG nanoparticles and the controls were measured using a nanoparticle analyzer (Nano-ZS90, Malvern Instruments Co., Ltd., Malvern, UK).

Evaluation of cytotoxicity
C57BL/6 mice (6-8 weeks old) were sacri ced by cervical dislocation, the tibiae of hind limbs and intact femurs were removed using surgical instruments. The bones were washed in PBS (pH 7.4) and bone marrow was ushed with PBS (pH 7.4) using a syringe. Clusters within the bone marrow were disaggregated using red blood cell lysis solution to yield a homogeneous cell suspension. The cells were seeded in a 100-mm bacteriological Petri dish in 20 mL of RPMI 1640 media containing 10% fetal bovine serum (FBS), 20 ng/mL GM-CSF, and 5 ng/mL IL-4 on day 0. Three days later, half of the media was replaced with fresh RPMI 1640 media containing the same concentrations of GM-CSF and IL-4. Cells were maintained in a humidi ed incubator at 37 °C and 5% CO 2 . Bone marrow-derived dendritic cell (BMDC) viability was measured by the MTT assay. Brie y, BMDCs (5 × 10 4 cells) were seeded in a 96-well plate and incubated with MSN-CD40/OVA/CpG nanoparticles and appropriate controls (OVA/CpG and MSN-IgG/OVA/CpG, MSN/OVA/CpG nanoparticles), diluted to a concentration of 12.5 μg/mL OVA, for 24 h. After removing the supernatant, 20 μL of MTT (0.5 mg/mL in fresh RPMI-1640) was added to each well for 4 h at 37 °C, after which the medium was removed and dimethyl sulfoxide was added to solubilize the formazan. The absorbance of each well was measured at 560 nm.

Evaluation of cellular uptake
The intracellular uptake of FITC-labeled OVA by mouse splenocytes was determined by ow cytometry. 2) and stained with murine anti-CD45, anti-B220, anti-CD19, anti-CD11c, anti-F4/80, and anti-CD11b mAb to gate B cells, DC, and macrophages. We also incubated immature BMDC (4 × 10 6 cells/wells on six-well plates) in the same way and, after washing with cold PBS (pH 7.2), the cells were collected and stained with anti-CD11c mAb to test their uptake ability by ow cytometry. To investigate the uptake mechanism, immature BMDC (4 × 10 6 cells/well) were planted in six-well plates at 37 °C in the presence of genistein (50 μg/mL) and chlorpromazine (10 μg/mL) for 1 h before incubation with uorescently labeled OVA in MSN-CD40/OVA-FITC/CpG nanoparticles.

Confocal imaging
To explore the localization of nanoparticles in DC, immature BMDC (4 × 10 6 cells/well) were plated onto 24-well plates at 37 °C, stained with LysoTrackerRed and Hoechst 33342, and xed with paraformaldehyde. The cells were then analyzed by confocal laser scanning microscopy (TCS SP8, Leica, Wetzlar, Germany).

Maturation and cytokine detection in vitro
Immature BMDCs (2 × 10 5 cells/well) were seeded onto a 96-well plate and incubated for 24 h with MSN-CD40/OVA/CpG nanoparticles and appropriate controls (OVA/CpG and MSN-IgG/OVA/CpG and MSN/OVA/CpG nanoparticles) at a nal OVA concentration of 12.5 μg /mL. The culture supernatant was removed and stained with anti-CD11c, anti-CD80, and anti-CD86 mAbs for 30 min on ice. The uorescence markers expressed on the surface of BMDCs were measured by ow cytometry. The culture medium was collected to measure TNFα levels using an ELISA kit.

In vitro cross-presentation and T cell priming
Immature BMDCs (1 × 10 6 cells/well) were seeded onto a 96-well U-bottom plate and incubated for 4 h with MSN-CD40/OVA/CpG nanoparticles and appropriate controls (OVA/CpG and MSN-IgG/OVA/CpG and MSN/OVA/CpG nanoparticles) at a nal OVA concentration of 12.5 μg/mL. CD8 + T cells were negatively isolated from the lymphocytes of OT-I mice using a mouse CD8 + T cell isolation kit and stained with 2 μM carboxy uorescein succinimidyl ester (CFSE) for 10 min, after which cells were washed twice with culture medium. Next, CD8 + T cells (1 × 10 5 cells/well) were seeded onto a 96-well U-bottom plate and cocultured with BMDC (1 × 10 4 cells/well) for 72 h. The culture medium was collected and the mixed cells were stained with anti-CD8 mAb for ow cytometry. The IFNγ concentration was also measured using an ELISA kit. 100 U/mL penicillin G, and 100 µg/mL streptomycin sulfate. Next, 2 × 10 5 B16-OVA melanoma cells were injected subcutaneously into the tail base of C57BL/6 mice, which were then immunized three times at 1week intervals (n = 5 per group). After 3 days, the mice were subcutaneously injected with saline, OVA/CpG, MSN/OVA/CpG, MSN-IgG/OVA/CpG, or MSN-CD40/OVA/CpG, as appropriate. The nal doses of OVA and CpG were 100 and 30 μg per mouse, respectively. The tumor size and body weight were measured every 2 days. Five days after the last immunization, the mice were sacri ced to detect tumorin ltrating T cells. Tumor tissues were cut into small pieces and stained with murine anti-CD45, anti-CD3, anti-CD4, and anti-CD8 mAb for ow cytometry. After the splenocytes and lymphocytes were cultured with brefeldin A and OVA 257−264 peptide at a nal concentration of 10 μg/mL, the cells were collected to measure intracellular IFNγ production by ow cytometry. The heart, liver, kidneys, and tumor were harvested and xed in 4% paraformaldehyde for histopathological examination.

Statistical analysis
Data are presented as the mean ± standard deviation (SD) unless otherwise stated. Student's t test was used to determine the statistical signi cance of differences among the groups. P < 0.05 was considered statistically signi cant.  (Fig. 1). Therefore, our results demonstrate that it is feasible to use MSN-CD40/OVA/CpG to deliver an antigen into DCs.

MSN-CD40/OVA-FITC/CpG improve internalization by DCs in vitro
The capability of splenocytes and BMDCs to internalize uorescently labeled OVA via MSN-CD40/OVA-FITC/CpG nanoparticles was tested by ow cytometry. The MSN-CD40/OVA-FITC/CpG nanoparticles signi cantly improved the uptake of OVA by different subsets of splenocytes (Fig. 2). The CD45 + CD11c + DC and CD45 + B220 + CD19b + B cells internalized MSN-CD40/OVA/CpG nanoparticles more e ciently than CD45 + CD11b + F4/80 + macrophages. The MSN-CD40/OVA-FITC/CpG nanoparticles also enhanced the uptake of OVA by BMDCs (Figs. 3A,B). To explore the mechanism involved in BMDC uptake of MSN-CD40/OVA-FITC/CpG nanoparticles, the cells were treated with genistein, a caveolae inhibitor, to inhibit endocytosis or chlorpromazine to inhibit clathrin-mediated endocytosis. The results showed that the uptake e ciency of MSN-CD40/OVA-FITC/CpG nanoparticles was reduced by chlorpromazine. In contrast, genistein elicited a non-signi cant reduction in FITC uorescence, which suggests that multiple endocytic pathways are involved in the internalization of CD40/OVA-FITC/CpG nanoparticles (Figs. 3C,D). Confocal uorescence microscopy clearly showed intracellular green uorescence, suggesting that FITClabeled OVA was taken up by the BMDCs. The red uorescence corresponding to LysoTracker Red further con rmed that MSN-CD40/OVA-FITC/CpG nanoparticles were internalized by BMDCs (Fig. 4). These results indicate that the MSN-CD40/OVA-FITC/CpG nanoparticles improved the uptake and delivery of CD40-targeting nanoparticles to DCs in vitro.

MSN-CD40/OVA/CpG nanoparticles enhanced the activation of DCs and antigen cross-presentation in vitro
The ability to promote DC maturation is an essential requirement of nanovaccines. Therefore, we evaluated the effects of the nanovaccine on DC maturation in vitro. The expression levels of CD80 and CD86 indicated that MSN-CD40/OVA/CpG nanoparticles more effectively induced BMDC maturation compared with MSN-IgG/OVA/CpG, MSN-IgG/OVA/CpG, MSN/OVA/CpG, and OVA/CpG nanoparticles (Figs. 5A,B). We also measured TNFα secretion into the culture medium by ELISA, and the results showed that the MSN-CD40/OVA/CpG nanoparticles enhanced cytokine release (Fig. 5C). The MTT assay showed that the MSN-CD40/OVA/CpG and other nanoparticles were not cytotoxic (Fig. 5D). To investigate the capacity of BMDCs on inducing CD8 + T cell responses, we isolated lymph node CD8 + T cells from OT-I mice and labeled the cells with 2 μM CFSE. Flow cytometry revealed that the MSN-CD40/OVA/CpG nanoparticles slightly induced CD8 + T cell proliferation, but markedly promoted CD8 + T cell activation, resulting in high IFNγ secretion (Fig. 6). These ndings demonstrate that the MSN-CD40/OVA/CpG nanoparticles can induce BMDC maturation and promote antigen-speci c CD8 + T cell functionality.

Effects of MSN-CD40/OVA/CpG nanoparticles on immune responses in vivo
To explore the effects of MSN-CD40/OVA/CpG nanoparticles on immune responses in vivo, we used C57BL/6 mice as the immune model. After three subcutaneous injections, the frequency of IFNγ-secreting activated cytotoxic CD8 + T cells of all splenocytes was determined by ow cytometry (Fig. 7A). The results showed that MSN-CD40/OVA/CpG nanoparticles induced intracellular secretion of IFNγ in splenocytes (Fig. 7B). The splenocytes were restimulated with OVA 257-264 peptide to acquire CTL effectors. The ELISA results revealed that MSN-CD40/OVA/CpG nanoparticles induced cytotoxic CD8 + T cells to secrete IFNγ (Fig. 7C). Because the lymphatic system is the main site where the DCs interact with T cells, we excised the lymph nodes from immunized mice and tested the DC surface molecules. The results showed that MSN-CD40/OVA/CpG nanoparticles induced the DC maturation markers MHC-II, CD80, and CD86 compared with the indicated controls (Figs. 7D,E).

Antitumor e cacy in vivo
Activation of tumor-associated antigen-speci c CD8 + T cells is vital to elicit a robust anti-tumor immune response. Therefore, in this study, C57BL/6 mice were subcutaneously injected with B16-OVA cells and treated with MSN-CD40/OVA/CpG nanoparticles or appropriate controls containing the same doses of OVA and CpG, as described in the Methods and Fig. 8A. There were no differences in the body weights of mice in each group (Fig. 8C), indicating no obvious toxicity of the nanoparticles. Tumor growth was inhibited in mice immunized with MSN-CD40/OVA/CpG nanoparticles compared with that in mice immunized with MSN-IgG/OVA/CpG nanoparticles, MSN-OVA/CpG nanoparticles, free OVA/CpG, or saline (Figs. 8B,D). We also excised the tumor from each mouse and stained it with antibodies for CD4 + and CD8 + T cells for ow cytometry. The results showed that MSN-CD40/OVA/CpG nanoparticles increased the frequency of CD45 + CD3 + CD8 + and CD45 + CD3 + CD4 + T in ltrating tumor cells (Figs. 9A,B). The spleen and lymph nodes were excised from tumor-bearing mice to detect intracellular IFNγ in splenocytes and lymphocytes (Figs. 9C,D). The MSN-CD40/OVA/CpG nanoparticles upregulated intracellular IFNγ production in splenocytes and lymphocytes to a greater extent than was observed in the other groups. Histopathological examination of the heart, liver, kidneys, and tumor ( Fig. 10)  inducing potent cytotoxic CD8 + T cell responses. The MSN-CD40/OVA/CpG nanoparticles detected by FT-IR and TEM have a particle size of about 120 nm. The DLS results showed that the dispersibility of the prepared MSN-CD40/OVA/CpG nanoparticles was good (Fig. 1). Because CD40 is expressed on DC, B cells, and monocytes, the MSN-CD40/OVA-FITC/CpG nanoparticles could target different subpopulations of splenocytes. Our experiments revealed that the nanoparticles were speci cally internalized by the DCs and B cells, but avoided non-speci c phagocytosis by macrophages (Fig. 2). This speci c targeting is a key advantage of the nanoparticles. B cells not only exert speci c humoral immune functions by producing antibodies, but are also important APCs. The B cell-mediated immune responses, which depends on the antigen, can be divided into T cell-dependent antigen (TD-Ag) immune responses and T cell-independent antigen (TI-Ag) immune responses. It was previously reported that an antigen-loaded calcium phosphate (CaP) nanovaccine effectively activates B cells. 43 The CaP nanovaccine, which relies on the function of the TLR ligand, can target B cells, promote their proliferation and differentiation, enhance and regulate the TI-Ag and TD-Ag responses, and enhance the humoral immune response. 44 However, because B cells display weaker phagocytic activity 45 and T cell activation, 46 than DC, B cells play a minor role in the effects of the nanovaccine.
To investigate the delivery of nanoparticles into DCs, OVA was labeled with uorescent dye (FITC) and used to form the MSN-CD40/OVA-FITC/CpG complex. The MSN-CD40/OVA-FITC/CpG nanoparticles were co-incubated with BMDCs, allowing us to determine the mean FITC uorescence in the cells. The mean uorescence of cells incubated with MSN-CD40/OVA-FITC/CpG nanoparticles was signi cantly different from that in the control groups (Figs. 3A,B). To validate the endocytic mechanism of MSN-CD40/OVA-FITC/CpG nanoparticles by BMDCs, we exposed the cells with the caveolar endocytosis inhibitor genistein 47 and the clathrin-mediated endocytosis inhibitor chlorpromazine. 48 The results showed that chlorpromazine (10 μg/mL) effectively inhibited BMDC uptake of MSN-CD40/OVA-FITC/CpG nanoparticles. Although the effect of genistein (50 μg/mL) was not statistically signi cant, ow cytometry showed a decrease in the mean FITC uorescence. Therefore, we concluded that BMDCs take up MSN-CD40/OVA-FITC/CpG nanoparticles through multiple pathways. TLR9 is localized in the endoplasmic reticulum rather than the cell surface. 49 Because the MSN-CD40/OVA/CpG nanoparticles enter DC via multiple pathways, they may also be useful to potentiate the activity of CpG oligodeoxynucleotides. [50][51][52] The MSN-CD40/OVA/CpG nanoparticles greatly enhanced the maturation of BMDCs in vitro. Incubating BMDCs with MSN-CD40/OVA/CpG nanoparticles for 24 h resulted in marked upregulation of the cell surface molecules CD80 and CD86 compared with the control conditions. ELISA also revealed increased expression of TNFα, and the MTT assay showed that the nanoparticles had no toxic effects (Fig. 5F). We also evaluated antigen-speci c CD8 + T cells from OT-I mice and stained them with CFSE. Flow cytometry showed that the MSN-CD40/OVA/CpG nanoparticles induced CD8 + T cell proliferation (Fig. 6A) and increased IFNγ secretion (Fig. 6B). These results indicate that the MSN-CD40/OVA/CpG nanoparticles promoted BMDC antigen cross-presentation, and induced antigen-speci c CD8 + T cell proliferation and activation.
When administered in vivo, the MSN-CD40/OVA/CpG nanoparticles induced signi cant DC maturation at the draining lymph node after three subcutaneous injections. The nanoparticles also induced the expression of the costimulatory markers CD80 and CD86, and of MHC-II (Figs. 7D,E). Furthermore, the nanoparticles stimulated the intracellular expression and secretion of IFNγ compared with the control conditions. Restimulation of splenocytes with OVA 257-264 also enhanced IFNγ secretion (Figs. 7B,C).
To investigate the antitumor effect of MSN-CD40/OVA/CpG nanoparticles, we established a B16-OVAbearing mouse model (Fig. 8A). Three subcutaneous injections of the nanoparticles resulted in a decrease in tumor volume without a change in the body weight of the treated mice (Figs. 8B,C). Flow cytometry revealed upregulation of tumor-in ltrating CD4 + T cells and CD8 + T cells together with increased intracellular IFNγ production (Figs. 9A,D). The systemic toxicity of anti-CD40 mAb can be reduced by using formulations that sequester the CD40 agonist in the tumor. 31,53 To test this, we harvested the heart, liver, kidney, and tumor tissues from the treated mice and histopathologic staining with hematoxylin and eosin revealed no obvious in ammation or adverse pathology (Fig. 9E). These results suggested that MSN-CD40 is a safe, potential vaccine carrier.

Conclusion
In conclusion, we used anti-CD40 mAb to target DCs by covalently attaching the mAb to the surface of MSNs, which were then encapsulated with OVA and the TLR9 agonist CpG. The resulting MSN-CD40/OVA/CpG nanoparticles were e ciently taken up by DCs to induce their DC and antigen presentation, activating CD8 + T cells in vitro and in vivo. We believe this represents a promising strategy to improve antigen cross-presentation, CTL immune activity, and antitumor immunotherapy.     Cell activity was not affected by the nanoparticles. The results are presented as means ± SD (n = 2). *P < 0.05, **P < 0.01, and ***P < 0.001.  and CD80 and CD86 (E) in the lymphocytes after immunization with MSN-CD40/OVA/CpG or the indicated controls. The results are presented as means ± SD (n = 5). *P < 0.05, **P < 0.01, and ***P < 0.001. Photographs of the tumor-bearing mice. The results are presented as means ± SD (n = 5). *P < 0.05, **P < 0.01, and ***P < 0.001.