Immunization with functionalized carbon nanotubes enhances the antibody response against mode antigen ovalbumin

Immunology Letters 178 (2016) 77–84

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Immunization with functionalized carbon nanotubes enhances the antibody response against mode antigen ovalbumin Xinfeng Zhu, Jiadi Sun, Yinzhi Zhang, Xiulan Sun ∗ State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China

a r t i c l e

i n f o

Article history: Received 30 March 2016 Received in revised form 7 July 2016 Accepted 8 August 2016 Available online 9 August 2016 Keywords: Carbon nanotubes Antibodies Complement system Cellular uptake Cytokine secretion

a b s t r a c t Carbon nanotubes (CNTs) have attracted considerable attention because of their potential application as a new nonvehicle. We have covalently conjugated the mode antigen ovalbumin (OVA) to functionalized multi-walled carbon nanotubes. Herein, we explored the underlying theoretical mechanisms of CNTs’ immunological adjuvant characterization. In vitro, the efficiency of cellular uptake of MWCNT-OVA into DC2.4 cells was improved over that of pure-antigen OVA. The costimulators (CD40/86), the major histocompatibility complex MHCII molecules, and the CD11c molecules were found to be upregulated. Further in vivo experiments established that the MWCNT-OVA group enhanced the IL-1␤, TNF-␣, and IL6 cytokine secretion, suggesting that MWCNT reinforced the immune response using different cytokine pathways. Anti-OVA antibodies after inoculation of MWCNT-OVA into mice were measured. The medium dose of MWCNTs conjugated with OVA induced the highest level of OVA-specific antibodies at day 82 and have a synergistic effect with the commercial Freund’s adjuvant. MWCNTs-KLH-MC-LR also induced higher levels of MC-LR-specific antibody than did KLH-MC-LR. MWCNTs also could activate the complement system which is closely related with humoral immunity. These results suggested that MWCNTs enhance the immune response and show excellent inherent characteristics to be applied as an adjuvant. © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Carbon nanotubes (CNTs) [1] are rolled hexagonal carbon networks capped by half fullerene molecules. Single-walled (SWCNTs), double-walled (DWCNTs), and multi-walled (MWCNTs) are three common carbon nanotube configurations. Due to their unique physical and chemical properties, carbon nanotubes are attracting increasing attention in the biological field as new vectors for the delivery of biomolecules such as peptides, proteins, and nucleic acid [2–6]. CNTs’ role in modulating immunological functions is one area of particular interest. CNTs have a relatively large surface area for chemical modification and can carry a large amount of antigen. The adjuvant effect of CNTs on immune responses has been shown to increase when CNTs’ size is decreased and surface area is increased. Conjugation of antigenic epitopes to nanoscale and submicroscale particulate scaffolds has shown that the particles improve immune responses [7]. Immunogens, such as tumor lysate protein [8] and viral peptides [9], conjugated to CNTs can cause strong antibody

∗ Corresponding author. E-mail addresses: [email protected] (X. Zhu), [email protected] (X. Sun).

responses in mice and no detectable cross reactivity to the CNTs was found. Peptide presentation by antigen presenting cells (APCs) is generally necessary for the generation of an immune response. Functionalized carbon nanotubes have been shown to cross cell membranes and to accumulate in the cytoplasm without being toxic to the cell [12]. CNT can be up taken by a wide variety of cell types and through several mechanisms, which appear to be particularly suited to deliver antigenic epitopes to APCs. Antigen delivery through nanoparticles also changes cellular trafficking and can acts as an intracellular depot of antigen, both of which activities enhance the immune response to the delivered antigen [13]. Nanoparticles alone can induce antibody production and cytokine secretion [14–16]. APCs such as macrophages and dendritic cells, phagocytose external materials and promote lymphocytes and other immune cells by releasing cytokines, initiating an adaptive immune response [2,17]. Hence, it is essential to further explore nanoparticles’ application to adjuvant activity in order to understand the effects of nanoparticles on cytokine release [18,19]. The humoral immunity is closely related with the complement system. The complement system consists of more than 30 species of protein which are widely found in serum, tissue fluid, and cell surface, and which participate in the innate immune system and

http://dx.doi.org/10.1016/j.imlet.2016.08.003 0165-2478/© 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

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adaptive immunity. The complement system could be able to identify, opsonize, and remove or kill foreign materials such as invading microorganisms and altered host cells. CNTs and functionalized CNTs can impact the immune system by activating the complement system [10,11]. However, there are few systematic explorations of CNTs’ effect on activation of specific immunity and the underlying theoretical mechanisms. In this paper, we focus on the immune response induced by MWCNTs. In vitro, we employed DC2.4, which are the most effective antigen presenting cells with which to study antigen uptake and antigen-presenting ability in cell-based experiments. In vivo, the levels of anti-OVA antibody production in mice were compared after immunization with MWCNT-ova and with MWCNTs alone. We asked whether MWCNT could act as complement activators. We found that antibody production is significantly increased in mice after immunization with MWCNT − OVA. To investigate the theoretical view of the NP-based adjuvant, we tried to build a bridge between the in vitro and in vivo performance. In addition, the antibody response induced by MWCNT co-administered with the conventional adjuvant Freund’s adjuvant was studied. In addition, the synthesis of a microcystin-LR-KLH- MWCNT was also made to verify the increased antibody response induced by MWCNTs for small molecule antigens. 2. Materials and methods 2.1. Materials and reagents Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from GIBCO. HRP-conjugated anti-mouse IgG were obtained from Jackson. Fluorescence-labeled anti-mouse anti-I-A[b] MHCII Ab, anti-mouse CD40 Ab, anti-mouse CD86 Ab, and anti-mouse CD11c Ab were purchased from eBioscience. CCK-8 reagent IL-1␤, TNF-␣ and IL-6 Quantikine ELISA kits were purchased from R&D Systems, Inc. (USA). Ovalbumin (OVA), carbodiimide (EDC), H2 SO4 , HNO3, N-Hydroxysuccinimide (NHS), Microcystins-LR (MC-LR), keyhole limpet hemocyanin (KLH), fluorescein isothiocyanate (FITC), and lipopolysaccharides (LPS) were purchased from Sigma–Aldrich. 2.2. Mice and cells 6–8 weeks old male BALB/c female mice were obtained from Jiangnan University (Wuxi, China).The mice were maintained under standard conditions according to institutional guidelines and monitored to be pathogen-free. Murine macrophage cell line RAW264.7 was obtained from the Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China), and cultured in a complete DMEM media supplemented with 10% (v/v) FBS, 100 g/mL streptomycin and 100 U/mL penicillin. The DC2.4 cells were from ATCC (American Type Culture Collection), which were cultured in RPMI1640 culture medium supplemented with 10% (v/v) FBS, 100 U/mL penicillin, and 100 g/mL streptomycin. All these cells were grown in a humidified incubator at 37 ◦ C, 5% CO2 . 2.3. Preparation of NP and antigen formulations Carbon nanotube (MWCNT) was obtained from Nanjing XFNANO Materials TechCo., Ltd. Outer average diameter was between 20 and 30 nm, and length was between 10 and 30 ␮m. Most studies were carried out using raw CNT, which generally contains microbial contaminants [20]. In order to eliminate the effects of microbial contaminants for the study, we ensured that all experiments were carried out in endotoxin-free conditions using ultra-pure grade reagents during both synthetic, in vitro and in vivo work. Ovalbumin was used as the immunogen.

Ova was conjugated to CNTs in the presence of a water-soluble carbodiimide (EDC) according to the process introduced by Kuiyang Jiang et al. [21], with some modifications. 10 mg CNT was suspended in a 10 mL mixture of 1:3 (V/V) HNO3 and H2 SO4 , and the SWCNT acid mixture was then subjected to sonication for 6 h. A nanotube mat was obtained after filtration using a 0.05 ␮m hydrophilized membrane and washed with pure grade water until filtrate became neutral, and then the nanotube was suspended in 15 mL MES buffer solution (50 mM, pH 6.0) by sonication. Then, 10 mL NHS (50 mgmL−1 ) in MES buffer was added to the above suspension. The mixture was sonicated for 30 min followed by addition of 5 mL fresh EDC (15 mgmL−1) in MES buffer. The mixture was stirred for 60 min and then, activated CNT solution was filtered through a 0.05 ␮m polycarbonate and rinsed thoroughly with MES buffer solution to remove excess EDC, NHS, and byproduct urea. The estered carbon nanotubes were redispersed in 10 mL of MES buffer and then 1.0 mL of a10 mg OVA in MES buffer was added. The reaction was carried out for 6 h. The nanotube suspension was centrifuged and washed with 50 mM MES buffer solution (pH 6.0) to remove unbound protein. The washed protein–nanotube conjugates were dispersed in 50 mM MES buffer solution. Microcystin-LR (MC-LR), KLH, and MWCNT were also conjugated. In a typical experiment, 2.5 mg of microcystins was dissolved in 0.5 mL of N-dimethyl formamide (DMF) followed by addition of freshly prepared EDC solution (3.75 mg of EDC in 0.5 mL DMF) and NHS solution (3.75 mg of NHS in 0.15 mL DMF). The reaction was kept at room temp for 30 min and then kept at 4 ◦ C overnight. The mixture was added slowly to 10 mg KLH which was dissolved in 5 mL of 0.1 M MES buffer. The estered carbon nanotubes were redispersed in 5 mL of MES buffer, and the mixture of KLH and MC-LR were added to estered carbon nanotubes. The reaction was carried out for 6 h. The nanotube suspension was centrifuged and washed with 50 mM MES buffer three times to remove unbound protein. 2.4. Characterization of conjugation To identify the characterization of OVA-MWCNT, OVA-MWCNT as well as MWCNT and OVA were scanned in the UV–vis spectrum from 200 nm to 400 nm. The protein concentration was measured by BCA method. The content of the MC-LR was determined by ELISA method to confirm the successful conjugation. 2.5. Cytotoxicity assay Cells were seeded at 104 cells/well (RAW264.7 cell and DC2.4) in 96-well plates. Cell viability was detected using a CCK-8 reagent. Cell were incubated with MWCNT-OVA for 48 h followed by addition of CCK-8 reagent to each well. After incubation at 37 ◦ C and 5% CO2 for 4 h, the absorbance at 450 nm was measured using a microplate reader. 2.6. In vitro cellular uptake In vitro studies were carried out using FITC labeled ova in DC2.4 cells. FITC labeled ova were prepared according to Stephanie Konnings et al. [22], with some modifications. First, 5 mg of ova was dissolved in 5 mL of carbonate buffer (0.01 M, pH 9.0). Next, 0.75 mg FITC dissolved in 0.75 mL DMSO was added to the ova solution. The mixture was gently stirred in the dark at 4 ◦ C for 8 h. Then the reaction was terminated by adding 10 ␮L 0.5 M NH4 Cl. Unbound FITC was removed by dialysis over three days. The resulting FITC-OVA solution was freeze-dried and subsequently stored protected from light at 4 ◦ C. Flow cytometry technique was used to investigate the effects of particles on antigen uptake, during which DC2.4 cells were seeded

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in a 6-well plate at a concentration of 5 × 105 cell/well for 12 h. Then FITC-OVA and FITC-OVA-MWCNT were added to the cells at a final ova concentration of 300 ␮g/mL in cell medium, and the cells were incubated for 4 h. Subsequently, cells were re-suspended and washed by PBS. These samples were analyzed on flow cytometer (Becton Dickinson). Cells without any treatment served as the blank control. Confocal laser-scanning microscope (CLSM) images were also taken. 2 × 105 /mL of DC2.4 cells were seeded in dish for 12 h, then the FITC-OVA and FITC-OVA-MWCNT were added to the cells at a final ova concentration of 300 ␮g/mL in cell medium, and the cells were incubated for 6 h. Cells were washed with PBS to remove the free particles, then were fixed in 3.7% paraformaldehyde for 30 min. The cell membranes and nuclei were stained with rhodaminephalloidin and DAPI, respectively, to give a visualization of the interaction between particles and cells [23]. 2.7. In vitro evaluation of the cell-surface molecules in macrophages RAW 264.7 cells and DC2.4 cells The effect of MWCNT on T cell recognition signals was analyzed in terms of the expression of the cell-surface molecules (CD11c, MHC II, CD40, CD86) on RAW 264.7 cells and DC2.4 cells in vitro. RAW 264.7 cells at a concentration of 4 × 105 cell/well (DC2.4 cell, 4 × 105 cell/well) were cultured in 6-well plates for 24 h and then incubated with different antigen formulations (20 ␮g/mL OVA and OVA-MWCNT) for 48 h at 37 ◦ C. Cells were treated with sterile PBS or lipopolysaccharides from E. coli (LPS, 50 ng/mL) as controls. Then cells in each group were washed and resuspended with PBS. The cells were washed and blocked with anti-mouse CD16/CD32 Ab, stained with fluorescence-labeled anti-mouse MHC Class II Ab, anti-mouse CD86 Ab, anti-mouse CD40 Ab, and anti-mouse CD11c Ab, at 4 ◦ C for 30 min. They were then washed with staining buffer, and analyzed with a BD FACSCalibur flow cytometer. 2.8. Cytokine production measurement For IL-1␤ measurement, RAW264.7 cells were first primed with 20 ng/mL LPS for 4 h. Then the cells were seeded in a 96-well cell culture plate at 104 cells/well. After seeding, the cells were treated with 20 ng/mL of LPS and various concentrations of MWCNTs for 24 h. The level of IL-1␤ was measured by ELISA. For IL-6 and TNF-␣ measurement, RAW264.7 cell were seeded in a 96-well cell culture plate at 10 4 cells/well, then were incubated with 20 ng/mL of LPS and MWCNTs at various concentrations for 24 h. The level of IL-6 and TNF-␣ were measured by ELISA. 2.9. Immunization and analysis of antibody responses Mice were randomly assigned to ten groups, and were injected subcutaneously (s.c.) with OVA alone, MWCNT-OVA, Freund’s adjuvant, MWCNT-KLH-MC-LR, and MWCNT-KLH-MC-LR for 14 d periodically. Additionally, to discover the synergistic effect between Freund’s adjuvant and MWCNT, mice were injected subcutaneously (s.c.) with OVA–MWCNT and Freund’s adjuvant (M group). After five times immunization, mice were sacrificed. All animals were bled through retro-orbital plexus 7 d post immunization. The anti-ova immunoglobulin G (IgG) titers of the individual mouse sera were determined using ELISA. The antibody titers were determined by considering as positive any OD450 absorbance value higher than 2.1 times the mean of the PBS alone group [24]. 2.10. Detection of complement 3 (C3) and C5a proteins C3 and C5a are measured by ELISA following the manufacturer’s protocols. Sera of J group mice which were immunized with OVA-

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MWCNT were collected at one week after the last immunization. The blood samples were clotted at room temperature and centrifuged at 4000 rpm for 20 min to separate the serum [25]. 2.11. Statistical analysis Statistical analysis was carried out using the student’s independent t-test. A value of p < 0.05 was considered to be significant. 3. Results and discussion 3.1. MWCNT- OVA conjugation characterization Fig. 1A shows the absorption peaks of OVA-MWCNT and OVA. As shown, OVA had an absorption peak at 280 nm and 210 nm. The carrier MWCNT had no absorption peak at 280 nm, and MWCNTOVA had an absorption peak at 250–280 nm. MWCNT-OVA samples had characteristic absorption peaks which show the unique pattern of proteins. The results above show that MWCNT were successfully conjugated with OVA. Fig. 1B shows the CD spectrum of MWCNTOVA and OVA alone, suggesting the protein secondary structure of OVA had changed after conjugating to SWCNT. Fig. 1C shows the image of the stable carboxylated MWCNT solution. Acid-treated MWCNT (Fig. 1D) and MWCNT-OVA (Fig. 1E) were visualized by TEM. As can be seen, the surface of MWCNT decorated by OVA molecules is different from the surface of acidtreated MWCNT, and the bundles of MWCNT decorated by OVA molecules is thicker, indicating that the protein conjugation was successful. On the other hand, the content of the MC-LR were 263.8 ␮g/L, detected by commercially available microcystin detection kit, indicating that MC-LR was conjugated to KLH. 3.2. Cellular uptake profiles of MWCNT-OVA The ability of nanoparticles to be taken up avidly by APCs is very important for their adjuvant effect [26]. To study the effect on cellular uptake of OVA, FITC-OVA and, FITC-OVAMWCNT were incubated with the professional APCs (DC2.4) for 6 h. Ultraviolet–visible spectra of OVA and FITC-OVA is shown in Fig. S1. FITC have the characteristic ultraviolet absorption peak at 495 nm. FITC-OVA also presented this peak, and red shift occurred. FACS analysis (Fig. 2A) showed that the fluorescent intensity of the FITC-OVA-MWCNT group was significantly higher than that in the pure-antigen FITC-OVA group. CLSM images (Fig. 2B–D) further provided visions that OVA-MWCNT accumulated in the cells to a large extent, compared to the FITC-OVA group. Studies have shown that the enhanced uptake in APCs was a possible stimulation mechanism of the adjuvant effect [26,27]. In this aspect, such an elevated antigen uptake would be a splendid property for MWCNT as a potent adjuvant. 3.3. MWCNT increase the expression of MHCII, CD40, and CD86 on RAW264.7 cells Interaction of several molecules such as MHC and CD86 molecules between T cells and APCs could activate T-cells. MHC II molecules present proteins for T cells that perform humoral response. CD40/CD86 are also known as co-stimulators for T cell activation and survival [28,29]. Additionally, we studied whether the MWCNT enhance cell maturation, and the expression of CD11c was analyzed. As shown in Fig. 3, in RAW 264.7 cells, OVA had little effect on the level of MHCII, CD40, CD86, and CD11c. OVA-MWCNT apparently enhanced the expression of MHCII after 48 h. Since exogenous antigens are generally loaded onto MHC II molecules for CD4+ T cells, upregulation of both MHCII and titer in vitro means the

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Fig. 1. MWCNT- OVA conjugation characterization. (A) Ultraviolet–visible spectra of OVA-MWCNT, SCNT, and OVA. (B) CD spectrum of free OVA and MWNT-OVA. (C) Image of stable carboxylated MWCNT solution. (C) TEM image of MWNT-COOH. (D) TEM image of MWNT –OVA conjugates.

Fig. 2. Effect of MWCNT on cellular uptake of FITC–OVA (green) in antigen presenting cells DC2.4. (A) Flow cytometric analysis (FACS) and (B–D) confocal-laser-scanning microscope (CLSM) images showing the cellular internalization of pure-antigen, OVA (B), FITC–OVA- MWCNT (C), and cell treated with PBS as control (A) (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

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Fig. 3. Effect of MWCNT on the expression of MHCII, CD40, and CD86 on RAW264.7 cells. The expression of the maturation markers MHCII, CD40, CD86 and CD11c on RAW264.7 cells treated with either medium (black line), OVA (red line), MWCNT-OVA (yellow line), or LPS (blue line) for 48 h. Gray background corresponds to the isotype control (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

OVA-MWCNT formulation enhanced CD4+ T mediated humoral immunity [30]. OVA-MWCNT apparently enhanced the expression of CD40 after 48 h. The expression of CD86 was slightly upregulated. The elevation of the aforementioned activation signals (MHC II molecules) and upregulated co-stimulators would make OVAMWCNT more powerful in provoking efficient T cell immunity. OVA-MWCNT apparently enhanced the expression of CD11c after 48 h, indicating that OVA-MWCNTs promote the maturation of APC. This is in agreement with previous studies, where APCs are shown to enhance the surface expression of CD40/86 and MHCII when exposed to CNTs in vitro and in vivo [31–33]. Interestingly, the expression of CD86 induced by cells treated with LPS slightly decreased compared with the untreated cells after 48 h, which is in agreement with J. Palomäki’s studies [33] that expression of CD86 on RAW 264.7 cells was lowered 48 h after LPS exposure. However, expression of CD86 after 48 h was increased. We compared the MHCII and CD40/86 levels of the experimental group’s DC2.4 cells, and found that OVA and OVA-MWCNT had no effects on the expression of MHCI/II, CD40, or CD86 on DC2.4 cells after 48 h. Stimulation with OVA-MWCNT did not reveal any remarkable changes in its expression (Fig. S2). 3.4. Inflammasome activation in RAW264.7 cells after exposure to MWCNT-OVA IL-1␤ is one of the important inflammatory mediators and a pro-inflammatory cytokine which plays an important role in the immune response. Functionalization of carbon nanoparticles can promote NLRP3 inflammasome activation. The inflammatory properties of MWCNT are related to their physicochemical characteristics and chemical surface functionalization [34]. Therefore, the production of IL-1␤ in RAW246.7 was investigated by ELISA to demonstrate whether or not the difference in antibody produc-

tion is due to inflammasome activation induced by different carbon nanotubes. Cytotoxicity in RAW246.7 after exposure to MWCNT-OVA for 24 h is shown in Fig. 4A. Cytotoxicity in DC2.4 after exposure to MWCNT-OVA for 24 h is shown in Fig. S3. As shown in Fig. 4B, IL-1␤ were assessed by ELISA. LPS was used to increases the amount of pro-IL-1, the immature state of IL-1. OVA had little effect on cell viability while cytotoxicity occurred in cells treated with MWCNT-OVA at a concentration of 30 ␮g/mL and 100 ␮g/mL. This is correlated with the production of IL-1␤ in cells treated with OVA and MWCNT-OVA. OVA evoked little IL-1␤ production while MWCNT-OVA induced increased IL-1␤ production at a concentration of 100 ␮g/mL. These results suggest that carbon nanotubes induce inflammasome activation. The production of tumor necrosis factor-␣ (TNF-␣) and IL-6 in RAW246.7 was investigated by ELISA to demonstrate whether or not the antibody production is due to inflammatory cytokine secretion induced by MWCNT. TNF-␣ is a pro-inflammatory cytokine mainly secreted by APCs and T cells. It is noteworthy that TNF-␣ is not induced via the inflammasome mediated immune response [35,36]. IL-6 is mainly produced by macrophages, T cells, and B cells. It is one of the key cytokines involved in immune regulation and inflammatory responses. Importantly, IL-6 can promote B cell differentiation into plasma cells and induce increased IL-2 expression and activate lymphocytes. We used an ELISA kit to detect TNF-␣ and IL-6 induction from RAW246.7 after exposure to MWCNT. As shown in Fig. 4C, MWCNT-OVA induced significantly higher levels of TNF-␣ at a concentration of 30 ␮g/mL and 100 ␮g/mL, although TNF-␣ secretion from cells treated with PBS and OVA alone did not have a significant increase. The induction of IL-6 showed similar trends to that of TNF-␣. Our in vitro experiments clearly demonstrate that MWCNT can induce the production of different kinds of cytokines from RAW264.7 at different concen-

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Fig. 4. Effect of MWCNT on inflammasome activation in RAW264.7 cells. (A) Cytotoxicity of MWCNT-OVA to RAW 264.7 cells at various concentrations after incubation for 24 h. (B) IL-1␤,(C)TNF-␣, and(D)IL-6 secretion from RAW 264.7 as an indication of inflammasome activation. Significant differences: *p < 0.05 vs control (mean ±SD).

Fig. 5. Anti-OVA antibody responses following immunization with OVA conjugated with MWCNT. Mice were subcutaneously injected with low dose OVA alone (A), low dose MWCNT-OVA (B), medium dose OVA alone (C), medium dose MWCNTOVA (D), high dose OVA alone (E), high dose MWCNT-OVA (F), Freund’s adjuvant (G group), OVA-MWCNT and Freund’s adjuvant (H group), MWCNT-KLH-MC-LR (I group), or MWCNT-KLH-MC-LR and Freund’s adjuvant (J group). The OVA-specific IgG titers and MC-LR-specific were detected at 37 d, 52 d, 67 d, and 82 d. Significant differences: *p < 0.05 vs control (mean ± SD), n = 8.

trations. These results suggest that carbon nanotubes’ induction of inflammatory cytokine secretion in RAW264.7 cells is related to the production of antibody, and that MWCNT enhance the immune response via different cytokine pathways. 3.5. Antibody responses in mice induced by different MWCNT-OVA Groups of eight mice were subcutaneously injected each time with 30 ␮g OVA/animal/dose of MWCNTs (low dose), 100 ␮g OVA/animal/dose of MWCNTs (medium dose), or 300 ␮g OVA/animal/dose of MWCNT (high dose); 30 ␮g OVA alone (low dose), 100 ␮g OVA alone (medium dose), or 300 ␮g OVA alone (high dose); or PBS as a negative control. OVA protein was coated to detect the OVA-specific IgG titers in mice by ELISA. As shown in Fig. 2, the IgG specific to OVA increased in mice immunized with all dose of the MWCNT-OVA, compared to mice immunized with all doses

of the OVA alone. Among the three dose levels of MWCNT-OVA, the medium does of MWCNT-OVA showed the highest antibody induction at the 82nd day. However, the high dose of MWCNT-OVA induced lower antibody titer than did the medium dose. The above results suggest that the MWCNT have an adjuvant effect. Compared with MWCNT conjugated to OVA, MWCNT conjugated to OVA injected with Freund’s adjuvant got higher titers, suggesting that carbon nanotubes and commercial Freund’s adjuvant have a synergistic effect. Additionally, MWCNT and MC-LR were conjugated to KLH,forming MWCNT-KLH-MC-LR,to testify to the enhanced humoral immune response of mouse injected with MWCNTKLH-MC-LR. MC-LR-OVA was coated to detect the MC-LR-specific IgG titers in mice by ELISA at 37th, 52nd, 67th, and 82nd day. Compared with KLH-MC-LR co-administered with Freund’s adjuvant, KLH-MC-LR conjugated to MWCNT induced higher MC-LR –specific IgG titer, which is 3.1 times the titer induced by KLH-MC-LR co-administered with Freund’s adjuvant. The 50% inhibition concentration (IC50 ) of antibody induced by KLHMC-LR co-administered with Freund’s adjuvant for MC-LR was 1.58 ± 0.1 ␮g/L. Except the cross-reactivity of MC-RR is about 21.6%, both MC-LF and MC-LW showed lower cross-reactivates (CR < 0.79%). 3.6. The activation of the complement system Moderate activation of the complement system promotes humoral immunity [37]. Complement components C3a, C5a, and C5b67 have chemokine activity and attract neutrophils and mononuclear macrophages to sites of inflammation. Complement receptor CR2 (CD21) can regulate B cell proliferation, differentiation, memory, and antibody production. Complement component C3b, C4b, iC3b combine with immunogen to make it easy to be taken up by phagocytic cells [38]. Therefore, complement activators may act as adjuvants. As described before, during activation of the complement, the third complement component C3 is enzymatically cleaved and C5a, which can recruit immune cells to the site of inflammation, is released. The variety of C3 and C5a reflect the activation of the complement system. We investigated whether MWCNTs could activate

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Fig. 6. Complement activation induced by subcutaneous injection of MWCNT-OVA. (A) C3 concetration of mice injected with OVA or MWCNT-OVA at the 2nd, 37th, 52nd, 67th, 82nd day. (B) C5a concetration of mice injected with OVA or MWCNT-OVA at the 37th, 52nd, 67th, 82nd day. Significant differences: *p < 0.05 vs control (mean ± SD), n = 8.

the complement system, which promotes antibody responses by detecting the levels of C3 and C5a in the serum of the mice. Mice were sacrificed after five times immunization. As shown in Fig. 6A, the levels of C3 in the serum of the mice immunized with MWCNTOVA are lower than that of the mice immunized with OVA alone. Meanwhile, as shown in Fig. 6B, C5a showed the opposite trend; the level of C5a induced by MWCNT-OVA increased compared with the control. OVA alone slightly induced the decreased C3 and increased C5a. However, the level of C3 induced by OVA was higher than that induced by MWCNT-OVA. The level of C5a induced by OVA was lower than that induced by MWCNT-OVA. The dynamic variation in the levels of C3 and C5a in the serum suggest that the MWCNT which are conjugated to OVA can activate the complement system. These findings are consistent with and may provide an explanation for our finding that CNTs enhance anti-OVA antibody response by immunizing with OVA coupled to nanotubes or mixed with nanotubes. Additionally, it is observed that much MWCNT-OVA aggregate at the injection site 90 days post subcutaneous injection (Fig. S4.) which may be related to the continuesly increase of C5a (Fig. 5). 4. Conclusions In the current study, we investigated humoral immune responses of mice injected subcutaneously with MWCNT-OVA. The middle dose of MWCNT-OVA induced the highest level of OVA-specific antibodies and have a synergistic effect with the commercial Freund’s adjuvant. We assume that varying degrees of increase of antibody titer are affected by the following three factors. First, differences in the level of uptake of these NPs resulted in differences in antibody production. Second, cytokine production by the APCs induced by different MWCNTs affect the titer. Finally, activation of the complement system induced by different MWCNT indirectly led to increase in titers. We carried out in vitro and in vivo studies to test our hypothesis. In vitro, DC2.4 cells uptake more f-MWCNT-OVA than pure-antigen OVA. CD40/86, MHCII, and CD11c molecules of RAW264.7 incubated with MWCNT-OVA increased, whereas MWCNT-OVA did not stimulate DC2.4 cell maturation. MWCNT-OVA induced the IL-1␤, TNF-␣, and IL-6 cytokine secretion from RAW264.7. During in vivo studies we found that MWCNT could activate the complement system. We speculate that the activation of the complement system, higher efficiency of cellular uptake, and cytokine secretion induced by MWCNT cause the high antibody titer. Acknowledgments This work has been supported by the NSFC-Guangdong province of China (U13012141), National Natural Science Foundation of

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