Lentinan-Modified Carbon Nanotubes as an Antigen Delivery System

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Lentinan-modified carbon nanotubes as an antigen delivery system modulate immune response in vitro and in vivo Jie Xing, Zhenguang Liu, Yifan Huang, Tao Qin, Ruonan Bo, Sisi Zheng, Li Luo, Yee Huang, Yale Niu, and Deyun Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04591 • Publication Date (Web): 14 Jul 2016 Downloaded from http://pubs.acs.org on July 22, 2016

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ACS Applied Materials & Interfaces

Lentinan-modified carbon nanotubes as an antigen delivery system modulate immune response in vitro and in vivo Jie Xing†, Zhenguang Liu†, Yifan Huang‡, Tao Qin‡, Ruonan Bo†, Sisi Zheng†, Li luo†, Yee Huang†, Yale Niu†, Deyun Wang*†,‡ †

Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing

Agricultural University, Nanjing 210095, PR China. ‡

College of Animal Science and Veterinary Medicine, Fujian Agriculture and Forestry

University, Fuzhou 350002, PR China.

ABSTRACT

Adjuvants enhance immunogenicity and sustain long-term immune responses. As vital components of vaccines, efficient adjuvants are highly desirable. Recent evidence regarding the potential of carbon nanotubes (CNTs) to act as a support material has suggested that certain properties, such as their unique hollow structure, high specific surface area and chemical stability, make CNTs desirable for a variety of antigen-delivery applications. Lentinan, a β-1, 3glucohexaose with β-1, 6-branches that is extracted from the mushroom Lentinus edodes, is an effective immunostimulatory drug that has been clinically used in Japan and China, and recent

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studies have proved that specific beta-glucans can bind to various immune receptors. In this research, we covalently attached lentinan to multi-walled carbon nanotubes (MWCNTs) and tested their ability to enhance immune responses as a vaccine delivery system. In vitro study results showed that the nanotube constructs could rapidly enter dendritic cells and carry large amounts of antigen. Moreover, maturation markers were significantly up-regulated versus the control. Thus, lentinan-modified multi-walled carbon nanotubes (L-MWCNTs) were regarded as an effective intracellular antigen depot and a catalyzer that could induce phenotypic and functional maturation of dendritic cells. Furthermore, compared with L-MWCNTs (35 µg/ml), a corresponding concentration of carboxylic carbon nanotubes (C-MWCNTs, 31.8 µg/ml) and an equivalent concentration of lentinan (3.2 µg/ml) did not remarkably influence the immune reaction in vitro or in vivo. Hence, we can hypothesize that the capability of L-MWCNTs was a consequence of the increased intracellular quantity of lentinan grafted onto the nanotubes. Overall, our studies demonstrated that L-MWCNTs significantly increased antigen accumulation in the cells and potentiated cellular and humoral immunity. In conclusion, L-MWCNTs constitute a potential vaccine delivery system to enhance immunogenicity for therapeutic purposes. KEYWORDS carbon nanotube; lentinan; adjuvant; immune response; OVA


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Introduction As an essential approach for preventing infectious diseases, vaccination has been widely accepted. Following scientific research on vaccine development, which has been strengthened globally by the World Health Organization, significant progress and many breakthroughs have been achieved over the past several decades. In the immunization process, the ingestion of antigens by antigen-presenting cells (APCs) is one of the key processes.1,2 However, the uptake of small soluble antigens, such as proteins and peptides, into cells is inadequate, and thus, these types of antigens are weakly immunogenic.3,4 Therefore, as vital components of vaccines, efficient adjuvants are highly desirable for improving humoral and cellular immune responses.5,6 Optimally designed vaccine adjuvants should be able to accelerate and intensify immunoreactions and modulate immunological responses with no cytotoxicity. Many limitations of commonly used immune adjuvants have been discovered (e.g., strong local stimulation, carcinogenesis and side effects).7 Their particular physicochemical properties and unique structure make carbon nanotubes (CNTs) useful for biomaterial applications.8 As a vaccine delivery system, the potential of CNTs is attributed to their non-immunogenicity, and as a support material, they have the ability to extend antigen-specific humoral and cellular immune responses over long periods of time.9,10 Among their many characteristics, it has already been proved that CNTs can penetrate cellular membranes and deliver biomolecules such as proteins,11 drugs12–14 and DNA15,16 into the cytoplasm without causing cell death.17–19 However, their carbon skeletons make CNTs very hydrophobic and insoluble, which quite limits the biomedical application of pure CNTs.20–22 One solution to this problem is chemical modification. Water solubility is increased when chemical molecules are grafted to the exterior wall of the nanotubes, and the application of CNTs in


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biomedicine becomes feasible. Although the biocompatibility of nanotubes is improved by functionalization, modification may modulate different cellular functions.23 In an earlier report, several pro-inflammatory genes in a monocytic cell line were activated by functionalized multiwalled carbon nanotubes, which were not cytotoxic to immune cells.24 As an immunological adjuvant, herbal polysaccharides can stimulate lymphocyte proliferation activity and enhance antibody titers.25 In recent years, the polysaccharide research field has focused on structural improvement and molecular modification, and polysaccharide complexes have shown stronger biological activities.7,26 Lentinan is a high molecular weight polysaccharide and a biological response modifier that can activate the immune system.27,28 Lentinan has been extensively studied because of its few side effects and bioavailability. Since 1985, lentinan has been clinically used in the treatment of cancer and combined with chemotherapy.29 In this study, we synthesized a novel adjuvant by covalently attaching lentinan to MWCNTs. The super-hydrophobic characteristic of the carbon nanotubes and the powerful immunological function of lentinan proved to be an excellent combination. The new adjuvant showed higher solubility and less cytotoxicity than C-MWCNTs, and it provided superior intracellular biostability, with an excellent depot effect that lentinan does not have. The aim of this work was to determine the physical and chemical properties of L-MWCNTs and then to investigate their influence on dendritic cells, in vitro and in vivo, as well as their effects on humoral and cellular immune responses.

Experimental Immobilization of biomolecules on the MWCNT surface C-MWCNTs (95 wt%, 9-10 nm in diameter, 1-2 µm in length) were purchased from SigmaAldrich (Nanocyl Inc. Sambreville Belgium). The amount of –COOH in the tube was 8 wt%,


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corresponding to approximately two functional groups for every 100 carbon atoms. N, Ndimethylformamide (Tianjin Bodi Chemical Technologyn Co., Ltd., TianJing, China) and lentinan (98%, Shanghai Jianglai Reagent Co., Ltd., Shanghai, China) were dried to remove any trace of water in advance. The C-MWCNTs (23 mg/ml) were first treated with thionyl chloride by using an ultrasonic bath for 1 h. DMF was then added to this mixture as catalyzer and refluxed for 15 h at 70 °C. After distillation under vacuum until the liquid became a solid, the obtained compound was placed in a vacuum drying oven for 3 h at 50 °C to remove the thionyl chloride. The nanotubes (23 mg/ml) and lentinan (8 mg /ml) were then treated with a mixture of DMF and pyridine (catalyzer) for 24 h at 110 °C. After centrifugation, the product was washed ultrasonically 5 times with excess DMAc/LiCl (95/5) solution and 3 times with warm water to remove the excess organic solvent and lentinan. The functionalized MWCNTs (L-MWCNTs) were then dried by vacuum freeze drying. The procedure of protein absorption to the MWCNTs was accelerated by using an ultrasonic cell disintegrator (JY92-II DN, Xinzhi Bio-technology and Science Inc., Ningbo, Zhejiang, China) for 30 min.30 To measure the content of the standard antigenic protein (OVA) attached to the L-MWCNTs, 10 mg of L-MWCNTs and 15 mg of OVA were placed in a 20-ml Erlenmeyer flask containing 10 ml of saline solution (pH 7.4) at 37°C and stirred with a magnetic stirrer. After incubation (0, 30, 60, 90, 120, 150 and 180 min), the upper fluid was collected and centrifuged at 12,000 rpm for 30 min at 4 °C, and then, the OVA concentration in the supernatant was assessed with a BCA Protein Assay Kit. Characterization of L-MWCNTs To examine the mechanism of the connection between the MWCNTs and lentinan, Fouriertransform infrared (FTIR) spectra were recorded using a 610-IR micro infrared spectrometer


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(Varian, U.S.A) in the wavelength range of 500~4000 cm-1 and confirmed with UV-scanning spectrophotometry at the wavelength of 190 ~ 250 nm, which was recorded by a Cary 5000 UVVis-NIR Spectrophotometer (Varian, U.S.A). The morphological examination of the L-MWCNTs was conducted with a Tecnai G2 F30 STWIN Transmission Electron Microscope (Philips, Netherlands) at 200 kV. The zeta potential of the nanotubes was measured by using a Zetasizer Nano ZS (Malvern, England) at 25 °C in PH 7.4. The amount of lentinan grafted onto the multi-walled carbon nanotubes was defined by thermogravimetric analysis (TGA). This examination was performed using a Pyris 1 TGA (PerkinElmer, U.S.A) between 20 °C and 500 °C at a heating rate of 20°C/min in flowing nitrogen. The grafting percentage could be calculated from the weight loss of the lentinan, CMWCNTs and L-MWCNTs. Cytotoxicity analysis of L-MWCNTs on splenocytes Based on previously described methods,31 splenocytes (2.5 × 106 cells/mL) were seeded at 100 µl per well in a 96-well plate and then incubated with L-MWCNTs at a series of concentrations (0 to 100 µM). After culture under a humidified atmosphere containing 5% CO2 at 37 °C for 48 h, 30 µL of MTT solution (5 mg/mL) was added to each well for 4 h of incubation. Then, the medium and MTT were replaced carefully, and 100 µL of DMSO solution was added. The absorbance was measured at 570 nm by using a microliter enzyme-linked immunosorbent assay reader (Bio-Rad, U.S.A). The results were expressed as the percentage of viable cells. The data of each concentration are presented from three replicates. Phenotypic analysis of bone marrow-derived dendritic cells


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The expression of key surface markers by bone marrow-derived dendritic cells (BMDCs) cultured for 48 h with L-MWCNTs (35 µg/ml), control C-MWCNTs (31.8 µg/ml), an equivalent amount of soluble lentinan (3.2 µg/ml), BMDCs cultured in medium alone as a blank control, and the dendritic cells cultivated in medium with LPS as a positive control (Sigma, 10 µg/ml) were determined. After collecting the treated BMDCs from the culture wells, the cells were washed with PBS. Then, the BMDCs were incubated for 30 min at 37°C with anti-CD11c monoclonal antibody (mAb) coupled with FTTC, anti-MHCⅡ mAb conjugated to phycoerythrin (PE), and anti-CD80-PE-Cy7 and anti-CD86-APC mAbs or the respective isotypes (BD Biosciences). Cells were analyzed by a BD FACSVerseTM flow cytometer. Antigen presentation assessment of BMDCs BMDCs were cultivated for 24 h with the constructs of FITC-labeled OVA alone and LMWCNTs+OVA complexes. Then, the treated cells were fixed with 4% paraformaldehyde for 15 min at 4°C and rinsed off with a gentle PBS wash. After nuclei were stained with DAPI for 5 min, DCs were washed twice with PBS. The experiment was observed using an LSM 710 laser scanning confocal microscope (Carl Zeiss, Germany). Immunization protocol Five-week-old male BALB/c mice purchased from the Comparative Medicine Centre of Yangzhou University were randomly divided into 5 groups with 24 mice in each group. Mice had free access to food and water and were kept under controlled temperature conditions and light–dark cycle. The mice were inoculated subcutaneously 3 times at a 2-week interval with the following five types of preparations: 200 µl PBS, 20 µg of OVA mixed with 3.2 µg of lentinan in 200 µl PBS, 20 µg of OVA mixed with 31.8 µg of C-MWCNTs in 200 µl PBS, 20 µg of OVA mixed with 35


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µg L-MWCNTs in 200 µl PBS, 20 µg of OVA dissolved in 200 µl PBS, and 20 µg of OVA formulated with Freund's complete adjuvant (FCA). Protein binding to the L-MWCNTs was accelerated by continuous ultrasonication for 30 min. The blood and spleens of vaccinated and control mice were collected at 7, 14, 21 and 28 days after the last immunization. Serum antibody analysis OVA-specific antibodies of sera from immunized mice were quantitatively measured by readyto-use Sandwich ELISA kits (R&D, Co., U.S.) according to the manufacturer's guidelines. Quantification of cytokine production by ELISA The cytokine levels of IL-4, IL-6, TNF-α, and IFN-γ were assayed by the ready-to-use Sandwich ELISA kits (R&D, Co., U.S.A) according to the manufacturer's guidelines. The absorbance at 450 nm was detected by a microliter enzyme-linked immunosorbent assay reader. Analysis of lymphocytes in the spleen At 7 days after the last immunization, the spleens of all groups were removed and homogenized with medium. The splenocytes (1 × 106 cells/mL) were plated onto a 24-well plate (1.5 mL/well) in triplicate and stimulated with OVA (50 µg/mL). Then, the cells were cultivated at 37 °C in a humid atmosphere with 5% CO2 for 72 h, and after that, the splenocytes were incubated for 30 min at 37 °C with anti-CD3e-PE-Cy5, anti-CD8-PE and anti-CD4-FITC mAbs or the respective isotypes (BD Biosciences). The percentage of CD3+ CD4+ and CD3+ CD8+ T cells was assessed by using fluorescence activated cell sorting (FACS). Analysis of dendritic cell activation and maturation in lymph nodes Sciatic and popliteal lymph nodes of vaccinated and control mice at 24 and 48 h after the first immunization were collected to determine DC activation in the lymph nodes. These tissues were homogenized into a single cell suspension, washed and stained with anti-CD11c–FITC, anti-


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MHC II-PE, anti-CD80-PE-Cy7and anti-CD86-APC, or the respective isotypes (BD Biosciences). All antibodies were used according to the manufacturer's guidelines. Dendritic cells were analyzed by a BD FACSVerseTM flow cytometer. Statistical analysis Experimental data were reported as the mean ± standard deviation (SD). Statistical significance for the experimental and corresponding control samples was evaluated using Student’s t-test and one-way analysis of variance (ANOVA); p-values less than 0.05 were considered statistically significant.

Results and discussion Characterization of L-MWCNTs and nanotube-formulated vaccines L-MWCNTs, a novel adjuvant integrating the advantages of carbon nanotubes and lentinan, have the potential to be a powerful and effective antigen delivery system. To further investigate whether this new nanocarrier has the ability to intensify the immune response, OVA was attached to the L-MWCNTs, as depicted in Figure 1a.


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Figure 1. Functionalization and physicochemical characterization of L-MWCNTs. (a) Schematic diagram of preparing the antigen delivery system. (b) Structure of lentinan. (c) FTIR transmission spectra of C-MWCNTs, L-MWCNTs and lentinan. (d) Absorbance spectra in the UV region for C-MWCNTs, L-MWCNTs and lentinan. (e, g) Representative TEM images of C-MWCNTs. (f, h) TEM images of L-MWCNTs. (i) TGA curves of C-MWCNTs, L-MWCNTs and lentinan. (j) The kinetics curve of OVA.

As shown in Figure 1c, the C-MWCNTs were detected by the presence of a peak at ≈ 1725 cm1

in the FTIR spectra, which was attributed to the stretching vibration of C = O in the carboxylic

group. The molecular C-C bond and the C-O stretching were situated at ≈1571 and ≈1154 cm-1, respectively.32 The lentinan spectrum showed strong wide band at ≈3413 cm-1, corresponding to O-H stretching of intramolecular or intermolecular hydrogen bonds and N-H stretching of –NH2. The peak at ≈2927 cm-1 was due to the C-H stretching vibration of methyne.33 The peak at ≈1024 cm-1 was assigned to the deformation vibration of the primary alcohol hydroxyl. The region at ≈1153 and ≈927 cm-1 corresponded to the symmetric and asymmetric vibrations of the ether linkages of the pyran ring polysaccharide (C-O-C). The characteristic absorption peak of furanose was situated at ≈850 cm-1, which was attributed to the deformation vibration of C-H in the anomerism of the α-D-glucose. The FTIR transmission spectra of L-MWCNTs showed a partial disappearance of the broad band at ≈1154 cm-1 of C-MWCNTs and a new peak at ≈1072 cm-1. That most likely occurred because the acyl chloride of the carbon nanotubes reacted with the primary alcohol hydroxyl of lentinan to form an ester bond, and the presence of peaks at ≈2913 and ≈856 cm-1 indicated that MWCNTs were functionalized with lentinan.


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The interaction between MWCNTs and lentinan was characterized by optical absorption spectroscopy (Figure 1d). The characteristic peak of lentinan at ≈198 nm was observed in its spectrum. A prominent absorption band at ≈203 nm was found in the spectrum of L-MWCNTs, and the red-shift of L-MWCNTs was quite possibly due to the structure of lentinan being changed when it was attached to the MWCNTs. These results were corroborated by the different decomposition temperatures of TGA. The surface chemistry of carbon nanotubes plays a decisive role in their properties.34 The morphologies of C-MWCNTs and L-MWCNTs were observed by TEM, and representative images are presented in Figure 1e, f, g and h. The sidewalls of C-MWCNTs were corrugated with carboxylated carbonaceous fragments. Compared with C-MWCNTs, there were many more attachments on the outer graphite sheets and inside the L-MWCNTs, which were lightly deformed and had a larger inner diameter. The TGA curves of C-MWCNTs, L-MWCNTs and lentinan were recorded and are displayed in Figure 1i. In the total heating process, the L-MWCNTs lost approximately 9.8 ± 0.9% by weight, whereas the C-MWCNTs and lentinan exhibited a weight loss of approximately 3.1 ± 0.2% and 73.8 ± 2.3%. The first weight loss interval below 130 °C for the three simples was because of the evaporation of absorbed water.33,35 The initial degradation temperatures of lentinan and L-MWCNTs were 194 °C and 150 °C. The different decomposition temperature was caused by the decomposition of lentinan, and the thermal degradation of L-MWCNTs showed that there was a certain amount of lentinan bounded to the MWCNTs, and the grafting ratio was 9.1%. In the release profile in Figure 1j, the kinetics curve of adsorption of the OVA model antigen on the L-MWCNTs shows that 0.2 mg OVA was absorbed by 1 mg of L-MWCNTs in 15 min,


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and a saturation of 0.45 mg of OVA absorbed by 1 mg of L-MWCNTs was reached after 150 min. This result indicated that for the 2:3 OVA/L-MWCNT weight ratio used in our formulation, even with lentinan grafted to the MWCNTs, there were still significant amounts of protein being adsorbed onto the surface of the nanotubes. The surface charge directly affects the stabilizing effect of the nanotubes, and the zeta potential of a biomolecule-MWCNT can be obtained through measurements at the edge of the carbon nanotube colloid. Colloidal particles with |ξ| > 15 mV are expected to be stable.36 Consequently, the ξ values of the biomolecule-MWCNTs should be at least comparable to the threshold. The ξ values of −20, −23 and −28 mV were estimated from the C-MWCNT, L-MWCNT and MWCNT+OVA dispersions, respectively, in physiological pH, indicating that all the biomolecule-MWCNTs disperse with high stability. For the L-MWCNT+OVA constructs, the average ξ value of −25 mV was between the ξ values of the L-MWCNT and MWCNT+OVA complexes, which indicated that both biomolecules were adsorbed onto the MWCNT surface and approached stable equilibrium. Cytotoxic effect of L-MWCNTs on splenocytes The viability of splenocytes exposed to L-MWCNTs was assessed by MTT assay. Splenocytes were incubated with 5 different concentrations of L-MWCNTs (10, 35, 60, 85 and 100 µg/ml) for 2 days (Figure 2a). This test revealed a dose-dependent decrease in the number of splenocytes cultured with L-MWCNTs. We observed no significant effect in viability up to a concentration of 35 µg/ml; therefore, this concentration was chosen for all subsequent assays.


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Figure 2. Uptake of L-MWCNT constructs by BMDCs. (a) Dose response plot of effect of L-MWCNTs on the viability of splenocytes was assessed by MTT assay. (b) The phenotype of BMDCs after culture with L-MWCNTs (35 µg/ml), CMWCNTs (31.8 µg/ml), soluble lentinan (3.2 µg/ml), a blank control and a positive control (LPS, 10 µg/ml) (c, d) Confocal images of BMDCs after treatment with OVA-FITC or LMWCNT+OVA for 24 h and nuclear staining with DAPI. The results are given as the mean ± SD (n = 3 different donors). *p < 0.05, **p < 0.01 compared to control.

Internalization of L-MWCNTs by mouse bone marrow-derived dendritic cells (BMDCs)


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To evaluate the maturation effect of L-MWCNTs, mouse bone marrow-derived dendritic cells (BMDCs) were chosen as a model. Based on the results of the cytotoxicity assay, we cultured BMDCs with the vaccine formulation’s concentration of L-MWCNTs, C-MWCNTs and soluble lentinan to investigate the influence on BMDCs. The phenotype of the BMDCs was evaluated based on the expression of MHCⅡand co-stimulatory molecules CD80 and CD86 as the maturation markers. The control C-MWCNTs did not modulate these maturation markers significantly, and lentinan simply enhanced the phenotype of CD80. In contrast, the up-regulated expression of MHCⅡ and CD80 indicated that L-MWCNTs could induce phenotypic and functional maturation of dendritic cells to a certain degree in vitro (Figure 2b). The cellular uptake of the nanotubes was examined by confocal fluorescence microscopy. Figure 2c and d showed confocal images of BMDCs after treatment with OVA-FITC or LMWCNT+OVA for 24 h. L-MWCNTs mainly distributed in the cytoplasm, especially near the nucleus, which indicated the phagocytic function of BMDCs. The colocalization of CNTs and the fluorescent protein confirmed the character of OVA attached to the L-MWCNTs, and the efficacy was significant. These results also proved that functionalization did not diminish the depot effect of the MWCNTs, and on the contrary, L-MWCNTs served as an efficient intracellular antigen depot. Furthermore, the images showed BMDCs loaded with the LMWCNT+OVA complex, which was visibly demonstrated by an enhancement of the fluorescence compared with cells only containing OVA, which indicated that L-MWCNTs are a productive antigen delivery system. L-MWCNTs+OVA induced antigen-specific immunity in mice To investigate the potential activity of L-MWCNTs on specific anti-OVA antibody responses in vivo, the mice were immunized with lentinan, carboxylic or lentinan-modified MWCNTs.


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Then, antibody IgG and IgG subclass titers were determined by ELISA. Lentinan immunization showed no remarkable difference from the group injected with OVA alone. For the C-MWCNT group, the amount of IgG was notably higher than that in the OVA group over the 14 days after the last immunization, and the complete formulation significantly enhanced the release of OVAspecific IgG during the whole 28 days with higher immunological intensity (Figure 3a). The immunoreactivity of the group immunized with L-MWCNTs+OVA constructs was almost equal to the group injected with FCA in the long term, but for IgG2a, L-MWCNTs were obviously more powerful than the other controls 28 days after final vaccination. Because the IgG2a antibody production reveals the Th1 immune response,37 we assumed that L-MWCNTs would induce a stronger Th1-biased immune response in the post period, even compared with FCA (Figure 3b).


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Figure 3. L-MWCNTs enhanced immune response in vivo. (a) ELISA detected the levels of OVA-specific IgG in serum of control and vaccinated mice. (b) ELISA detected the levels of IgG subclasses titers IgG1 and IgG2a in sera of control and vaccinated mice.

Cytokine levels in serum Cytokines in the serum were determined. Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-6) in the serum 7 and 28 days after the final immunization were measured by ELISA. Figure 3c and d demonstrates that the effect of L-MWCNTs on cytokine production is almost superior to FCA and better than that of other groups. Furthermore, the data proved that the new vaccine formulation could generate unremitting immune responses over long periods of time. These indicators suggested that L-MWCNTs could obviously enhance both the Th1 and Th2 immuneresponse. Notably, the secretion of some cytokines was significantly suppressed by the C-MWCNTs in prophase. This effect of inhibiting the Th1 and Th2 response could be because


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C-MWCNTs contain more metallic ions. These results indicated that L-MWCNTs have relatively minimal adverse effects in vivo.


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Figure 3. (c, d) L-4, IL-6, TNF-α, and IFN-γ levels in the serum 7 and 28 days after the last immunization were measured by ELISA. Values were presented as the mean ± SD of quadruplicates. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the OVA group. #p < 0.05, ##p < 0.01, ###p < 0.001 compared to control.

The activation of lymphocytes in secondary lymphoid tissues To examine the effect of L-MWCNTs on the cellular immune function of mice, we detected the percentage of CD4+and CD8+ T cells in the spleen 7 days after the last immunization. As Figure 4a and b reveal, L-MWCNTs stimulated higher percentages of both CD4+ and CD8+ T lymphocytes compared with OVA alone, whereas FCA failed to do so. L-MWCNTs significantly improved the percentage of CD4+ and CD8+, which indicates that L-MWCNTs can generate a powerful cellular immune response in vivo.


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Figure 4. Effects of L-MWCNTs on peripheral lymphoid organs. (a, b) Flow cytometry assays to detect the percentage of CD4+ cells and CD8+ cells in splenocytes removed from the mice 7 days after the last immunization that were injected with OVA and L-MWCNTs+OVA. (c, d) The phenotype of DCs in sciatic and popliteal lymph nodes at 24 and 48 h after the first immunization. Each experiment was repeated three times and is expressed as the mean± SD. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the OVA group. #p < 0.05, ##p < 0.01, ###p < 0.001 compared to control.


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We further examined the activation markers of dendritic cells in the lymph node. The expression of CD80 showed no obvious difference among the vaccine formulations and other groups at 24 h except control and just following primary immunization (Figure 4c). However, all three markers of the mice injected with L-MWCNTs+OVA displayed a significantly higher frequency than the negative control at 48 h, and the others were nearly indistinguishable, suggesting the promotion of the activation and maturation of dendritic cells in these lymph nodes (Figure 4d).

Conclusions In this study, we synthesized L-MWCNTs as a novel adjuvant by using covalent bonds to combine the advantages of two materials. For carbon nanotubes, L-MWCNTs showed higher solubility and lower cytotoxicity, and due to lentinan, the compound had excellent intracellular biostability with a long-acting depot effect. Our results demonstrated that L-MWCNTs were able to reinforce humoral and cellular immunity. This improvement of performance is most likely because the nanotubes prolong retention in the immunocytes while containing a large number of antigens, and lentinan acts as an immunopotentiating agent. The stimulatory side-effects and the toxicological of MWCNTs on the healthy mice had been investigated. The results showed that there was no obvious accumulation and injury of MWCNTs observed in the liver, spleen, kidney, or heart. 38 Therefore, L-MWCNTs did not presented any adverse reactions, which indicating the latent capacity for continue to develop. Given these encouraging results, L-MWCNTs are an excellent vaccine delivery system with good development prospects.


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ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author *Deyun Wang, Ph.D.; College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, P R China; E-mail: [email protected]; Tel: 0086-25-84395203; Fax: 0086-2584398669. Funding Sources National Natural Science Foundation of China (Grant No. 31372472), Special Fund for Agroscientific Research in the Public Interest (Grant Nos. 201303046, 201403051) Notes The authors have no conflicts of interest to declare.

ACKNOWLEDGMENT The project was supported by the National Natural Science Foundation of China (Grant No. 31372472), Special Fund for Agro-scientific Research in the Public Interest (Grant Nos. 201303046, 201403051) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We are grateful to all the staff in the Institute of Traditional Chinese Veterinary Medicine of Nanjing Agricultural University for their assistance with experiments. REFERENCES (1) Savina, A.; Amigorena, S. Phagocytosis and Antigen Presentation in Dendritic Cells. Immunol. Rev. 2007, 219, 143–156. (2) Batista, F. D.; Harwood, N. E. The Who, How and Where of Antigen Presentation to B Cells. Nat. Rev. Immunol. 2009, 9, 15–27.


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Lentinan-Modified Carbon Nanotubes as an Antigen Delivery System

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