Five-week-old female ICR mice (Grade II) weighing 18–22 g were purchased from Zhejiang Chinese Medical University Animal Research Center (Hangzhou, China) and acclimatized for one week prior to use. Rodent laboratory chow and tap water were provided ad libitum and maintained under controlled conditions with a temperature of 24 ± 1 °C, humidity of 50 ± 10%, and a 12/12 h light/dark cycle. All procedures related to the animals and their care conformed to the internationally accepted principles as found in the Guidelines for Keeping Experimental Animals issued by the government of China.

Chitosan (CS) was obtained from the Chitosan Company of Pan’an, Zhejiang, China (degree of deacetylation, 95%; average molecular weight, 220 kDa). Ovalbumin (OVA), concanavalin A (Con A), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lipopolysaccharide (LPS), RPMI-1640 medium, and rabbit anti-mouse IgG peroxidase conjugate were purchased from Sigma Chemical Co., Saint Louis, MO, USA; goat anti-mouse IgG1, IgG2a and IgG2b peroxidase conjugate were from Southern Biotech. Assoc., Birmingham, AL, USA; Quil A was kindly provided by BrenntagNordic A/S, Denmark. Fetal calf serum (FCS) was provided by Hangzhou Sijiqing Corp., Hangzhou, Zhejiang, China. Cytokines (IL-2, IL-10, IFN-γ) detecting ELISA kits were from Rapidbio Lab., West Hills, CA, USA. Trizol was from Invitrogen, China; revert Aid™ M-MuLV reverse transcriptase was from Fermentas, USA; diethylpyrocarbonate (DEPC) and ribonuclease inhibitor were from Biobasic, Canada; oligo (dT)₁₈ were from Sangon, China.

Human leukemia K562 cell lines, sensitive to natural killer (NK) cells, were purchased from the Institute of Cell Biology, Chinese Academy Sciences, Shanghai, China. They were maintained in the logarithmic phase of growth in RPMI 1640 medium supplemented with 2 mM l-glutamine (Sigma), 100 IU/mL penicillin, 100 g/mL streptomycin (Sigma), and 10% fetal calf serum at 37 °C under humidified air with 5% CO₂.

Chitosan (CS) was obtained from the Chitosan Company of Pan’an (Zhejiang Province, China). The degree of deacetylation was about 95% as determined by elemental analysis, and the average molecular weight of the chitosan was 220 kDa as determined by viscometric methods [34]. Chitosan nanoparticles were prepared and characterized as previously described [35]. Briefly, Chitosan was dissolved at 0.5% (w/v) with 1% (v/v) acetic acid (HOAc) and then raised to pH 4.6–4.8 with 10 N NaOH. CNP were formed by coacervation between positively charged chitosan (0.5%, w/v) and negatively charged sodium tripolyphosphate (0.25%, w/v). Nanoparticles with different mean size were obtained by adjusting the volume ratio of chitosan to tripolyphosphate solution. Nanoparticles were purified by centrifugation at 9000 g for 30 min. Supernatants were discarded and chitosan nanoparticles were extensively rinsed with distilled water to remove any NaOH residues, and freeze dried before further use or analysis. The freeze-dried chitosan nanoparticles were suspended in water for characterization or use for other experiments. Particle size distribution and the zeta potential of chitosan nanoparticles were determined using Zetasizer Nano-ZS90 (Malvern Instruments). The analysis was performed at a scattering angle of 90°at a temperature of 25 °C using samples diluted to different concentration with de-ionized distilled water. Atomic force microscopy (AFM, SPM-9500J3) was used for visualization of the chitosan nanoparticles deposited on silicon substrates operating in the contact mode. AFM imaging was performed using Si₃N₄ probes with a spring constant of 0.06 N/m.

A stock CNP suspension or CS solution with a concentration of 3 mg/mL was prepared. The CNP was sterilized by passing it through a 0.22 μm Millipore filter, CS was autoclaved to remove any contaminant and then analyzed for endotoxin level by a gel-clot Limulus amebocyte lysate assay (Zhejiang A and C Biological, Zhejiang, China). The endotoxin level in the stock soln. was less than 0.5 EU/mL.

Five-week-old female ICR mice were divided into five groups, each consisting of six mice. Animals were injected twice subcutaneously on the back with CNP at a single dose of 0.15, 0.3, 0.75, 1.5 mg in 0.5 mL saline solution at weekly intervals, and monitored daily for 14 days. Saline-treated animals were included as control and the toxicity was assessed by lethality, local swelling and loss of hair at the site of injection.

Five-week-old female ICR mice were divided into six groups, each consisting of six mice. Animals were immunized subcutaneously with OVA (25 μg) alone or with OVA (25 μg) dissolved in saline containing QuilA (10 μg), or CS (50 μg) or CNP (12.5, 50, 200 μg) on day 1. The boosting injection was given 2 weeks later. Saline-treated animals were included as controls. Splenocytes and sera were collected 2 weeks after the secondary immunization for measurement of OVA-specific antibody, natural killer (NK) cell activity and proliferation assay.

OVA-specific IgG, IgG1, IgG2a, and IgG2b antibodies in sera were detected by an indirect ELISA. In brief, microtiter plate wells (Nunc) were coated with 100 μL OVA solution (25 μg/mL in 50 mM carbonate–bicarbonate buffer, pH 9.6) for 24 h at 4 °C. The wells were washed three times with PBS containing 0.05% (v/v) Tween 20 (PBS/Tween), and then blocked with 5% FCS/PBS at 37 °C for 2 h. After three washings, 100 μL of a series of diluted sera samples (initial dilution 1:50) or 0.5% FCS/PBS as control were added to triplicate wells. The plates were then incubated for 2 h at 37 °C, and then washed three times. Aliquots of 100 μL of rabbit anti-mouse IgG horseradish peroxidase conjugate diluted 1:10,000, goat anti-mouse IgG1 peroxidase conjugate 1:8000, IgG2a peroxidase conjugate 1:8000, and IgG2b peroxidase conjugate 1:8000 with 0.5% FCS/PBS were added to each plate. The plates were further incubated for 2 h at 37 °C. After washing, the peroxidase activity was assayed as follows: 100 μL of substrate solution (10 mg of O-phenylenediamine and 37.5 μL of 30% H₂O₂ in 25 mL of 0.1 M citrate–phosphate buffer, pH 5.0) was added to each well. The plate was incubated for 10 min at 37 °C, and enzyme reaction was terminated by adding 50 μL/well of 2 N H₂SO₄. The optical density was measured in an ELISA reader at 490 nm, where sets of sera samples have been subjected to within and between group comparisons, ELISA assays were performed on the same day for all of the samples.

Spleen collected from the OVA-immunized mice under aseptic conditions, in Hank’s balanced salt solution (HBSS, Sigma), was minced using a pair of scissors and passed through a fine steel mesh to obtain a homogeneous cell suspension. The erythrocytes were lysed with ammonium chloride (0.8%, w/v). After centrifugation (380× g at 4 °C for 10 min), the pelleted cells were washed three times in PBS, and resuspended in complete medium. Cell numbers were counted with a hemocytometer by trypan blue dye exclusion technique. Cell viability exceeded 95%. The activity of NK cells was measured as previously described [36]. Briefly, K562 cells were used as target cells and seeded in 96-well U-bottom microtiter plate (Costar) at 1 × 10⁵ cells/well in RPMI 1640 complete medium. Splenocytes prepared were used as the effector cells, and were added at 5 × 10⁶ cells/well to give E/T ratio 50:1. The plates were then incubated for 20 h at 37 °C in 5% CO₂ atmosphere. 50 μL of MTT solution (2 mg/mL) was added to each well and the plate was incubated for another 4 h and subjected to MTT assay. Three kinds of control measurements were performed: Target cells control, blank control and effector cells control. NK cell activity was calculated as the following equation: NK activity (%) = (ODT − (ODS − ODE))/ODT × 100, where ODT is the optical density value of target cells control; ODS is the optical density value of test samples; and ODE is the optical density value of effector cells control.

Spleen collected from the OVA-immunized mice under aseptic conditions, in Hank’s balanced salt solution (HBSS, Sigma), was minced using a pair of scissors and passed through a fine steel mesh to obtain a homogeneous cell suspension, and the erythrocytes were lysed with ammonium chloride (0.8%, w/v). After centrifugation (380× g at 4 °C for 10 min), the pelleted cells were washed three times in PBS, and resuspended in complete medium. Cell numbers were counted with a haemocytometer by trypan blue dye exclusion technique. Cell viability exceeded 95%. Splenocyte proliferation was assayed as previously described [37]. Briefly, splenocytes from each mouse were seeded into four wells of a 96-well flat-bottom microtiter plate at 5 × 10⁶ cell/mL in 100 μL of complete medium. Con A (final concentration 5 μg/mL), LPS (final concentration 10 μg/mL), OVA (final concentration 30 μg/mL), or medium were then added, giving a final volume of 200 μL. The plates were incubated at 37 °C in a humid atmosphere with 5% CO₂. After 44 h, 50 μL of MTT solution (2 mg/mL) was added to each well and incubated for further 4 h. The plates were centrifuged (1400× g, 5 min) and the untransformed MTT was removed carefully by pipetting. 150 μL of a DMSO working solution (192 μL DMSO with 8 μL 1 N HCl) was added to each well, and the absorbance was evaluated in an ELISA reader at 570 nm with a 630 nm reference after 15 min. The stimulation index (SI) was calculated based on the following formula: SI = the absorbance value for mitogen-cultures divided by the absorbance value for non-stimulated cultures.

Splenocytes (5 × 10⁵ cells/well) from the immunized mice prepared as described before were incubated with ConA (final concentration 5 μg/mL) in 24-well culture plates at 37 °C in 5% CO₂. After 48 h, the plate was centrifuged at 1400× g for 5 min and culture supernatants were collected for the determination of INF-γ, IL-2, IL-10 levels. The presence of INF-γ, IL-2, IL-10 in the cultured supernatants of splenocytes were determined using the mouse ELISA kits (Rapidbio Lab., West Hills, CA, USA).

Splenocytes from the immunized mice prepared as described before were seeded into 24-well lat-bottom microtiter plate (Nunc) at 5 × 10⁶ cell/mL in 1 mL complete medium, then ConA (final concentration 5 μg/mL) was added giving a final volume of 2 mL (triplicate wells). The plates were incubated at 37 °C in a humidified atmosphere 5% CO₂. After 12 h treatment, cells were collected by centrifugation (380× g at 4 °C for 10 min), and washed with ice-cold PBS, then subjected to RNA extraction. Cells were lysed in 0.8 mL of Trizol reagent (Invitrogen, China) and the total RNA was isolated according to the manufacture’s protocol. The concentration of total RNA was quantified by determining the optical density at 260 nm. The total RNA was used and reverse transcription was performed by mixing 2 μg of RNA with 0.5 μg oligo (dT)₁₈ primer in a DEPC-treated tube. Nuclease-free water was added giving a final volume of 12.5 μL. This mixture was incubated at 70 °C for 5 min and chilled on ice for 2 min. Then, a solution containing 4 μL of M-MuLV 5×reaction buffer, 2 μL of 10 mM dNTP, 20 U of ribonuclease inhibitor, and DEPC-treated water was added, giving a final volume of 19 μL, and the tubes were incubated for 5 min at 37 °C. The tubes then received 200 U of M-MuLV reverse transcriptase and were incubated for 60 min at 42 °C. Finally, the reaction was stopped by heating at 70 °C for 10 min. The samples were stored at −20 °C until further use.

As shown in Table 1, the primers were used to amplify cDNA fragments (381-bp IL-2 fragment, 460-bp IFN-γ fragment, 324-bp IL-10 fragment and 564-bp GAPDH fragment). Amplification was carried out in total volume of 50 µL containing 4 µL (10 µM) of each cytokine-specific primers, 5 μL of 10× PCR buffer, 4 μL of MgCl₂ (25 mM), 4 μL of dNTPs (2.5 mM), 2 µL of transcribed cDNA, and 0.25 µL of Taq DNA polymerase. PCR was performed for 33 (IL-2), 32 (IFN-γ), 30 (IL-10) or 28 (GAPDH) cycles using a MyCycler (Bio-Rad, Hercules, CA) with the following program of denaturation at 94 °C for 5 min, following by indicated cycles of 94 °C for 30 s, annealing at 58 °C (GAPDH), 60 °C (IL-2), 62 °C (IL-10), 63 °C (IFN-γ) for 30 s, and elongation at 72 °C for 30 s, and a final extension step at 72 °C for 10 min. Semi-quantitative RT-PCR was performed using GAPDH as a house keeping gene to normalize gene expression for the PCR templates. The PCR products were studied on a 1.5% agarose gel and the amplified bands were visualized using Gel DOC2000 (Bio-Rad, USA) after staining with GoldView. The size of the amplification fragments was determined by comparison with a standard DNA marker. The relative level of cytokine expression is calculated for 100 copies of the GAPDH house keeping gene following the formula: n = 100 × (the intensity of cytokine gene expression band/the intensity of GAPDH band).

Data were expressed as mean ± standard deviations (S.D.) and examined for their statistical significance of difference with ANOVA and a Tukey post hoc test by using SPSS 16.0. P-values of less than 0.05 were considered statistically significant.

As shown in Figure 1A,B, chitosan nanoparticles regularly formed and well distributed in acetic acid (HOAc)/sodium tripolyphosphate solution (pH 5.5) were used in this study. The mean size and size distribution of each batch of nanoparticle suspension was analyzed using the Zetasizer analysis (Figure 1C,D). The size distribution profile represents a typical batch of nanoparticles with a mean diameter of 83.66 nm and a narrow size distribution ranging from 63.16 to 101.70 nm (polydispersity index <1), and shows that the surfaces of chitosan nanoparticles have a positive surface charge of about 35.43 mV. These excellent characteristics are of benefit to the stabilization and penetration capability of CNP, enabling CNP to easily penetrate through capillary and epithelial tissue.

The endotoxin level in a stock CNP with a concentration of 3 mg/mL was measured to be less than 0.5 endotoxin units (EU)/mL. Therefore, the CNP samples used in this study were excluded from endotoxin contamination. When the animals were administered subcutaneously twice ranging from 0.15 to 1.5 mg at weekly intervals, no harm was observed. Local swelling or loss of hair was not observed in mice at the test doses. The results suggested that the safety dose of CNP used for animals was at least up to 60 mg/kg.

The OVA-specific IgG, IgG1, IgG2a and IgG2b antibody levels in the serum were measured by the indirect ELISA method two weeks after the last immunization (Figure 2). As a positive control, QuilA elicited the highest IgG and IgG isotypes levels. The serum IgG and IgG1 levels in mice immunized with OVA were significantly enhanced by CNP compared with the control groups (OVA and CS) (P < 0.05). Moreover, significant enhancements in OVA-specific IgG2a and IgG2b levels were observed in mice immunized with CNP compared with the OVA and CS control group (P < 0.05), and there were significant differences among CNP at all doses and QuilA (P < 0.05).

The effects of CNP on natural killer (NK) cell activity in OVA-immunized mice were shown in Figure 3. CNP and Quil A significantly enhanced the killing activity of NK cell in the OVA-immunized mice (P < 0.05). The findings indicated that CNP could promote lytic activity of NK cells in mice immunized with OVA.

The effects of CNP and QuilA on splenocyte proliferative responses to ConA, LPS and OVA stimulation are shown in Figure 4. Mice immunized with OVA plus CNP or QuilA had higher splenocyte proliferative response to ConA, LPS and OVA than the mice injected with OVA alone (P < 0.05). Mice immunized with OVA plus CNP also had higher ConA-, LPS- and OVA-stimulated splenocyte proliferative response than the mice injected with OVA plus CS (P < 0.05).

The contents of cytokines IFN-γ, IL-2 and IL-10 in the supernatants from cultured splenocytes in the mice immunized with OVA-CNP were significantly higher than those in OVA and OVA-CS control mice (P < 0.05) (Figure 5). QuilA enhanced significantly the production of cytokine IL-10 (Th 2 type immune response), IFN-γ and IL-2 (Th 1 type immune response) (P < 0.05) in the supernatants from cultured splenocytes in the mice immunized with OVA. These results suggested that CNP significantly enhanced the production of the Th1 and Th2 cytokines in OVA-immunized mice, and CNP markedly improved the adjuvant activity of chitosan in the OVA-immunized mice.

As shown in Figure 6 and Table 2, CNP and QuilA not only significantly increased the mRNA expression of Th1 cytokines IL-2 and IFN-γ (P < 0.05), but also enhanced that of Th2 cytokines IL-10 in splenocytes from the immunized mice (P < 0.05). Therefore, the findings suggested that CNP up-regulated the gene expression of Th1/Th2 cytokines in splenocytes from the immunized mice.