Curdlan sulfate- O-linked quaternized chitosan nanoparticles: potential adjuvants to improve the immunogenicity of exogenous antigens via intranasal vaccination

Abstract

Introduction: The development of ideal vaccine adjuvants for intranasal vaccination can provide convenience for many vaccinations. As an ideal intranasal vaccine adjuvant, it should have the properties of assisting soluble antigens to pass the mucosal barrier and potentiating both systemic and mucosal immunity via nasal administration.

Methods: By using the advantages of polysaccharides, which can promote both T-helper 1 and 2 responses, curdlan sulfate (CS)-O-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride (O-HTCC) nanoparticles were prepared by interacting CS with O-HTCC, and the adjuvancy of the nanoparticles was investigated.

Results: The results showed that the polysaccharide-based nanoparticles induced the proliferation and activation of antigen-presenting cells. High protein-loading efficiency was obtained by testing with the model antigen ovalbumin (Ova), and the Ova adsorbed onto the cationic CS/O-HTCC complexes was taken up easily by the epithelium. To evaluate the capacity of the Ova/CS/O-HTCC nanoparticles for immune enhancement in vivo, we collected and analyzed immunocytes, serum, and mucosal lavage fluid from intranasally vaccinated mice. The results showed that Ova/CS/O-HTCC nanoparticles induced activation and maturation of antigen-presenting cells and provoked the proliferation and differentiation of lymphocytes more significantly compared to the immunization of Ova mixed with aluminum hydroxide gel. Furthermore, CS/O-HTCC evoked a significantly higher level of Ova-specific antibodies.

Conclusion: Therefore, these results suggest that CS/O-HTCC nanoparticles are ideal vaccine adjuvants for soluble antigens used in intranasal or mucosal vaccination.

Keywords: O-linked quaternized chitosan; adjuvant; curdlan sulfate; immune response; nanoparticle; nasal mucosa immunization; ovalbumin.

Figure 1

Preparation of CS and O-HTCC. Notes: (A) Synthetic route of CS; (B) synthetic route of O-HTCC; a, chitosan; b, N-benzylidene chitosan; c, O-quaternary aminonium-N-benzylidene chitosan; and d, O-HTCC. Abbreviations: CS, curdlan sulfate; O-HTCC, O-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride.

Figure 2

Physicochemical properties of the complexes. Notes: (A) Transmission electron microscopy imaging of the complexes; (B) size distribution of the complexes; (C) ζ-potential distribution of the complexes. Abbreviations: CS, curdlan sulfate; O-HTCC, O-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride; Ova, ovalbumin.

Figure 3

In vitro release of the complexes. Notes: (A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis of Ova released from the complexes: lane 1, Ova; lanes 2–9, Ova release from 0 to 48 hours. (B) Analysis of Ova release in the complexes (n=3). Abbreviations: Ova, ovalbumin; CS, curdlan sulfate; O-HTCC, O-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride.

Figure 4

Uptake ability of FITC-Ova-loaded complexes. Notes: (A) Fluorescence microscopy analysis of FITC-Ova uptake (400×); (B) quantification of cell uptake of FITC-Ova (n=3); (C) representative flow cytometry results of cell uptake ability. Abbreviations: FITC, fluorescein isothiocyanate; CS, curdlan sulfate; O-HTCC, O-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride; Ova, ovalbumin.

Figure 5

Effect of Ova-related complexes on lymphocytes. Notes: (A) Spleen-lymphocyte proliferation; (B) T-lymphocyte transformation; (C) B-lymphocyte transformation; (D) flow cytometry results of proportion of CD3⁺CD4⁺ lymphocytes; (E) flow cytometry results of proportion of CD3⁺CD8⁺ lymphocytes; (F) quantification of proportion of CD3⁺CD4⁺ lymphocytes; (G) quantification of proportion of CD3⁺CD8⁺ lymphocytes; (H) proportion of CD4⁺CD69⁺ lymphocytes. *P<0.05, **P<0.01, ***P<0.001 compared to PBS controls; ^#P<0.05, ^##P<0.01, ^###P<0.001 compared to Ova controls; ^!P<0.05, ^!!P<0.01, ^!!!P<0.001 compared to Ova + AL controls (n=3). Abbreviations: CS, curdlan sulfate; O-HTCC, O-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride; Ova, ovalbumin; AL, Alhydrogel.

Figure 6

Effect of Ova-related complexes on antigen-presenting cells. Notes: (A) Macrophage proliferation; (B) macrophage phagocytosis; (C) proportion of F4/80⁺ macrophages; (D) proportion of CD11c⁺ splenocytes; (E) proportion of CD11c⁺CD40⁺ splenocytes; (F) proportion of CD11c⁺ CD86⁺ splenocytes; (G) proportion of CD11c⁺ MHCII⁺ splenocytes. *P<0.05, **P<0.01, ***P<0.001 compared to PBS control; ^#P<0.05, ^##P<0.01, ^###P<0.001 compared to Ova control; ^!P<0.05, ^!!P<0.01, ^!!!P<0.001 compared to the Ova + AL control (n=3). Abbreviations: CS, curdlan sulfate; O-HTCC, O-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride; Ova, ovalbumin; AL, Alhydrogel; MHC, major histocompatibility complex.

Figure 7

Expression level of cytokines. Notes: (A) IFNγ concentration; (B) IL4 concentration. *P<0.05, **P<0.01 compared to PBS control; ^#P<0.05 compared to Ova control (n=3). Abbreviations: CS, curdlan sulfate; O-HTCC, O-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride; Ova, ovalbumin; AL, Alhydrogel.

Figure 8

Humoral immunoresponse of immunized mice. Notes: (A) Relative expression of IgG in serum; (B) relative expression of IgA in serum; (C) expression of subtype antibodies in serum. *P<0.05, **P<0.01, ***P<0.001 compared to PBS control; ^#P<0.05, ^##P<0.01, ^###P<0.001 compared to Ova control; ^!P<0.05, ^!!P<0.01 compared to Ova + AL control (n=8). Abbreviations: CS, curdlan sulfate; O-HTCC, O-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride; Ova, ovalbumin; AL, Alhydrogel.

Figure 9

Mucosal immunoresponse of immunized mice. Notes: (A) Expression of secretory IgA in saliva; (B) expression of secretory IgA in vaginal lavage fluid. ***P<0.001 compared to PBS control; ^###P<0.001 compared to Ova control; ^!!P<0.01, ^!!!P<0.001 compared to the Ova + AL control (n=8). Abbreviations: CS, curdlan sulfate; O-HTCC, O-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride; Ova, ovalbumin; AL, Alhydrogel.

Scheme 1

CS/O-HTCC nanoparticles as a potential mucosal vaccine adjuvant. Notes: Cationic CS/O-HTCC nanoparticles carry Ova to penetrate the nasal mucosal layer and facilitate the activation and proliferation of APCs. Then, the activated APCs trigger the secretion of cytokines and present the processed antigens to lymphocytes, which induce the proliferation and differentiation of these cells to evoke a high level of Ova-specific antibodies. Upon intranasal administration, the CS/O-HTCC nanoparticles boost a high expression level of secretory IgA (sIgA) in both saliva and the vagina to induce mucosal immunoresponses. Abbreviations: CS, curdlan sulfate; O-HTCC, O-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride; Ova, ovalbumin; APCs, antigen-presenting cells; T_H2, T-helper 2.

Scheme 2

Preparation of empty and Ova-loaded nanoparticles. Abbreviations: Ova, ovalbumin; CS, curdlan sulfate; O-HTCC, O-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride.