Antigen-conjugated Quaternized Chitosan Nanoparticles For Nasal Immunization

The frequent outbreak of respiratory infectious diseases such as 2005 H5N1 avian influenza and 2009 influenza A (H1N1) caused enormous economic loss and great social panic. How to prevent these diseases remains an arduous task for medical researchers. Almost all viruses and bacteria causing respiratory infectious diseases infect through the large surface area of mucosa. The nasal mucosa is often the first point of contact for inhaled antigens, and therefore it serves as an ideal vaccination site. Nasal vaccination can induce nasal mucosal immunity, distant mucosal immunity as well as systemic immunity, making it suitable for preventing respiratory infectious diseases.Quaternized chitosan has been widely used as nasal immunization carriers due to its good biocompatibility, easy penetration across mucosal epithelia and easy recognition by antigen presenting cells. However, there are several problems in preparing antigen-encapsulated quaternized chitosan nanoparticles, such as low encapsulation efficiency, burst release and incomplete release, resulting in unsatisfying immune responses. So we designed a novel antigen-conjugated quaternized chitosan nanoparticle carrier to overcome these problems. This nanoparticle carrier possesses the following advantages:① simple preparation and high use ratio of antigens (>95%); ②antigens are conjugated on the surface of nanoparticles and are easy to be recognized and uptaken by macrophages;③ antigens are covalently conjugated with nanoparticles and are not easy to fall off, which is beneficial for transport of antigens to cervical lymoh nodes, resulting in enhanced mucosal and systemic immune responses. Moreover, there are limited enzymes in nasal cavity, leading to slight damage of antigens exposed on the surface of nanoparticles after nasal administration.In the first part, ovalbumin (OVA) conjugated quaternized chitosan nanoparticles (OVA-NP) were prepared and characterized. Water soluble quaternized chitosan (TMC) was synthesized through the free radical polymerization of chitosan and N-trimethylaminoethylmethacrylate chloride, and maleimide-TMC (Mal-TMC) was synthesized through the reaction of TMC and N-Succinimidyl 3-maleimidopropionate. The structures of TMC and Mal-TMC were confirmed by 1H NMR and FT-IR. The yield of TMC and Mal-TMC was 78.7±3.5% and 93.3±2.6%, respectively, and the degree of quaternization of TMC was 30.4±1.1%. Blank nanoparticles were prepared by ionic crosslinking of TMC and sodium tripolyphosphate. The particle size of blank nanoparticles was 140 nm. OVA was eazily conjugated to blank nanoparticles through the reaction of maleimide group and thiol group. The particle size and Zeta potential of OVA-NP was 140.9 nm and 10.3 mV, respectively. OVA-NP were stable at low temperature and were not damaged in nasal mucosa homogenate.In the second part, targeting of OVA-NP to cervical lymph nodes was investigated. Calu-3 monolayers were used to study the transport of OVA-NP across nasal mucosa, and no significant difference was observed between the transport amounts of OVA-NP and OVA solution. RAW 264.7 macrophages were used to study the recognition and uptake of OVA-NP by immunocytes, and cellular uptake of OVA-NP was 6.7 times higher than that of OVA solution and significantly higher than that of antigen-encapsulated TMC nanoparticles (NPe). Biodistribution of OVA-NP after nasal administration was investigated by 125I labeling method and compared with that of NPe, OVA/TMC physical mixture, OVA/TMC conjugates and OVA solution. OVA-NP showed better targeting profiles to the superficial and deep cervical lymph nodes than all the other groups, and the AUC0-4 h of OVA-NP in the two tissues was 1.7 and 2.5 times higher than that of OVA solution and 1.4 and 1.4times higher than that of NPe, respectively. The lymph node targeting indices of OVA-NP in superficial and deep cervical lymph nodes was 2.5 and 3.3 times higher than that of OVA solution and 1.5 and 1.5 times higher than that of NPe.In the third part, the systemic and mucosal immune responses of OVA-NP were investigated after nasal vaccination. Aluminium hydroxide precipitated OVA induced strong systemic immune responses after intramuscular injection (I.M.), but mucosal immune responses were not detected. After three nasal immunizations of OVA-NP, serum IgG, IgGl, IgG2a and IgA were significantly increased, which were 5.3-24.8 times higher than that of NPe and even higher than that of the I.M. group. The slgA levels in the nasal washes, lung washes, saliva and vaginal washes of the OVA-NP group were also significantly increased, which were 1.3-17.9 times higher than that of the NPe group. These results demonstrated that OVA-NP induced strong immune responses after nasal administration and could serve as an effective carrier for nasal vaccination.In the fourth part, the mechanisms of OVA-NP inducing immune responses after nasal administration were investigated. In vitro co-incubation experiments demonstrated that OVA-NP could stimulate Raw 264.7 macrophages to release IL-1β and IL-6 and stimulate splenocytes of mice to release IL-2 and IFN-y, which may be related with the immunoadjuvant effects of OVA-NP. After nasal administration, OVA-NP were transported across the nasal mucosa mainly via glands and entered nasal associated lymphoid tissue (NALT) via M cells, and then drained to cervical lymph nodes to induce immune responses. IgA plasma cells were visualized by the immunofluorescence labeling method, and the OVA-NP group showed the most abundant IgA plasma cells, which were mainly located in the surrounding tissues of glands in the lamina propria. NALT participates in the mucosal and systemic immune responses, but it is not the main effector site. The primary role of NALT is to take up nanoparticles and then drain them to deep cervical lymph nodes.In the fifth part, in vitro and in vivo toxicity of OVA-NP was evaluated. High concentrations of OVA-NP influenced the energy metabolism of Calu-3 cells and increased the cell membrane permeability, which may be a result of chitosan-induced perturbation of cell membrane. OVA-NP did not increase the amounts of active oxygen and malondialdehyde in cells, indicating that OVA-NP will not induce lipid peroxidation injury. OVA-NP showed no obvious influence on toad palate mucosal cilia movement. After nasal administration of OVA-NP for three times, nasal mucosa of rats was intact with bushy cilia, suggesting little toxicity of OVA-NP to the nasal mucosa. OVA-NP did not induce hemolysis of rabbit erythrocyte. These results demonstrated that OVA-NP is a safe carrier for nasal vaccination.In the sixth part, H1N1 influenza antigen conjugated TMC nanoparticles (H1N1-NP) with a particle size of 140 nm were prepared. The preparation process and enzyme in nasal mucosa showed slight influence to the hemagglutination activity of antigen. After nasal immunization of mice for three times, H1N1-NP induced comparable systemic antibody responses to the intramuscular group and higher mucosal immune responses than all the other groups, suggesting that conjugation of H1N1 influenza antigen on TMC nanoparticles can significantly increase its nasal immunogenicity. NALT participated in the mucosal and systemic immune responses of OVA-NP, as evidenced by the same change trend of slgA and IgG secreted by NALT with those in mucosa and serum. The hemagglutination inhibition test demonstrated that H1N1-NP induced protective antibodies against H1N1 influenza virus. H1N1-NP showed no obvious influence on nasal epithelia and cilia, proving it to be a safe nasal vaccine. In conclusion, antigen-conjugated TMC nanoparticles are effective and safe carriers for nasal vaccination.