The Construction Of Novel Nano-carrier And Its Application On Effective Transcutaneous Immunization

Objective:To prepare two types of flexible liposomes and chitosan nanoparticle, and to determine their efficacies for the transcutaneous delivery of antigen and the immune response induced in vivo, for further anti-tumor therapy as effective ranscutaneous vaccines.Methods:1. ovalbumin(OVA)-liposome based transcutaneous vaccines were prepared with the methods of reverse phase evaporation and film dispersion, while OVA loaded chitosan-TPP graft nanoparticle was prepared by crosslinking of low molecular weight chitosan with polyanion sodium TPP. The nano-scale carriers were evaluated by determination of particle sizes, morphology, zeta potential and in vitro release.2. Using solution as control, accumulative penetration amount of different NaFl liposome formulations were examined in Franz diffuse cell at different time under the principle of random and comparison. The drug distribution in the stratum corneum and the viable skin were detected by fluorospectrophotometer. The influence of preparation method on the structure of OVA was determined by SDS-PAGE. The mechanism of liposomes and chitosan nanoparticle penetrating into the skin were identified by fluorescence microscope after making the frozen slice of the pig skin.3. The factors including influence of different adjuvants, the removal of the stratum corneum in the transcutaneous immunization were also testified for the optimized animal model.4. By detecting the serum IgG levels, the efficacy of the OVA loaded flexible liposome and nanoparticle in activating the immune responses upon transcutaneous delivery was investigated. The protection of the nano-scale system from stratum corneum barrier was evaluated.5. The ability of langerhans cells in migratiing to lymph nodes was testified compared to the ovalbumin loaded chitosan nanoparticle and free ovalbumin solution, which was conducive to analyze the mechanism of the immune response elcited by the ovalbumin loaded chitosan nanoparticle.6. The anti-tumor transcutaneous vaccine was established applied by gp100 protein loaded chitosan nanoparticle, vaccine protection treament effect in mice beared melanoma solid tumor was evaluated.Results:Two flexible liposomes which well encapsulated ovalbumin were prepared by film dispersion or reverse-phase evaporation methods. Chitosan grafted tripolyphosphate (TPP) nanoparticle were prepared by crosslinking of low molecμlar weight chitosan with polyanion sodium TPP. The sizes of prepared flexible liposomes and chitosan nanoparticle ranged from 200 to 400 nanometers.The nanosale system can protect the antigen from degradation and the antigen-loaded nanocarrier penetrated in the skin can keep a high antigen concentration and content. In vivo, the fluorescence-labeled liposome and nanoparticles were detected in the ducts of the hair follicles, sweat glands, sebaceous glands, etc. indicating that the felxible liposome can penetrate the skin barrier through the skin appendages pathway. Upon transcutaneous administration, both OVA encapsulated flexible liposomes and chitosan nanoparticle elicited strong immune responses, especially OVA loaded chitosan nanoparticle which has raised the similar anti-OVA IgG titer to that of positive control (OVA solution was administrated by subcutaneous injection with Al(OH)3 as an adjuvants). The co-administration imiquimod with the OVA loaded nano-formulation expressed a signifcant enhancement on the transcutaneous immune responses.The liposome-based transcutaneous vaccine could overcome the stractum corneum barrier. The nanoparticle-based transcutanoues vaccine showed higher immune effect on intact skin compared to the liposome-based transcutanoues vaccine, however on the stratum corneum removed skin, the chitosan nanopaticle based vaccine could elicit enhanced immune effect. To further determine the mechanism of chitosan nanoparticle in transcutaneous immunization, the langerhans cells were isolated by the immunomagnetic beads method. For the capture of antigen by langerhans cells, the FITC-OVA nanopaticle represented a stronger cell uptaking ability than the FITC-OVA solution group, the same result was obtained in the in vivo migration capability to lymph nodes. The anti-tumor therapeutic effect after transcutaneous vaccination of gp100 loaded chitosan nanoparticles was testified, and the survival of tumor-bearing mice was increased.Conclusions:Results of the study highlight the nanoscaled formulations, both flexible liposome and chitosan nanoparticle, can protect the antigen from degradation, the antigen-loaded nanocarrier penetrated in the skin can keep a high antigen concentration and content, which can be viewed as promising carriers for the transcutaneous delivery of antigen-proteins.The stratum corneum barrier had a weaker influence on liposome based transcutaneous vaccine, which the negative impact of stratum corneum barrier on the conventional transcutaneous vaccine was overcomed. Chitosan nanoparicle showed an advantageous carrier in inducing the immune response compared to the flexible liposome, however the stractum corenum could still influence the efficiency of immune response. The antigen uptake and presentation capability of langerhans cell was influenced by the carrier category, antigen loaded chitosan nanoparticle could be better uptaken and antigen-presenting cells could be easier migrated to the surrounding lymph nodes, which was the key factor in transcutaneous immunization. With imiquimod as an effective transcutaneous immunization enhancer, chitosan nanoparticle showed the potential for transcutaneous vaccine development. Chitosan nanoparticle based vaccines have a promising vaccine effect for tumor immunotherapy and clinical therapy potential.