Chitosan as adjuvant and particle forming excipient in a nano-in-microparticulate dry powder for nasal and pulmonary vaccine delivery

In this work, a dry powder nano-in-microparticle (NiM) vaccine formulation was developed by using a very gentle two-step-production method involving, first, an ionic gelation step to achieve primary nanoparticles followed by a spray drying step to gain the final dry powder vaccine formulation. The biopolymer chitosan was chosen as adjuvant and particle forming excipient because of its mucoadhesive and adjuvant properties. At first, different chitosan qualities were analysed with respect to their polymer properties like the molecular weight and the degree of deacetylation. Subsequently, an in vivo study (in cooperation with the University of Otago, Dunedin, New Zealand) was conducted in order to evaluate the adjuvant activity of three different chitosan qualities after s.c. administration. For this, two of the chitosans used were spray dried prior to the application and then administered as PBS soluble hydrochloride salts, the third quality was used as received and thus given as a PBS chitosan suspension. All three qualities were given together with the model antigen ovalbumin and also without it as placebo formulations. As a result of this study Chitosan from Sigma was identified as the chitosan with the highest adjuvant activity. Parallel to this study, a nanoparticle production method was developed. For this, chitosan was dissolved in acidic medium and then the bile salt derivative sodium deoxycholate was added under stirring to induce the gelation process. A method was developed to obtain nanoparticles with about 250 nm in diameter. Particles of this size are known to be taken up well by DCs specialised cells, called M-cells, which are located in the mucosal membranes. Particulate antigens induce both a strong systemic and also a local immune response on the mucosal membranes. The nanoparticles were combined with two model antigens, bovine serum albumin and ovalbumin, that were associated with the particles during the particle forming process. The particle forming process was further investigated regarding the relationship between the pH value of the chitosan solution and the resulting particle size as well as the antigen loading efficiency. These experiments were performed in order to enable the user to produce tailored particles in the different size ranges, if desired. In addition, the influence of the molecular weight of the polymer on the size of the generated particles was analysed. After that, freeze and spray drying with different stabilising excipients were examined in order to transfer the nanosuspension into a dry powder formulation being capable for direct respiratory administration. Finally, it was found that adding a 10% mannitol solution to the nanosuspension followed by spray drying led to the best dry powder NiM formulation regarding handling and powder properties. Subsequently, protein stability in the final formulation was verified using SDS-PAGE and circular dichroism analysis. The results showed that the antigen (OVA) was not affected by the production process. The obtained powder was further analysed regarding its deposition profile inside the nasal cavity of adults and children using two nasal cast models and a nasal powder dispenser. In order to determine the respirable fraction the Twin Stage Glass Impinger with two dry powder inhalers were used. Obtained data showed that the nasal deposition of the NiM formulation inside the adults nasal cast model was satisfactory, while, due to the anatomy, the amount of NiM formulation deposited in the anterior regions of the child model was unacceptably high and thus the obtained deposition profile failed to match the desired one. Regarding the pulmonary delivery it was found that the produced powder showed a very high capsule and device retention. This problem was solved by adding 1% magnesium stearate to the spray dried powder and blending for 3 h. Nevertheless, the resulting respirable fraction remained little too low. Finally, a second in vivo as well as another in vitro study were performed. In these studies the produced NiM formulation was administered intranasally to mice and the immune response measured as expression of CD4+ and CD8+ transgenic (OVA specific) cells was analysed via flow cytometry. In addition, the cell toxicity and the cellular uptake of the NiM formulation were investigated. In these studies it was found that the NiM formulation was taken up by dendritic cells in a satisfactory extent of about 50%. Unfortunately, the magnitude of the immune response induced by the NiM formulation was rather modest and not significantly higher than those induced by the antigen-free placebo NiM formulation. Based on the obtained results, in future work it should be examined if a second adjuvant can be added to the existing formulation in order to increase the immune response. This new formulation should then be tested again in an in vivo study regarding its immunological potential. In order to ensure to induce an appropriate immune response in further studies the amount of antigen in the formulation should also be increased to about 2%. But for this, the NiM formulation which was developed in this work is certainly a good basis for further development and optimisation


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