In a previous study, different sizes of bilosome (niosomes containing bile acid salt, for use as a mucosal delivery system) with influenza haemagglutinin antigen were shown to have an influence on the Th1/Th2 immune response in immunised mice. In that study, the size of the vesicles could not be finely controlled. Since then, improved manufacturing equipment has enabled better control over the preparation of vesicles. The aim of this project was to establish if different sizes of niosomes administered parenterally had an impact on the immune responses induced by influenza antigen in mice. Large niosomes were prepared using a thin film hydration technique followed by a mini-extruder to control the size, whereas small niosomes were manufactured using a Nanoassemblr (microfluidic technology). Albumin was used as a model antigen to study the stability of the niosomes with antigen, stored at 4 and 25°C. Influenza antigen was then entrapped in the niosomes and tested in vivo. Parenteral vaccination of 3 groups of mice (small niosomes, large niosomes and unentrapped formulation) was carried out in weeks 0, 2, 4. Serum was prepared (a pre-bleed in week -1, then in weeks 0, 2, 4, 6 and at post-mortem) and tested for IgG1 and IgG2a antibody levels. Consequently, spleen cells from mice were analysed in a proliferation assay, stimulated with concanavalin A and influenza antigen and cytokines in the supernatant measured by enzyme linked immunoassay.;The results showed that small niosomes (122.1±0.3 nm for albumin, 157.9±1.8 nm for influenza antigen) were more uniform with a narrower polydispersity compared with the large niosomes (352.9±0.9 nm for albumin, 388.8±10.0 nm for influenza antigen). Both types of vesicle (large and small niosomes) with influenza antigen were larger than vesicles with albumin because of the larger molecular weight of the protein. The small niosomes had a zeta potential of -18.7±2.0 mV (albumin) and -14.6±1.5 mV (influenza HA) compared with the large niosomes -28.4±1.9 mV (albumin) and -16.6±2.1 mV (influenza HA). Both sizes of niosomes were more stable at 25°C compared with 4°C due to the fact that this temperature was above the phase transition temperature which caused surfactant in the formulation to exist in a liquid state.Niosomes play a key role as an adjuvant to help raise high antibody immune responses from the resazurin proliferation assay. The results showed stimulation of cells from animals treated with large and small niosomes were more responsive than unentrapped formulation.Although cytokine analysis showed that the large niosomes induced the high level of IFN-ɣ compared with unentrapped formulation, there is no conclusive evidence that size makes any difference to antibody levels. Early on the large produces higher antibody levels, and the small produces slightly less, but the difference is not significant. IL-4 and IL-5 cytokine analysis is required to confirm the Th2 response and IFN-ɣ is needed to be repeated to confirm the preference of Th1 response of large niosomes.
|Date of Award||20 Apr 2017|
- University Of Strathclyde
|Supervisor||Valerie Ferro (Supervisor) & Alexander Mullen (Supervisor)|