Drug Delivery

Objectives The successful development of a parenteral liposome based vaccine requires the fabrication of a stable, sterile and freeze dried product. One of the major concerns in the process is the design of a pathogen free formulation, which can either be developed using a continuous aseptic process or end sterilized. Of the plethora of the sterilization techniques available, gamma radiation has shown to be a convenient, promising method for freeze dried liposomes (Strensrud et al 1999). The aim of the current work was to establish the influence of exposing a freeze-dried cationic liposome-based vaccine to 25KGy gamma radiation so as to produce a sterile product. Methods Liposomal formulations consisting of DDA and TDB (1.25 mg/mL and 250 mg/mL, respectively) were prepared by the lipid hydration method. The freeze-dried formulations were exposed to gamma radiation to a dose of 25 KGy. Validation was carried out by exposing strips impregnated with Bacillus pumilus spores (2.5 × 10 CFU) to gamma radiation followed by culturing the strips in trypton soya media. Chemical stability was studied by NMR spectra using a Bruker AC250 spectrometer at H (250.1 MHz). Physico-chemical analysis, including: vesicle size, surface charge, viscosity, pH and antigen release profiles, was also conducted. Results The results from culturing the sterilized strips indicate that the exposure to gamma radiation was an effective method in completely eradicating the growth of bacterial spores. Positive controls confirmed that the media used supported the spore growth. NMR scans before and post gamma sterilization were similar, suggesting the absence of any degradation products. The H NMR spectrum for the DDA lipid presents a sharp peak (1.2 ppm) representing the methylene groups present in the long alkyl chain. The peak at 0.84 ppm represents the end methyl groups attached to the long carbon chain. The chemical shift observed at 3.37 ppm represents both methyl as well as the methylene groups present surrounding the nitrogen atom. Similarly, no significant changes in the various physico-chemical parameters measured were detected. Previously the damage caused to liposomes by exposure to gamma radiation has been attributed to two factors: direct influence of radiation on the bilayer and an indirect effect caused by the action of the reactive species that are generated by gamma radiation on the bilayer (Zuidam et al 1995); however, the composition of the formulation may also control degradation (Stensrud et al 1999). Conclusions The physico-chemical stability of the freeze dried DDA liposomes can therefore be due to the presence of cationic nitrogen at the head group which resists any radiation induced damage unlike the negatively charged DSPG liposomes which showed a change in the phospholipids concentration post radiation (Stensrud et al 1999). Additionally the development of a freeze dried product with low levels of moisture content better also adds to the stability. Gamma sterilization therefore provides the opportunity to develop sterile and chemically stable vaccine formulations.