Diphtheria and tetanus toxoid microencapsulation into conventional and end-group alkylated PLA/PLGAs

The feasibility of biodegradable polyester microspheres (MS) for single injection vaccines will greatly depend on the toxoid stability within the MS exposed to in vivo conditions. This study examined the effects of polymer type and co-encapsulated additives on diphtheria (Dtxd) and tetanus (Ttxd) toxoid entrapment and stability. The co-encapsulated stabilizers influenced significantly the entrapment of Dtxd and Ttxd in PLA/PLGA MS. Typically, 5% BSA or trehalose decreased the amount of Dtxd entrapped in spray-dried MS, whereas BSA increased the entrapment in coacervated MS. Further, the entrapment of Dtxd decreased as a function of polymer hydrophobicity in spray-dried MS. Without additives, approx. 64, 43 and 16% entrapment efficiency of ELISA-reactive antigen was obtained for 14-17 kDa PLGA 50:50, PLGA 75:25 and PLA, respectively. The novel end-group stearylated 1-PLAs were only processed by coacervation. Satisfactory entrapment of 30-60% Dtxd was obtained. Here, albumin was a prerequisite for toxoid encapsulation, as BSA-free formulations produced strong toxoid precipitation. Furthermore, protein burst release increased with the more hydrophobic polymers, with Dtxd, Ttxd and the co-encapsulated BSA following a similar pattern and magnitude. This investigation also revealed that the method of protein extraction from the microspheres (O/W-partition or polymer hydrolysis) as well as the analytical methods (HPLC or ELISA) strongly influenced the determined amount of encapsulated toxoid and BSA. In conclusion, the study revealed the complexity of antigen microencapsulation when using different preparation and analytical techniques, as well as different types of materials.