Herein, we describe the detailed development of a simple and effective method to microencapsulate vaccine antigens in poly(lactic-co-glycolic acid) (PLGA) by simple mixing of preformed active self-microencapsulating (SM) PLGA microspheres in a low concentration aqueous antigen solution at modest temperature (10-38 °C). Co-encapsulating protein-sorbing vaccine adjuvants and polymer plasticizers were used to "actively" load the protein in the polymer pores and facilitate polymer self-healing at a temperature>the hydrated polymer glass transition temperature, respectively. The microsphere formulation parameters and loading conditions to provide optimal active self-healing microencapsulation of vaccine antigens in PLGA was investigated. Active self-healing encapsulation of two antigens, ovalbumin and tetanus toxoid (TT), in PLGA microspheres was adjusted by preparing blank microspheres containing different vaccine adjuvants (aluminum hydroxide (Al(OH)₃) or calcium phosphate). Active loading of vaccine antigen in Al(OH)₃-PLGA microspheres was found to: a) increase with an increasing loading of Al(OH)₃ (0.88-3 wt.%) and addition of porosigen, b) decrease when the inner Al(OH)₃/trehalose phase to 1 mL outer oil phase and size of microspheres was respectively >0.2 mL and 63 μm, and c) change negligibly by PLGA concentration and initial incubation (loading) temperature. Encapsulation of protein sorbing Al(OH)₃ in PLGA microspheres resulted in suppression of self-healing of PLGA pores, which was then overcome by improving polymer chain mobility, which in turn was accomplished by coincorporating hydrophobic plasticizers in PLGA. Active self-healing microencapsulation of manufacturing process-labile TT in PLGA was found to: a) obviate micronization- and organic solvent-induced TT degradation, b) improve antigen loading (1.4-1.8 wt.% TT) and encapsulation efficiency (~97%), c) provide nearly homogeneous distribution and stabilization of antigen in polymer, and d) provide improved in vitro controlled release of antigenic TT.
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