Purpose To investigate the effect of γ-irradiation of poly(lactic-co-glycolic acid) (PLGA)/Al(OH)3/0 or 5 wt% diethyl phthalate (DEP) IRAK2 microspheres for active self-healing encapsulation of vaccine antigens. after γ-irradiation. Antigen-sorbing capacity encapsulation efficiency and Tg of the polymer were also not adversely affected. When DEP-loaded microspheres were irradiated at 0.2 MRad/h some PLGA pores healed during irradiation and PLGA healing during encapsulation was suppressed. The molecular weight of PLGA was slightly reduced when DEP-loaded microspheres were irradiated at the same dose rate. These trends were not observed at 0.37 Indocyanine green MRad/h. Gamma irradiation slightly increased TT initial burst release. Apart from the slightly higher polymer molecular weight decline caused by higher irradiation dose in case of DEP-loaded microspheres the small increase in total irradiation dose from 1.8 to 2.5 MRad had insignificant effect on the polymer and microspheres properties analyzed. Conclusion Gamma irradiation is usually a plausible approach to provide a terminally sterilized self-healing encapsulation PLGA excipient for vaccine delivery. release of antigenic vaccines (1 2 4 6 7 15 19 Further PLGA microspheres administered by a parenteral route have to meet the pharmacopoeial requirements of sterility. Preparation of vaccine loaded PLGA microspheres with organic solvents under an aseptic environment involves high production costs (2 4 22 Therefore terminal sterilization of finished product is preferred since it is easier from a technological point of view and more economical than aseptic processing (25-29). The commonly employed terminal sterilization methods are by steam dry heat ethylene oxide gas electron beam irradiation and γ-irradiation (15 23 25 29 Among them dry heat and steam sterilization is carried out at high temperature and can cause significant degradation to protein antigens and hydrolysis of PLGA microparticles; and ethylene oxide is not applicable due to its toxic residues (15 23 25 29 36 Thus γ-irradiation is usually a preferred method of terminal sterilization for injectable PLGA formulations due to its high efficiency negligible thermal effects and absence of post-sterilization treatment of the samples (e.g. aeration of the samples to remove toxic residues and microbial testing to verify sterility are required after ethylene oxide sterilization) (39). However terminal γ-irradiation of finished PLGA formulations has been found to cause undesirable effects on stability of encapsulated vaccine or therapeutic protein polymer properties and antigen/protein release behavior (15 23 26 30 36 37 Encapsulation of vaccine antigens in PLGA microspheres under moderate conditions is critical to avoid the damage during preparation and to obtain controlled release. Indocyanine green Recently we reported a new self-healing based method by which encapsulation of vaccine antigens in PLGA microspheres was achieved by simple mixing of preformed aluminum hydroxide (Al(OH)3)-PLGA-hydrophobic plasticizer microspheres with low concentration (0.64-1 mg/mL) vaccine antigen solution at 10-38° C (22 40 The new method obviated vaccine antigen instability commonly observed with the double emulsion-solvent evaporation method; and also exhibited high loading (1-1.8 wt%) and encapsulation efficiency (~97%) of antigens. This method Indocyanine green resulted in polymer stabilization and long-term release of stable/immunoreactive antigens (22 40 It was hypothesized that active self-healing encapsulation after sterilization of preformed Al(OH)3-PLGA-0 and 5 wt% hydrophobic plasticizer microspheres would decrease production costs to make single-dose vaccination economically feasible. In addition this encapsulation paradigm opens-up the possibility to encapsulate vaccine antigens at the point-of- care. In this study we investigated the effect of terminal γ-irradiation sterilization on pre-formed PLGA microspheres that are used for active self-healing encapsulation of tetanus toxoid (TT) and other alum-adsorbing antigens. To test this concept polymer properties (glass transition heat (Tg) molecular weight and self-healing of PLGA) active self-encapsulation kinetics (loading capacity and encapsulation efficiency) and release characteristics were evaluated before and after γ-irradiation of Al(OH)3-PLGA-0 or 5 wt% hydrophobic plasticizer microspheres. The Indocyanine green effect of γ-irradiation Indocyanine green dose and dose rate on the aforementioned properties was also studied. MATERIALS AND METHODS Materials Tetanus toxoid (3120 Lf/mL) was received from Serum Institute of India Ltd. (Pune India). Al(OH)3 adjuvant bovine serum albumin (BSA) poly(vinyl alcohol) (PVA) (80% hydrolyzed) succinic.