european journal of pharmaceutical sciences 28 ( 2 0 0 6 ) 179–188 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/ejps Influences of excipients on in vitro release and in vivo performance of tetanus toxoid loaded polymer particles Yogesh K. Katare, Amulya K. Panda ∗ National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India article info Article history: Received 7 September 2005 Received in revised form 24 January 2006 Accepted 28 January 2006 Published on line 3 March 2006 Keywords: Vaccine delivery Stabilizers Entrapment efficiency Tetanus toxoid Antibody titers abstract Protein instability during microencapsulation has been one of the major hurdles of biodegradable polymer particles-based vaccine delivery systems. In the present work, effect of serum albumin, sucrose and sodium bicarbonate on surface morphology, entrapment efficiency, in vitro release and in vivo performance tetanus toxoid (TT) loaded PLA particles were investigated. Use of serum albumin as well as high concentration of protein anti- gen (∼60 mg/ml) helped in protecting the immunoreactivity of the antigen during primary emulsification step of particle formulation. Incorporation of sucrose in the internal aqueous phase led to the reduction in encapsulation efficiency of TT from 43.8 ± 4.3% to 27.3 ± 3.6% in PLA particles and resulted with formation of particles having irregular surface character- istics. Addition of sodium bicarbonate along with sucrose during primary emulsion led to slight improvement in encapsulation efficiency of TT (34.3 ± 3.2%) but affected the in vivo performance in terms of serum anti-TT antibody titers from single point immunization. Restoration of osmotic balance by adding equivalent amount of sucrose in external aque- ous phase helped in preventing multiple emulsion instability and subsequently improved the encapsulation efficiency of TT to 63.1 ± 4.2%. Maximum entrapment efficiency of TT up to 69.2 ± 5.1% was achieved when serum albumin, sucrose and sodium bicarbonate were used in internal aqueous phase and sucrose was used in the external aqueous phase. Poly- mer particles entrapping tetanus toxoid along with optimal stabilizers showed burst release of immunoreactive antigen (>40% in early period) and elicited high and sustained anti-TT antibody titers from single point intramuscular immunization. Anti-TT antibody titers were further enhanced upon immunization of admixture of PLA particles and alum. Choice and use of stabilizers during particle formulation thus need careful considerations not only to protect the immunoreactivity of the antigen, but also to produce stable, uniform particles for optimal in vivo performances. © 2006 Elsevier B.V. All rights reserved. 1. Introduction Biodegradable microparticles based on polylactide-co- glycolide and polylactide polymers have been investigated as delivery systems for therapeutic proteins, antigens and DNA vaccines (Hanes et al., 1997; Cleland et al., 2001; Panyam ∗ Corresponding author. Tel.: +91 11 267 035 09; fax: +91 11 261 621 25. E-mail address: amulya@nii.res.in (A.K. Panda). and Labhasetwar, 2003; O’Hagan et al., 2004; Jiang et al., 2005). Multiple emulsion solvent evaporation method is most frequently used for the formulation of protein/antigen entrapped polymer particles because of its simplicity and versatility (Couvreur et al., 1997; Vila et al., 2002). For antigens, these polymeric particulate system holds the potential for the 0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.01.010