International Journal of Pharmaceutics 311 (2006) 1–10 Alginate microspheres prepared by internal gelation: Development and effect on insulin stability Catarina M. Silva a,∗ , Ant ´ onio J. Ribeiro b , Isabel Vit´ oria Figueiredo c , Alexandra Rocha Gonc ¸alves d , Francisco Veiga a a Laborat´ orio de Tecnologia Farmacˆ eutica, Faculdade de Farm´ acia, Universidade de Coimbra, Rua do Norte, 3000-295 Coimbra, Portugal b Laborat´ orio de Tecnologia Farmacˆ eutica, Instituto Superior de Ciˆ encias da Sa ´ ude-Norte, Rua Central de Gandra, 1317, 4585-116 Gandra, Paredes, Portugal c Laborat´ orio de Farmacologia, Faculdade de Farm´ acia, Universidade de Coimbra, Rua do Norte, 3000-295 Coimbra, Portugal d Departamento de Qu´ ımica, Faculdade de Ciˆ encias e Tecnologia, Universidade de Coimbra, 3004-535 Coimbra, Portugal Received 8 July 2005; received in revised form 11 October 2005; accepted 15 October 2005 Available online 26 January 2006 Abstract Recombinant human insulin was encapsulated within alginate microspheres by the emulsification/internal gelation technique with the objective of preserving protein stability during encapsulation procedure. The influence of process and formulation parameters was evaluated on the morphology and encapsulation efficiency of insulin. The in vitro release of insulin from microspheres was studied under simulated gastrointestinal conditions and the in vivo activity of protein after processing was assessed by subcutaneous administration of extracted insulin from microspheres to streptozotocin-induced diabetic rats. Microspheres mean diameter, ranging from 21 to 287 m, decreased with the internal phase ratio, emulsifier concentration, mixer rotational speed and increased with alginate concentration. Insulin encapsulation efficiency, near 75%, was not affected by emulsifier concentration, mixer rotational speed and zinc/insulin hexamer molar ratio but decreased either by increasing internal phase ratio and calcium/alginate mass ratio or by decreasing acid/calcium molar ratio and alginate concentration. A high insulin release, above 75%, was obtained at pH 1.2 and under simulated intestinal pH a complete dissolution of microspheres occurred. Extracted insulin from microspheres decreased hyperglycemia of diabetic rats proving to be bioactive and showing that encapsulation in alginate microspheres using the emulsification/internal gelation is an appropriate method for protein encapsulation. © 2006 Elsevier B.V. All rights reserved. Keywords: Alginate; Bioactivity; Insulin; Internal gelation; Microspheres 1. Introduction Despite the major advances that have occurred, since insulin introduction in 1922, relating to production, purification and pharmaceutical formulation, insulin-replacement therapy is far from ideal. The optimal method of insulin delivery must be safe, should provide insulin to diabetic patients in a way that will correct the metabolic abnormalities of diabetes mellitus, and must be psychologically and socially acceptable (Brange and Langkjaer, 1997). Although oral administration of insulin is of greatest interest, its low bioavailability has not permit- ted to achieve an efficient formulation. The entrapment within ∗ Corresponding author. Tel.: +351 239 855085; fax: +351 239 855099. E-mail address: catarinasilva@ci.uc.pt (C.M. Silva). microparticulate drug delivery systems, using various kinds of polymers and techniques has been extensively investigated (Damge et al., 1988; Morishita et al., 1992; Mathiowitz et al., 1997; Lowman et al., 1999; Agarwal et al., 2001) to increase the poor permeability across intestinal epithelia and avoid destruc- tion by proteolytic intestinal enzymes. However, in the devel- opment of new protein delivery methods, there are several key points that have to be considered, namely, the impact of manu- facturing on the integrity of the protein (Cleland et al., 2001). During the formation of microspheres, the protein molecules are often exposed to heat, mechanic stirring, adsorption onto matrix polymer, exposure to organic solvents and surface ten- sion, which may result in a different molecular deterioration of the protein, such as molecular breakdown, denaturation and aggregation (Li et al., 2000). Thus, a mild encapsula- tion method avoiding stress conditions should be selected to 0378-5173/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijpharm.2005.10.050