Carbohydrate Polymers 98 (2013) 1397–1408 Contents lists available at ScienceDirect Carbohydrate Polymers jo ur nal homep age: www.elsevier.com/locate/carbpol Ultrasonication of insulin-loaded microgel particles produced by internal gelation: Impact on particle’s size and insulin bioactivity Ana C. Santos a , Joana Cunha b,c , F. Veiga a , A. Cordeiro-da-Silva b,d , Antonio J. Ribeiro a,∗ a Centro de Estudos Farmacêuticos, Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal b IBMC – Instituto de Biologia Molecular e Celular, University of Porto, 4150-180 Porto, Portugal c Institute for the Biomedical Sciences Abel Salazar and Faculty of Medicine, University of Porto, 4050-343 Porto, Portugal d Faculty of Pharmacy, University of Porto, 4050-343 Porto, Portugal a r t i c l e i n f o Article history: Received 3 December 2012 Received in revised form 13 June 2013 Accepted 27 June 2013 Available online 23 July 2013 Keywords: Hydrogels Particle size Physical stability Ultrasound Emulsion In vitro models a b s t r a c t Alginate-dextran sulfate (ADS) microgel has been used to protect insulin from gastrointestinal attack and as a carrier to promote insulin permeation through intestinal epithelium. The throughput of ADS submicron particles generation by emulsification/internal gelation is limited by its wide size distribution. The aim of this work was to study the recovery protocol influence on ADS particles through the deter- mination of its impact on particles’ size distribution and bioactivity. ADS particles showed a wide and multimodal distribution, characterized by a high aggregation phenomenon. In an attempt to reverse particles’ tendency to aggregate and to homogenize particle size ADS populations were submitted to ultrasonication, while particle size distribution, physical and chemical stability, and the bioactivity of entrapped insulin were investigated. After ultrasonication a narrower particle population shifted to the nanoscale, with higher physical stability and significant insulin bioactivity was obtained. Emulsification internal/gelation followed by ultrasonication constituted a valid strategy to obtain ADS particles at the submicron range, with high stability and without significantly compromising insulin bioactivity, so offer- ing promises, under previously well established conditions, to evaluate impact of ADS particle’s size on biopharmaceutical and pharmacokinetics phases. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction Microgels have gained considerable attention in recent years as one of the most promising nanoparticulate drug delivery sys- tems owing to their unique potentials via combining microgel’s hydrophilicity and extremely high water content with a nanosize scale. Particles’ size is a keyrole in biomedical applications, with given influence on biorecognition (Hwang, Truong & Sim, 2012), biodistribution (Hirn et al., 2011), bioadhesion (Hasani, Pellequer & Lamprecht, 2009), biocompatibility (Akbar, Mohamed, Whitehead & Azzawi, 2011), and biodegradation (Cui, Hunter, Yang, Chen & Gan, 2011). Smaller particles may penetrate cells by passing directly through the cell membrane or infiltrate between cells, translocate to new sites and to the blood/lymph and thereby target organs away Abbreviations: ADS, alginate/dextran sulfate; NPs, nanoparticles; LD, laser diffractometry; CD, circular dichroism; DMEM, Dulbecco’s modified eagle medium; FBS, fetal bovine serum; Cryo-SEM, cryo-scanning electron microscopy; ANOVA, one way analysis of variance; LOD, limit of detection; DLS, dynamic light scattering; ELS, electrophoretic light scattering; PI, polydispersity index; w/o, water/oil. ∗ Corresponding author. Tel.: +351 239488400; fax: +351 239488503. E-mail address: aribeiro@ff.uc.pt (A.J. Ribeiro). from their portal of entry (Geiser et al., 2005). Controlled release experiments with hydrophobic dexamethasone and hydrophilic vitamin C used as encapsulant models showed that for these small molecular drugs, the loading amount was mainly determined by the surface area of the nanoparticles (NPs), and the subsequent release of the drug dramatically decreased with the increasing of the particle size (Gan et al., 2012). Therefore, emulsification/internal gelation technique, initially conceived for microparticles prepara- tion (Poncelet, Lencki, Beaulieu, Halle, Neufeld & Fournier, 1992), rapidly spurred interest in extending it into the submicron range. Recent works concerning insulin delivery systems have shown good characteristics in terms of insulin chemical stability and bioac- tivity maintenance (Luo et al., 2012; Zhang et al., 2010), however most of them are for subcutaneous injection (Al-Tahami, Oak & Singh, 2010; Chen, Wu, Guo, Xin & Li, 2011; Oak & Singh, 2011) and use synthetic instead of biopolymers. Insulin-loaded alginate- dextran sulfate (ADS) particles prepared by emulsification/internal gelation constitute a promising controlled delivery system for oral route (Reis, Veiga, Ribeiro, Neufeld & Damge, 2008). The objective of insulin entrapment into the polymeric matrix is to minimize protein denaturation, stabilizing and maintaining its physiological activity during both particle manufacturing and drug release (Reis, Ribeiro, Houng, Veiga & Neufeld, 2007; Silva, Ribeiro, Figueiredo, 0144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carbpol.2013.06.063