Chitosan-coated alginate micro-particles delivery of active principles through conventional pelleted food - A study in Tilapia (Oreochromis niloticus) E. Giacomello a , G. Sava b,c , F. Vita b , N. Delhom d , P. Mahl d , A. Bergamo c, ⁎ ,1 a Dept. of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy b Dept. of Life Sciences, University of Trieste, Trieste, Italy c Callerio Foundation Onlus, Trieste, Italy d Virbac, 13ème Rue LID, BP 27, Carros 06511 Cedex, France. abstract article info Article history: Received 20 July 2020 Received in revised form 14 September 2020 Accepted 20 September 2020 Available online 26 September 2020 Keywords: Lysozyme Oral delivery Oreochromis niloticus Chitosan-coated microparticles Gut uptake Antibiotics Vaccines The search for alternatives to antibiotics in aquaculture has focused on the use of vaccines for immune- prophylaxis. The purpose of this study was to examine the feasibility and characteristics of chitosan-alginate mi- croparticles for the oral delivery of immune-prophylactics to finfish. The microparticles, which incorporate fluorescent-labelled lysozyme, were produced by spray-drying method; their structural properties and uptake from the gastrointestinal tract of Tilapia (Oreochromis niloticus) were assessed by microscopy. The main findings show that the microparticles are able to retain their content in an acidic environment and to release it later in slightly alkaline conditions such as those found in the intestines. Moreover, both the microencapsulation proce- dure and the biopolymers used have no deleterious impact on the lysozyme lytic activity, which is maintained after the protein has been released from the microparticles. Administered in vivo in Tilapia by medicated food, the microparticles transit unaffected through the stomach, and reach the anterior intestines, in particular the villum sectum and the basal lamina of epithelial cells, 2 and 4 h after feeding. Overall, the evidence obtained here supports the potential of these chitosan-alginate microparticles as agents for oral immune-prophylaxis in the management of fish diseases. © 2020 Elsevier B.V. All rights reserved. 1. Introduction Global fish production peaked at about 171 million tonnes in 2016, with aquaculture representing 47% of the total. With capture fishery production relatively static since the late 1980s, aquaculture has been responsible for the continuing impressive growth in the supply of fish for human consumption. Between 1961 and 2016, the average annual increase in global fish consumption (3.2%) outpaced population growth (1.6%) [1]. Despite its rapid expansion, infectious agents, including virus, bacteria, fungi and parasites negatively affect the aquaculture industry [2]. The traditional use of antibiotics has been criticized due to the po- tential development of antibiotic-resistant bacteria, presence of antibi- otic residues in aquatic products, destruction of microbial populations in the aquatic microenvironment and suppression of the aquatic animal's immune system [3,4]. Because of these reasons, the use of an- tibiotics has been limited in the European Union, prompting many sci- entists and industry to search for an alternative to antibiotics. As a consequence, the development and implementation of preventive strat- egies for the management of disease in aquaculture through immune- prophylaxis, particularly through the use of vaccines, has received great impetus and proved to be effective in managing diseases in aqua- culture [5–7]. The mammalian Gut Associated Lymphoid Tissue (GALT) comprises Peyer's patches, M cells, and lymphocytes distributed in the layer of the gut mucosa. While fish GALT lacks Peyer's patches and M cells, it pre- sents Intra Epithelial Lymphocytes (IELs) and epithelial cells with pino- cytic activity. Accordingly, several works provided evidence that the distal section of the gastrointestinal tract in teleosts is capable of antigen uptake and translocation of these antigens to macrophages and lym- phocytes in the lamina propria and systemic lymphoid organs [8,9]. This phenomenon, widely accepted and demonstrated, constitutes the International Journal of Biological Macromolecules 165 (2020) 82–92 Abbreviations: BCA, bicinchoninic acid; BW, body weight; EC 50 , concentration that causes 50% lysis of Micrococcus; FITC, fluorescein isothiocyanate; GALT, gut associated lymphoid tissue; GI, gastrointestinal; HPMC, hydroxypropylmethyl cellulose; IELs, intra- epithelial lymphocytes; MPs, microparticles; PBS, phosphate buffered saline; PFA, parafor- maldehyde; RT, room temperature; SEM, scanning electron microscopy; Tris, tris(hy- droxymethyl)methylamine.. ⁎ Corresponding author at: Dept of Life Sciences, University of Trieste, Via A. Fleming, 22, 34127 Trieste, Italy. E-mail addresses: egiacomello@units.it (E. Giacomello), nathalie.delhom@virbac.com (N. Delhom), philippe.mahl@virbac.com (P. Mahl), abergamo@units.it (A. Bergamo). 1 Present address: Dept. of Life Sciences, University of Trieste, Via A. Fleming, 22, 34,127 Trieste, Italy. https://doi.org/10.1016/j.ijbiomac.2020.09.162 0141-8130/© 2020 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac