Research paper DODAB:monoolein liposomes containing Candida albicans cell wall surface proteins: A novel adjuvant and delivery system Catarina Carneiro a , Alexandra Correia b , Tony Collins a , Manuel Vilanova b,c , Célia Pais a , Andreia C. Gomes a,e , M. Elisabete C.D. Real Oliveira d,e , Paula Sampaio a,⇑ a Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal b IBMC – Instituto de Biologia Molecular e Celular, Porto, Portugal c Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal d Centre of Physics (CFUM), University of Minho, Braga, Portugal e NanoDelivery I&D in Biotechnology, Biology Department, Braga, Portugal article info Article history: Received 27 July 2014 Accepted in revised form 29 November 2014 Available online 10 December 2014 Keywords: DODAB:MO liposomes Nanoparticles Adjuvant activity Antigen delivery systems Candida albicans proteins Liposome endocytosis Humoral and cellular immune responses Antigen protection abstract We describe the preparation and characterization of DODAB:MO-based liposomes and demonstrate their adjuvant potential and use in antigen delivery. Liposomes loaded with Candida albicans proteins assem- bled as stable negatively charged spherical nanoparticles with a mean size of 280 nm. High adsorption efficiency (91.0 ± 9.0%) is attained with high lipid concentrations. The nanoparticles were non-toxic, avidly taken up by macrophage cells and accumulated in membrane rich regions with an internalization time of 20 min. Immunized mice displayed strong humoral and cell-mediated immune responses, producing antibodies (IgGs) against specific cell wall proteins, Cht3p and Xog1p. DODAB:MO-based liposomes loaded with C. albicans proteins have an excellent immunogenic potential and can be explored for the development of an immunoprotective strategy against Candida infections. Ó 2014 Elsevier B.V. All rights reserved. 1. Introduction Delivery systems and adjuvants are frequently used in vaccine formulations to ensure efficient antigen delivery and induction of an adequate immune response. In fact, delivery systems have been applied in marketed vaccines for over 70 years to protect and carry antigens as well as to act as adjuvants for molecules with low immunogenicity [1,2]. Currently, many options for delivery systems are available, including liposomes, polymer-based micro- and nanoparticles, virosomes, immune stimulating com- plexes (ISCOMS) and many more [3–6]. Of these, liposomes can also act as immunological adjuvants [7] and are the most widely investigated delivery system for phagocyte-targeted therapies [8,9]. Liposomes offer a flexible system for manipulation and enable the preparation of vesicles with varying lamellarities, physical characteristics, adsorption efficiencies, and antigen encap- sulation abilities [3]. In addition, liposomes are biodegradable and biocompatible and may be functionalized to provide cell targeting specificity and antigen protection [10,11]. Cationic liposomes have been used extensively as drug and vac- cine delivery systems [12,13]. Various types of cationic lipids may be used in the preparation of liposomes, varying in the charge of the hydrophilic head group, the number and/or length of fatty acid chains (tail) and/or their degree of saturation. The cationic surfactant dioctadecyldimethylammonium bromide (DODAB) is a synthetic amphiphilic lipid composed of a hydrophilic positively charged dimethylammonium group (head) attached to two hydro- phobic 18-carbon alkyl chains (tail). In aqueous buffers, DODAB molecules self-assemble into closed liposomal vesicular bilayers [14]. DODAB cationic vesicles have been used as carriers in drug delivery [15,16] as well as adjuvants for vaccination, displaying http://dx.doi.org/10.1016/j.ejpb.2014.11.028 0939-6411/Ó 2014 Elsevier B.V. All rights reserved. Abbreviations: ISCOMS, immune stimulating complexes; DODAB, dioctadecyl- dimethylammonium bromide; PBS, phosphate buffer saline; TDB, trehalose 6,6 0 - dibehenate; MO, 1-monooleoyl-rac-glycerol; CWSP, cell wall surface proteins; DTT, dithiothreitol; HBSS, Hanks’ balanced salt solution; DEMEM, Dulbecco’s Modified Eagle’s Medium; FBS, foetal bovine serum; NBT, nitroblue tetrazolium; BCIP, 5- bromo-4-chloro-3-indolylphosphate; MW, molecular weight; vDODAB, DODAB molar fraction; DTS, Dispersion Technology Software; Cryo-SEM, cryogenic scan- ning electron microscopy; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazo- lium bromide; ELISA, enzyme-linked immunosorbent assay; ADS, antigen delivery systems; PDI, polydispersivity . ⇑ Corresponding author. Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, 4710-057 Braga, Portugal. Tel.: +351 253601546; fax: +351253604319. E-mail address: psampaio@bio.uminho (P. Sampaio). European Journal of Pharmaceutics and Biopharmaceutics 89 (2015) 190–200 Contents lists available at ScienceDirect European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb