European Journal of Pharmaceutical Sciences Design and Influence Of

European Journal of Pharmaceutical Sciences 37 (2009) 563–572 Contents lists available at ScienceDirect European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps Design and influence of ␥-irradiation on the biopharmaceutical properties of nanoparticles containing an antigenic complex from Brucella ovis Raquel Da Costa Martins a, Carlos Gamazo b, Juan Manuel Irache a,∗ a Department of Pharmacy and Pharmaceutical Technology, University of Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain b Department of Microbiology and Parasitology, University of Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain article info abstract Article history: Despite vaccination campaigns, brucellosis is still one of the most common bacterial zoonosis in the Received 25 February 2009 world. This work describes the development of a novel formulation strategy to the delivery of the Bru- Received in revised form 3 April 2009 cella ovis antigenic extract (HS) into ovine mucosal surfaces. Thus, HS was entrapped in conventional Accepted 3 May 2009 and mannosylated poly(anhydride) nanoparticles by the solvent displacement method, and the resulting Available online 12 May 2009 nanosystems were ␥-irradiated to accomplish the sterilization required for the ophthalmic administration route. Sterilization, at either 10 kGy or 25 kGy, did not modify the size, morphology and antigen content Keywords: of the nanoparticles. Similarly, the integrity and antigenicity of the entrapped antigen were not affected Brucellosis ␥ ␥ Nanoparticles by -irradiation. The 25 kGy -irradiation dose seemed to influence negatively the HS release from the Vaccine carriers. However, and in accordance with the Pearson’s correlation, all the release patterns followed a ␥-Irradiation similar tendency. Furthermore, the stability of the vaccine systems on lachrymal and nasal ovine fluids, Sterilization showed that ␥-irradiation had no significant effects on the vaccine systems. Since all the vaccine systems accomplished the pharmacopoeial biological tests required for ␥-irradiation doses under 25 kGy, these results are highly suggestive for the use of HS loaded poly(anhydride) nanoparticles as an efficient vaccine delivery system for brucellosis immunoprophylaxis, especially for ophthalmic administration. © 2009 Elsevier B.V. All rights reserved. 1. Introduction ovine brucellosis, which involves either subcutaneous or ocular administration routes (Blasco, 1997). Nevertheless, among all the Brucellosis is one of the major contagious bacterial zoonosis drawbacks of the attenuated strains, Rev1 displays high resid- worldwide (Boschiroli et al., 2001) and some Brucella spp. has being ual virulence for both animals and humans, produce abortions, considered as biological warfare agents (level B, classification by the infertility, and it is resistant to streptomycin (Blasco, 1997; Blasco United States Centers for Disease Control) (Greenfield et al., 2002). and Diaz, 1993). Another inconvenience in the use of this vac- Brucella spp. may affect sheep, goats, rams, cattle, swine, dogs, cine is the long persistence in the immunized animal, due to rodents and several other animals (Cutler et al., 2005; Whatmore the high level persistence of circulating antibodies (against the et al., 2008). Humans become infected by coming in contact with smooth lipopolysaccharide), which interfere with serological diag- animals or contaminated animal products causing a range of long- nosis, avoiding the differentiation between the infected animals lasting or chronic symptoms (Young, 1995). and the vaccinated ones (Schurig et al., 2002). Other disadvan- Ovine brucellosis is an infectious disease caused by both Brucella tage of Rev1 is that its use is not allowed in countries officially ovis and Brucella melitensis, and it causes important losses in ovine free from B. melitensis such as Canada and USA, most of Northern industry (abortion, infertility and mortality). Taking in account that Europe, Australia and New Zealand. Recently, another commer- the human disease demands complex and protracted treatment cial vaccine available against ovine brucellosis has been proposed schedules that still are not always effective, and that the global (Schurig et al., 1991). This vaccine is based on the live attenuated dissemination of animal brucellosis is not achieved, eradication of Brucella abortus RB51, however, although does not interfere with this zoonosis will only be feasible with an adequate animal vaccine. the serological diagnosis, it has been proven to be not so effec- Nowadays, the live attenuated vaccine B. melitensis Rev1 is tive against B. ovis in rams as Rev1, being neither able to protect the most effective immunoprophylactic system available against nor reduce the severity of the infection or the development of genital lesions (Jimenez de Bagues et al., 1995). In addition, RB51 also offers a number of drawbacks including rifampicin resistance ∗ and lack of effectiveness when prevalence is high (Moriyon et al., Corresponding author. Tel.: +34 948 425600; fax: +34 948 425619. E-mail address: [email protected] (J.M. Irache). 2004). 0928-0987/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2009.05.002 564 R. Da Costa Martins et al. / European Journal of Pharmaceutical Sciences 37 (2009) 563–572 Subcellular vaccines display important advantages to face the BCATMProtein Assay Reagent Kit was from Pierce (Rockford, USA). classical live attenuated vaccines handicaps. In this context, the hot Acrylamide CriterionTMXT Precast gels (18 Comb, 30 ␮L, 1 mm) saline (HS) subcellular antigenic extract from B. ovis, which has been and the Silver Stain kit were obtained from Bio-Rad Labora- probed to be highly immunogenic (Blasco et al., 1993; Gamazo et tories (California, USA). Peroxidase-conjugate rabbit anti-sheep al., 1989), is mainly composed by the major membrane antigens immunoglobulin G (heavy and light chain specific) (RaSh H+L) was like outer membrane proteins (OMPs) and rough lipopolysaccha- purchased from Nordic Immunological Laboratories (Tilburg, The ride (R-LPS). However, due to its non-replicant nature, adequate Netherlands). PVDF (polyvinylidene fluoride papers, pore size of adjuvants have to be associated to the HS extract, in order to 0.45 ␮m) sheets were from Schleicher & Schuell (Germany) and promote cellular mediated immunity, specifically Th1 type (Araya 4-chloro-1-naphtol from Merck (Germany). The rainbow coloured and Winter, 1990; Oliveira and Splitter, 1995). Poly(␧-caprolactone) protein molecular weight marker was from Amersham Pharmacia microparticles containing HS (HS-PEC) were found to be a protec- Biotech (Freiburg, Germany). All other chemicals were of analytical tive vaccine in mice and ram models (Estevan et al., 2006; Munoz grade and supplied from Merck (Madrid, Spain). et al., 2006; Murillo et al., 2001). Moreover, the lack of interference in the B. melitensis diagnostic tests and the intrinsic avirulence and 2.2. Methods innocuousness, converted HS-PEC microparticles as an attractive anti-Brucella vaccine candidate. 2.2.1. Extraction and characterization of the HS On the other hand, it is well known that the mucosal administra- The hot saline antigenic complex (HS) was obtained from the tion route can mimic the bacteria behaviour and generate immunity strain B. ovis REO 198 by the hot saline method described previously at the major portals of pathogen entry. In addition, this type of (Gamazo et al., 1989). To get a large and homogenous batch of cells administration can also be safer with less adverse effects and facil- for extractions, a thawed vial of stock suspension was streaked onto itates its dispensation and application. Regarding the imitation of BAB (Blood Agar Base no. 2, Difco, Detroit, USA) plates, and 24 h the Brucella infection and colonization patterns, the antigens deliv- cultures were inoculated into a 10 L bioreactor (B. Braun Biotech, ery to mucosal surfaces is of remarkable concern, as it has been Germany), and incubated at 37 ◦C for 48 h. Live cells were sus- shown by the ocular administration of Rev1 vaccine (Blasco, 1997). pended in saline solution (0.85% NaCl, 10 g packed cells per 100 mL), The need of an adequate adjuvant capable of increase the mucosal and heated in flowing steam for 15 min. Following centrifugation immune response and protection without the need of booster at 12,000 × g for 15 min, the supernatant was dialyzed for 5 days ◦ doses lead us to purpose the use of poly(anhydride) nanoparticles. at 4 C against several changes of deionized water (dH2O). The dia- These controlled release systems have been proposed as adju- lyzed material was ultracentrifuged for 3 h at 60,000 × g and the vants since they: (i) protect the loaded antigen from enzymatic pellet (HS) washed in dH2O, freeze-dried and stored at room tem- degradation, (ii) prolong immune response, (iii) enhance the anti- perature. gen delivery to the mucosal associated lymphoid tissue (MALT) Total protein was determined by the BCATM Protein Assay and (iv) exhibit a strong bioadhesive performance (Almeida and method (Smith et al., 1985), with bovine serum albumin as stan- Alpar, 1996; Arbos et al., 2003; Gomez et al., 2007; Motwani et al., dard. The analysis for 2-keto-3-deoxyoctonate (KDO, exclusive 2008). To exploit the potential of these systems, mannosamine was marker of LPS) corrected for 2-deoxyaldoses was performed by added to the nanoparticles as a specific target ligand. The effec- the method of Warren modified by Osborn (Osborn, 1963). The tiveness

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