Carbohydrate Polymers 83 (2011) 1854–1861 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Chitosan microparticles loaded with exotoxin A subunit antigen for intranasal vaccination against Pseudomonas aeruginosa: An in vitro study Shahrouz Taranejoo a , Mohsen Janmaleki a , Mohammad Rafienia b,∗ , Mahdi Kamali c , Maysam Mansouri c a Nanomedicine and Tissue Engineering Research Center, Shahid Beheshti University (M.C.), Taleghani Hospital, Parvaneh St., Velenjak, 1985717443 Tehran, Iran b Medical Physics and BioMedical Engineering Department, School of Medicine, Biosensor Research Center, Isfahan University of Medical Sciences, 81744-176, Isfahan, Iran c Nanobiotechnology Research center, Baghiatallah University of Medical Sciences, Mollasadra St., Vanak, 1435916471 Tehran, Iran article info Article history: Received 12 April 2010 Received in revised form 30 July 2010 Accepted 22 October 2010 Available online 30 October 2010 Keywords: Chitosan Microparticles Spray drying Catalytic domain of exotoxin A (PEIII) Pseudomonas aeruginosa abstract Chitosan microparticles (CMs) were prepared with tripolyphosphate by spray-drying. Effects of polymer molecular weight, sonication power, cross-linking time and concentration of TPP on release profiles of catalytic or third domain pseudomonas exotoxin A (PEIII) and morphology of CMs were evaluated. The mean particle sizes of CMs were in the range from 1.09–1.46 m and antigen loading efficiencies were more than 59%. As the molecular weight of chitosan increased, microparticles had a more spherical shape and a smooth surface. An increase in sonication power and decrease in cross-linking time resulted microparticles morphology changes. Approximately 60–80% of PEIII released from microparticles within the first few hours. The release of antigen is increased significantly by raising the sonication power more than 45 W. When the cross-linking time extended from 15 to 60 min, the release of PEIII significantly reduced. The release of PEIII from the microparticles increased when concentration of TPP was raised. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Pseudomonas aeruginosa, a gram-negative bacterial pathogen found mostly in water reservoirs, causes severe nosocomial and community acquired infections at a variety of body sites includ- ing the urinary tract, surgical or burn wounds, the cornea and the lower respiratory tract (Driscoll, Brody, & Kollef, 2007; Hauser & Rello, 2003). Patient groups at risk for acquisition of P. aeruginosa infections include those with hereditary diseases such as cystic fibrosis, paraplegic and burn patients, ones hospitalized in inten- sive care units and those undergoing mechanical ventilation, and patients immunosuppressed by certain diseases like cancer and AIDS. Although antibiotic therapy has considerably improved in the management of infectious diseases in general, many P. aeruginosa infections cannot be fully treated or eradicated by the application of anti-pseudomonal drugs and can thus establish chronic infec- tions. Thus, vaccine development against P. aeruginosa may indeed be useful. Designs of P. aeruginosa vaccine are based on lipopolysac- charide, polysaccharide and polysaccharide-conjugate vaccines. Some other important methods include Flagella vaccines, outer membrane protein vaccines, killed whole cell and live-attenuated vaccines. While many experimental vaccines and monoclonal anti- bodies have been tested in preclinical trials, only a few have reached ∗ Corresponding author. Tel.: +98 3117922480; fax: +98 3116688595. E-mail address: m rafienia@med.mui.ac.ir (M. Rafienia). clinical phases and none of them has obtained approval. The lack of efficient P. aeruginosa vaccines may be overcome by using con- trolled release drug or gene delivery systems (Doring & Pier, 2008). In relation to the different types of infection caused by P. aeruginosa-localized on mucosal surfaces such as the airways or systemic infection in the blood stream, one of the potential routes of administration of the vaccines is inhalation that could enhance their effectiveness. Concerning different routes of drug admin- istration, intranasal drug delivery has many benefits, such as a large epithelial surface area produced by several microvilli, a porous endothelial membrane, and the ability to induce mucosal as well as systemic immunity. However, there are still limitations of nasal immunization, including limited diffusion of macromolecules across the mucosal barrier, enzymatic degradation within nasal secretions, and rapid clearance from nasal cavity, resulting in rapid systemic drug absorption (Kang et al., 2007). In terms of peptide and protein delivery, natural polymers such as collagen, alginate and chitosan show great potential in use of vaccine delivery systems for pulmonary and intranasal delivery. Chitosan is a linear polysaccharide that is composed of randomly distributed d-glycosamine and N-acetyl-glycosamine units linked in a ˇ(1 → 4) manner. Chitosan is used in multiple biomedical and pharmaceutical applications because of its bioavailability, nontox- icity, biocompatibility, biodegradability, high charge density, etc. (Kang et al., 2007). It has been shown that bioavailability of drug and carrier is raised by opening the tight junctions of epithelial cell layers and also reducing the rate of mucociliary clearance 0144-8617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbpol.2010.10.051