Cationic submicron emulsions for pulmonary DNA immunization Maytal Bivas-Benita a, * , Marion Oudshoorn a , Stefan Romeijn a , Krista van Meijgaarden b , Henk Koerten c , Hans van der Meulen c , Gregory Lambert d , Tom Ottenhoff b , Simon Benita e , Hans Junginger a , Gerrit Borchard a a Leiden/Amsterdam Center for Drug Research, Division of Pharmaceutical Technology, P.O. Box 9502, 2300 RA Leiden, The Netherlands b Leiden University Medical Center, Department of Immunohematology and Blood Transfusion, P.O. Box 9600, 2300 RC Leiden, The Netherlands c Leiden University Medical Center, Department of Molecular Cell Biology, P.O. Box 9503, 2300 RA Leiden, The Netherlands d Novagali Pharma, Genavenir IV, 1 rue Pierre Fontaine, 91000 Evry, France e Department of Pharmaceutics, School of Pharmacy, The Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem, Israel Received 18 May 2004; accepted 3 August 2004 Available online 15 September 2004 Abstract Pulmonary immunization against inhaled pathogens such as Mycobacterium tuberculosis would induce local and systemic immune responses and protect from entry and dissemination of the pathogen. The aim of this study was to evaluate cationic submicron emulsion as a potential carrier for DNA vaccines to the lung. DNA loaded emulsions were 128–152 nm in size and retained positive zeta potential above +40 mV during 3 months of storage. Loading efficiency was above 99%, DNA was protected from DNase I degradation up to 60 min and was stable in presence of 75% fetal calf serum (FCS). The plasmid DNA was detected in the endo-lysosomal compartment of the human bronchial cell line, Calu-3, 6 h after application. No cytotoxic effect on these cells was observed. Human dendritic cells were matured in presence of DNA loaded emulsion, although to a lesser extent than DNA solution indicating slower release and lower exposure to unmethylated CpG sequences. These results indicate that cationic submicron emulsions are potential DNA vaccine carriers to the lung since they are able to transfect pulmonary epithelial cells, which possibly induce cross priming of antigen presenting cells and directly activate dendritic cells, resulting in stimulation of antigen specific T-cells. D 2004 Elsevier B.V. All rights reserved. Keywords: Cationic submicron emulsions; Pulmonary delivery; Tuberculosis DNA vaccines; Calu-3 cells; Dendritic cells 1. Introduction Mucosal vaccination is an evolving field of interest, especially in viral and bacterial diseases where the mucosal tissue is the portal of entry of the 0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2004.08.008 * Corresponding author. Tel.: +31 71 5274245; fax: +31 71 5274565. E-mail address: m.bivas@lacdr.leidenuniv.nl (M. Bivas-Benita). Journal of Controlled Release 100 (2004) 145 – 155 www.elsevier.com/locate/jconrel GENE DELIVERY