Poly(D,L-lactide-co-glycolide) (PLGA) microparticles containing plasmid DNA (pDNA) have potential uses as vaccine delivery systems. Nevertheless, the established double emulsion and solvent evaporation method used to produce them is characterised by a low encapsulation efficiency (about 20%) and nicks the supercoiled DNA. The aim of this work was to develop an encapsulation process to optimise the overall encapsulation efficiency and the supercoiled DNA content, to obtain a carrier suitable for mucosal delivery of DNA vaccines. Our strategy was to reduce the global negative charge of DNA which was unfavourable to its incorporation into the polymer by condensing it with cationic poly(aminoacids) which were previously reported to improve cell transfection. In this study, after characterisation of the compaction of DNA plasmid encoding for a Green Fluorescent Protein, we demonstrated that resulting complexes were successfully encapsulated into PLGA microparticles presenting a mean size around 4.5 microns. The preliminary step of complexation enhances the yield of the process by a factor 4.1 and protects the supercoiled form. In a bacteria transformation assay, we demonstrated that extracted pDNA (naked or complexed) remained in a transcriptionally active form after encapsulation. Bovine macrophages in culture phagocytosed microparticles loaded with uncomplexed/complexed with poly(L-lysine) pDNA. The production of the Green Fluorescent Protein demonstrated that these carriers could deliver intact and functional plasmid DNA probably by escaping from lysosomal degradation.