142 Introduction here are an estimated 500,000 new cases of Visceral leishmaniasis (VL) and more than 50,000 deaths from the disease each year, a toll that is surpassed among the parasitic diseases only by malaria (Chappuis et al., 2007). VL is a systemic disease caused by the obligate intra-macrophage protozoa Leishmania donovani and L. infantum. Current drugs, such as the pentavalent antimonials, liposomal amphotericin B and miltefosin have several disadvantages in terms of tolerability, therapeutic regimen, duration of treatment, speciic- ity and patient compliance. Moreover, resistance to the irst-line treatment with Pentostam ® or Glucantime ® has emerged in the past decades (Chappuis et al., 2007; Piscopo and Azzopardi, 2007). he development of new chemical entities for combating VL remains necessary, and plant extracts are a valuable source of innovative lead structures. RESEARCH ARTICLE Intracellular drug delivery in Leishmania-infected macrophages: Evaluation of saponin-loaded PLGA nanoparticles H. Van de Ven 1 , M. Vermeersch 2 , R.E. Vandenbroucke 3,4 , A. Matheeussen 2 , S. Apers 5 , W. Weyenberg 1 , S.C. De Smedt 3 , P. Cos 2 , L. Maes 2 , and A. Ludwig 1 1 University of Antwerp, Laboratory of Pharmaceutical Technology and Biopharmacy, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Universiteitsplein 1, CDE, Antwerpen (Wilrijk), 2610 Belgium, 2 University of Antwerp, Laboratory of Microbiology, Parasitology and Hygiene (LMPH), Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Groenenborgerlaan 171, Antwerp, 2020 Belgium, 3 Ghent University, Laboratory of General Biochemistry and Physical Pharmacy, Department of Pharmaceutics, Harelbekestraat 72, Ghent, 9000 Belgium, 4 Department for Molecular Biomedical Research (DMBR), Molecular Mouse Genetics, Ghent University-VIB, Belgium, and 5 University of Antwerp, Laboratory of Pharmacognosy and Pharmaceutical Analysis, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Universiteitsplein 1, Antwerp (Wilrijk), 2610 Belgium Abstract Drug delivery systems present an opportunity to potentiate the therapeutic efect of antileishmanial drugs. Colloidal carriers are rapidly cleared by the phagocytic cells of the reticuloendothelial system (RES), rendering them ideal vehicles for passive targeting of antileishmanials. This paper describes the development of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) for the antileishmanial saponin β-aescin. NPs were prepared using the combined emulsiication solvent evaporation/salting-out technique. Confocal microscopy was used to visualise the internalisation and intracellular traicking of luorescein- and nile red-labelled PLGA NPs in J774A.1 macrophages infected with GFP-transfected Leishmania donovani. The in vitro activity of aescin and aescin-loaded NPs on L. infantum was determined in the axenic model as well as in the ex vivo model. The developed PLGA NPs were monodispersed with Z ave <300 nm, exhibited negative zeta potentials and had relatively high drug loadings ranging from 5.80 to 8.68% w/w PLGA. The luorescent NPs were internalised by the macrophages and traicked towards the lysosomes after 2 h in vitro incubation. Co-localisation of the NPs and the parasite was not shown. A two-fold increase in activity was observed in the ex vivo macrophage model by encapsulating β-aescin in PLGA NPs (IC 50 , 0.48–0.76 µg/mL vs. 1.55 ± 0.32 µg/mL for the free drug). Keywords: Leishmaniasis, J774A.1 macrophages, PLGA nanoparticles, saponins, simplex mixture design, co-localisation, GFP-transfected L. donovani Address for Correspondence: H. Van de Ven, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Universiteit Antwerpen, Universiteitsplein 1, B-2610 Antwerpen, Belgium. Tel.: +32 (0)3 265 27 13. E-mail: helene.vandeven@ua.ac.be; helene.vandeven@yahoo.com (Received 07 March 2011; revised 04 May 2011; accepted 17 May 2011) Journal of Drug Targeting, 2012; 20(2): 142–154 © 2012 Informa UK, Ltd. ISSN 1061-186X print/ISSN 1029-2330 online DOI: 10.3109/1061186X.2011.595491