International Journal of Pharmaceutics 381 (2009) 130–139 Contents lists available at ScienceDirect International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm Pharmaceutical Nanotechnology Relevance of the colloidal stability of chitosan/PLGA nanoparticles on their cytotoxicity profile Noha Nafee a , Marc Schneider a,b,∗ , Ulrich F. Schaefer a , Claus-Michael Lehr a a Biopharmaceutics & Pharmaceutical Technology, Saarland University, Saarbrücken, Germany b Pharmaceutical Nanotechnology, Saarland University, Saarbrücken, Germany article info Article history: Received 4 December 2008 Received in revised form 8 April 2009 Accepted 16 April 2009 Available online 18 May 2009 Keywords: PLGA nanoparticles Chitosan Cytotoxicity Colloidal properties MTT assay LDH assay In vitro tests abstract The application of nanoparticles on a sub-cellular level necessitates an in depth study of their biocompat- ibility. However, complete characterization of the particles under the physiological conditions relevant for biological evaluation is still lacking. Our goal is therefore to evaluate the possible toxicity aspects of chitosan-modified PLGA nanoparticles on different cell lines and relate them to the parameters affecting the colloidal stability of the nanoparticles. The impacts of different factors such as nanoparticle concen- tration, exposure time, chitosan content in the particles and pH fluctuations on the cell viability were investigated. Meanwhile, the colloidal stability of the particles in cell culture media was checked by mea- suring their size and charge as well as visualizing the particles in media by scanning force microscopy (SFM). A slight shift in the pH of the culture medium to the acidic side allows the protonation of chitosan; thus the increased positive surface charge induced membrane damage (∼50% increase in LDH released). Besides, cell viability is reduced by 15% in the absence of serum; serum in the culture medium forms a protective shell around the particles; such interaction influences the surface charge of the particles and was found to be a function of chitosan content in the particles. In conclusion, there is an undeniable impact of cell type, medium, presence/absence of serum on the colloidal state of the particles that consequently influence their interaction with the cells. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Nanomedicine, a new attractive term frequently applied nowa- days that implies for the medical application of nanotechnology as an alternative to the classical drug formulations. In the last decade, an increasing number of investigations concerning the use of nanoscale structures for drug and gene delivery purposes have been developed (Jin and Ye, 2007; Azarmi et al., 2008). Despite the significant scientific interests and promising potential in numerous applications, the safety aspects of nanoparticulate systems remain a growing concern as the processing of nanoparticles in biologi- cal systems could lead to unpredictable effects. In addition, due to the greater surface area-to-volume ratio for nanoscale mate- rial, the toxicity could differ from a similar bulk material (Xia et al., 2006). Indeed dealing with metal-based nanoparticles for drug delivery is much more crucial; therefore, a new sub-discipline of ∗ Corresponding author at: Pharmaceutical Nanotechnology, Saarland University, Campus A4 1, D-66123 Saarbrücken, Germany. Tel.: +49 681 302 2438; fax: +49 691 302 4677. E-mail address: Marc.Schneider@mx.uni-saarland.de (M. Schneider). nanotechnology called nanotoxicology has emerged (Fischer and Chan, 2007). One of the main goals in nanomedicine is the use of body- friendly and biodegradable materials and polymeric excipients. Poly(d,l-lactide-co-glycolide) (PLGA) is a biodegradable, synthetic polymer frequently used in drug/gene delivery (Panyam and Labhasetwar, 2003). The slight negative surface charge of PLGA nanoparticles (PLGA NP) tends to limit their interaction with the negatively charged plasmids and their intracellular uptake. There- fore, attempts have been made to modify the surface of PLGA NP using cationic polymers such as chitosan (Nafee et al., 2007; Ravi Kumar et al., 2004) retrieved from biological sources. Chitosan has been shown to be relatively safe (Corsi et al., 2003; Lee et al., 2001). Moreover, chitosan is approved as a food additive in Japan, Italy and Finland and as a wound dressing in the USA (Illum, 1998) and is widely used in drug delivery owing to its biocompatibility, mucoad- hesive and permeability enhancing properties (Dodane et al., 1999). Nowadays, chitosan and its derivatives, e.g., trimethyl chitosan and thiolated chitosan gained a great interest as non-viral transfection reagents (Issa et al., 2005; Amidi et al., 2007; Martien et al., 2007; Hohne et al., 2007; Hwang et al., 2008). However, the derivatization and degree of deacetylation was sometimes found to influence the safety of the polymer (Kean et al., 2005; Guggi et al., 2004). Other 0378-5173/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijpharm.2009.04.049