Research paper Charged nanoparticles as protein delivery systems: A feasibility study using lysozyme as model protein Cuifang Cai a,b , Udo Bakowsky a , Erik Rytting a , Andreas K. Schaper c , Thomas Kissel a, * a Department of Pharmaceutics and Biopharmacy, Philipps Universita ¨ t of Marburg, Marburg, Germany b School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, China c Department of Geosciences and Materials Science Center, Philipps Universita ¨ t of Marburg, Marburg, Germany Received 4 July 2007; accepted in revised form 8 October 2007 Available online 12 October 2007 Abstract The aim of this study was to investigate the feasibility of negatively charged nano-carriers (nanoparticles), consisting of poly- mer blends of poly(lactide-co-glycolide) (PLGA) and poly(styrene-co-4-styrene-sulfonate) (PSS), to improve the loading capacity and release properties of a positively charged model protein, lysozyme, through an adsorption process. Nanoparticles were pre- pared by a solvent displacement method and characterized in terms of size, f-potential, morphology, as well as loading capacity of model protein lysozyme. Morphology of these particles was investigated using transmission electron microscopy (TEM), scan- ning electron microscopy (SEM) and atomic force microscopy (AFM). The loading capacity of lysozyme was evaluated as a func- tion of polymer blend ratio, protein concentration, pH, and ionic strength; in vitro release profiles were also studied. The results show that negatively charged nanoparticles were obtained using polymer blends of PLGA and PSS, characterized by increased net negative surface charge with increasing ratios of PSS. Moreover, protein loading capacity increased as function of PSS/PLGA ratio. Increased pH facilitated the adsorption process and improved the loading capacity. Maximum loading efficiency was achieved at salt concentrations of 50 mM. In vitro release of lysozyme from the polymer blend nanoparticles was dependent on drug loading and full bioactivity of lysozyme was preserved throughout the process. These findings suggest that this is a fea- sible method to prepare nanoparticles with high surface charge density to efficiently adsorb oppositely charged protein through electrostatic interactions. Ó 2007 Elsevier B.V. All rights reserved. Keywords: Charged nanoparticles; Lysozyme; Electrostatic interaction; Protein delivery; Charge density 1. Introduction Recent years have seen increasing interest in polymeric nanoparticles as carriers for hydrophilic macromolecules such as proteins, vaccines, and polynucleotides [1,2]. Numerous investigations have shown that nanoparticles can not only improve the stability of therapeutic agents against enzymatic degradation and control the release of therapeutic agents, but they can also be delivered to distant target sites either by localized delivery using a catheter- based approach with a minimal invasive procedure, or they can be conjugated to a biospecific ligand which could direct them to the target tissue or organ [1,3]. For an effective nanoparticulate delivery system, the nanoparticle size and loading must be adjusted carefully, and protein stability during preparation and release must be ensured. Depend- ing on the preparation method, drugs or antigens can either be entrapped in the polymer matrix, encapsulated in a liquid core, surrounded by a shell-like polymer membrane, or bound to the particle surface by adsorption [4]. Some reported methods for preparing nanoparticles from 0939-6411/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejpb.2007.10.005 * Corresponding author. Department of Pharmaceutics and Biophar- macy, Philipps Universita ¨t of Marburg, Ketzerbach 63, D-35032 Mar- burg, Germany. Tel.: +49 6421 282 5881; fax: +49 6421 282 7016. E-mail address: kissel@staff.uni-marburg.de (T. Kissel). www.elsevier.com/locate/ejpb Available online at www.sciencedirect.com European Journal of Pharmaceutics and Biopharmaceutics 69 (2008) 31–42