Research Paper Development of a Novel Method for the Preparation of Thiolated Polyacrylic Acid Nanoparticles Melanie Greindl 1 and Andreas Bernkop-Schnu ¨ rch 1,2 Received February 20, 2006; accepted May 31, 2006; published online August 9, 2006 Purpose. To develop a novel method for the preparation of thiolated polyacrylic acid nanoparticles via ionic gelation. Materials and Methods. In a first step nanoparticles were generated by ionotropic gelation of polyacrylic acid (PAA) of three different molecular weights (100, 240 and 450 kDa) and various cations including Ca 2+ , Mg 2+ , Zn 2+ , Al 3+ and Fe 3+ . Via in vitro characterization of the particles (particle size, size distribution and zeta potential) the optimal preparation conditions were established. Taking into consideration, that thiolated polyacrylic acid (PAA-Cys) displays higher mucoadhesive and permeation enhancing properties than unmodified PAA, PAA-Cys nanoparticles were produced in the same manner with Ca 2+ , as the most promising results concerning particle size and stability of particles could be achieved with this ionic crosslinker. The nanoparticles were stabilized via the formation of inter- and intrachain disulfide bonds within these particles due to oxidation with H 2 O 2 . Ca 2+ was removed proximately by the addition of EDTA and exhaustive dialysis. Results. Using the preparation method described above PAA-Cys nanoparticles of a mean diameter of about 220 nm (PAA 100 -Cys), 250 nm (PAA 240 -Cys) and 295 nm (PAA 450 -Cys) can be generated. In comparison to PAA nanoparticles ionically crosslinked with Ca 2+ , the removal of the crosslinker Ca 2+ from PAA-Cys particles led to a nearly three-fold decrease in the zeta potential, from about j7 up to j20 mV. Apart from this advantage, covalently crosslinked PAA-Cys nanoparticles were more firm as they remained stable when incubated in hydrochloride solution, whereas ionically crosslinked particles dissolved at pH lower than 5. Conclusions. This novel nanoparticulate delivery system seems to be a promising vehicle for the administration of therapeutic proteins, genes and antigens via mucosal membranes. KEY WORDS: calcium; crosslinking; nanoparticles; polyacrylic acid; thiomer. INTRODUCTION In recent years polymer nanoparticles have been widely investigated as a carrier for drug delivery. For non-invasive drug administration particulate delivery systems offer the advantage of providing a prolonged residence time on mucosal membranes (1) and the possibility to reach greater mucosal surface areas leading to a comparatively higher drug uptake (2). Although the efficacy of such systems has already been demonstrated in various clinical trials (3), it is believed that the full potential of non-invasive nanoparticulate deliv- ery systems has by far not been reached. Nanoparticulate delivery systems for non-invasive administration are based on various polymers such as polyacrylates (4), PLGA (5) or chitosans (6). Among these polymers polyacrylic acid offers the advantage of high mucoadhesive properties because of the formation of non-covalent bonds such as hydrogen bonds, ionic interactions and van der Waals forces or physical interpenetration effects of polymer chains and mucus (7,8). These mucoadhesive properties of PAA were even signifi- cantly further improved by the immobilization of thiol groups on the polymer. Grabovac et al. (9), for instance, could show that the mucoadhesive properties are even 20-fold improved due to the immobilization of thiol groups on the polymer (10). These strongly improved mucoadhesive properties are based on the formation of disulfide bonds between the thiolated polymer and cysteine-rich subdomains of the mucus gel layer (11). Accordingly, thiolated PAA nanoparticles should display comparatively higher mucoadhesive proper- ties than unmodified PAA nanoparticles being advantageous for the mucosal administration of various drugs. In order to benefit from the high mucoadhesive proper- ties of thiolated PAA on the one hand and the advantages of nanoparticulate delivery systems such as an increase of the adhesive force or a prolongation of the GI transit time on the other hand, it was the aim of this study to develop a novel method for the preparation of thiolated polyacrylic acid nanoparticles. The strategy pursued to achieve that goal is outlined in Fig. 1. In a first step nanoparticles were produced 2183 0724-8741/06/0900-2183/0 # 2006 Springer Science + Business Media, Inc. Pharmaceutical Research, Vol. 23, No. 9, September 2006 ( # 2006) DOI: 10.1007/s11095-006-9087-1 1 Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University Innsbruck, Innrain 52, Josef-Mo ¨ ller- Haus, 6020 Innsbruck, Austria. 2 To whom correspondence should be addressed. (e-mail: andreas. bernkop@uibk.ac.at)