In situ gelling properties of chitosan-thioglycolic acid conjugate in the presence of oxidizing agents Duangkamon Sakloetsakun, Juliane M.R. Hombach, Andreas Bernkop-Schnu ¨ rch * Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 52, Josef Mo ¨ller Haus, A-6020 Innsbruck, Austria article info Article history: Received 8 May 2009 Accepted 29 July 2009 Available online 21 August 2009 Keywords: In situ gelling Chitosan-TGA/oxidizing agent systems Sol–gel transition Oxidizing agents Thiomers abstract The rheological behaviour of chitosan-thioglycolic acid conjugate (chitosan-TGA) in the presence of four oxidizing agents was investigated. Chitosan-TGA was synthesized via amide bond formation between the primary amino group of chitosan and the carboxylic acid group of thioglycolic acid. The sol–gel phase transition of the polymer was determined by rheological measurements. Moreover, cytotoxicity of the gel in combination with each oxidizing agent was evaluated utilizing LDH and MTT assay. The modified chitosan displayed 1053  44 mmol/g thiol groups. Results of rheological studies showed that 1% (m/v) chitosan-TGA without any oxidizing agents became gel within 40 min. In contrast, when the oxidizing agents hydrogen peroxide, sodium periodate, ammonium persulfate and sodium hypochlorite were added, respectively, gelation took place within a few minutes. Within 20 min, hydrogen peroxide having been added in a final concentration of 25.2 nmol/L increased dynamic viscosity of 1% (m/v) chitosan-TGA up to 16,500-fold. This can be explained by the formation of inter- and/or intramolecular disulfide bonds which were indirectly verified via the decrease in thiol groups. Additionally, evidence of an increase in cross-linking of thiolated chitosan as a function of time was provided by frequency sweep measure- ments. Furthermore, viability of Caco-2 cells having been incubated with chitosan-TGA/oxidizing agent systems assessed by MTT assay was 70–85% and the percentage of LDH release was only in case of the chitosan-TGA/ammonium persulfate system significantly (p < 0.05) raising compared to the negative control. According to these results, chitosan-TGA/oxidizing agent combinations might be a promising novel in situ gelling system for various pharmaceutical applications such as a controlled drug release carrier or for tissue engineering. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction In the 1990s the concept of in situ gelling polymers was pio- neered in order to prolong the residence time of liquidly applied formulations such as eye drops [1], as scaffold material in tissue engineering [2,3] and as coating material in wound healing [4]. So far, however, in situ gelling polymers have in many cases not reached their full potential due to an insufficient increase in viscosity and/or a too long time period needed for sol–gel transition. Among in situ gelling polymers, thiolated polymers or so-called thiomers and in particular thiolated chitosans turned out to be a promising alternative to well-established polymers such as poloxamers or certain polysaccharides [5,6]. They are obtained by the covalent attachment of thiol groups to various polymer back- bones and possess in situ gel forming properties owing to the formation of inter- and/or intramolecular disulfide bonds at pH- levels above 5 [7]. The thiolation of chitosan with iminothiolane HCl, for instance, yielded chitosan-4-thiobutylamidine (chitosan- TBA) that showed an even more than 3200-fold increase in dynamic viscosity within six hours [8]. Chitosan-thioglycolic acid (chitosan-TGA) and chitosan-thioethylamidine (chitosan-TEA) representing two other in situ gelling thiolated chitosan derivatives exhibited a 168- and 6190-fold increase in elastic modulus within 6 h, respectively [9,10]. However, the increase in viscosity is for various applications still not high enough and the phase transition time of these three thiomers is considerably long. It was therefore the aim of this study to generate a new system of thiolated chitosan to further increase the dynamic viscosity and to essentially reduce the reverse volume phase time. As compara- tively high degrees of thiolation can be achieved by a simple one- step coupling reaction [11], chitosan-TGA was chosen as test candidate in this study. Oxidizing agents are likely promising compounds to enhance the dynamic viscosity and consequently to reduce the time of phase transition. In this study, hydrogen peroxide (H 2 O 2 ), sodium periodate (NaIO 4 ), ammonium persulfate * Corresponding author. Tel.: þ43 512 507 5371; fax: þ43 512 507 2933. E-mail address: andreas.bernkop@uibk.ac.at (A. Bernkop-Schnu ¨ rch). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.07.060 Biomaterials 30 (2009) 6151–6157