Chitosan–thioglycolic acid conjugate: An alternative carrier for oral nonviral gene delivery? Ronny Martien, 1 Brigitta Loretz, 1 Marlene Thaler, 2 Sayeh Majzoob, 3 Andreas Bernkop-Schnu¨ rch 1 1 Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University of Innsbruck, Innrain 52, 6020 Innsbruck, Austria 2 Institute of Zoology, Leopold-Franzens-University of Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria 3 Department of Pharmaceutics, Tehran University of Medical Sciences, Tehran 14174, Iran Received 5 May 2006; revised 21 September 2006; accepted 25 September 2006 Published online 30 January 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31135 Abstract: Regarding safety concerns, nonviral gene deliv- ery vehicles that have the required efficiency and safety for use in human gene therapy are being widely investi- gated. The aim of this study was to synthesize and evalu- ate a thiolated chitosan to improve the efficacy of oral gene delivery systems. Thiolated chitosan was synthesized by introducing thioglycolic acid (TGA) to chitosan via am- ide bond formation mediated by a carbodiimide. Based on this conjugate, nanoparticles with pDNA were generated at pH 4.0 and 5.0. Cytotoxicity of the thiolated chitosan/ pDNA nanoparticles on Caco-2 cells was evaluated. The diameter of thiolated chitosan/pDNA nanoparticles was in the range of 100–200 nm. The zeta potential was deter- mined to be 5–6 mV. Due to stability toward nucleases, the transfection rate of thiolated chitosan/pDNA nanopar- ticles was fivefold higher than that of unmodified chito- san/pDNA nanoparticles. Lactate dehydrogenase tests for thiolated chitosan/pDNA (pH 4.0 and 5.0) showed that (3.79 6 0.23)% and (2.9 6 0.13)% cell damage. According to these results, thiolated chitosan represents promising excipients for preparation DNA nanoparticles in nonviral gene delivery system. Ó 2007 Wiley Periodicals, Inc. J Biomed Mater Res 82A: 1–9, 2007 Key words: nonviral vector; thiolated chitosan; thioglycolic aid (TGA); gene delivery; transfection rate INTRODUCTION Safe and efficient delivery of DNA into cells is still a dominant task in today’s biotechnological research. Currently, gene delivery to cells can be accomplished by using viral and nonviral vectors. 1 Statistics cover- ing all clinical trials worldwide in gene delivery reveal that viral vectors are still the clear number one, being used in more than 70% of all protocols. 2 Regarding safety concerns, nonviral gene delivery vehicles that have the required efficiency and safety for use in human gene therapy are being widely investigated as possible alternatives. 3 Intestinal epi- thelium is considered to be an attractive site for oral delivery of therapeutic genes. One of the most com- pelling factor is its ease of access via luminal route, which would allow direct in vivo gene transfer by oral administration. 4 The intestine could be an alter- native target for the treatment of many metabolic and nutritional defects 5 and a target for gene delivery in vaccination strategies. 6 The most promising nonviral gene delivery sys- tems thus far comprise ionic complexes formed between DNA and polycationic polymers, 7 such as chitosan. Chitosan is relatively low toxic biodegrad- able polycationic polymer 8 and provides a strong electrostatic interaction with the negative charge of DNA to form nanoparticles. 9 These properties make chitosan a good candidate for nonviral gene deliv- ery. 10 Thiolated chitosan as a new derivative of chitosan has been synthesized. 11 These thiolated poly- mers or so-called thiomers are hydrophilic polymers such as chitosan derivatized with thiol groups on their side chains. Based on thiol exchange reactions and/or a simple oxidation process, disulfide bonds are formed of inter- and intramolecular disulfide bonds within the thiomer itself; matrix nanoparticles and particulate delivery systems show strong cohe- sive properties, resulting in comparatively higher stability. Particles will be disintegrated inside the cell (reduction condition) and release their drug or DNA. 11 Like chitosan, thiomer is relatively low toxic. 12 Thiolated chitosan displays enzyme inhibi- Correspondence to: A. Bernkop-Schnu¨ rch; e-mail: andreas. bernkop@uibk.ac.at Contract grant sponsor: Austrian Federal Ministry for Education, Science, and Culture ' 2007 Wiley Periodicals, Inc.