DNA Gel Particles: Particle Preparation and Release Characteristics M. Carmen Mora ´n,* ,† M. Grac ¸ a Miguel, † and Bjo ¨rn Lindman †,‡ Chemistry Department, Coimbra UniVersity, 3004-535 Coimbra, Portugal, and Physical Chemistry 1, Lund UniVersity, P. O. Box 124, 22100 Lund, Sweden ReceiVed March 7, 2007 Aqueous mixtures of oppositely charged polyelectrolytes undergo associative phase separation, resulting in coacervation, gelation, or precipitation. This phenomenon has been exploited here to form DNA gel particles by interfacial diffusion. We report the formation of DNA gel particles by mixing solutions of DNA (either single-stranded (ssDNA) or double-stranded (dsDNA)) with solutions of cationic surfactant CTAB and solutions of the protein lysozyme. Swelling, surface morphology, and DNA release determinations indicate different interaction of ssDNA and dsDNA with both the surfactant and the protein. By using CTAB and lysozyme as the base material, the formation of a DNA reservoir hydrogel, without adding any kind of cross-linker or organic solvent, was demostrated. Introduction A novel nonviral vector for gene therapy is recognized as successful if it is biocompatible, capable of interacting with DNA, forms sufficiently small particles that can be formulated reproducibly, endocytosed, able to protect the complexed DNA from degradation during transport, and capable of delivering DNA to the target tisssue in sufficient quantity. 1-3 Regarding this goal, some authors have developed a way to deliver DNA to the target cells by encapsulating it into a controlled-release system. For this purpose, poly(lactic-co-glycolic acid) (PLGA) microspheres have been used. 4-6 However, the problems encountered in reaching this goal were related not only to the microencapsulation technologies but also to the intrinsic nature of polyesters. Indeed, the PLGA microencapsulation technologies imply the use of organic solvents and high-energy sources, thus leading to a significant degradation of the encapsulated macro- molecule during the course of the PLGA hydrolysis. Following the identification of these problems, a number of strategies aimed at either modifying the encapsulation techniques or using new encapsulation materials were explored. Chitosan microspheres containing plasmid DNA were prepared by the complex coacervation method in the absence 7 or presence of a DNA condensing agent. 8 Interestingly, interactions between oppositely charged sur- factants and polyelectrolytes in aqueous solutions can lead to associative phase separation, where the concentrated phase assumes the form of a viscous liquid, gel, or precipitate. This behavior has been exploited to form gel particles, which have been prepared by the dropwise addition of a cellulose-based polycation solution (chitosan, N,N,N-trimethylammonium- derivatized hydroxyethyl cellulose (Amerchol JR-400)) 9-12 to anionic (sodium dodecyl sulfate (SDS), sodium perfluoro- octanoate (FC7)) and catanionic (cetyltrimethylammonium bromide (CTAB)/sodium perfluorooctanoate (FC7)) 13 surfactant solutions. The goal of this study is to investigate the formation of DNA gel particles at water/water emulsion-type interfaces by mixing DNA (either single- (ssDNA) or double-stranded (dsDNA)) with the cationic surfactant cetyltrimetrylammonium bromide (CTAB) or the protein lysozyme. Particles were characterized for swelling, dissolution behavior, surface morphology, and DNA release. The originality of this work consists of forming DNA reservoir hydrogels without adding any kind of cross-linker or organic solvent. Methods Materials. Deoxyribonucleic acid (DNA) from salmon testes (in the salt form, 1000 base pairs) was purchased from Sigma and used as received. The DNA concentrations were determined spectro- photometrically considering that for an absorbance of 1 at 260 nm a solution of dsDNA has a concentration of 50 μg/mL and a solution of ssDNA has a concentration of 40 μg/mL. 14 All DNA concentrations are presented in molarity per phosphate group (i.e., molarity per negative charge). The ratios of absorbance at 260 and 280 nm of the stock solutions were found to be between 1.8 and 1.9, which suggested the absence of proteins. 15 Cetyltrimethylammonium bromide (CTAB), obtained from Sigma, was recrystallized with acetone and ethanol. Lysozyme from chicken egg white (molecular mass 14.3 kDa) was purchased from Sigma and used as received. All experiments were performed using Millipore Milli-Q deionized water (18.2 MΩ cm resistivity). 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