Influence of Chitosan Structure on the Formation and Stability of


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Biomacromolecules 2005, 6, 3357-3366

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Influence of Chitosan Structure on the Formation and Stability of DNA-Chitosan Polyelectrolyte Complexes Sabina P. Strand,*,† Signe Danielsen,‡,§ Bjørn E. Christensen,† and Kjell M. Vårum† NOBIPOL, Department of Biotechnology, and Biophysics and Medical Technology, Department of Physics, The Norwegian University of Science and Technology, NTNU, NO-7491 Trondheim, Norway Received June 3, 2005; Revised Manuscript Received August 10, 2005

The interactions between DNA and chitosans varying in fractional content of acetylated units (FA), degree of polymerization (DP), and degree of ionization were investigated by several techniques, including an ethidium bromide (EtBr) fluorescence assay, gel retardation, atomic force microscopy, and dynamic and electrophoretic light scattering. The charge density of the chitosan and the number of charges per chain were found to be the dominating factors for the structure and stability of DNA-chitosan complexes. All high molecular weight chitosans condensed DNA into physically stable polyplexes; however, the properties of the complexes were strongly dependent on FA, and thereby the charge density of chitosan. By employing fully charged oligomers of constant charge density, it was shown that the complexation of DNA and stability of the polyplexes is governed by the number of cationic residues per chain. A minimum of 6-9 positive charges appeared necessary to provide interaction strength comparable to that of polycations. In contrast, further increase in the number of charges above 9 did not increase the apparent binding affinity as judged from the EtBr displacement assay. The chitosan oligomers exhibited a pH-dependent interaction with DNA, reflecting the number of ionized amino groups. The complexation of DNA and the stability of oligomerbased polyplexes became reduced above pH 7.4. Such pH-dependent dissociation of polyplexes around the physiological pH is highly relevant in gene delivery applications and might be one of the reasons for the high transfection activity of oligomer-based polyplexes observed. Introduction Self-assembly of polycations (PC) and polyanions (PA) yielding polyelectrolyte complexes (PEC) through a cooperative system of electrostatic interactions is widely exploited both in nature and in technological applications. Among many systems studied, the condensation of DNA by PC has received considerable attention due to its application in nonviral gene delivery.1-3 Although the collapse of extended DNA chains into compact particles seems striking, it is just one example of PA-PC interactions leading to a transition from the extended to the compacted state, driven by the overall increase in the entropy of the system due to the released counterions. Regardless of the nature of the condensing agent, the resulting compact forms adopt a limited range of morphologies, generally a mixture of toroids, rods, and globules, although in different relative amounts.4-7 The prominent toroidal morphology of condensed DNA particles is not restricted to DNA but has also been observed for other polyanions with sufficiently large persistence length, such as xanthan.8 The development of safe and effective vectors for delivery of therapeutic genes remains a central challenge in the field of gene therapy. The safety concerns associated with viral * Corresponding author. E-mail: [email protected] † NOBIPOL, Department of Biotechnology. ‡ Biophysics and Medical Technology, Department of Physics. § Present address: Department of Radiation Oncology, University Hospital of St. Olav, 7006 Trondheim, Norway.

vectors have triggered increasing interest toward vectors based on cationic polymers.9-12 Despite the similar principles of formation and the similar morphology of complexes formed by different PC, the size, stability, and colloidal properties of the complexes strongly depend on the PC used and, likewise, their performance as nonviral gene delivery systems.3,11,13,14 In addition to the chemical structure of the repeating unit of the PC, other factors such as molecular weight, polymer/DNA mixing ratio, and details of preparation have been shown to impact transfection efficiency.14-17 Chitosans, a family of linear binary polysaccharides consisting of (1 f 4)-β-linked 2-acetamido-2-deoxy-Dglucose (GlcNAc) and its de-N-acetylated analogue (GlcN), have emerged as a biocompatible alternative to synthetic polycations, suitable for in vivo gene delivery to mucosal tissues.17-23 Besides biodegradability24 and low toxicity,17,25,26 chitosan also offers an advantage inherent to synthetic PC; its properties may be tuned through the fraction of acetylated units (FA), degree of polymerization (DP), and its polydispersity, as well as the pH-dependent degree of ionization. Tailoring of chitosans with respect to FA, DP, and polydispersity provides a tool for controlling the functional properties of chitosans.27 In the field of gene delivery, the FA and DP of chitosan are reported to affect the transfection efficiency of DNAchitosan polyplexes.17-19,28 Recently, the low molecular weight chitosan-based polyplexes appeared especially promising, showing a 120-260-fold higher gene expression in

10.1021/bm0503726 CCC: $30.25 © 2005 American Chemical Society Published on Web 09/10/2005

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Biomacromolecules, Vol. 6, No. 6, 2005

Strand et al.

Table 1. Chitosan and Chitosan Oligomers Used in This Study fraction of acetylated units (FA)

intrinsic viscosity η (mL/g)

degree of polymerization (DP)

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