Ultrapure chitosan oligomers as carriers for corneal gene transfer Eytan A. Klausner a, * , Zhong Zhang a , Robert L. Chapman a , Richard F. Multack b, c , Michael V. Volin d a Department of Pharmaceutical Sciences, Midwestern University Chicago College of Pharmacy, Downers Grove, IL 60515, USA b Department of Surgery, Midwestern University Chicago College of Osteopathic Medicine, Downers Grove, IL 60515, USA c Department of Ophthalmology, Advocate Medical Group, 4001 Vollmer Road, Olympia Fields, IL 60461, USA d Department of Microbiology and Immunology, Midwestern University Chicago College of Osteopathic Medicine, Downers Grove, IL 60515, USA article info Article history: Received 28 August 2009 Accepted 12 October 2009 Available online 30 October 2009 Keywords: Ultrapure chitosan oligomers Chitosan-DNA nanoparticles Corneal gene therapy Non-viral gene transfer Cornea abstract NOVAFECT chitosans are ultrapure chitosan oligomers that were recently marketed as carriers for non- viral gene therapy. There are no reports on systematic design and improvement of formulations based on NOVAFECT chitosans for gene delivery. Therefore, we have designed and characterized chitosan-DNA nanoparticles based on NOVAFECT. We found that the size of oligomeric chitosan-DNA nanoparticles is small, 98.2  4.4 nm. Zeta potential measurements of oligomeric chitosan-DNA nanoparticles exhibited a strong positive charge 44.1  3.5 millivolt. In vitro transfection studies demonstrated the ability of oligomeric chitosan-DNA nanoparticles to effectively transfect COS-7 cells. In rat corneas, injection of a select formulation of oligomeric chitosan-DNA nanoparticles into the stroma showed that (a) luciferase gene expression was 5.4 times greater than following administration of polyethylenimine-DNA nano- particles; and (b) the cells that express the transgene, green fluorescent protein, were keratocytes (corneal fibroblasts). This study lays the foundation for evaluating oligomeric chitosan-DNA nano- particles as pharmaceuticals for corneal gene therapy, a promising approach for the treatment of acquired and inherited corneal diseases that otherwise lead to blindness. Oligomeric chitosan-DNA nanoparticles can also be evaluated for the treatment of ocular diseases outside of the cornea, and for various additional gene therapy applications. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Non-viral gene vectors are biomaterials, which are frequently based on cationic polymers [1] complexed with negatively charged DNA molecules [2,3]. The nature of the polymer used is important since it affects the delivery of the gene, which is still a major hurdle for successful gene therapy [4,5]. A commonly used polymer in non-viral gene transfer is chitosan [6,7], a cationic biopolymer derived from chitin by partial deacetylation [8,9] that can sponta- neously form nanoparticles with DNA [10]. A major advantage of chitosan as a gene carrier is its low toxicity [11,12]. In order to obtain enhanced gene transfer that can lead to successful gene therapy, it is important to use a type of chitosan for which efficiency was tested in terms of structural characteristics, such as molecular weight and degree of deacetylation [13]. At present, there is no consensus regarding the desired characteristics of chitosan to be used for gene therapy [14,15]. Additional studies that identify different types of chitosan for gene transfer are still needed. Artursson et al. designed ultrapure chitosan oligomers that are fully deacetylated and have a low molecular weight, e.g. about 1.3–9.5 kDa. The low molecular weight of the chitosans was proposed to overcome undesirable viscosity-enhancing properties of high molecular weight chitosans, and to form more easily dissociated complexes with DNA [16]. Indeed, strong complexation between high molecular weight chitosan and DNA was suggested to inhibit the release of DNA from the complex [17,18]. These chitosan oligomers were further processed to obtain highly defined oligomers with a narrow molecular weight distribution. In mouse lungs, chitosan-DNA nanoparticles based on such oligomeric chitosan showed comparable transfection efficiency [16] and improved safety [19] over polyethylenimine- DNA nanoparticles (the gold standard in non-viral gene therapy [20]); and improved transfection efficiency over DNA nano- particles based on polymeric chitosan [16]. NOVAFECT chitosans are commercially available chitosans based on the design reported by Arturrson et al. [16]. A NOVAFECT test kit contains 2 oligomeric chitosans, NOVAFECT O 15 and NOVAFECT O 25, and a polymeric chitosan, NOVAFECT G 214, used as a reference. * Corresponding author. Midwestern University, 555 31st Street, AH 373, Downers Grove, IL 60515 USA. Tel.: þ1 630 515 7104; fax: þ1 630 515 6958. E-mail address: eklausner@midwestern.edu (E.A. Klausner). 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.2009.10.031 Biomaterials 31 (2010) 1814–1820