Ultrapure chitosan oligomers as carriers for corneal gene transfer
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 spontaneously 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 nanoparticles 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. Currently, to the best of our knowledge there are no reports about systematic design of formulations based on NOVAFECT chitosans as carriers for gene delivery.
In order to assess the potential of NOVAFECT chitosans for gene therapy we designed chitosan-DNA nanoparticles based on different NOVAFECT chitosans and with various ratios of nitrogen atoms from the polycation to phosphate groups of the DNA backbone (N/P ratios). We characterized the nanoparticles for their size, zeta potential, and ability to complex DNA and transfect cells in culture. In addition, we compared the ability of various formulations of NOVAFECT-DNA nanoparticles to express the luciferase gene in the rat corneas following intrastromal injection.
We used corneal gene transfer because enhancement of gene delivery in this model is anticipated to increase the chances of success of corneal gene therapy, an approach that has potential in treating corneal diseases [21], [22], [23], which are a major cause of blindness worldwide [24]. The eye, a delicate organ, was previously shown to tolerate chitosan nanoparticles very well [25]. To further understand the performance characetristics of chitosan oligomers for corneal gene therapy we used fluorescence microscopy as a method to identify the type of corneal cells that express the transgene following intrastromal injection.
Section snippets
Materials
NOVAFECT chitosans were purchased from NovaMatrix/FMC (Sandvika, Norway). The NOVAFECT kit contained NOVAFECT O 15, molecular weight 5.7 kDa, degree of deacetylation 99%, acetate salt; NOVAFECT O 25, molecular weight 7.3 kDa, degree of deacetylation 99%, acetate salt; and NOVAFECT G 214, molecular weight 340 kDa, degree of deacetylation 92%, glutamate salt. NOVAFECT chitosans are ultrapure and have endotoxin levels ≤ 0.05 EU/mg. gWiz-luc and gWiz-GFP plasmids were purchased from Genlantis (San
Measurements of size and zeta potential of chitosan-DNA nanoparticles
The average size of chitosan-DNA nanoparticles is shown in Fig. 1A. It is noted that the type of chitosan used affects the size and the correlation between N/P ratio and size of the nanoparticles: Sizes of nanoparticles based on oligomeric chitosans (ranging from 74.4 ± 8.3 to 98.2 ± 4.4 nm) are generally smaller than sizes of nanoparticles based on polymeric chitosan (ranging from 112.6 ± 9.1 to 171.5 ± 15.1 nm). Sizes of nanoparticles based on chitosan oligomers are not substantially affected by N/P
Discussion
This study shows the potential of chitosan oligomers as carriers in corneal gene transfer. A formulation of chitosan-DNA nanoparticles, based on NOVAFECT O 15 with N/P ratio of 20 led to significantly greater transgene expression in comparison to administration of polyethylenimine-DNA nanoparticles (Fig. 4), the gold standard in non-viral gene therapy [27].
The method that we used for the preparation of chitosan-DNA nanoparticles, modified from Artursson et al. [16], led to the formation of
Conclusion
Here we introduce a simple method for the preparation of chitosan-DNA nanoparticles based on commercially available chitosan oligomers that are ultrapure, highly deacetylated and have a low molecular weight distribution. Under certain experimental conditions, a formulation of oligomeric chitosan-DNA nanoparticles with enhanced transfection efficiency was identified. This study lays the foundation for evaluating chitosan oligomers as carriers in corneal gene therapy, ocular gene therapy and
Acknowledgements
We declare no conflict of interests. We wish to thank Brian Zanotti from the Department of Microbiology and Immunology of Midwestern University College of Osteopathic Medicine for technical assistance with the preparation of the fluorescence micrographs. This work was supported by a Research Stimulation Grant of Midwestern University Chicago College of Pharmacy to Eytan A. Klausner, by a Research Grant from the Midwest Eye-Banks to Michael V. Volin, and by the Department of Pharmaceutical
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