Original Article
Preclinical development and ocular biodistribution of gemini-DNA nanoparticles after intravitreal and topical administration: Towards non-invasive glaucoma gene therapy

https://doi.org/10.1016/j.nano.2014.05.010Get rights and content

Abstract

Gene therapy could offer improvement in the treatment of glaucoma compared to the current standard of lowering intraocular pressure. We have developed and characterized non-viral gemini surfactant-phospholipid nanoparticles (GL-NPs) for intravitreal and topical administration. Optimized GL-NPs (size range 150-180 nm) were biocompatible with rat retinal ganglion (RGC-5) cells with > 95% viability by PrestoBlue™ assay. GL-NPs carrying Cy5-labeled plasmid DNA demonstrated distinct trafficking behavior and biodisposition within the eye in vivo after intravitreal or topical application with respect to pathways of movement and physicochemical stability. After intravitreal injection in mice, GL-NPs localized within the nerve fiber layer of the retina, whereas after topical application, GL-NPs were located in several anterior chamber tissues, including the limbus, iris and conjunctiva. GL-NPs were thermodynamically stable in the vitreous and tear fluid and were trafficked as single, non-aggregated particles after both types of administration.

From the Clinical Editor

In this paper, the development and characterization of non-viral gemini surfactant-phospholipid nanoparticles is reported with the goal of establishing a gene delivery system that addresses glaucoma in a non-invasive fashion. The authors found that after topical application, the concentration of these nanoparticles was higher in anterior chamber-related components of the eye, whereas intra-vitreal administration resulted in accumulation in the retinal nerve fibre layer.

Graphical Abstract

Non-viral trifunctional gemini surfactant-phospholipid nanoparticles carrying Cy5-labeled plasmid DNA demonstrated excellent thermodynamic stability in the vitreous and tear fluid and distinct trafficking behavior and biodisposition within the eye in vivo after intravitreal or topical application with respect to pathways of movement and physicochemical stability. After intravitreal injection in mice, localized within the nerve fiber layer of the retina, whereas after topical application, GL-NPs were located in several anterior chamber tissues, including the limbus, iris and conjunctiva.

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Section snippets

Background

Glaucoma is the second leading cause of blindness with 60.5 million patients worldwide in 2010, a figure that is expected to increase to 79.6 million by 2020.1 Risk factors of glaucoma include intraocular pressure (IOP) beyond the normal range of 10-21 mmHg,2, 3 vascular disorders that lead to reduced perfusion of the optic nerve head (ONH) (see review by Yanagi et al4), and certain biomechanical features of the ONH and retina.5, 6 IOP management, which is currently the main treatment for

Gemini nanoparticle formulation and optimization

Gemini NPs based on the pH-sensitive gemini surfactant 12-7NH-12 were prepared as described earlier.25 NPs (PG + L) were assembled in two steps: the plasmid (pCMV-tdTomato, Clontech Laboratories Inc., Mountain View, CA) and 12-7NH-12 gemini surfactant were mixed at room temperature for 15 min to form plasmid–gemini (PG) complexes at different cationic:anionic (ρ ±) charge ratios. Helper lipid vesicles (1 mM DOPE [Avanti Polar Lipids, Alabaster, AL] vesicles, prepared using the thin-film method25)

Physicochemical characterization and transfection efficiency of NPs

The two-step NP assembly process is illustrated in Figure 1, A and the parameters optimized during the formulation development process in Figure 1, B. Physicochemical parameters deemed relevant for intraocular performance such as surface charge, size, morphology and NP assembly technique are shown in Table 1. Particle size of PGLDOPE NPs ranged from 158.7 ± 4.7 nm to 220.1 ± 7.0 nm with a surface charge (ζ potential) of >+30 mV for all charge ratio conditions. The effect of charge ratio on

Discussion

The development of non-viral gene delivery systems for ophthalmic administration remains challenging due to several factors. The anatomy and physiology of the eye, limit particles and molecules to pass to the back of the eye (see review by Alqawlaq et al28). The tear film covers corneal and conjuctival layers, and limits the bioavailability of applied vectors due to the high tear turnover rate as well as the lacrimal and nasolacrimal drainage. Tissue barriers, namely the cornea, conjuctiva,

Acknowledgments

The research in this paper was supported by operating and equipment grants from the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation and the Ontario Research Fund. The generous support of the Canada Research Chairs Program the Canada Foundation for Innovation and the Ontario Research Fund is also gratefully acknowledged (M. Foldvari). J Sivak is the TWH and TGH Foundation Glaucoma Research Chair.

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