Non-viral gemini surfactant-phospholipid nanoparticles for topical gene delivery to the retina

Glaucoma is a group of optic nerve degenerative diseases, which leads to gradual and permanent vision loss. Recent developments in the field of gene therapy have proposed increasingly promising treatments for glaucoma, in the form of delivery of neuro-protective or neuro-regenerative genes to the retina. Despite these developments, there are concerns related to the biocompatibility and invasiveness of common gene delivery systems, since they are commonly mediated by viral gene carriers and invasive administration methods. Non-viral gene delivery systems offer a safe and increasingly efficient alternative to deliver therapeutic genes to the retina. An example of these systems is gemini-phospholipid nanoparticles (GL-NPs), which have been successfully used to deliver genes in similarly challenging anatomical settings, such as the skin. The objective of this thesis is to demonstrate the potential of GL-NPs, as candidate gene delivery vehicles for topically administered genes, targeted to the retina. The dicationic gemini surfactant, 12-7NH-12 was used, along with the helper lipids, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), to prepare various types of GL-NPs, and assess their transfection efficiency in the rat retinal ganglion cell line (RGC-5). The transfection efficiency was evaluated using flow cytometry, as a function of several physical and chemical parameters of GL-NPs. These include a range of charge ratios (5:1 to 15:1 ρ±), helper lipid composition (several DOPE: DPPC ratios), order of assembly (plasmid-gemini + lipid versus gemini-lipid + plasmid), and manufacturing method of helper lipid vesicles (thin film versus high pressure homogenization method). Size and zeta (ζ) potential characterization of GL-NPs was carried out in parallel, using dynamic light scattering, to relate the physical parameters of GL-NPs to their respective transfection efficiency. A comprehensive toxicological evaluation was undertaken to assess the extent of GL-NP’s toxicity in RGC-5 cells, using the resazurin-based PrestoblueTM cell toxicity assay. Optimized GL-NPs were used to induce expression of the brain derived neurotrophic factor (BDNF) in RGC-5 cells, and were assessed in terms of their capacity to induce neurite outgrowth. Quantification of neurite outgrowth was carried out by measuring average neurite length in RGC-5 cells, by confocal microscopic imaging of immunostained neurites. Furthermore, confocal microscopic studies were carried out to assess the extent of GL-NP’s corneal permeation in a 3-D human corneal epithelial (HCE) model. A parallel toxicological evaluation was completed to ensure GL-NP’s biocompatibility with the corneal epithelial cells. Finally, GL-NP biodistribution pattern and gene transfer capacity was assessed in a mouse model, following topical and intravitreal administration. The transfection efficiency in RGC-5 cells, which ranged between 2.1 ± 0.3% and 14.5 ± 1.4%, was highly dependent on GL-NP’s charge ratio, helper lipid composition, order of assembly, and manufacturing technique. GL-NPs at 10:1 ρ± charge ratio, assembled with homogenized DOPE (25%)-DPPC (75%) helper lipid vesicles, in the plasmid-gemini + lipid order, mediated the highest transfection efficiency in RGC-5 cells. These GL-NPs had a size of 222.8 ± 4.2 nm and a ζ potential of +33.5±2.9 mV. Optimized GL-NPs were highly biocompatible with both RGC-5 and HCE model cells, with viability values ranging between 94.8 ± 6 % to 100 ± 3.4 %. Assessment of corneal permeation showed that GL-NPs were able to bind to the corneal epithelial surface and achieve a moderate permeation depth (35-40 μm), following topical application in the HCE model. Intravitreal injection of the non-viral GL-NPs in mice has successfully led to their localization within the nerve fiber layer (NFL) of the retina. Finally, GL-NPs were non-invasively delivered to several anterior chamber tissues, including the limbus, the iris and conjunctiva, following topical administration. GL-NPs offer several advantageous features critical to topical and intravitreal ocular administration of gene carriers, including in vitro corneal binding and effective biodistribution following in vivo topical and intravitreal administration, high biocompatibility, and a highly tunable transfection efficiency. The current data presents 12-7NH-12 GL-NPs as a promising candidate for ocular gene therapy applications.1 yea