Hyaluronic acid coating of gold nanoparticles for intraocular drug delivery: Evaluation of the surface properties and effect on their distribution.

Due to the unique anatomical structure of the eye, ocular drug delivery is a promising delivery route for the treatment of several ocular diseases, such as the ocular neovascularization that contributes to diabetic retinopathy. This disease is triggered by inflammation, retinal ischemia, and/or deposits of advanced-glycation end-products (AGEs), as well as increased levels of vascular endothelial growth factor (VEGF), interleukins, or reactive oxygen species (ROS). Gold has unique antioxidant and antiangiogenic properties and can inhibit angiogenic molecules. Furthermore, gold nanoparticles (GNPs) are not only biocompatible, they are easy to synthesize, they absorb and scatter visible light, and they can be made with precise control over size and shape. GNPs are an excellent candidate for ocular drug delivery because they can be conjugated to an extraordinarily diverse array of different biomolecules, and surface functionalization can improve the mobility of GNPs across the physiological barriers of the eye, such as the vitreous humour or the inner limiting membrane. For this purpose, we employed low molecular weight hyaluronan (HA) to increase the mobility of the nanoparticles as well as target them to HA receptors that are expressed in different cells of the eye. In this study, the combination of gold and HA enhanced the stability of the whole carrier and promoted their distribution across ocular tissues and barriers to reach the retina. Moreover, analysis in vitro, ex vivo, and in ovo revealed the protective and antiangiogenic effect of GNPs as inhibitors of AGEs-mediated- retinal pigment epithelial cell death and neovascularization. We demonstrated that conjugation with HA enhances GNP stability and distribution due to a specific CD44 receptor interaction. The capacity of HA-GNPs to distribute through the vitreous humour and their avidity for the deeper retinal layers ex vivo, suggest that HA-GNPs are a promising delivery system for the treatment of ocular neovascularization and related disorders.

Structural recovery of the retina in a retinoschisin-deficient mouse after gene replacement therapy by solid lipid nanoparticles.

X-linked juvenile retinoschisis (XLRS) is a retinal degenerative disorder caused by mutations in the RS1 gene encoding a protein termed retinoschisin. The disease is an excellent candidate for gene replacement therapy as the majority of mutations have been shown to lead to a complete deficiency of the secreted protein in the retinal structures. In this work, we have studied the ability of non-viral vectors based on solid lipid nanoparticles (SLN) to induce the expression of retinoschisin in photoreceptors (PR) after intravitreal administration to Rs1h-deficient mice. We designed two vectors prepared with SLN, protamine, and dextran (DX) or hyaluronic acid (HA), bearing a plasmid containing the human RS1 gene under the control of the murin opsin promoter (mOPS). In vitro, the nanocarriers were able to induce the expression of retinoschisin in a PR cell line. After injection into the murine vitreous, the formulation prepared with HA induced a higher transfection level in PR than the formulation prepared with DX. Moreover, the level of retinoschisin in the inner nuclear layer (INL), where bipolar cells are located, was also higher. Two weeks after vitreal administration into Rs1h-deficient mice, both formulations showed significant improvement of the retinal structure by inducing a decrease of cavities and PR loss, and an increase of retinal and outer nuclear layer (ONL) thickness. HA-SLN resulted in a significant higher increase in the thickness of both retina and ONL, which can be explained by the higher transfection level of PR. In conclusion, we have shown the structural improvement of the retina of Rs1h-deficient mice with PR specific expression of the RS1 gene driven by the specific promoter mOPS, after successful delivery via SLN-based non-viral vectors.

Solid lipid nanoparticle-based vectors intended for the treatment of X-linked juvenile retinoschisis by gene therapy: In vivo approaches in Rs1h-deficient mouse model.

X-linked juvenile retinoschisis (XLRS), which results from mutations in the gene RS1 that encodes the protein retinoschisin, is a retinal degenerative disease affecting between 1/5000 and 1/25,000 people worldwide. Currently, there is no cure for this disease and the treatment is based on the application of low-vision aids. The aim of the present work was the in vitro and in vivo evaluation of two different non-viral vectors based on solid lipid nanoparticles (SLNs), protamine and two anionic polysaccharides, hyaluronic acid (HA) or dextran (DX), for the treatment of XLRS. First, the vectors containing a plasmid which encodes both the reporter green fluorescent protein (GFP) and the therapeutic protein retinoschisin, under the control of CMV promoters, were characterized in vitro. Then, the vectors were subretinally or intravitreally administrated to C57BL/6 wild type mice. One week later, GFP was detected in all treated mice and in all retinal layers except in the Outer Nuclear Layer (ONL) and the Inner Nuclear Layer (INL), regardless of the administration route and the vector employed. Finally, two weeks after subretinal or intravitreal injection to Rs1h-deficient mice, GFP and retinoschisin expression was detected in all retinal layers, except in the ONL, which was maintained for at least two months after subretinal administration. The structural analysis of the treated Rs1h-deficient eyes showed a partial recovery of the retina related to the production of retinoschisin. This work shows for the first time a successful RS1 gene transfer to Rs1h-deficient animals using non-viral nanocarriers, with promising results that point to non-viral gene therapy as a feasible future therapeutic tool for retinal disorders.

Solid Lipid Nanoparticles as Non-Viral Vectors for Gene Transfection in a Cell Model of Fabry Disease.

Here, we demonstrate the ability of solid lipid nanoparticle-based non-viral vectors to increase the α-galactosidase A levels of the IMFE1 cell line, an in vitro model for target cells in Fabry disease. For this purpose, vectors containing the pR-M10-αGal A plasmid, which encodes the α-galactosidase A enzyme, were prepared; the in vitro transfection efficacy was studied in IMFE1 cells, and the results were confirmed by RT-PCR. The cellular uptake of the vectors, intracellular disposition of the plasmid, and probable endocytosis pathways of the nanoparticles were also analyzed. The vectors used for the studies carried protamine (P-DNA-SLN), dextran and protamine (D-P-DNA-SLN), or hyaluronic acid of two different molecular weights and protamine (HA150-P-DNA-SLN or HA500-P-DNA-SLN). The new formulations, which presented a particle size in the range of nanometers (from 218 nm to 348 nm) and a positive superficial charge, were able to increase α-galactosidase A activity up to 4-fold in comparison to non treated IMFE1 cells. The most efficient vectors were those that included HA, and no differences due to changes in the molecular weight of HA were detected. The observed lack of colocalization with each of the four different Nile Red-labeled vectors and transferrin or cholera toxin appears to indicate that clathrin- and caveolae-independent pathways may be involved in their cellular uptake. Additionally, colocalization with LysoTracker indicated that the formulations were exposed to lysosomal activity, which may be responsible for the release of the plasmid from the vector. In conclusion, we reveal the potential of SLN-based vectors to efficiently transfect an immortalized Fabry patient cell line.