Dextran and Protamine-Based Solid Lipid Nanoparticles as Potential Vectors for the Treatment of X-Linked Juvenile Retinoschisis

Abstract

The goal of the present study was to analyze the potential application of nonviral vectors based on solid lipid nanoparticles (SLN) for the treatment of ocular diseases by gene therapy, specifically X-linked juvenile retinoschisis (XLRS). Vectors were prepared with SLN, dextran, protamine, and a plasmid (pCMS-EGFP or pCEP4-RS1). Formulations were characterized and the in vitro transfection capacity as well as the cellular uptake and the intracellular trafficking were studied in ARPE-19 cells. Formulations were also tested in vivo in Wistar rat eyes, and the efficacy was studied by monitoring the expression of enhanced green fluorescent protein (EGFP) after intravitreal, subretinal, and topical administration. The presence of dextran and protamine in the SLN improved greatly the expression of retinoschisin and EGFP in ARPE-19 cells. The nuclear localization signals of protamine, its ability to protect the DNA, and a shift in the entry mechanism from caveola-mediated to clathrinmediated endocytosis promoted by the dextran, justify the increase in transfection. After ocular administration of the dextran-protamine-DNA-SLN complex to rat eyes, we detected the expression of EGFP in various types of cells depending on the administration route. Our vectors were also able to transfect corneal cells after topical application. We have demonstrated the potential usefulness of our nonviral vectors loaded with XLRS1 plasmid and provided evidence for their potential application for the management or treatment of degenerative retinal disorders as well as ocular surface diseases.

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“…It is also used in some eye drops as a lubricant, and in certain intravenous fluids to solubilize other factors, e.g. iron (=iron dextran such as Cosmofer) 14 .…”

Biomateriais para formulações de base nanotecnológica visando terapia gênica ocular

“…101 Nanopartículas lipídicas sólidas pré-formadas contendo DOTAP foram propostas para associar complexos de protamina e pDNA, 102 e protamina com sulfato de dextrano e pDNA. 103 O uso de protamina parece impactar algumas das principais características físico-químicas das partículas, como tamanho e potencial de superfície. Entretanto, a capacidade de transfecção do pDNA é de cerca de 30-44% em cé-lulas de retina (ARPE-19) em comparação com os 5-7% relativos às mesmas nanopartículas lipídicas sólidas sem protamina.…”

“…A grande maioria das células transfectadas pertence a gânglios da retina, sendo quase negligenciável a transfecção em células do epitélio da retina. 103 O uso de protamina parece alterar o mecanismo de internalização de nanopartículas lipídicas sólidas contendo o lipídio catiônico DOTAP em células ARPE-19, através de um mecanismo envolvendo principalmente caveolina/rafts de lipídios para internalização por clatrina. 102 Uma alta densidade de aminoácidos básicos como lisina ou arginina (como é o caso da protamina) favorece o transporte até o núcleo celular através do complexo de poros nuclear.…”

Physicochemical properties of polymers: An important system to overcome the cell barriers in gene transfection

“…The use of elements modulating these pathways improves transfection 78 . Chlorpromazine (an inhibitor of the clathrin pathway) could partially inhibit the internalization of PEI carrying a spliceshifting oligonucleotide, in contrast with methyl-β-cyclodextrin (CD; an inhibitor of lipid rafts or caveolae) which could prevent the internalization almost completely.…”