RESUMO
The present study aimed to evaluate the incorporation of protamine into niosome/DNA vectors to analyze the potential application of this novel ternary formulation to deliver the pCMS-EGFP plasmid into the rat retina. Binary vectors based on niosome/DNA and ternary vectors based on protamine/DNA/niosomes were prepared and physicochemically characterized. In vitro experiments were performed in ARPE-19 cells. At 1:1:5 protamine/DNA/niosome mass ratio, the resulted ternary vectors had 150 nm size, positive charge, spherical morphology, and condensed, released, and protected the DNA against enzymatic digestion. The presence of protamine in the ternary vectors improved transfection efficiency, cell viability, and DNA condensation. After ocular administration, the EGFP expression was detected in different cell layers of the retina depending on the administration route without any sign of toxicity associated with the formulations. While subretinal administration transfected mainly photoreceptors and retinal pigment epithelial cells at the site of injection, intravitreal administration produced a more uniform distribution of the protein expression through the inner layers of the retina. The protein expression in the retina persisted for at least one month after both administrations. Our study highlights the flattering properties of protamine/DNA/niosome ternary vectors for efficient and safe gene delivery to the rat retina.
Assuntos
DNA/metabolismo , Técnicas de Transferência de Genes , Lipossomos/uso terapêutico , Protaminas/metabolismo , Retina/metabolismo , Animais , Linhagem Celular , DNA/química , Técnica Indireta de Fluorescência para Anticorpo , Técnicas In Vitro , Lipossomos/farmacologia , Masculino , Microscopia de Fluorescência , Plasmídeos/metabolismo , Protaminas/química , Ratos , Ratos Sprague-Dawley , Retina/citologia , Tomografia de Coerência Óptica , Transfecção/métodosRESUMO
We designed niosomes based on three lipids that differed only in the polar-head group to analyze their influence on the transfection efficiency. These lipids were characterized by small-angle X-ray scattering before being incorporated into the niosomes which were characterized in terms of pKa, size, zeta potential, morphology and physical stability. Nioplexes were obtained upon the addition of a plasmid. Different ratios (w/w) were selected to analyze the influence of this parameter on size, charge and the ability to condense, release and protect the DNA. In vitro transfection experiments were performed in HEK-293, ARPE-19 and MSC-D1 cells. Our results show that the chemical composition of the cationic head-group clearly affects the physicochemical parameters of the niosomes and especially the transfection efficiency. Only niosomes based on cationic lipids with a dimethyl amino head group (lipid 3) showed a transfection capacity when compared with their counterparts amino (lipid 1) and tripeptide head-groups (lipid 2). Regarding cell viability, we clearly observed that nioplexes based on the cationic lipid 3 had a more deleterious effect than their counterparts, especially in ARPE-19 cells at 20/1 and 30/1 ratios. Similar studies could be extended to other series of cationic lipids in order to progress in the research on safe and efficient non-viral vectors for gene delivery purposes.
Assuntos
Lipídeos/química , Transfecção , Sobrevivência Celular/efeitos dos fármacos , DNA/administração & dosagem , DNA/química , DNA/genética , Estabilidade de Medicamentos , Células HEK293 , Humanos , Lipídeos/síntese química , Lipídeos/toxicidade , Lipossomos , Tamanho da PartículaAssuntos
Doenças dos Bovinos/genética , Suscetibilidade a Doenças/veterinária , Predisposição Genética para Doença , Mycobacterium avium subsp. paratuberculosis/fisiologia , Paratuberculose/genética , Polimorfismo de Nucleotídeo Único , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Suscetibilidade a Doenças/microbiologia , Feminino , Estudos de Associação Genética/veterinária , Haplótipos , Paratuberculose/microbiologia , Reação em Cadeia da Polimerase/veterináriaRESUMO
The development of novel non-viral delivery vehicles is essential in the search of more efficient strategies for retina and brain diseases. Herein, optimized niosome formulations prepared by oil-in water (o/w) and film-hydration techniques were characterized in terms of size, PDI, zeta potential, morphology and stability. Three ionizable glycerol-based cationic lipids containing a primary amine group (lipid 1), a triglycine group (lipid 2) and a dimethylamino ethyl pendent group (lipid 3) as polar head-groups were part of such niosomes. Upon the addition of pCMS-EGFP plasmid, nioplexes were obtained at different cationic lipid/DNA ratios (w/w). The resultant nioplexes were further physicochemically characterized and evaluated to condense, release and protect the DNA against enzymatic digestion. In vitro experiments were performed to evaluate transfection efficiency and cell viability in HEK-293, ARPE-19 and PECC cells. Interestingly, niosome formulations based on lipid 3 showed better transfection efficiencies in ARPE-19 and PECC cells than the rest of cationic lipids showed in this study. In vivo experiments in rat retina after intravitreal and subretinal injections together with in rat brain after cerebral cortex administration showed promising transfection efficiencies when niosome formulations based on lipid 3 were used. These results provide new insights for the development of non-viral vectors based on cationic lipids and their applications for efficient delivery of genetic material to the retina and brain.
Assuntos
Córtex Cerebral/metabolismo , Vetores Genéticos/química , Lipossomos/química , Propanolaminas/farmacologia , Retina/metabolismo , Transfecção/métodos , Ureia/análogos & derivados , Animais , Cátions , Linhagem Celular , Células Cultivadas , DNA/administração & dosagem , DNA/genética , Estabilidade de Medicamentos , Genes Reporter , Vetores Genéticos/administração & dosagem , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HEK293 , Hipocampo/citologia , Hipocampo/embriologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Injeções Intraoculares , Injeções Intravítreas , Lipossomos/administração & dosagem , Masculino , Neurônios/citologia , Propanolaminas/administração & dosagem , Propanolaminas/síntese química , Ratos , Ratos Sprague-Dawley , Epitélio Pigmentado da Retina/citologia , Ureia/administração & dosagem , Ureia/síntese química , Ureia/farmacologiaRESUMO
Niosomes represent a recent promising approach for gene delivery purposes. We elaborated on a novel niosome formulation based on the 2,3-di(tetradecyloxy)propan-1-amine cationic lipid, combined with squalene and polysorbate 80 to evaluate the transfection efficiency in rat retinas. Niosomes prepared by the solvent emulsification-evaporation technique were mixed with the pCMSEGFP plasmid to form lipoplexes which were characterized in terms of morphology, size, surface charge, and DNA condensation, protection and release. In vitro studies were conducted to evaluate transfection efficiency, viability and internalization mechanism in HEK-293 and ARPE-19 cells. The efficacy of the most promising formulation was evaluated in rat eyes by monitoring the expression of the EGFP after intravitreal and subretinal injections. Lipoplexes at 15/1 ratio were 200nm in size, 25mV in zeta potential and exhibited spherical morphology. At this ratio, niosomes condensed and protected the DNA from enzymatic digestion. Lipoplexes successfully transfected HEK-293 and specially ARPE-19 cells, without affecting the viability. Whereas lipoplexes entered mainly retinal cells by clathrin-mediated endocytosis, HEK-293 cells showed a higher caveolae-dependent entry. After ocular administration, the expression of EGFP was detected in different cells of the retina depending on the administration route. This novel niosome formulation represents a promising approach to deliver genetic material into the retina to treat inherited retinal diseases.