RESUMEN
BACKGROUND: Nanoparticle interactions with cellular membranes and the kinetics of their transport and localization are important determinants of their functionality and their biological consequences. Understanding these phenomena is fundamental for the translation of such NPs from in vitro to in vivo systems for bioimaging and medical applications. Two CdSe/ZnS quantum dots (QD) with differing surface functionality (NH2 or COOH moieties) were used here for investigating the intracellular uptake and transport kinetics of these QDs. RESULTS: In water, the COOH- and NH2-QDs were negatively and positively charged, respectively, while in serum-containing medium the NH2-QDs were agglomerated, whereas the COOH-QDs remained dispersed. Though intracellular levels of NH2- and COOH-QDs were very similar after 24 h exposure, COOH-QDs appeared to be continuously internalised and transported by endosomes and lysosomes, while NH2-QDs mainly remained in the lysosomes. The results of (intra)cellular QD trafficking were correlated to their toxicity profiles investigating levels of reactive oxygen species (ROS), mitochondrial ROS, autophagy, changes to cellular morphology and alterations in genes involved in cellular stress, toxicity and cytoskeletal integrity. The continuous flux of COOH-QDs perhaps explains their higher toxicity compared to the NH2-QDs, mainly resulting in mitochondrial ROS and cytoskeletal remodelling which are phenomena that occur early during cellular exposure. CONCLUSIONS: Together, these data reveal that although cellular QD levels were similar after 24 h, differences in the nature and extent of their cellular trafficking resulted in differences in consequent gene alterations and toxicological effects.
Asunto(s)
Autofagia/efectos de los fármacos , Compuestos de Cadmio/toxicidad , Puntos Cuánticos/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Selenio/toxicidad , Sulfuros/toxicidad , Compuestos de Zinc/toxicidad , Compuestos de Cadmio/análisis , Compuestos de Cadmio/metabolismo , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Puntos Cuánticos/análisis , Puntos Cuánticos/metabolismo , Compuestos de Selenio/análisis , Compuestos de Selenio/metabolismo , Sulfuros/análisis , Sulfuros/metabolismo , Compuestos de Zinc/análisis , Compuestos de Zinc/metabolismoRESUMEN
The development of biotechnological pharmaceutics, like macro- and nanocarriers, can benefit greatly from studying their characteristics in situ using advanced fluorescence microscopy methods. While choosing the optimal labeling method for visualizing the carrier or its cargo is crucial, it seldom receives attention. The possibility that high labeling densities alter the intracellular processing of the molecule is considered, but how and at which point this interference happens is not yet studied. The aim of this study was to elucidate the effect of labeling density on the cellular trafficking of labeled pDNA. Due to the drastic effect on expression levels for higher labeling densities, we tried to determine at which steps in the intracellular processing labeled pDNA behaves different than its nonlabeled counterpart. Therefore, different labeling densities, up to the manufacturer's recommended density, were tested. It was found that the cellular uptake remains unaffected, while the affinity for lipids is increased, which affects dissociation from the lipid-based complex and may affect endosomal escape. Also, nuclear injections clearly demonstrated that transcription is affected. The information and methodology, included in this work, could be helpful in determining if the labeling method and density used yields biological relevant results for the intended research question.
Asunto(s)
Plásmidos/metabolismo , Transfección/métodos , Transporte Biológico/fisiología , Endosomas/metabolismo , Células HeLa , Humanos , Lípidos/química , Liposomas/química , Liposomas/metabolismo , Microscopía Fluorescente , Plásmidos/química , Plásmidos/genética , Reacción en Cadena de la PolimerasaRESUMEN
Intravitreal administration of nanomedicines could be valuable for retinal gene therapy, if their mobility in the vitreous and therapeutic efficacy in the target cells can be guaranteed. Hyaluronic acid (HA) as an electrostatic coating of polymeric gene nanomedicines has proven to be beneficial on both accounts. While electrostatic coating provides an easy way of coating cationic nanoparticles, the stability of electrostatic complexes in vivo is uncertain. In this study, therefore, we compare electrostatic with covalent coating of gene nanocarriers with HA for retinal gene therapy via intravitreal administration. Specifically, DOTAP:DOPE/plasmid DNA lipoplexes coated with HA are evaluated in terms of intravitreal mobility using a previously optimized ex vivo model. We find that both electrostatic and covalent HA coating considerably improve the mobility of the lipoplexes in the vitreous humor of excised bovine eyes. In addition we evaluate in vitro uptake and transfection efficiency in ARPE-19 cells. Contrary to PEGylated lipoplexes it is found that HA coated lipoplexes are efficiently internalized into ARPE-19 cells. Covalent HA-coated lipoplexes had an 8-fold increase of transgene expression compared to the uncoated lipoplexes. We conclude that covalent HA-coating of gene nanomedicines is a promising approach for retinal gene therapy by intravitreal administration.
Asunto(s)
Ácidos Grasos Monoinsaturados/química , Ácido Hialurónico/química , Compuestos de Amonio Cuaternario/química , Retina/efectos de los fármacos , Animales , Cationes , Bovinos , Línea Celular , Supervivencia Celular , ADN/administración & dosificación , Sistemas de Liberación de Medicamentos , Colorantes Fluorescentes/química , Terapia Genética , Humanos , Inyecciones Intravítreas , Liposomas , Nanopartículas , Fosfatidiletanolaminas/química , Plásmidos , Polietilenglicoles , Electricidad Estática , Propiedades de Superficie , Transfección , Cuerpo Vítreo/metabolismoRESUMEN
Retinal gene therapy could potentially affect the lives of millions of people suffering from blinding disorders. Yet, one of the major hurdles remains the delivery of therapeutic nucleic acids to the retinal target cells. Due to the different barriers that need to be overcome in case of topical or systemic administration, intravitreal injection is an attractive alternative administration route for large macromolecular therapeutics. Here it is essential that the therapeutics do not aggregate and remain mobile in the vitreous humor in order to reach the retina. In this study, we have evaluated the use of hyaluronic acid (HA) as an electrostatic coating for nonviral polymeric gene nanomedicines, p(CBA-ABOL)/pDNA complexes, to provide them with an anionic hydrophilic surface for improved intravitreal mobility. Uncoated polyplexes had a Z-averaged diameter of 108nm and a zeta potential of +29mV. We evaluated polyplexes coated with HA of different molecular weights (22kDa, 137kDa and 2700kDa) in terms of size, surface charge and complexation efficiency and noticed their zeta potentials became anionic at 4-fold molar excess of HA-monomers compared to cationic monomers, resulting in submicron ternary polyplexes. Next, we used a previously optimized ex vivo model based on excised bovine eyes and fluorescence single particle tracking (fSPT) microscopy to evaluate mobility in intact vitreous humor. It was confirmed that HA-coated polyplexes had good mobility in bovine vitreous humor, similar to polyplexes functionalized with polyethylene glycol (PEG), except for those coated with high molecular weight HA (2700kDa). However, contrary to PEGylated polyplexes, HA-coated polyplexes were efficiently taken up in vitro in ARPE-19 cells, despite their negative charge, indicating uptake via CD44-receptor mediated endocytosis. Furthermore, the HA-polyplexes were able to induce GFP expression in this in vitro cell line without apparent cytotoxicity, where coating with low molecular weight HA (22kDa) was shown to induce the highest expression. Taken together our experiments show that HA-coating of nonviral gene complexes is an interesting approach towards retinal gene therapy by intravitreal administration. To our knowledge, this is the first time electrostatic HA-coating of polyplexes with different molecular weights has been evaluated in terms of their suitability for intravitreal delivery of therapeutic nucleic acids towards the retina.
Asunto(s)
ADN/química , Terapia Genética , Ácido Hialurónico/química , Línea Celular , ADN/administración & dosificación , Humanos , Receptores de Hialuranos/metabolismo , Inyecciones Intravítreas , Nanomedicina , Plásmidos , Polímeros/química , Retina/metabolismoRESUMEN
Therapeutic application of nucleic acids requires their encapsulation in nanosized carriers that enable safe and efficient intracellular delivery. Before the desired site of action is reached, drug-loaded nanoparticles (nanomedicines) encounter numerous extra- and intracellular barriers. Judicious nanocarrier design is highly needed to stimulate nucleic acid delivery across these barriers and maximize the therapeutic benefit. Natural polysaccharides are widely used for biomedical and pharmaceutical applications due to their inherent biocompatibility. At present, there is a growing interest in applying these biopolymers for the development of nanomedicines. This review highlights various polysaccharides and their derivatives, currently employed in the design of nucleic acid nanocarriers. In particular, recent progress made in polysaccharide-assisted nucleic acid delivery is summarized and the specific benefits that polysaccharides might offer to improve the delivery process are critically discussed.
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Nanopartículas/química , Ácidos Nucleicos/química , Polisacáridos/química , Animales , Química Farmacéutica , Portadores de Fármacos/química , HumanosRESUMEN
AIM: To develop a robust assay to evaluate and compare the intravitreal mobility of nanoparticles in the intact vitreous body. MATERIALS & METHODS: Excised bovine eyes were prepared to preserve the fragile structure of the vitreous humor, while permitting high-resolution fluorescence microscopy and single-particle tracking analysis of intravitreally injected nanoparticles. This assay was validated by analyzing polystyrene beads and further employed to evaluate gene nanomedicines composed of poly(amido amine)s and plasmid DNA. RESULTS: The assay was able to distinguish immobilized cationic nanoparticles from mobile PEGylated nanoparticles. PEGylation of the polyplexes resulted in a drastic improvement of their mobility. CONCLUSION: An ex vivo eye model is presented for studying nanoparticle mobility in intact vitreous humor by single-particle tracking microscopy. These results give important guidelines for developing gene- and drug-delivery nanomedicines that are compatible with intravitreal administration.
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Microscopía Fluorescente/métodos , Nanopartículas/administración & dosificación , Nanopartículas/análisis , Cuerpo Vítreo/ultraestructura , Animales , Bovinos , ADN/administración & dosificación , Inyecciones Intravítreas , Nanomedicina , Plásmidos/administración & dosificación , Polietilenglicoles/administración & dosificación , Polietilenglicoles/análisisRESUMEN
In the field of nanomedicine, ample attention has been paid to the development of nanocarriers for the intracellular delivery of therapeutic cargo, such as nucleic acids for gene therapy. The efficiency with which these non-viral carriers deliver their payload at the required intracellular site of action remains low. Despite extensive research on cellular attachment, endocytosis and intracellular trafficking of nanocarriers, clear-cut rules for the design of effective nanocarriers to improve nucleic acid transfer are still lacking. This is mainly caused by the cell type-dependence of this highly dynamic cellular processing, and to the lack of reliable methods to study these events. For these reasons there is a strong demand for the development and standardization of such methods in order to better understand the intracellular dynamics of nanomedicine processing and validate cellular and intracellular targeting strategies. This review aims at providing an overview of the different processes that are currently known to be involved in the cellular processing of nanomedicines, with a focus on cellular internalization mechanisms, as this has received a great deal of attention in the last couple of years. Furthermore, the intracellular hurdles that need to be overcome to allow efficient NA transfer will be critically discussed. In addition, an overview will be given of various methodologies that have been applied to unravel these cellular processing mechanisms, with a discussion on their strengths and weaknesses.