RESUMEN
Efficient and safe delivery systems for siRNA therapeutics remain a challenge. Elevated secreted protein, acidic, and rich in cysteine (SPARC) protein expression is associated with tissue scarring and fibrosis. Here we investigate the feasibility of encapsulating SPARC-siRNA in the bilayers of layer-by-layer (LbL) nanoparticles (NPs) with poly(L-arginine) (ARG) and dextran (DXS) as polyelectrolytes. Cellular binding and uptake of LbL NPs as well as siRNA delivery were studied in FibroGRO cells. siGLO-siRNA and SPARC-siRNA were efficiently coated onto hydroxyapatite nanoparticles. The multilayered NPs were characterized with regard to particle size, zeta potential and surface morphology using dynamic light scattering and transmission electron microscopy. The SPARC-gene silencing and mRNA levels were analyzed using ChemiDOC western blot technique and RT-PCR. The multilayer SPARC-siRNA incorporated nanoparticles are about 200 nm in diameter and are efficiently internalized into FibroGRO cells. Their intracellular fate was also followed by tagging with suitable reporter siRNA as well as with lysotracker dye; confocal microscopy clearly indicates endosomal escape of the particles. Significant (60%) SPARC-gene knock down was achieved by using 0.4 pmole siRNA/µg of LbL NPs in FibroGRO cells and the relative expression of SPARC mRNA reduced significantly (60%) against untreated cells. The cytotoxicity as evaluated by xCelligence real-time cell proliferation and MTT cell assay, indicated that the SPARC-siRNA-loaded LbL NPs are non-toxic. In conclusion, the LbL NP system described provides a promising, safe and efficient delivery platform as a non-viral vector for siRNA delivery that uses biopolymers to enhance the gene knock down efficiency for the development of siRNA therapeutics.
Asunto(s)
Silenciador del Gen , Técnicas de Transferencia de Gen , Nanopartículas/química , Osteonectina/genética , ARN Interferente Pequeño/metabolismo , Endocitosis , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Humanos , Espacio Intracelular/metabolismo , Masculino , Nanopartículas/ultraestructura , Proteínas de Neoplasias/metabolismo , Osteonectina/antagonistas & inhibidores , Osteonectina/biosíntesis , Osteonectina/ultraestructura , Tamaño de la Partícula , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Proteínas Ribosómicas/metabolismo , Electricidad EstáticaRESUMEN
In recent years colloidal particles and capsules, layer-by-layer (LbL) coated with biocompatible polyelectrolytes, have received much attention as drug-delivery systems. In this study an LbL-assembled, biopolymer-based multilayer system was established as a combined transporter and sensor for monitoring intracellular degradation and processing. CaCO(3) cores were functionalized with fluorescein isothiocyanatelabelled poly(allylamine hydrochloride) (FITC-PAH). This pH-sensitive fluorescent dye allows identifying the location of these LbL-coated particles in cell compartments of different pH, like the endo-lysosome and cytoplasm. The labelled core was then coated with consecutive layers of protamine (PRM) and dextran sulfate (DXS). Finally, plasmid DNA (pEGFP-C1) as a reporter agent for drug release in the cytoplasm was integrated into the biocompatible and degradable PRM/DXS multilayer. The system was tested regarding its long-term stability and interaction with HEK 293T/17 cells. These multifunctional microparticles allow the simultaneous investigation of particle localization and processing within cells, and should thus provide a valuable tool for studying and improving the controlled LbL-based release of active agents into cells.
Asunto(s)
Compartimento Celular , Portadores de Fármacos/química , Plásmidos/administración & dosificación , Transporte Biológico , Carbonato de Calcio , Materiales Biocompatibles Revestidos , Coloides/química , Sulfato de Dextran/química , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Microscopía Confocal , Protaminas/químicaRESUMEN
Multifunctional colloidal microparticles allow the integration of various active agents as well as reporter molecules into one system without interfering combining delivery and sensing functions. In this study, calcium carbonate particles were functionalized with fluorescein isothiocyanate-labeled poly(allylamine hydrochloride) (FITC-PAH) allowing particle localization in cell compartments of different pH. Plasmid DNA (pEGFP-C1 and pDsRed1-N1) as a reporter agent for drug release in the cytoplasm and rhodamine-B-isothiocyanate-labeled protamine (RITC-PRM) were integrated into biocompatible and biodegradable PRM/DXS multilayers. The uptake and processing of the particles by HEK293T/17 cells were investigated via flow cytometry and confocal laser scanning microscopy. The presented data show a clear correlation between the fluorescence intensity of the FITC-labeled core, that is, the particle localization after cellular uptake, and the expression of fluorescent proteins by the cells without further cell staining. In conclusion, this particle design allows the simultaneous study of particle location and processing to monitor the transport and release of active agents and should thus be an invaluable tool for the study and design of nano- and microcarrier systems.