RESUMEN
Since recombinant viral vectors have been associated with serious side effects, such as immunogenicity and oncogenicity, synthetic delivery systems represent a realistic alternative for achieving efficacy in gene therapy. A major challenge for non-viral nanocarriers is the optimization of transgene expression in the targeted cells. This goal can be achieved by fine-tuning the chemical carriers and the adding specific motifs to promote cellular penetration. Our study focuses on the development of novel folate-based complexes that contain varying quantities of folate motifs. After controlling for their physical properties, neutral folate-modified lipid formulations were compared in vitro to lipoplexes leading to comparable expression levels. In addition, no cytotoxicity was detected, unlike what was observed in the cationic controls. Mechanistically, the delivery of the transgene appeared to be, in part, due to endocytosis mediated by folate receptor targeting. This mechanism was further validated by the observation that adding free folate into the medium decreased luciferase expression by 50%. In vivo transfection with the folate-modified MM18 lipid, containing the highest amount of FA-PEG(570)-diether co-lipid (w:w; 90:10), at a neutral charge ratio, gave luciferase transgene expression. These studies indicate that modification of lipids with folate residues could enhance non-toxic, cell-specific gene delivery.
Asunto(s)
Células Epiteliales/metabolismo , Ácido Fólico/química , Liposomas/química , Nanopartículas/química , Transfección/métodos , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Receptor 1 de Folato/metabolismo , Ácido Fólico/toxicidad , Células HeLa , Humanos , Liposomas/toxicidad , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Microscopía Fluorescente , Nanopartículas/toxicidad , Cavidad Nasal/metabolismo , Plásmidos/química , Plásmidos/genética , Plásmidos/metabolismo , Reproducibilidad de los Resultados , Tráquea/metabolismoRESUMEN
We demonstrate the utility of a microfluidic platform in which water-in-oil droplet compartments serve to miniaturize cell lysate assays by a million-fold for directed enzyme evolution. Screening hydrolytic activities of a promiscuous sulfatase demonstrates that this extreme miniaturization to the single-cell level does not come at a high price in signal quality. Moreover, the quantitative readout delivers a level of precision previously limited to screening methodologies with restricted throughput. The sorting of 3 × 10(7) monodisperse droplets per round of evolution leads to the enrichment of clones with improvements in activity (6-fold) and expression (6-fold). The detection of subtle differences in a larger number of screened clones provides the combination of high sensitivity and high-throughput needed to rescue a stalled directed evolution experiment and make it viable.
Asunto(s)
Evolución Molecular Dirigida , Técnicas Analíticas Microfluídicas , Sulfatasas/metabolismo , Escherichia coli/metabolismo , Fluoresceína/síntesis química , Fluoresceína/química , Fluoresceína/metabolismo , Hidrólisis , Cinética , Técnicas Analíticas Microfluídicas/instrumentación , Miniaturización , Aceites/química , Plásmidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfatasas/genética , Agua/químicaRESUMEN
We have previously shown that synthetic archaeal lipid analogues are useful vectors for drug/gene delivery. We report herein the synthesis and gene transfer properties of a series of novel di- and tetraether-type archaeal derivatives with a poly(ethylene glycol) (PEG) chain and further equipped with a folic acid (FA) group. The synthetic strategy and the purification by dialysis ensured complete removal of free FA. The lipids were mixed with a conventional glycine betaine-based cationic lipid and the resulting formulations were tested in transfection assays after complexation with plasmid DNA. All four novel co-lipids afforded efficient in vitro gene transfection. Moreover, the FA-equipped derivatives permitted ligand/receptor-based targeted transfection; their activity was inhibited when free FA was added to the transfection medium. These novel archaeal derivatives equipped with FA-PEG moieties may thus be of great interest for targeted in vivo transfection.
Asunto(s)
Ácido Fólico/química , Vectores Genéticos/síntesis química , Vectores Genéticos/genética , Lípidos/síntesis química , Lípidos/genética , Transfección/métodos , Archaea/química , ADN/química , Ácido Fólico/genética , Genes Reporteros , Vectores Genéticos/química , Células HeLa , Humanos , Lípidos/química , Liposomas/química , Conformación Molecular , Polietilenglicoles/química , EstereoisomerismoRESUMEN
A synthetic route for the preparation of symmetrical and unsymmetrical archaeal tetraether-like analogues has been described. The syntheses are based upon the elaboration of hemimacrocyclic tetraether lipid cores from versatile building blocks followed by simultaneous or sequential introduction of polar head groups. Functionalizations of the tetraether lipids with neutral lactose or phosphatidylcholine polar heads and cationic glycine betaine moieties were envisaged both to increase membrane stability and to exhibit interactions with charged nucleic acids. Additionally, mannose and lactose triantennary clusters designed as multivalent ligands for selective interaction with lectin-type receptors were also efficiently synthesized for active cell/tissue targeting.
