RESUMEN
The synthesis and properties of gemini surfactants of the type (R(1)(CO)-Lys(H)-NH)2(CH2)n are reported. For a spacer length of n = 6, the hydrophobic acyl tail was varied in length (R(1) = C8, C10, C12, C14, C16, and C18) and, for R(1) = C18, the degree of unsaturation. For R(1)(CO) = oleoyl (C18:1 Z) the spacer length (n = 2-8) and the stereochemistry of the lysine building block were varied; a 'half-gemini' derivative with a single oleoyl tail and head group was also prepared. The potential of the gemini surfactants to transfer polynucleotides across a cell membrane was investigated by transfection of HeLa cells with beta-galactosidase, both in the presence and absence of the helper lipid DOPE. Oleoyl was found to be by far the best hydrophobic tail for this biological activity, whereas the effect of the lysine stereochemistry was less pronounced. The effect of an optimum spacer length (n = 6) was observed only in the absence of helper lipid. The most active surfactant, i.e. the one with oleoyl chains and n = 6, formed liposomes with sizes in the range of 60-350 nm, and its lipoplex underwent a transition from a lamellar to a hexagonal morphology upon lowering the pH from 7 to 3.
Asunto(s)
Técnicas de Transferencia de Gen , Liposomas/química , Lisina/química , Tensoactivos/química , Cationes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Lípidos/química , Liposomas/síntesis química , Liposomas/ultraestructura , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Modelos Químicos , Estructura Molecular , Fosfatidiletanolaminas/química , Plásmidos/genética , Dispersión del Ángulo Pequeño , Espectrometría de Masa por Ionización de Electrospray , Tensoactivos/síntesis química , Transfección/métodos , Difracción de Rayos X , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
The superior surfactant properties of cationic gemini surfactants are applied to the complex problem of introducing genes into cells. Of almost 250 new compounds tested, of some 20 different structural types, a majority showed very good transfection activity in vitro. The surfactant is shown to bind and compact DNA efficiently, and structural studies and calculations provide a working picture of the "lipoplex" formed. The lipoplex can penetrate the outer membranes of many cell types, to appear in the cytoplasm encapsulated within endosomes. Escape from the endosome--a key step for transfection--may be controlled by changes in the aggregation behavior of the lipoplex as the pH falls. The evidence suggests that DNA may be released from the lipoplex before entry into the nucleus, where the new gene can be expressed with high efficiency.