Evaluation of liposome populations using a sucrose density gradient centrifugation approach coupled to a continuous flow system.
A method for the evaluation of liposome size populations using sucrose density gradient centrifugation coupled with a continuous flow system is presented. Liposomes, prepared using different methods (rapid solvent evaporation, rehydration, and detergent removal) and modified by assaying several procedures (shaking, sonication and extrusion) were evaluated according to the type of liposome, size and polydispersity. The preparation of liposomes was carried out in the presence of the fluorophor cresyl violet. Extracts of the liposomes were homogenised and centrifuged at 20,073 x g at 4 degrees C for 30 min using sucrose density gradient centrifugation programmes, which provide efficient liposome separation in different sizes. The results of the separation procedure were tested by aspiration of the extracts into a continuous flow system in which the liposomes were disrupted by the continuous mixing with a Triton X-100 solution, prior to their translation to the detector. The luminescence provided by the liberation of the encapsulated fluorophor indicates the distribution of liposomes in each density gradient stage. Three zones were obtained: zone alpha, containing giant unilamellar and multivesicular vesicles, zone beta, with large and medium size liposomes, and zone gamma, which contained small size liposomes. The precision of the separation zones obtained, expressed as RSD%, was lower than 5.6% in all instances. The method provides a relative rapid way to evaluate the liposome polydispersity and size after using conventional methods of synthesis and mechanical modifications.
Sánchez-López
V
V
Department of Analytical Chemistry, Faculty of Sciences, University of Córdoba, Marie Curie Annex Building, Campus of Rabanales, E-14071 Córdoba, Spain.
Fernández-Romero
J M
JM
Gómez-Hens
A
A
D048588:Benzoxazines; D002499:Centrifugation, Density Gradient / Q000295:instrumentation* / Q000379:methods*; D003902:Detergents / Q000737:chemistry; D005470:Fluorometry; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000032:analysis* / Q000737:chemistry; D010078:Oxazines; D010316:Particle Size; D012997:Solvents / Q000737:chemistry; D013395:Sucrose
D048588:Benzoxazines; D003902:Detergents; D008055:Lipids; D008081:Liposomes; D010078:Oxazines; D012997:Solvents; C028911:cresyl violet; D013395:Sucrose