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http://arbutusbio.com/docs/Lipos ... or_Distribution.pdf 里面介绍了一种采用荧光染料的方法： Encapsulation The pharmacology of a liposomal formulation of NA will be largely determined by the extent to which the NA is encapsulated inside the liposome bilayer(s). Encapsulated NA will be protected from nuclease degradation, while those that are merely associated with the surface of a liposome will be less protected. Encapsulated NA shares the extended circulation lifetime and biodistribution of the intact liposome, while those that are surface associated will adopt the pharmacology of naked NA once they disassociate from the liposome surface. For this reason encapsulation must be accurately determined. An acceptable method is the use of a membrane-impermeable fluorescent dye exclusion assay. This method requires a dye that has enhanced fluorescence when associated with NA. Specific dyes are available for the quantitative determination of plasmid DNA, single-stranded deoxyribonucleotides, and single- or double-stranded ribonucleotides. Encapsulation is determined by adding the dye to a liposomal formulation, measuring the resulting fluorescence and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent. Detergentmediated disruption of the liposomal bilayer releases the encapsulated NA, allowing it to interact with the membrane-impermeable dye. NA encapsulation is calculated as E = (Io-I)/Io, where I and Io refer to the fluorescence intensities before and after the addition of detergent [55]. Although other methods have been used to determine the liposomal encapsulation of NAs, including nuclease protection assays, chromatographic separation [43], density gradient ultracentrifugation [103], and capillary electrophoresis [104], this method is the most accurate, rapid, and cost-effective. Methods that rely on nuclease protection or chromatographic separation often fail to differentiate encapsulated NA from that which is merely surface associated or trapped in lipid–NA aggregates. |
8楼2020-01-16 16:27:01
