title: Preparation of small amounts of sterile siRNA-liposomes with high entrapping efficiency by dual asymmetric centrifugation (DAC).
abstract: Liposomal formulation of siRNA is an attractive approach for improving its delivery in vivo, shielding the RNA from nucleases and promoting tumor targeting. Here, the production of very small batch sizes of siRNA-liposomes by using the "dual asymmetric centrifugation (DAC)" technique was investigated. This new technique combines rapid and sterile liposome preparation with very high entrapping efficiencies. DAC is here presented in conjunction with a non-destructive microscale analysis based on double fluorescence labeling, which enables monitoring of siRNA integrity during the liposomal preparation. Integrity is reflected in spatial proximity of the dyes, which results in measurable fluorescence resonance energy transfer (FRET). The combination of DAC and the sensitive FRET analysis allows the handling of batch sizes down to 20 mg of conventional liposomes (CL) and sterically stabilized liposomes (SL). These were prepared in common 2 ml reaction tubes and loaded with calcein or labeled siRNA. Liposome sizes were 79+/-16 nm for CL and 109+/-9 nm for SL loaded with siRNA. Trapping efficiencies ranged from 43 to 81%, depending on batch size, enclosed compound, and liposome composition. FRET monitoring showed that the siRNA remained intact throughout DAC and that liposomal formulations protected the siRNA from nucleases. siRNA-liposomes remained stable for at least 3 months.
authors.affiliation: Tumor Biology Center, Department of Clinical Research, Breisacher Strasse 117, Freiburg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Hirsch
authors.forename: Vittorio
authors.initials: V
authors.lastname: Ziroli
authors.forename: Mark
authors.initials: M
authors.lastname: Helm
authors.forename: Ulrich
authors.initials: U
authors.lastname: Massing
mesh_terms: D002498:Centrifugation; D002851:Chromatography, High Pressure Liquid; D004339:Drug Compounding / Q000295:instrumentation / Q000379:methods*; D004355:Drug Stability; D005452:Fluoresceins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D008081:Liposomes; D010316:Particle Size; D034741:RNA, Small Interfering / Q000008:administration & dosage*; D013050:Spectrometry, Fluorescence; D013242:Sterilization
chemical_list: D005452:Fluoresceins; D008081:Liposomes; D034741:RNA, Small Interfering; C007740:fluorexon