[Preparation of cationic liposomes and its role in enhancing cellular uptake of antisense oligonucleotides].
OBJECTIVE
To prepare the liposomes which protect antisense oligodeoxynucleotides (ASON) against nuclease degradation and delivery ASON into cytoplasmic efficiently.


METHODS
A cationic derivative of cholesterol, 3 beta-[N-(N',N'-dimethylaminoethan)-carbamoyl] cholesterol (DC-Chol) was synthesized and used to prepare cationic liposome. The characteristics of liposomes/ASON complexes including size, drug loaded efficiency and structure were investigated. Cellular uptake of fluorescence labled ASON (FAM-ASON) under different condition was determined by flow cytometric analysis. Denatured polyacryamide gel electrophoresis (DPGE) was used to analyze the role of liposomes in protecting ASON.


RESULTS
The mean values of preliposomes and liposomes/ASON complexes size were 185.7 and 228.2 nm, respectively. Cationic liposomes showed a high adsorption capacity for ASON. When the +/- charge ratio exceeded 2:1, more than 90% of the ASON was loaded into liposomes. Agarose gel electrophoresis showed three different existence of ASON in liposomes formulation: free, absorbed and encapsulated types. Concerning cellular uptake, DC-Chol liposomes indicated high efficient effect of increasing cellular uptake of ASON. Compared with free ASON, the total fluorescence intensity in cytoplasma was significantly enhanced. The level of increasing was largely depended on +/- charge ratio. The cellular uptake of FAM-ASON decreased in the presence of serum. The cellular total fluorescence intensity in 10% and 30% fetal bovine serum of cultured medium were only 22.3% and 15.5% as that of serum-free media, respectively. DPGE confirmed that free ASON was rapidly degraded by DNase I while ASON encapsulated into liposomes was efficiently protected.


CONCLUSIONS
The cationic DC-Chol liposomes are shown to be promising carriers to deliver ASON into cytoplasma.
Chen
Hai-liang
HL
College of Pharmaceutical Sciences, First Affiliated Hospital, College of Medical Sciences, Zhejiang University, Hangzhou 310031, China.
Liang
Wen-quan
WQ
Shao
Jun-bin
JB
Chen
Zhi
Z
D002784:Cholesterol / Q000031:analogs & derivatives* / Q000494:pharmacology*; D004337:Drug Carriers; D016503:Drug Delivery Systems; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes / Q000494:pharmacology*; D009101:Multiple Myeloma / Q000473:pathology; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000097:blood / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004337:Drug Carriers; D008081:Liposomes; D016376:Oligonucleotides, Antisense; C070648:3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol; D002784:Cholesterol