In vivo delivery of phosphorothioate oligonucleotides into murine retina.
OBJECTIVE
To determine the fate of phosphorothioate oligonucleotides (S-ODNs), which are commonly used for antisense strategy, in murine retina in vivo with the use of fluorescein isothiocyanate (FITC)-labeled S-ODNs, and to evaluate our fusogenic liposome system that may facilitate the delivery of S-ODNs.


METHODS
The FITC-labeled S-ODNs were encapsulated in liposomes, which were then coated with the envelope of inactivated hemagglutinating virus of Japan (HVJ; Sendai virus) by fusion (HVJ liposomes). Intravitreal injection of naked FITC-labeled S-ODNs or of the HVJ liposomes was done in ICR mice. After fixation, cryosections and flat-mounted retinas were prepared and examined by fluorescence microscopy.


RESULTS
Injection of naked FITC-labeled S-ODNs at 3 micromol/L exhibited weak fluorescence in 13% of the cells in the ganglion cell layer. When the concentration was increased to 30 micromol/L, high fluorescence was seen in 59% of cells in the ganglion cell layer at this time. This fluorescence diminished within a day. In contrast, injection of HVJ liposomes containing FITC-labeled S-ODNs at 3 micromol/L resulted in high fluorescence in 44% of the cells in the ganglion cell layer at 1 hour, and this fluorescence lasted for up to 3 days. This treatment also resulted in high fluorescence within retinal vessel walls, and weak fluorescence in photoreceptor cells.


CONCLUSIONS
Intravitreally injected S-ODNs were rapidly eliminated from neural retina, and the use of HVJ liposomes could improve the delivery of S-ODNs. This method may be a potentially useful system for the antisense-based therapies for retinal diseases.
Hangai
M
M
Department of Ophthalmology and Visual Science, Graduate School of Medicine, Kyoto University, Japan.
Tanihara
H
H
Honda
Y
Y
Kaneda
Y
Y
D000818:Animals; D016503:Drug Delivery Systems; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D007267:Injections; D008081:Liposomes; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000493:pharmacokinetics*; D018108:Respirovirus; D012165:Retinal Ganglion Cells / Q000378:metabolism*; D013873:Thionucleotides / Q000008:administration & dosage / Q000493:pharmacokinetics*; D014760:Viral Fusion Proteins / Q000493:pharmacokinetics; D014822:Vitreous Body
D005456:Fluorescent Dyes; D008081:Liposomes; D016376:Oligonucleotides, Antisense; D013873:Thionucleotides; D014760:Viral Fusion Proteins; D016650:Fluorescein-5-isothiocyanate