Preparation of liposome-encapsulated clodronate

Three-sn-phosphatidylcholine (from egg yolk) 172 mg, cholesterol 16 mg, and α-tocopherol 0.5 mg were dissolved in 2 mL of methanol-chloroform solution (methanol: chloroform = 1: 2) in a round-bottomed flask. After evaporating organic solvents, the lipid film was hydrated with 1 mL of 200 mg/mL disodium clodronate tetrahydrate (Tokyo Chemical Industry, Tokyo, Japan) or 9.57 mM PBS for the vehicle control. Suspensions were freeze-thawed 5 times by transferring between liquid nitrogen and a water bath at 40 °C to increase the inclusion rate in liposomes⁴². Liposomes were extruded 21 times through a 0.4-μm pore polycarbonate filter using a LiposoFast extruder (Avestin, Mannheim, Germany). Liposome sizes obtained by a FPAR-1000 particle analyzer (Otsuka Electronics, Osaka, Japan) were confirmed to be 293.4 ± 19.6 nm (clodronate) or 555.9 ± 57.2 nm (PBS). The encapsulating rate of clodronate in liposomes was estimated to be 70%, as previously described⁴². Suspensions were stored at 4 °C and used within 2 weeks.