Cholesterol

Liposomes containing (dichloromethylene)bisphosphonic acid (clodronate; Sigma-Aldrich, St. Louis, MO, USA) were prepared according to the method of Van Rooijen et al. (13 (link)). In brief, 43 mg of L-α-phosphatidylcholine (Sigma-Aldrich) and 4 mg of cholesterol (Nacalai Tesque) were dissolved in chloroform and dried under reduced pressure. The lipid membranes were hydrated in 5 mL of PBS containing 0.7 M clodronate and sonicated with a tip-type sonicator (US-300; Nihonseiki Kaisha Ltd, Tokyo, Japan). clodronate that was not encapsulated in liposomes was removed by ultracentrifugation, and the liposome pellet was resuspended in 2 mL of PBS.

Fludrocortisone (FD), aldosterone (Aldo), spironolactone (Spi), methyl green and diaminobenzidine (all from Sigma-Aldrich, St. Louis, MO, USA), cresyl violet acetate (MP Biomedicals, Illkirch, France) and Luxol Fast Blue (Chroma-Gesellschaft Schmidt and Co., Stuttgart, Germany) were used in the present study. Cholesterol, lithium carbonate, pentobarbital sodium and sodium hydroxide were purchased from (Nacalai Tesque (Kyoto, Japan).

Dipalmitoylphosphatidylcholine (NOF Corporation, Tokyo, Japan), dipalmitoyl phosphatidylethanolamine (NOF Corporation), dipalmitoyl phosphatidylglycerol sodium salt (NOF Corporation), and cholesterol (Nacalai Tesque, Inc., Kyoto, Japan) were mixed in a round bottom flask at a molar ratio of 15:15:30:40, dissolved in a chloroform/methanol (2:1, v/v) mixture, and then evaporated at 37°C using a rotary evaporator to produce a thin hemispherical lipid film. To produce anionic LPs with a size of ~100 nm, the film was hydrated with buffer containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 4.0) and 120 mM (NH₄)₂SO₄ at 60°C. The freeze–thaw cycle was repeated five times using a liquid nitrogen bath. The crude solution of LPs was subjected to a Lipex extruder (Northern Lipids, Vancouver, BC, Canada) equipped with a polycarbonate filter (pore size, 200 nm) four times at 60°C, and twice with a filter (pore size, 50 nm). The solution was further sonicated for 10 minutes on ice using an Astrason ultrasonic disruptor (Misonix, Farmingdale, NY, USA).