Furthermore, we examined the antitumor effects of AG73–Dox in vitro and in vivo. 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoylphosphatidylethanolamine-methoxy-polyethyleneglycol (DSPE–PEG2000–OMe),

and 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine–polyethyleneglycol–maleimide (DSPE–PEG2000–Mal) were purchased from NOF Corporation (Tokyo, Japan). Doxorubicin (Dox) was purchased from Everolimus datasheet SIGMA-Aldrich Co. (St. Louis, MO, USA). For cell culture, Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Kohjin Bio Co., Ltd. (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from Equitech Bio Inc. (Kerrville, TX, USA). All other materials were used without further purification. Liposomes composed of DSPC and DSPE–PEG2000–OMe at a molar ratio of 94:6 were prepared by a reverse-phase evaporation method. In brief, all reagents were dissolved in 1:1 (v/v) chloroform/diisopropylether. Three hundred millimolar citrate buffer (pH 4.0) was then added to the lipid solution, and the mixture was sonicated and evaporated at 65 °C. The organic solvent was completely removed, and the size of the liposomes was adjusted to approximately 150 nm using extruding equipment and sizing filters (pore sizes: 100 and 200 nm, Nuclepore Track-Etch Membrane, Whatman plc, UK). For the fluorescent labeling of the lipid

membrane, 1,1-dioctadecyl-3,3,3,3-tetramethyl-indocarbocyanine PCI-32765 chemical structure perchlorate (DiI) was also added (1 mol% of total C-X-C chemokine receptor type 7 (CXCR-7) lipids). After the sizing, the liposomes were passed through a 0.45-μm pore-size filter (Syringe filter, ASAHI TECHNOGLASS Co., Chiba, Japan) for sterilization. The Dox-encapsulating

liposomes were prepared by a remote loading method with a pH gradient [4] and [19]. In brief, liposomes were passed through a Sephadex G-50 (GE Healthcare UK Ltd., Buckinghamshire, England) spin column that was equilibrated with N-[2-hydroxyethyl]piperazine-NV-[2-ethanesulfonic acid] (HEPES)-buffered saline (HBS; 20 mM HEPES, 150 mM NaCl, pH 7.5) to exchange the external buffer. The eluted liposomes had a transmembrane pH gradient with pH 4.0 inside and pH 7.5 outside the liposomes. The eluted liposomes were incubated with Dox (at a Dox:lipid molar ratio of 1:5) at 65 °C for 30 min. To remove the unencapsulated Dox, the mixture was passed through a Sephadex G-50 spin column. The Dox-encapsulating liposomes were stored at 4 °C until use. The lipid concentration was measured using Phospholipid C test Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and the Dox concentration was determined by measuring the absorbance at 480 nm (Infinite M1000, TECAN, Männedorf, Switzerland) in a 1% Triton X-100 solution and comparing the absorbance value to the standard curve.