The Cys–AG73 peptide (CGG–RKRLQVQLSIRT) and a scrambled Cys–AG73T control peptide (CGG–LQQRRSVLRTKI) were synthesized manually using the 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase strategy and were prepared in the COOH terminal
amide form and purified by reverse-phase high-performance liquid chromatography. Dox-encapsulating liposomes composed of DSPC, DSPE–PEG2000–OMe, and DSPE–PEG2000–Mal at a molar ratio of 94:4:2 were prepared by a reverse-phase evaporation method and a remote loading method. For coupling, AG73 peptide at a molar ratio of 2-fold DSPE–PEG2000–Mal was added to the Dox-encapsulating liposomes, and the mixture was incubated for 24 h at 4 °C to conjugate the cysteine of the Cys–AG73 peptide with the maleimide of the Dox-encapsulating liposomes using a thioether bond. Dinaciclib solubility dmso The resulting AG73 peptide-conjugated CDK inhibitor Dox-encapsulating liposomes (AG73–Dox) were passed through a Sephadex G-50 spin column to remove any excess peptide. AG73–Dox was modified with
6 mol% PEG and 2 mol% peptides. The particle size and zeta potential of the liposomes were measured using NICOMP 380 ZLS (Particle Sizing Systems, Santa Barbara, CA, USA). 293T human embryonic kidney carcinoma cells that stably overexpressed syndecan-2 (293T-Syn2) and murine colorectal carcinoma cells (colon26) were cultured in DMEM that was supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), and puromycin (0.4 or 100 μg/ml) at 37 °C in a humidified 5% CO2 atmosphere. Male BALB/c mice (6 weeks old) were purchased from Tokyo Laboratory Animals Science Co., Ltd. (Tokyo, Japan). Animal use and relevant experimental procedures were approved by the Tokyo University of Pharmacy and Life Science Committee on the Care and Use of Laboratory Animals. The
intracellular uptake of liposomes was determined by flow cytometry analysis . 293T-Syn2 cells (1×105 cells/well) were seeded in a 24-well plate and incubated for 48 h at 37 °C in 5% CO2. Colon26 cells (5×104 cells/well) were also seeded in a 24-well plate and incubated for 24 h at 37 °C in 5% CO2. The medium was then replaced with Dox-encapsulating liposomes (Dox–PEG), AG73–Dox, or Dox-encapsulating Non-specific serine/threonine protein kinase AG73T peptide-modified liposomes (AG73T–Dox) that were diluted with culture medium for a final Dox concentration of 20 μg/ml. The plates were incubated for 1 h at 37 °C. The medium was removed; subsequently, each cancer cell line was washed with phosphate-buffered saline (PBS), and the cell samples were examined by flow cytometry using an FACScan (Becton Dickinson, San Jose, CA, USA). Cell-associated Dox was excited with an argon laser (488 nm). Data were collected in 10,000 gated events and analyzed with the CELL Quest software program. 293T-Syn2 cells (2 or 5×104 cells/well) were seeded in a 24-well plate and incubated for 48 h at 37 °C in 5% CO2.