For immunohistochemistry, the tumor section was blocked with 10% lapine serum in TBS for 30 min at 4 °C and incubated with anti-mouse CD31 antibody (BD Pharmingen) overnight at 4 °C. Subsequently, the section was incubated with Alexa Fluor 488 rabbit anti-rat IgG (Molecular Probes) for 1 h at room temperature. Each tumor and organ section was then mounted with VECTASHIELD Hard Set Medium with DAPI (VECTOR LABORATORIES, INC, USA) and fluorescently observed with BZ-8100 (KEYENCE, Japan). First, we sought to prepare Dox-encapsulating AG73 peptide-modified liposomes (AG73–Dox). As shown in Table 1, the mean particle diameter

of Dox–PEG, AG73–Dox, or AG73T–Dox ranged from 130 to 170 nm with a relatively narrow distribution (Fig. 1). The zeta potential of the Dox-encapsulating liposomes was slightly negative selleck compound in value. The efficiency for the remote loading of Dox into the liposomes was 90–95% with a drug/lipid ratio of 1:5 (molar ratio). The encapsulated efficiency also remained unchanged even in the case of peptide modification.

After one month of storage at 4 °C, the encapsulated efficiency was less than 5%. To examine the selective cellular uptake of Dox transfected by liposomes via the syndecan-2 receptor, the amount of Dox uptake into cancer cells including syndecan-2 overexpressing ZD1839 price cells was evaluated by flow cytometry analysis. As shown in Fig. 2, the cellular uptake of AG73–Dox on both 293T-Syn2 and colon26 was higher

than that of Dox–PEG or AG73T–Dox. The laminin-derived AG73 peptide is known as a ligand for syndecans, and it has been reported that syndecan-2 is highly expressed in various cancer cells [3], [5], [8], [24] and [26]. In addition, the AG73 peptide has been shown to bind to the heparin sulfate side chains of syndecan [8]. Therefore, to verify that AG73–Dox can bind to syndecan-2 on the surface of cancer cells, the cancer cells were treated with AG73–Dox and heparin (Fig. 3). Our data showed that the cellular uptake of AG73–Dox was down-regulated by the treatment with heparin. However, the cellular uptake of Dox–PEG or AG73T–Dox was not down-regulated by the treatment (data not shown). Therefore, these results suggested that AG73–Dox could effectively target cancer cells via the syndecan-2 receptor. To further elucidate the intracellular uptake of Dox, the intracellular localization of Dox VAV2 was evaluated after the treatment of cells with Dox–PEG, AG73–Dox, AG73T–Dox, or free Dox using confocal microscopy. As shown in Fig. 4, the cells treated with AG73–Dox showed a strong red fluorescence, whereas cells treated with Dox–PEG or AG73T–Dox showed a faint red fluorescence. The intracellular localization of Dox in the cells treated with AG73–Dox could be observed on the surface of the cell membrane and in the cytoplasm with a slight colocalization in the nuclei. However, these localizations after the treatment with AG73–Dox were blocked by heparin.