However, there was no significant difference between the Dox–PEG group and the free Dox group, whereas the tumor growth inhibition efficacy of the AG73–Dox group was better than that of the free Dox group (P<0.05). When the see more antitumor effect of Dox–PEG was compared with that of AG73–Dox, there was no significant difference in antitumor effect. We also monitored the body weights of the tumor-bearing mice to assess any side effects of AG73–Dox. The body weight change during the tumor treatment was not observed in the AG73–Dox group. To evaluate the targeting effect of AG73 peptide-modified liposomes in vivo, we examined the intratumoral

localization of AG73 peptide-modified liposomes. As shown in Fig. 8, DiI-labeled PEG liposomes (PEG-L), which did not

contain Dox, were leaked from intratumoral vessels and diffused in the tumor tissue, whereas DiI-labeled AG73 peptide-modified liposomes (AG73-L) were mainly bound to intratumoral vessels and were partially extravasated in the tumor. The AG73 peptide is a ligand for syndecan-2. Syndecan-2 is also highly expressed in human vascular endothelial cells [17]. Moreover, the cellular uptake of AG73–Dox in human umbilical vein endothelial cells was higher than that of Dox–PEG or AG73T–Dox, which was observed using flow cytometry (data not shown). In this study, there is little difference between antitumor effect of Dox–PEG and that of AG73–Dox. However, it seems that AG73–Dox PLX3397 purchase tend to target not only tumor tissue but also intratumoral

vessels ( Fig. 7 and Fig. 8). To assess whether AG73-L is tumor selectivity, we injected DiI-labeled PEG-L or AG73-L and observed various other organs (heart, liver, spleen, and kidney) using fluorescence microscopy. As shown in Fig. 9, although the fluorescence intensity of AG73-L was slightly high in the heart compared to that of PEG-L, there was little difference between PEG-L and AG73-L in the other organs. Recently, to enhance the therapeutic effect of Dox, the drug delivery field has focused its attention on designing nanoparticles that are capable of releasing a drug efficiently when exposed to a specific triggering mechanism [1] and [2]. Such triggers include pH, light, ultrasound, enzymatic action, and heat [13]. Among the trigger-sensitive nanoparticle Sitaxentan formulations that have been developed, ultrasound-sensitive liposomes (bubble liposomes) could function as a novel gene delivery tool by exposure to ultrasound [25] and [18]. Therefore, it is necessary to optimize the AG73–Dox for in vivo application by changing the peptide modification ratio or PEG ratio. Furthermore, the combination of AG73–Dox with bubble liposomes and ultrasound may enable enhancement of the therapeutic effect. In this study, doxorubicin-encapsulating AG73 peptide-modified liposomes (AG73–Dox) were developed to increase the intracellular uptake of anticancer drugs and to achieve an improved therapeutic effect specifically against tumors.