4),

which suggested that the Palbociclib chemical structure Dox localized in the endosomes [12]. Thus, these results suggested that AG73–Dox could enhance the intracellular uptake of Dox compared with Dox–PEG or AG73T–Dox. To examine the therapeutic efficiency of AG73–Dox, the cytotoxicity of Dox-encapsulating liposomes against cancer cells was evaluated using a WST assay. The cytotoxicity studies were initially performed with empty liposomes on the cancer cells to determine whether the liposomes themselves contributed to the cytotoxicity. The concentration of lipids tested was matched to the amount of lipids used in the drug-encapsulating formulations. At the concentration of lipids used, the cell viability was more than 80% that of the control (data not shown). These data indicated that the empty liposomes alone BLU9931 cost did not have a significant cytotoxicity. As shown in Fig. 5, the cell viability was dependent on the concentration of Dox. Furthermore, the cytotoxicity of AG73–Dox was more sensitive than that of Dox–PEG or AG73T–Dox against both types of cancer cells. In particular, the cytotoxicity of AG73–Dox against 293T-Syn2 was notably higher in comparison with that against colon26. This result may be due to the fact that the ability and

sensitivity of the intracellular uptake of free Dox into the cancer cells were distinct from those of 293T-Syn2 and colon26, as shown by confocal microscopy (Fig. 4), as it has been reported that colon26 exhibits a constitutive expression of mdr1a and mdr1b that encodes the drug efflux transporter P-glycoprotein [16]. To understand the drug delivery and release behaviors of AG73–Dox, the cells were washed after the treatment of AG73–Dox

and placed into fresh medium for further incubation at 37 °C. They were imaged using confocal microscopy at various time points after washing. As shown in Fig. 6, Dox was diffused through the 293T-Syn2 in a time-dependent manner and then was colocalized with the nuclei. This result could be due to the release Fossariinae of Dox from AG73–Dox inside endosomes with a lower pH. In addition, at 24 h post-incubation, we observed fragmentation of the nuclei, which is characteristic of apoptosis (indicated by white arrows in Fig. 6). Moreover, at 48 h post-incubation, we observed increased fragmentation of the nuclei. Therefore, these results suggested that after the cellular uptake of AG73–Dox, Dox was slowly released from AG73–Dox and was subsequently transferred to nuclei, which led to cytotoxicity (Fig. 5). Next, as AG73–Dox showed higher cellular uptake and cytotoxicity against cancer cells compared to Dox–PEG, the antitumor efficacy of AG73–Dox in vivo was evaluated in colon26 tumor-bearing mice at a dose of 2 mg Dox/kg body weight. As shown in Fig. 7, the Dox–PEG group had suppressed tumor growth compared with the free Dox group.