Figure 1. (a) Illustration of the tumor targeting and lysosome escape of BCPB-B-DOX. The abundant PBA groups of BCPB-B-DOX are conductive to tumor targeting and lysosome escape and can greatly improve the antitumor efficacy of the prodrug. (b) Synthesis and drug loading of the CPBs.

Figure 2. MTT assays for (a) blank brushes and (b) drug-loaded brushes against CT26 cells after 24 h incubation. (c) IC50 calculated from MTT assay data. (d) Confocal laser scanning microscopy (CLSM) images and (e) mean fluorescence intensities measured by flow cytometry of the CT26 cells after 2 h incubation with the FITC-labeled BCPB-B-DOX and BCPB-DOX at 37 °C, respectively. Scale bars = 20 μm. ***P < 0.001 compared with BCPB-DOX group.

Figure 3. (a) Colocalization observation by CLSM of the FITC-labeled brushes (green) and LysoTracker (red) in CT26 cells. Scale bars = 20 μm. (b) Evolution with time of the Pearson’s correlation coefficients between the signals from the FITC-labeled CPBs and LysoTracker. (c) GeneOntology (GO) pathway cellular component (CC) analysis of BCPB-B in CT26 cells.

Figure 4. (a) Transcellular transfer study of the FITC-labeled BCPB-B and BCPB in CT26 cells. The left diagram illustrates the general experimental procedures. The cells on coverslips (I) were coincubated with the FITC-labeled CPBs for 4 h, washed with PBS, and imaged by CLSM. Thereafter, coverslips (I) were coincubated with coverslips (II) bearing fresh cells in fresh culture medium for 12 h. After repeating  the above process, coverslips (III) were obtained. The right picture shows the CLSM images of the cells on the coverslips (I), (II), and (III), respectively. Scale bars = 20 μm. (b) CLSM images of the optical slices through the centers of HepG2MCs incubated jointly with the FITC labeled BCPB-B and RBITC-labeled BCPB for different periods (left) and the corresponding fluorescence plate quantification data of the MCs (right). Scale bars = 100 μm.

Figure 5. (a) NIRF images of the H22 tumor-bearing mice and (b) mean fluorescence intensities of the tumors at different time points after tailvein injection of the NIR-797-labeled BCPB-B-DOX and BCPB-DOX, respectively. The tumor region is circled by a red dotted line. (c) NIRF images and (d) mean fluorescence intensities of the tumors and organs excised at 168 h after injecting the labeled BCPB-B-DOX and BCPB-DOX. (e) DOX concentrations in plasma versus time after tail vein injection of BCPB-B-DOX and BCPB-DOX. DOX concentrations in different tissues at different time points after tail-vein injection of (f) BCPB-B-DOX and (g) BCPB-DOX; data are presented as mean ± SD (n = 3). (h) AUC of DOX accumulation in tumors in BCPB-B-DOX and BCPB-DOX groups. (i) CLSM images of the frozen sections of the tumors from the mice at 96 h after tail-vein injection of the FITC-labeled BCPB-B and BCPB, respectively. Scale bars = 100 μm.

Figure 6. (a) Illustration of antitumor study schedule with one dose treatment of 4 mg/kg DOX equivalent. (b) Relative tumor volume and (c) survival rate of the H22 tumor-bearing mice after one dose treatment with different protocols indicated. *P < 0.05, compared with BCPB-DOX group from the 5th day. (d) Illustration of antitumor study schedule with multiple intravenous administrations with each dose of 4 mg/kg DOX equivalent. (e) Relative tumor volume of the H22 tumor bearing mice treated by five doses with different protocols indicated, and photographs of the sarcomas excised from the mice on the 13th day after the first treatment. *P < 0.05, compared with BCPB-DOX group from the 5th day. (f) Body weight change of the H22 tumor bearing mice treated by five doses with different protocols indicated. Data are presented as mean ± SD (n = 8).