br Table IC M CPT concentration of
Table 2 IC50 (μM, CPT concentration) of NPs against CT-26 cells.
Incubation time CUL-CPT-NPs CS-CPT-NPs
treated with CUL-CPT-NPs at the time points of 0.5, 1 and 3 h, re-spectively. These findings suggest that surface functionalization with CS is liable to endow NPs with colon cancer-targeted capacity. Moreover, we found that although the fluorescence signals in PEG300 remarkably increased with the incubation time (Fig. 3a), the percentages of blue fluorescence-positive cells were 79.8% and 81.6% after the treatment with CS-CPT-NPs for 1 h and 3 h, respectively (Fig. 3b). These results were consistent with our previous study . It was found that the
fluorescence intensities of cells with the treatment of NPs could increase even without increases in the percentages of fluorescence-positive cells. To further confirm the CD44-mediated cellular uptake of NPs, we evaluated the cell internalization eﬃciency of CS-CPT-NPs in the pre-sence of free CS. Fig. 3b indicated that the cellular uptake eﬃciencies of CS-CPT-NPs significantly decreased in the medium supplemented with free CS, revealing that CS-CPT-NPs were taken up into CT-26 cells via CD44-mediated endocytosis.
3.4. In vitro anti-colon cancer activities of NPs
To evaluate the anti-colon cancer activities of NPs, we treated CT-26 cells with these NPs for 24 h (Fig. 4a) and 48 h (Fig. 4b), respectively. We found that CUL-CPT-NPs and CS-CPT-NPs showed anti-colon cancer activities as a function of drug concentration and co-incubation time.
Fig. 5. In vivo anti-colon cancer activities of CUL-CPT-NPs and CS-CPT-NPs. (a) Changes in body weight in diﬀerent treatment groups. Mouse body weight was normalized to day 0 body weight (expressed as a percentage). (b) Tumor growth profiles, (c) tumor images and (d) tumor weights in diﬀerent groups with the treatment of CUL-CPT-NPs or CS-CPT-NPs. Statistical significance was assessed using ANOVA followed by a Bonferroni post-hoc test (*P < 0.05 and **P < 0.01). Each point represents the mean ± S.E.M. (n = 5).
Furthermore, the anti-colon cancer activities of CS-CPT-NPs were sig-nificantly stronger than that of CUL-CPT-NPs, which might be attrib-uted to the improved cellular uptake of CS-functionalized NPs. To further quantitatively characterize the anti-cancer activities of NPs, we obtained IC50 values of these two types of NPs based on MTT results. As summarized in Table 2, the IC50 values for CUL-CPT-NPs were 6.312 μM and 0.655 μM at 24 h and 48 h, respectively, which was 6.2- and 3.1-fold higher than the corresponding IC50 values for CS-CPT-NPs. To further quantitatively compare the pro-apoptosis eﬀects of CUL-CPT-NPs and CS-CPT-NPs, we treated CT-26 cells with these two types of NPs, and stained these cells with an Annexin V-FITC/PI Apoptosis De-tection Kit. As seen in Fig. 4c and Fig. S2, the percentages of viable cells decreased with increasing the NP-treated time. Moreover, CS-CPT-NP-treated cells had 1.6-, 1.2- and 1.5-fold higher percentages of apoptotic cells than CUL-CPT-NP-treated cells. The above results clearly suggest that CS-CPT-NPs have much stronger in vitro anti-cancer activities than CUL-CPT-NPs.
3.5. Hemolysis assay
The blood compatibility is a critical parameter for in vivo application of nanotherapeutics . As presented in Fig. S3a,b, there was no distinguishable evidence of hemolysis for erythrocytes treated with CS-CPT-NPs under the CPT concentration of 100 μM, which was sig-nificantly higher than the concentrations applied in cell experiments. Additionally, in vivo hemocompatibility results (Fig. S3c) indicated that all the tested blood parameters, including red blood cells (RBC), he-moglobin (HGB), mean cell hemoglobin (MCH) and mean corpuscular