br Table br The main
The main pharmacokinetic parameters after a single intravenous injection. SNPs and EBNPs were given to SD rats at the LA-SN38 dose of 8 mg/kg, respectively (n = 5).
Parameter t1/2 Cmax AUC0-24h MRT Vz CL
only 34.93% of SN38 released after 24 h. This result suggests that EBNPs released slowly and has no burst release compared to SNPs which released 61.79% at the same time point. Furthermore, EBNPs were found to be stable when stored at 4℃, with no visible aggregations formed during several days.
All the above results confirmed that PEO-PBO as a material could be loaded with LA-SN38, and form EBNPs with small size and narrow distribution. More importantly, the obtained EBNPs effectively con-trolled the release rate of SN38.
3.2. In vitro cytotoxicity and apoptosis assay of EBNPs
Colon cancer is one of the most common malignant tumors. The antitumor activity of EBNPs was evaluated with human colon cancer cell lines HT-29 and HCT-116 by CCK8 assay. As shown in Fig. 2, EBNPs showed high antitumor activity in vitro comparable to CPT-11 and SNPs. The IC50 (half maximal inhibitory concentration) values of EBNPs in HT-29 and HCT-116 were 0.63 and 5.56 μg/mL, respectively, which was ˜3- and 14-fold higher than that of SNPs, and much higher than that of CPT-11(Table 1). These data indicate that EBNPs could effectively enter NPI-0052 and release SN38 to exert antitumor effects.
An Annexin V-FITC and PI double-staining experiment was carried out to investigate the apoptosis induced by different formulations in HCT-116 cells. The ratio of HCT-116 cells undergoing early and late apoptosis was increased with EBNPs treated (Fig. 2E). The total per-centage of apoptotic cells was the sum of cells in early and late apop-tosis. After 24 h of treatment, compared with PBS group (9.66% of cells were apoptotic), 38.33%, 30.01%, 19.64% and 9.66% of cells were
apoptotic at equivalent SN38 for EBNPs, SNPs, and CPT-11, respec-tively. This result is consistent with cytotoxicity assays in vitro, de-monstrating EBNPs were an effective drug delivery system for the transport of drugs into cells.
3.3. Uptake of EBNPs in HCT-116 cells and macrophages
The macrophage is a critical element in the defense system. Particulate drug carriers, including liposomes, NP, micelle can be re-cognized as a foreign object by the mononuclear phagocyte system (MPS) cells and are rapidly removed from circulation . The ideal drug carrier should be able to reduce the recognition of phagocytic system and prolong their circulation in the blood system. To investigate whether EBNPs can improve the uptake of LA-SN38 by cancer cells and reduce uptake by macrophage cells, we conducted the uptake experi-ments in the human colorectal cancer cells of HCT-116 and macro-phages cells of RAW264.7. Cells were treated with EBNPs and SNPs (100 μg/mL, SN38 equivalent) for the designed time, respectively. Then the cells were collected, washed, lysed, extract and quantified by HPLC (Fig. 2D and E). In contrast to SNPs, EBNPs significantly facilitated HCT-116 cells uptake (Fig. 2D). In parallel, for macrophage RAW264.7 cells, EBNPs was obviously reduced uptake compared to SNPs (Fig. 2E). EBNPs used PEO-PBO as a polymer material, the PEO block is non-toxic, highly hydrated and can effectively prevent the adsorption of proteins and adhesion of cells . Overall, these results demonstrated that the EBNPs could avoid the phagocytosis by macrophages and facilitate the uptake by tumor cells. Furthermore, EBNPs may have the potential to prolong blood circulation and targeted tumor site.
Fig. 4. In vivo evaluation of EBNPs in tumor-bearing mice. Tumor volume (A) and body weight analysis of mice bearing HCT-116 tumor treated by various formulations (SN38 equivalent, 10 mg/kg) (n = 8). *p < 0.05, **p < 0.01. Arrows represent the intravenous injections. C) H&E and PCNA staining analysis of the tumors after being excised at the end of the study. (D) Histopathology of liver, lung, and kidney of mice in the respective groups dissected at the end of the study.
3.4. Pharmacokinetic study
The pharmacokinetic profile was used to quantitatively record the metabolic process of drugs in the body and the crucial pharmacokinetic parameters were obtained . Fig. 3A showed the mean plasma concentration-time profiles of SN38 after intravenous injected of EBNPs through SD rat tail vein at the LA-SN38 dose of 8 mg/kg with SNPs as contrast. The concentration versus time data was fitted using a non-compartmental model, and the main pharmacokinetic parameters of formulations are summarized in Table 2. EBNPs were more slowly cleared in the blood circulation after intravenous administration