br compared with SNPs In addition SNPs
compared with SNPs. In addition, SNPs was eliminated much faster than EBNPs during the first 2 h, which might be due to premature burst release as seen in in vitro release experiments. Compared with SNPs, the EBNPs showed a longer half time (t1/2), a higher area under the concentration-time curve (AUC), a smaller apparent volume of dis-tribution (Vz), a slower clearance rate (CL) and a longer mean residence time(MRT), indicating that EBNPs had longer circulation characteristics in the blood. The surface of EBNPs consists of the hydrophilic PEO segment. PEO prevents the opsonization of particle surface with pro-teins, which lead to the clearance of drug carriers by phagocytic U0126 . These results strongly suggest that EBNPs could reduce the
clearance rate of LA-SN38 and thereby extend its cycle time in the blood.
3.5. In vivo and ex vivo biodistribution studies
PEO-PBO NPs have been shown to reduce liver clearance and en-hance tumor targeting . Herein, we used PEO-PBO as polymer material to load LA-SN38 and prepare EBNPs, with the purpose of in-creasing the accumulation and penetration of the NPs at the tumor site. In vivo imaging is a noninvasive near-infrared fluorescence imaging in antitumor studies and is widely used to investigate the fate of a drug delivery system in live mice . Mice bearing HCT-116 tumors were intravenously injected with DiR-loaded EBNPs (at DiR equivalent dose of 1 mg/kg). After 2, 6, and 24 h post-injection, whole body imaging of mice was performed, respectively. As shown in Fig. 3B, fluorescence was mainly distributed in the tumor, and the fluorescence intensity was strengthened with the prolongation of time within 24 h, indicating that EBNPs had the advantage of targeting tumor and accumulated in tumor over time. In order to see the distribution of each organ more clearly, tumors and major organs were also separated for ex vivo imaging after the whole-body imaging. As shown in Fig. 3C and D, tumor tissues had the strongest fluorescence intensity compared to other organs. After 2 h, the fluorescence of other organs gradually decreased, but the fluores-cence of the tumor site gradually accumulated. These results were consistent with in vivo imaging study reported previously , and correlate well with uptake and pharmacokinetic data. Based on the above results, we concluded that EBNPs had the advantage of prolonging blood circulation and avoiding phagocytosis by the phago-cytic system, thereby targeting tumors .
3.6. In vivo antitumor efficacy
EBNPs were further evaluated in HCT-116 tumor-bearing mice. PBS, CPT-11, SNPs, and EBNPs were intravenously injected through a tail vein when the tumor volume reached ˜100 mm3. As shown in Fig. 4A, EBNPs were more effective in tumor inhibition compared to SNPs group and CPT-11group (P < 0.01). Furthermore, H&E and PCNA evaluation were used to assess the antitumor efficacy at the end of the study (Fig. 4C). Cell apoptosis patterns in tumor tissues both through H&E and PCNA evaluation were consistent with the results of tumor in-hibitory study. In H&E evaluation, the saline control group tumor had the histological features of rapid tumor growth, including the chro-matin cell nuclei, the cytoplasm was sparse, the nuclei were large, and the tumor cells were closely arranged . In contrast, the tumors of mice treated with CPT-11, SNPs and EBNPs all exhibited varying de-grees of shrinkage of tumor cells, most of the necrotic tissue was uni-formly stained red and cell separation occurred. The most obvious shrinkage of tumor cells was in the tumors of the EBNPs treated group, which indicated that the highest level of tumor necrosis was achieved after EBNPs treated. The PCNA evaluation also exhibited the lowest level of proliferating cell nuclear antigen in EBNPs treated tumors. All the results demonstrated EBNPs is a promising drug delivery system for treating colorectal tumor.
Toxicity assessment is very important for delivery systems used in vivo. Therefore, we monitored the change of body weight during the study and carried out the histopathological analysis of the main organ by H&E staining at the end of the study. As indicated in Fig. 4B, the body weight of EBNPs showed no significant change duration of the study, implying that there is no obvious systemic toxicity associated with this treatment. Furthermore, the histopathological analysis results (Fig. 4D) indicated that all formulations treatment groups had no ob-vious pathological symptoms compared to the saline group, which further confirmed the safety of EBNPs. All the results indicated that EBNPs were well tolerated at the tested dose. Colloids and Surfaces B: Biointerfaces 181 (2019) 822–829