Marizomib br were used and mL of DiR loaded EBNPs
were used and 0.2 mL of DiR-loaded EBNPs (100 μg/mL of DiR) were injected via tail vein. At the designed time point (2 h, 6 h and 24 h), the mice were anesthetized and fluorescent images were detected using an Clairvivo OPT in vivo imaging system of (SHIMADZU Corporation, Kyoto, Japan). After imaging, the mice were euthanized and major organs including hearts, livers, spleens, lungs, kidneys, and tumors were excised. The fluorescence signal intensities of tissues at different time point were also measured. r> 2.11. In vivo antitumor efficacy
HCT-116 tumor-bearing mice with ˜200 mm3 of tumor volume were randomly divided into four groups, i.e., PBS, CPT-11, SNPs, and EBNPs, with each group having eight mice.
The treatment was repeated via the tail vein every 3 days for a total of 4 injections at a dose of 10 mg/kg (SN38 equivalent). The mice were weighed and their tumors were measured with calipers every 2 or 3 days, the volume was calculated according to the formula: vo-lume = [length × (width)2]/2. Mice were sacrificed when the tumor exceeded a predetermined threshold of 1500 mm3. Tumors and main organs were excised and fixed in 4% paraformaldehyde in phosphate-buffered saline for histological assay. The fixed tumors and organs were embedded in paraffin, sections were stained with hematoxylin and eosin (H&E) and evaluated by microscopy. The tumor sections were also probed with Marizomib against proliferating cell nuclear antigen (PCNA) to detect the degree of cell proliferation.
2.12. Statistical analysis
Data are presented as the means ± standard deviation (S.D). Significant differences were determined using the Student’s t-test. P-value < 0.05 was considered statistically significant, while P-value < 0.01 was considered highly significant.
Fig. 2. In vitro cytotoxicity of EBNP and other formulations. Human colon cancer cell lines HT-29(A) and HCT-116 (B) were used. The cell viability was determined using CCK8 assay after 48 h treatment. (C) Apoptosis of HCT-116 cells analyzed by FCM using Annexin V/PI double staining kit after 24 h treatment (5 μM, SN38 equivalent). Bottom left quadrants, viable cells; bottom right quadrants, early apoptotic cells; top right quadrants, late apoptotic cells; top left quadrants, necrotic cells. Drug uptake in HCT-116 (D) and RAW264.7 (E) cells uptake efficiency efficacy of formulations (100 μg/ml, SN38 equivalent) over 12 h. Statistically significant differences are marked as *p < 0.05, **p < 0.01.
Cell line IC50 value (μg/ml)
CPT-11 SNPs EBNPs
3. Results and discussion
3.1. Preparation s and characterization of EBNPs
Various derivatives of SN38 have been synthesized in order to in-crease the hydrophilicity of SN38 [19–21]. Wang et al. have synthe-sized LA-SN38 and demonstrated that it could self-assemble to form NPs . At the same time, a liposomal system was used to load LA-SN38. The results confirmed that the anti-tumor effect of LA-SN38 was im-proved through formulation modification . Polymer NPs with a core-shell structure could effectively encapsulate drug into the hydro-phobic core, and protect the drug from hydrolysis and prolong drug circulation in the blood [23–25]. Moreover, the passive accumulation of polymer NPs in the solid tumor can be achieved by the EPR effect
[26–28]. PEO-PBO is a newly developed material. Wang et al. firstly used PEO-PBO to encapsulate PTX, which proved that PEO-PBO was superior to PEG-PLA, and PEO-PBO material could reduce liver clear-ance, thereby enhancing tumor targeting . Therefore, we chose PEO-PBO as drug delivery material to encapsulate LA-SN38 and pro-duce EBNPs. PEO-PBO is an amphiphilic material. When the organic phase was dripped into water phase, the hydrophilic segment PEO faced to the water, the hydrophobic segment PBO formed to a core, then the hydrophobic drug LA-SN38 is transferred and entrapped into the hy-drophobic core, the outer PEO layer is equivalent to a shell. When the organic solvent was removed, the drug-loaded nanoparticles having a stable core-shell structure were finally formed. To study the physico-chemical properties of EBNPs, the particle size, morphology, EE, and LC were analyzed.
In vitro release experiment was performed to confirm that EBNPs formulations had lower SN38 release rates compared to SNPs . As shown in Fig. 1C, EBNPs slowly released SN38 in dialysis media, with
Fig. 3. In vivo behaviors of EBNPs after in-travenous administration. (A) Pharmacokinetics of SNPs and EBNPs after intravenous injection (n = 5) of (SN38 equivalent, 10 mg/kg) from 0 to 24 h (n = 5). (B) Fluorescence imaging of DiR-loaded EBNPs in HCT-116 tumor-bearing mice. Fluorescence imaging (C) and the quantification of the mean fluorescent intensity (D) of dissected organs by IVIS imaging (n = 3).