Validation of the optimum formula.

The goal of this study was the formulation and optimization by statistical means of bilosomal formulations of axitinib (AXT) in order to improve its anticancer efficacy in a targeted manner. A central composite rotatable design was employed Using Design-Expert® software. The formulation factors were cholesterol, span 60, and sodium deoxy cholate (SDC) amounts (mg), whereas the dependent responses were Entrapment efficiency (EE%), Vesicles’ size (VS), and Zeta potential (ZP). The design expert software was utilized to perform the numerical optimization process. The optimized bilosomal formulation was assessed using differential scanning calorimetry (DSC), X-ray diffraction (XRD), transmission electron microscope (TEM), in-vitro release study, short-term stability study, and in-vitro cell proliferation assay and flow cytometry on MCF-7 breast and OV-2774 ovarian cancer cell lines. The optimized formulation was found to be composed of 19.999, 111.869 and 15 mgs of cholesterol, span 60, and SDC, respectively with a desirability of 0.753. EE%, VS, and ZP were predicted to be 88.4977%, 594.592 nm, and −44.2354 mV, respectively. The validation process on the optimized formula demonstrated that the variation from the predicted responses was less than 5%. The DSC and XRD studies revealed that AXT was entrapped within the bilosomal vesicles. The optimized AXT bilosomal formulation exhibited spherical non-aggregated nanovesicles in TEM images. Furthermore, it improved AXT release when compared to AXT suspension. According to stability experiments, the optimum bilosomal formulation was stable for thirty days. The cytotoxicity of the optimized bilosomal formulation was enhanced on the MCF-7 breast and OV-2774 ovarian cancer cell lines compared to AXT suspension even at lower concentrations. Flow cytometry showed that AXT loaded BSMs made a significant increase in the percentage of apoptotic cells in MCF-7 and OV-2774 cells, respectively. Molecular docking suggests that axitinib and SDC decreased the activation of the caspase-8 receptor on the surface of ovarian and breast cancer, which consequently led to an increase in anticancer activity. So, BSMs might be regarded a promising carrier of AXT to target ant treat breast and ovarian cancers.本研究旨在通过统计学手段开发并优化阿昔替尼（axitinib, AXT）双质体制剂，以靶向提升其抗癌疗效。本研究借助Design-Expert®软件，采用中心复合旋转设计（central composite rotatable design）开展实验。考察的处方因素为胆固醇、司盘60（Span 60）与脱氧胆酸钠（sodium deoxy cholate, SDC）的投加量（单位：mg），考察的响应指标包括包封率（Entrapment efficiency, EE%）、囊泡粒径（Vesicles’ size, VS）与Zeta电位（ZP）。随后借助Design-Expert®软件完成数值优化流程。对优化得到的双质体制剂，采用差示扫描量热法（differential scanning calorimetry, DSC）、X射线衍射（X-ray diffraction, XRD）、透射电子显微镜（transmission electron microscope, TEM）、体外释放实验、短期稳定性实验，以及针对MCF-7乳腺癌细胞与OV-2774卵巢癌细胞系的体外细胞增殖实验与流式细胞术进行表征与评价。最终优化得到的处方组成为：胆固醇19.999 mg、司盘60 111.869 mg、脱氧胆酸钠15 mg，综合满意度为0.753。预测得到的包封率、粒径与Zeta电位分别为88.4977%、594.592 nm与-44.2354 mV。对优化处方的验证实验结果显示，实测响应值与预测值的偏差小于5%。差示扫描量热法与X射线衍射实验结果表明，阿昔替尼成功包埋于双质体囊泡内部。透射电子显微镜图像显示，优化后的阿昔替尼双质体制剂呈现无聚集的球形纳米囊泡结构。此外，与阿昔替尼混悬液相比，该制剂可显著提升阿昔替尼的体外释放效果。稳定性实验结果表明，最优双质体制剂可稳定保存30天。相较于阿昔替尼混悬液，优化后的双质体制剂在低浓度下即可提升对MCF-7乳腺癌细胞与OV-2774卵巢癌细胞的细胞毒性。流式细胞术结果显示，载阿昔替尼双质体制剂可分别显著提升MCF-7与OV-2774细胞的凋亡率。分子对接实验结果表明，阿昔替尼与脱氧胆酸钠可下调卵巢癌与乳腺癌细胞表面的半胱天冬酶-8（caspase-8）受体活化水平，进而增强抗癌活性。综上，双质体纳米囊泡（BSMs）可作为阿昔替尼的潜在递送载体，用于靶向治疗乳腺癌与卵巢癌。