Cubosomal nanoformulation increase <i>in vitro</i> dissolution and anticancer activity of Fisetin in A549 lung cancer cells

<b>Aim:</b> To prepare fisetin (FIS) cubosomal nanoformulation to increase aqueous solubility and anticancer activity. <b>Methods:</b> Top-down method using glyceryl monooleate (GMO) and Pluronic F-127. <b>Results:</b> Optimized using 2% GMO and 1% Pluronic F-127, reported 93.07 nm particle size, 80.10% drug entrapment, and reports more than 50% enhanced <i>in vitro</i> drug release than native FIS. MTT assay reports IC₅₀ Values of FIS 16.59 μg/ml and optimized cubosomal FIS nanoformulation (FISCUB) 12.18 μg/ml. The colony numbers observed in clonogenic assay for FISCUB were 8.33 ± 0.58 and FIS 11.67 ± 1.15. In flow cytometry study, apoptotic cells in FISCUB and FIS-treated A549 cells were found to be 33.4 and 6.83% respectively. <b>Conclusion:</b> A stable cubosomal nanoformulation of FIS showed enhanced aqueous solubility and anticancer activity. Fisetin (FIS) is a plant secondary metabolite from the class of flavonoids reported promising therapeutic potential. It possesses the problem of low aqueous solubility and bioavailability that restricts its utilization of maximum therapeutic benefits against its anticancer and other uses. The solubility and bioavailability issue can be solved using nanoformulations and many researchers have reported novel nanoparticulate drug-delivery systems and other phytoformulations. Using a top-down method, we prepared and optimized a novel FIS cubosomal formulation, which is a liquid dispersion of crystalline cubic nanoparticles of an amphiphilic lipid glyceryl monooleate. The Design expert^® software, a 3²-full factorial design was used for optimization. The particle size, entrapment efficiency, viscosity, pH and polydispersity index were tested. The previously reported HPLC analytical method was used for FIS estimation. The cubosomal formulation batch B2 prepared using 2% lipid (GMO) and 1% Pluronic F-127 was an optimized batch. High-resolution transmission electron microscopy and x-ray diffraction study were performed for the cubosomal particle characteristic study. <i>In vitro</i> release study confirms that the FIS release was more in the prepared cubosomal formulation. In the 3 month stability study, optimized batch B2 was found to be stable after 3 months. We observed transmission electron microscope images and confirmed the cubic structures of particles present in the nanorange concerning their size. The <i>in vitro</i> study of the optimized cubosomal formulation was carried out to study the effect of cubosomal formulation on cellular absorption and cytotoxicity studies. In the <i>in vitro</i> study, MTT, Clonogenic, Flow cytometry assay, and acridine orange-ethidium bromide staining were performed. The optimized cubosomal formulation reported more cytotoxicity results than the native FIS. In the FIS cubosomal nanoformulation study, we have prepared, and optimized cubosomal FIS nanoformulation with a simple procedure and a smaller number of ingredients for better delivery of fisetin.

**研究目的**：制备非瑟酮（fisetin, FIS）立方脂质体纳米制剂，以提升其水溶性与抗癌活性。
**实验方法**：采用自上而下法，以单油酸甘油酯（glyceryl monooleate, GMO）与泊洛沙姆F-127（Pluronic F-127）为原料制备制剂。
**实验结果**：本制剂最优配方为2% GMO与1% 泊洛沙姆F-127，所得纳米粒粒径为93.07 nm，药物包封率达80.10%，体外药物释放量较原生非瑟酮提升50%以上。MTT细胞活性实验结果显示，原生非瑟酮的半数抑制浓度（IC₅₀）为16.59 μg/ml，优化后的立方脂质体非瑟酮纳米制剂（FISCUB）则为12.18 μg/ml。集落形成实验结果显示，FISCUB组的细胞集落数为8.33±0.58，原生非瑟酮组为11.67±1.15。流式细胞术检测结果表明，经FISCUB与原生非瑟酮处理的A549细胞中，凋亡细胞占比分别为33.4%与6.83%。
**研究结论**：本研究所制备的稳定非瑟酮立方脂质体纳米制剂，可显著提升药物水溶性与抗癌活性。

非瑟酮（FIS）属于黄酮类植物次生代谢物，已被证实具有良好的治疗潜力。但其水溶性与生物利用度较低，限制了其在抗癌及其他治疗领域中最大疗效的发挥。纳米制剂可有效解决上述溶解度与生物利用度问题，目前已有众多学者报道了新型纳米粒给药系统及其他植物源制剂。本研究采用自上而下法，制备并优化了一种新型非瑟酮立方脂质体制剂：该制剂是以两亲性脂质单油酸甘油酯为基质的结晶性立方纳米粒液体分散体系。本研究采用3²全因子设计，借助Design-Expert®软件完成制剂优化。考察指标包括粒径、包封率、黏度、pH值与多分散性指数。采用已报道的高效液相色谱（HPLC）法对非瑟酮进行含量测定。采用2%脂质（GMO）与1%泊洛沙姆F-127制备的B2批立方脂质体制剂为最优批次。通过高分辨透射电子显微镜与X射线衍射实验，对立方脂质体纳米粒的形貌与结构进行表征。体外释放实验结果证实，所制备的立方脂质体制剂的非瑟酮释放量更高。3个月稳定性考察结果显示，最优批次B2在放置3个月后仍保持稳定。透射电子显微镜图像证实，所得纳米粒为立方结构，粒径处于纳米尺度范围内。针对最优立方脂质体制剂开展体外实验，以考察其对细胞摄取与细胞毒性的影响。体外实验涵盖MTT细胞活性实验、集落形成实验、流式细胞术检测以及吖啶橙-溴化乙锭双染色实验。实验结果表明，最优立方脂质体制剂的细胞毒性显著高于原生非瑟酮。本研究通过简易的制备流程与更少的辅料种类，成功制备并优化了非瑟酮立方脂质体纳米制剂，以实现非瑟酮的高效递送。