Box–Behnken design of thermo-responsive nano-liposomes loaded with a platinum(IV) anticancer complex: evaluation of cytotoxicity and apoptotic pathways in triple negative breast cancer cells

Herein, thermo-responsive liposomes (TLs) loaded with Asp (Asp/TLs) were produced by self-assembling DPPC, DSPE-PEG2000, and cholesterol. The preparation variables were optimized using the Box–Behnken design (BBD). The optimized Asp/TLs exhibited an average particle size of 114.05 ± 1.56 nm, PDI of 0.15 ± 0.015, zeta potential of −15.24 ± 0.65 mV, and entrapment efficiency (EE%) of 84.08 ± 2.75%. In addition, under physiological conditions, Asp/TLs showed spherical shape, outstanding stability and thermo-triggered the release of Asp at 38 °C, reaching the maximum Asp release at 40 °C. The MTT assay showed that the optimal Asp/TLs exhibited the highest cytotoxic activity upon exposure to mild hyperthermia (40 °C) against the invasive triple-negative breast cancer cell line (MDA-MB-231) when compared to other preparations. The IC50 of Asp/TLs (40 °C) was estimated at 0.9 μg mL−1, while that of free Asp (40 °C) was 3.83 μg mL−1. As such, the optimal Asp/TLs were shown to increase the cytotoxic activity of Asp by 4-fold upon exposure to mild hyperthermia. The IC50 values of Asp and Asp/TLs without exposure to 40 °C were 6.6 μg mL−1 and 186 μg mL−1, respectively. This indicated that Asp was released only when placed at 40 °C. The apoptosis assay revealed that Asp/TLs (40 °C) caused a remarkable increase in the percentage of cell population among both the late apoptosis and necrosis quartiles, as well as a significant decline in the viable cell quartile (P ≤ 0.001) when compared to Asp (40 °C). Asp/TLs (40 °C) and Asp (40 °C) could stimulate the intrinsic apoptosis pathway by upregulating the apoptotic genes Bak and Bax, while downregulating the anti-apoptotic genes, BCL-xL and BCL-2. The free Asp (40 °C) increased the gene expression of Bak and Bax by 4.4- and 5.2-folds, while reducing the expression of BCL-xL and BCL-2 by 50% and 73%, respectively. The optimal Asp TLs (40 °C) manifested more potent effects as demonstrated by the upregulation of Bak, Bax, and P53 by 5.6-, 7.2-, and 1.3-folds, as well as the downregulation of BCL-xL and BCL-2 by 70% and 85%, respectively. As such, the optimal Asp TLs (40 °C) treatment displayed the most potent cytotoxic profile and induced both apoptosis and necrosis in MDA-MB-231.

本研究所制备的含Asp（Asp/TLs）的热响应脂质体（TLs）是通过自组装二棕榈酰磷脂酸（DPPC）、聚乙二醇2000的DSPE和胆固醇而得。采用Box–Behnken设计（BBD）优化了制备变量。优化的Asp/TLs表现出平均粒径为114.05 ± 1.56纳米，PDI为0.15 ± 0.015，ζ电位为−15.24 ± 0.65毫伏，以及包封效率（EE%）为84.08 ± 2.75%。此外，在生理条件下，Asp/TLs呈现球形，具有卓越的稳定性，并在38°C的温度下触发Asp的热释放，于40°C时达到Asp释放的最大值。MTT试验表明，相较于其他制剂，在40°C的轻度高温下，优化的Asp/TLs对侵袭性三阴性乳腺癌细胞系（MDA-MB-231）展现出最高的细胞毒性活性。Asp/TLs在40°C下的半数抑制浓度（IC50）估计为0.9 μg mL−1，而游离Asp的IC50为3.83 μg mL−1。因此，优化的Asp/TLs在轻度高温下能够将Asp的细胞毒性活性提高4倍。未经40°C处理的Asp和Asp/TLs的IC50值分别为6.6 μg mL−1和186 μg mL−1。这表明Asp仅在40°C时释放。凋亡试验揭示，与40°C下的Asp相比，Asp/TLs（40°C）在晚凋亡和坏死四分位数中细胞群体的百分比显著增加，同时存活细胞四分位数的显著减少（P ≤ 0.001）。Asp/TLs（40°C）和Asp（40°C）可通过上调凋亡基因Bak和Bax，下调抗凋亡基因BCL-xL和BCL-2来刺激内在凋亡途径。游离Asp（40°C）将Bak和Bax的基因表达上调了4.4-和5.2倍，同时将BCL-xL和BCL-2的表达分别降低了50%和73%。优化的Asp TLs（40°C）表现出更显著的效果，表现为Bak、Bax和P53的上调幅度分别为5.6-、7.2-和1.3倍，以及BCL-xL和BCL-2的下调幅度分别为70%和85%。因此，优化的Asp TLs（40°C）治疗显示出最强的细胞毒性特征，并在MDA-MB-231细胞中诱导凋亡和坏死。