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

Abstract

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 (<i>P</i> ≤ 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 <i>Bak</i> and <i>Bax</i>, while downregulating the anti-apoptotic genes, <i>BCL-xL</i> and <i>BCL-2</i>. The free Asp (40 °C) increased the gene expression of <i>Bak</i> and <i>Bax</i> by 4.4- and 5.2-folds, while reducing the expression of <i>BCL-xL</i> and <i>BCL-2</i> by 50% and 73%, respectively. The optimal Asp TLs (40 °C) manifested more potent effects as demonstrated by the upregulation of <i>Bak</i>, <i>Bax</i>, and <i>P53</i> by 5.6-, 7.2-, and 1.3-folds, as well as the downregulation of <i>BCL-xL</i> and <i>BCL-2</i> 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.