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 degrees C, reaching the maximum Asp release at 40 degrees C. The MTT assay showed that the optimal Asp/TLs exhibited the highest cytotoxic activity upon exposure to mild hyperthermia (40 degrees C) against the invasive triple-negative breast cancer cell line (MDA-MB-231) when compared to other preparations. The IC50 of Asp/TLs (40 degrees C) was estimated at 0.9 mu g mL(-1), while that of free Asp (40 degrees C) was 3.83 mu g 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 degrees C were 6.6 mu g mL(-1) and 186 mu g mL(-1), respectively. This indicated that Asp was released only when placed at 40 degrees C. The apoptosis assay revealed that Asp/TLs (40 degrees 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 degrees C). Asp/TLs (40 degrees C) and Asp (40 degrees 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 degrees 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 degrees 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 degrees C) treatment displayed the most potent cytotoxic profile and induced both apoptosis and necrosis in MDA-MB-231.

Automated summary

In this study, thermo-responsive liposomes (TLs) loaded with Asp (Asp/TLs) were prepared and optimized. The optimized Asp/TLs exhibited desirable physicochemical properties and high encapsulation efficiency. Asp/TLs showed the ability to release Asp under elevated temperatures and displayed high cytotoxic activity against invasive triple-negative breast cancer cells. The induction of apoptosis was achieved through the regulation of apoptotic gene expression.