Polymeric Nanoparticles Potentiate the Anticancer Activity of Novel PI3Kα Inhibitors Against Triple-Negative Breast Cancer Cells

Abstract

Background: Dysregulation in phosphoinositide-3-kinase alpha (PI3Kα) signaling is implicated in the development of various cancers, including triple-negative breast cancer (TNBC). We have previously synthesized a series of N-phenyl-6-chloro-4-hydroxy-2-quinolone-3-carboxamides as targeted inhibitors against PI3Kα. Herein, two drug candidates, R7 and R11, were selected to be further investigated as a nanoparticle (NP) formulation against TNBC. Methods: R7 and R11 were entrapped in D-α-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS) polymeric NPs by nanoprecipitation. Following their physicochemical characterization, the anticancer activity of the compounds and their NP formulations was evaluated in the TNBC cell line MDA-MB-231 by conducting viability, uptake, and apoptosis assays, as well as penetration assays in a multicellular tumor spheroid model. Results: The NPs exhibited a particle size of 100-200 nm, excellent drug loading efficiencies, and sustained release under physiologic conditions. Viability assays revealed superior potency for the NP formulations, with IC₅₀ values of 20 µM and 30 µM for R7- and R11-loaded NPs, respectively, compared to the free compounds, which exhibited IC₅₀ values of 280 µM and 290 µM for R7 and R11, respectively. These results were attributed to the inherent antiproliferative activity of TPGS, as evidenced by the cytotoxicity of the drug-free NPs, as well as the enhanced cellular uptake enabled by the NP vehicle, as demonstrated by fluorescence microscopy imaging and flow cytometry measurements. Further investigations showed that the NPs promoted apoptosis via a mitochondrial-dependent pathway that involved the activation of proapoptotic caspases. Moreover, the NP formulations enhanced the penetration ability of the free compounds in multicellular tumor spheroids, causing a time- and concentration-dependent disruption of the spheroids. Conclusions: Our findings highlight the important role nanotechnology can play in improving the biopharmaceutical properties of new drug candidates and facilitating their in vivo translation.

Keywords: PI3Kα; TPGS; polymeric nanoparticles; protein kinases; triple-negative breast cancer.

Figure 1

(A) Structure of R7 and R11 compounds; (B) Structure of TPGS; (C) Preparation of R7- and R11-loaded TPGS NPs by nanoprecipitation.

Figure 2

Representative intensity-weighted particle size distributions of (A) R7 NP2 and (B) R11 NP2.

Figure 3

Characterization of R7 and R11 NPs by (A) UV–Vis and (B) FT-IR spectroscopy; (C) In vitro release of R7 and R11 from their respective NPs in PBS pH 7.4, 37 °C (mean ± SD; n = 3); (D) Stability of R7 and R11 NPs upon incubation with RPMI 1640 medium supplemented with 10% FBS for 1 h at 37 °C. Results are expressed as the fold increase in size relative to fresh NPs (mean ± SD; n = 3).

Figure 4

Viability (mean ± SD; n = 5) of MDA-MB-231 cells upon treatment with the free compounds R7 and R11, their corresponding NPs, blank (drug-free) NPs diluted to equivalent concentrations as the drug-loaded NPs, and doxorubicin HCl (DOX) for 72 h.

Figure 5

Uptake of C6-labeled NPs by MDA-MB-231 cells. (A) Fluorescence microscopy images and (B) flow cytometry histograms of cells incubated with free coumarin 6 (C6) or an equivalent concentration of C6-loaded NPs for 1 h compared to the control; (C) Cell-associated fluorescence obtained from flow cytometry measurements expressed as the fold increase in fluorescence intensity compared to the control (mean ± SD; n = 3; * p < 0.05).

Figure 6

Proapoptotic activity of the free compounds R7 and R11, their corresponding NPs, and blank (drug-free) NPs in MDA-MB-231 cells. (A) JC-1 mitochondrial membrane potential assay results expressed as the JC-1 monomer/aggregate ratio normalized to the control (untreated) cells (mean ± SD; n = 3); (B) Activity of caspase-3, (C) caspase-8, and (D) caspase-9 upon treating the cells with IC₅₀ concentrations of each material for 24 h. Results are expressed as the expression level of each caspase normalized to the control (untreated) cells (mean ± SD; n = 3). * p < 0.05, ** p < 0.01, and **** p < 0.0001 compared to the control; ^# p < 0.05 and ^#### p < 0.0001 comparing R7 NPs and free R7; ^$$$$ p < 0.0001 comparing R11 NPs and free R11; ^& p < 0.05 and ^&&&& p < 0.0001 comparing R7 NPs and R11 NPs to blank NPs.

Figure 7

Merged red and green fluorescence microscopy images of MDA-MB-231 cells stained with JC-1 following treatment with IC₅₀ concentrations of R7 and R11, their corresponding NPs, and blank (drug-free) NPs for 24 h.

Figure 8

Effect of R7- and R11-loaded NPs on multicellular MDA-MB-231 spheroids compared to the free compounds and blank NPs. The spheroids were imaged at 0 and 72 h after treatment with 10 and 100 µM of each material (scale bar = 200 µM).