Docetaxel-Loaded Methoxy poly(ethylene glycol)-poly (L-lactic Acid) Nanoparticles for Breast Cancer: Synthesis, Characterization, Method Validation, and Cytotoxicity

Abstract

This study aimed to synthesize and characterize DTX-mPEG-PLA-NPs along with the development and validation of a simple, accurate, and reproducible method for the determination and quantification of DTX in mPEG-PLA-NPs. The prepared NPs were characterized using AFM, DLS, zetasizer, and drug release kinetic profiling. The RP-HPLC assay was developed for DTX detection. The cytotoxicity and anti-clonogenic effects were estimated using MTT and clonogenic assays, respectively, using both MCF-7 and MDA-MB-231 cell lines in a 2D and 3D culture system. The developed method showed a linear response, high precision, accuracy, RSD values of ≤2%, and a tailing factor ≤2, per ICH guidelines. The DTX-mPEG-PLA-NPs exhibited an average particle size of 264.3 nm with an encapsulation efficiency of 62.22%. The in vitro drug kinetic profile, as per the Krosmeyers-Peppas model, demonstrated Fickian diffusion, with initial biphasic release and a multistep sustained release over 190 h. The MTT assay revealed improved in vitro cytotoxicity against MCF-7 and MDA-MB-231 in the 2D cultures and MCF-7 3D mammosphere cultures. Significant inhibitions of the clonogenic potential of MDA-MB-231 were observed for all concentrations of DTX-mPEG-PLA-NPs. Our results highlight the feasibility of detecting DTX via the robust RP-HPLC method and using DTX-mPEG-PLA-NPs as a perceptible and biocompatible delivery vehicle with greater cytotoxic and anti-clonogenic potential, supporting improved outcomes in BC.

Keywords: breast cancer; docetaxel; mPEG-PLA; nanoparticles; validation method.

Figure 1

Particle size distribution and morphological analysis. (A) Topography image of B-NPs. (B) Differential phase micrographs of B-NPs. (C) Topography images of DTX-mPEG-PLA-NPs. (D) Differential phase micrographs of DTX-mPEG-PLA-NPs. (E) DLS of B-NPs, the cumulative particle size distribution curve, and D80 average particle size of B-PNs—198.7 nm. (F) DLS of DTX-mPEG-PLA-NPs, the cumulative particle size distribution curve, and D80 average particle size of DTX-mPEG-PLA-NPs—264.3 nm.

Figure 2

In vitro drug release kinetics. (A) In vitro drug release profiles of Free-DTX at pH 5.5 and 7.4 and DTX-mPEG-PLA-NPs. (B) Korsmeyer–Peppas mathematical model of drug release kinetics at pH 7.4. (C) Korsmeyer–Peppas mathematical model of drug release kinetics at pH 5.5.

Figure 3

Cell viability MTT assay. (A) Micrograph of MCF-7 2-D monolayer culture. (B) Micrograph of MDA-MB-231 monolayer culture. (C–E) Mammosphere generation, micrograph of 3D culture from day 7 to day 15. (F) Percent cell viability of MCF-7 cells against DTX-mPEG-PLA-NPs and Free-DTX in 2D culture. (G) Percent cell viability of MDA-MB-231 cells against DTX-mPEG-PLA-NPs and Free-DTX in 2D culture. (H) Percent cell viability of MCF-7 cells against DTX-mPEG-PLA-NPs and Free-DTX in 3D mammosphere culture. (I,J) Micrographs of MCF-7 3-D mammospheres after 15 days of incubation with Free-DTX (I) and DTX-MPEG-PLA-NPs (J). IC50 half maximum inhibitory concentration, (Scale bar; (A–E) and (I,J): 200 μm). Results were represented as mean ± SD (n = 3). Asterisk (*) represents the significant alpha values.

Figure 4

Clonogenic assay. (Ai–vi) Crystal violet staining of MDA-MB-231 cells after 21 days of incubation with DTX-mPEG-PLA-NPs and Free-DTX in 2D culture. (Aii–iv) Inhibition of the clonogenic potential of MDA-MB-231 cells at day 21 against 0.1, 6, and 12 μg/mL concentrations of Free-DTX. (Av–vii) Inhibition of the clonogenic potential of MDA-MB-231 cells at day 21 against 0.1, 6, and 12 μg/mL concentrations of DTX-MPEG-PLA-NPs. (B) Survival fraction of MDA-MB-231 cells after treatment with 0.1, 6, and 12 μg/mL concentrations of DTX-mPEG-PLA-NPs and Free-DTX. A represents the figure panel, while Roman number (i or ii) represents the number of figures in panel A. Asterisk (*) represents the significant alpha values.