Tailoring micellar nanocarriers for pemetrexed in breast cancer: design, fabrication and in vitro evaluation

Abstract

Aim: To investigate the pemetrexed encapsulated polymeric mixed micelles (PMMs) against breast cancer treatment.Methods: We meticulously optimized the formulation and conducted extensive characterizations, including photon correlation spectroscopy for micellization, advanced analytical techniques and in vitro cell line assessments.Results: The PMM exhibited favorable characteristics, with a spherical morphology, hydrodynamic particle size of 19.58 ± 0.89 nm, polydispersity index of 0.245 ± 0.1, and a surface charge of -9.70 ± 0.61 mV. Encapsulation efficiency and drug payload reached 96.16 ± 0.37% and 4.5 ± 0.32%, respectively. Cytotoxicity analysis indicated superior efficacy of the PMM over the drug solution.Conclusion: The PMM formulation exhibited controlled release of the drug, and demonstrated enhanced cytotoxicity against breast cancer cells, highlighting its therapeutic promise.

Keywords: TPGS; breast cancer; pemetrexed; pluronics; polymeric mixed micelles.

Graphical abstract

Figure 1.

Characterization studies. (A) Critical micellar concentration determination using dynamic light scattering, and (B) correlation between KCPS, hydrodynamic size and concentration. Scanning electron microscope images of blank-PMM (C) and lyophilized Pem-PMM (D). TEM images of blank PMM (E) and Pem-PMM (F). All experiments were performed in triplicate. KCPS: Kilo counts per second; PMM: Polymeric mixed micelle; Wt: Weight.

Figure 2.

Fourier transform infrared analysis, powder x-ray diffraction and differential scanning calorimetric analysis of formulation components. (A) Fourier transform infrared spectra of Pem, blank PMM and Pem-PMM. (B) Powder x-ray diffraction diffractograms of blank PMM, Pem-PMM, physical mixture, and Pem. (C) DSC thermogram of Pem. (D) DSC thermogram of Pluronic F127. (E) DSC thermogram of Pem-PMM. All the experiments were performed in triplicate. DSC: Differential scanning calorimetry; Pem: Pemetrexed; Pem-PMM: Pemetrexed-loaded polymeric mixed micelle; PMM: Polymeric mixed micelle.

Figure 3.

1D 500 MHz nuclear magnetic resonance spectra. (A) 1D ¹H NMR spectrum of Pem. (B) 1D ¹H NMR overlay spectra of Pem, Pem-PMM and blank PMM in D₂O. (C) 1D ¹H (partial) spectrum illustrating the Pem and Pem-PMM in D₂O. (D) 1D selective rotating-frame Overhauser effect spectroscopy spectra showing (i) partial ¹H spectrum of Pem-PMM and (ii) partial ¹H NMR selective rotating-frame Overhauser effect spectroscopy illustrating the irradiation of peak at 8.35 of Pem-PMM spectra in H₂O + D₂O. All the experiments were performed in triplicate (n = 3). NMR: Nuclear magnetic resonance; Pem: Pemetrexed; Pem-PMM: Pemetrexed-loaded polymeric mixed micelle; PMM: Polymeric mixed micelle.

Figure 4.

Characterization studies. (A) Partial ¹H-¹H 2D rotating-frame Overhauser effect spectroscopy spectrum illustrating cross-peaks in the Pem-PMM in H₂O + D₂O. (B) Stability analysis of Pem-PMM solution showing the effect of size and PDI for 7 days. (C) Particle size distribution of reconstituted Pem-PMM. (D) Overlay of powder x-ray diffraction analysis of lyophilized Pem-PMM formulation at 1 month and 3 months. All the experiments were performed in triplicate. PDI: Polydispersity index; Pem-PMM: Pemetrexed-loaded polymeric mixed micelle.

Figure 5.

In vitro drug-release studies. (A) Pem solution at pH 4.0, 6.8 and 7.4. (B) Pem-PMM at pH 4.0, 6.8 and 7.4. (C) Higuchi kinetics at pH 4.0, 6.8 and 7.4. (D) Hemolysis assay of the Pem, blank PMM and Pem-PMM. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of Pem, Pem-PMM and blank on MCF-7 cells at (E) 24 h and (F) 48 h. (G) Cell uptake studies of control, FITC and FITC-loaded PMM in MCF-7 cells. The data experiments were performed in triplicate and represented as mean ± standard deviation. */^#represents p ≤ 0.05; **/^##represents p ≤ 0.01; ***/^###represents p ≤ 0.001; ****represents p ≤0.0001. conc.: Concentration; FITC: Fluorescein isothiocyanate; Pem: Pemetrexed; Pem-PMM: Pemetrexed-loaded polymeric mixed micelle; PMM: Polymeric mixed micelle.

Figure 6.

Qualitative apoptosis assay in MCF-7 cells. (A) Ethidium bromide/acridine orange dual staining of cells (10× magnification) after treatment with Pem-PMM. (B) DAPI staining of cells (40× magnification) after treatment with Pem-PMM. The yellow arrows indicate the morphological changes and nuclear fragmentation of the cells. The yellow boxes represent the few apoptotic cells at higher magnification. (C) Reactive oxygen species/2′,7′-dichlorofluorescein diacetate assay postincubation of MCF-7 cells (10× magnification) with Pem-PMM. All the experiments were performed in triplicate. DAPI: 4′-6-diamino-2-phenylindole; Pem-PMM: Pem-loaded polymeric mixed micelle.

Figure 7.

In vitro analysis of Pem-PMM in MCF-7 cells. (A) Morphological observations of MCF-7 cells treated with low dose, IC₅₀ and high dose of Pem-PMM. The inhibitory effect of Pem-PMM on cell migration activity of MCF-7 cells. (B) Images of migrated MCF-7 cells, scale bar = 100 μm. Quantification of cell migration of MCF-7 cells after treatment with a low dose and IC₅₀ of Pem-PMM. (C) Percentage wound area. (D) Percentage inhibition. The data experiments were performed in triplicate and represented as mean ± standard deviation.***p < 0.001; ****p < 0.0001 represents significant value. Pem-PMM: Pem-loaded polymeric mixed micelle.