Harnessing Potential of ω-3 Polyunsaturated Fatty Acid with Nanotechnology for Enhanced Breast Cancer Therapy: A Comprehensive Investigation into ALA-Based Liposomal PTX Delivery

Abstract

Our hypothesis posited that incorporating alpha-linolenic acid (ALA) into liposomes containing Paclitaxel (PTX) could augment cellular uptake, decrease the therapeutic dosage, and alleviate PTX-related side effects. Our investigation encompassed characterization of the liposomal formulation, encompassing aspects like particle size, surface morphology, chemical structure, drug release kinetics, and stability. Compatibility studies were performed through Fourier transform infrared spectroscopy (FTIR). By utilizing the Box-Behnken design (BBD), we developed ALA-based liposomes with satisfactory particle size and entrapment efficiency. It is noteworthy that ALA incorporation led to a slight increase in particle size but did not notably affect drug entrapment. In vitro drug release assessments unveiled a sustained release pattern, with ALA-PTX liposomes demonstrating release profiles comparable to PTX liposomes. Morphological examinations confirmed the spherical structure of the liposomes, indicating that substituting ALA with phosphatidylcholine did not alter the physicochemical properties. Cellular uptake investigations showcased enhanced uptake of ALA-based liposomes in contrast to PTX liposomes, likely attributed to the heightened fluidity conferred by ALA. Efficacy against MCF-7 cells demonstrated concentration-dependent reductions in cell viability, with ALA-PTX liposomes exhibiting the lowest IC50 value. Morphological analysis confirmed apoptotic changes in cells treated with all formulations, with ALA-PTX liposomes eliciting more pronounced changes, indicative of enhanced anticancer efficacy.

Keywords: cancer; cell culture; liposome; paclitaxel; α-linolenic acid.

Figure 1

The compatibility of all compounds that were used for the development of liposomes by FTIR. The figure shows no major changes in peak shifting and functional groups.

Figure 2

Surface morphology of (A) Placebo, (B) ALA-Liposome, (C) PTX-Liposome, and (D) ALA-PTX-Liposome determined by using scanning electron microscopy of liposomes.

Figure 3

Surface morphology and internal structure of (A) Placebo, (B) ALA-Liposome, (C) PTX-Liposome, and (D) ALA-PTX-Liposome determined by using transmission electron microscopy of liposomes.

Figure 4

Pilot illustration of cumulative drug release profiles for PTX-Lipo and ALA-PTX-Lipo using the dialysis method in PBS (pH 7.4) at various time intervals up to 48 h.

Figure 5

The figure demonstrates the percentage of cell viability after 24 h of incubation at half the IC50. Statistical significance was determined at * p  <  0.05, ** p  <  0.01, *** p  <  0.001, and comparisons were made with the control.

Figure 6

(A) Internalization and distribution of liposomes in cells. Caumarine-6 dye was internalized into the cytoplasm (green), and DAPI staining (blue) labelled the nucleus of the cell. The merged panel indicates the internalization of liposomes into both the cytoplasm and the nucleus(magnification 20×). (B) Plot showing the ALA-Lipo and ALA-PTX-Lipo cell uptake comparison in quantitative data.

Figure 7

The DAPI-stained nuclei of MCF-7 cells are shown in the figure with changes in their morphology. Furthermore, DAPI staining following treatment with PTX-Lipo, ALA-Lipo, and ALA-PTX-Lipo demonstrated VC (viable cells), CC (chromatin condensation), and NF (nuclear fragmentation), indicated by an arrow, in comparison to the control group.

Figure 8

(A) The figure illustrates apoptotic morphological changes in MCF-7 cells stained with AO/EtBr. Additionally, treatment with PTX-Lipo, ALA-Lipo, and ALA-PTX-Lipo in AO/EtBr staining revealed early apoptotic regions marked by yellow areas and late apoptotic nuclei appearing bright orange compared with the control group. (B) The quantitative data also showed notable variations among groups. VC: viable cells; LA: late apoptosis. An inverted fluorescent microscope with 20× magnification was used for capturing the pictures. Statistical significance was determined at *** p  <  0.001, and comparisons were made with the control.

Figure 9

(A) JC-1 staining was performed on MCF-7 cells to evaluate mitochondrial membrane potential and pictures were taken with 20× magnification. Treatment with PTX-Lipo, ALA-Lipo, and ALA-PTX-Lipo increased green fluorescence of the JC-1 monomer, indicating a lower mitochondrial potential compared with the control group, which exhibited red fluorescence indicative of high mitochondrial potential. (B) The qualitative data also showed notable variation among the groups. Statistical significance was determined at * p < 0.05, ** p < 0.01, and comparisons were made with the control.