Inhibition of EGFR1 in Triple Negative Breast Cancer Cells Using siRNA Loaded with Fe₃O₄ Magnetic Nanoparticles

Triple-negative breast cancer (TNBC) is known as the most common cancer among women in the world. Although overexpression of the EGFR1 gene is observed in more than 70% of patients, medications based on inhibitors of this receptor are ineffective. This is due to the fact that the protein remains active, even after administration with inhibitory drugs. We used siRNA to silence the EGFR1 gene in this study. The siRNA was packaged in a magnetic nanoparticle (MNP) that was functionalized with poly ethyleneimine (PEI), PEGylated folic acid (FA-PEG), and β-cyclodextrin (βCD). Multi-functionalized FA-PEG/βCD functionalized PEI-coated iron oxide magnetic nanoparticles (Fe₃O₄@CD-PEI/FA-PEG) were produced by post-surface modification. This multi-functionalized magnetic nanocarrier was structurally verified using scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), atomic force microscopy (AFM), and vibrating sample magnetometry (VSM). Small interfering RNAs (siRNA) were used as a precise tool in gene therapy in this study to remove EGFR1 gene mRNA. siRNA molecules are about 21 nucleotides that cause cleavage in the complementary sequence of the target mRNA. The capacity to deliver siRNAs against EGFR1 to the breast cancer cell line (MD-MBA-231) was evaluated. According to flow cytometry, siRNA was efficiently delivered to the cells, with an absorption rate of 62%. In addition, confocal microscopy confirmed the cell uptake and endosomal escape. Also, the expression level of the EGFR1 gene was measured by the quantitative PCR method, which showed a 78% knockdown of this gene at the mRNA level.

The fate of the siRNA@[Fe₃O₄@βCD-PEI/FA-PEG] complex after endocytosis. (1) siRNA@[Fe₃O₄@βCD-PEI/FA-PEG] entering the cell (early endosome) through endocytosis. (2) Entry of siRNA@[Fe₃O₄@βCD-PEI/FA-PEG] into the late endosome. (3a) Endosomal escape and cargo survival (siRNA). (3b) Lysosomal digestion pathway. (4) Release of siRNA to cytosol and interaction with RISC after rupture of endosome membrane. (5) mRNA degradation and EGFR1 gene silencing