Evaluate the effects of NLC lipid nanoparticles functionalized with a chitosan-folic acid-methylene blue conjugate on inducing apoptosis in breast cancer cells and inhibiting angiogenesis: An in silico and in vitro investigation

Nanotechnology-based drug delivery systems, including chitosan (CS) and nanostructured lipid carriers (NLCs), offer enhanced therapeutic targeting for breast cancer. This study develops CS-folate-modified NLCs (MB-NCF-NPs) encapsulating methylene blue (MB) to improve tumor-specific delivery and efficacy. Molecular docking (MOE 2024) and dynamics (GROMACS) assessed MB's binding to cancer-associated proteins (PDB IDs: 4u15 , 1EER , 3POS , 6YR9 ). NLCs were synthesized using oleic acid/glyceryl monostearate, surface-modified with CS-folate, and characterized via DLS, FESEM, and FTIR. Encapsulation efficiency was quantified spectrophotometrically. Cytotoxicity (MTT assay), apoptotic gene expression (qPCR for Caspases 3,8,9), and anti-angiogenic effects (CAM assay) were evaluated across MCF-7, AGS, A549, and HFF cell lines. Statistical significance ( p < 0.05) was determined using SPSS. MB exhibited the strongest binding to the A549-linked M3-mT4L receptor (binding energy: −6.1 kcal/mol), weak/no affinity for Estrogen Receptor, Calreticulin, and FAK. Molecular dynamics confirmed stable interactions (minor SASA/Rg fluctuations). MB-NCF-NPs displayed optimal size (84.19 nm), zeta potential (+19.42 mV), and high encapsulation (95.7 %). Cytotoxicity was concentration-dependent, with IC50 values of 12.3 μg/mL (MCF-7), 15.1 μg/mL (AGS), and 18.4 μg/mL (A549), versus >50 μg/mL for HFF. Apoptosis induction was evidenced by upregulated caspase activity (3.8–5.2-fold). CAM assays revealed reduced angiogenesis (35 % vessel inhibition) and embryonic growth disruption. MB-NCF-NPs demonstrate selective cytotoxicity and anti-angiogenic effects via targeted MB delivery, attributed to folate receptor-mediated uptake. Stable nanostructure properties, coupled with low normal cell toxicity, highlight their potential as precision therapies for breast cancer. Further in vivo validation is warranted to translate these findings.