Development and In Vitro Evaluation of Kaempferol-Loaded Liposomes in LN-229 Glioblastoma Cells: Potency, Selectivity, and Modulation of ROS-Dependent Cytotoxicity

Abstract Glioblastoma multiforme has a median survival of 12–15 months despite multimodal treatment, due to blood-brain barrier impermeability limiting CNS drug bioavailability. Liposomal nanoformulations circumvent this barrier while offering tunable surface chemistry and amphiphilic drug-loading capacity. Here, we report encapsulation of kaempferol, a dietary flavonoid, within phosphatidylcholine-cholesterol liposomes (Lip-KPF) and evaluate cytotoxic activity against LN-229 glioblastoma cells relative to free kaempferol (KPF) and puromycin aminonucleoside (Puro-A). LCMS confirmed 56% encapsulation efficiency; dynamic light scattering yielded a zeta potential of −15.32 ± 0.83 mV, mean particle size of 200.44 ± 12.22 nm, and PDI of 0.29 ± 0.06, with 31P NMR confirming a multilamellar structure. Lip-KPF potency against LN-229 cells increased progressively across 24–72 h, peaking at 72 h (19.880 μM) with significantly lower IC50 values than free KPF (p < 0.001), while maintaining lower astrocyte toxicity. Selectivity indices for Lip-KPF exceeded 1 at every time point, widening over time. Cell death was confirmed by TUNEL assay and phase-contrast microscopy. RT-qPCR revealed concurrent BAX and TP53 upregulation alongside CASP3/CASP7 suppression in both groups, with no significant difference between KPF and Lip-KPF; ELISA corroborated CASP3 suppression, consistent with a possible caspase-independent death mechanism. In a preliminary antioxidant rescue assay, pretreatment did not significantly alter KPF-induced cytotoxicity but significantly increased it in Lip-KPF-treated cells, indicating a cytotoxic mechanism independent of the ROS pathway contributing to free KPF’s activity. These findings support Lip-KPF as a candidate for continued preclinical investigation in LN-229 glioblastoma cells.