Therapeutic Evaluation of Kaempferol-loaded Liposomes in Hepatocellular Carcinoma through i-NOS/e-NOS/p53/Caspase-Mediated Apoptotic Pathway

The clinical application of kaempferol (KP), a promising anticancer flavonoid, is limited by its poor bioavailability, rapid metabolism, and inadequate tissue distribution. To overcome these limitations, KP-loaded nanoliposomes (KP-LNPs) were developed, and their anti-hepatocellular carcinoma (HCC) potential was investigated. KP-LNPs were formulated and characterized using Fourier-transform infrared (FTIR) spectroscopy, atomic force microscopy (AFM), transmission electron microscopy (TEM), zeta potential, and particle size analysis. The anti-HCC effects and cellular internalization of KP-LNPs were assessed in vitro using HepG2 cells. Additionally, ELISA was performed to evaluate the concentration changes related to the mitochondrial apoptotic proteins i/e-NOS, p53 and Caspase-3/9. The optimized KP-LNP formulation exhibited a uniform average particle size of 141.4 ± 1.12 nm, with a polydispersity index (PDI) of 0.141 ± 0.02 and a zeta potential of − 28.57 ± 0.47 mV, indicating good stability. TEM and AFM analyses confirmed a spherical morphology, while FTIR spectra showed no interactions between KP and the lipid components. The entrapment efficiency of KP was 83%. Cell cytotoxicity assays using HepG2 cells revealed that KP-LNPs exhibited significantly higher anticancer activity than free KP, as evidenced by enhanced cytotoxicity. ELISA analysis indicated that KP-LNPs promoted apoptosis by upregulating apoptotic proteins (e-NOS, p53, and caspases) and downregulating the anti-apoptotic protein (i-NOS). The developed KP-LNP significantly enhances KP’s therapeutic efficacy of KP against HCC by triggering the i-NOS/e-NOS-dependent p53/Caspase mitochondrial apoptotic pathway. These results indicate that KP-LNP is a promising candidate for enhancing HCC treatment. Liposomes are potentially active hydrophobic and hydrophilic molecules because of their high biocompatibility, biodegradability, and minimal immunogenicity. Phosphatidylcholine, a zwitterionic and membrane-mimetic lipid, is commonly used owing to its stability and physiological compatibility. The anti-HCC effects of KP-LNPs were assessed using HepG2 cells. KP-LNPs induced apoptosis through the i/e-NOS/p53/Caspase-mediated apoptotic pathway.