Chitosan-encapsulated linoleic acid from Leucaena leucocephala (Lam.) de Wit. inhibits FOXL2 mutation in MNU-induced ovarian granulosa cell tumors in Wistar rats

Abstract

One of the biggest challenges in drug delivery is delivering drugs to their sites of action to avoid the systemic side effects that are common with most antineoplastic drugs. This study investigates the potential of linoleic acid (LA) isolated from Leucaena leucocephala extract, encapsulated in chitosan nanoparticles (LACS2), and targeting FOXL2 gene mutation in methyl nitrosourea (MNU)-induced ovarian granulosa cell tumors in Wistar rats. Glutaraldehyde was used as a crosslinker to conjugate chitosan with linoleic acid, which was further mediated with folate. Conjugation was confirmed using ¹H NMR, FTIR, DSC, and SEM. The NPs were characterized for morphology, yield, in vitro drug release, particle size, and surface charge using SEM, DLS, and Zeta sizer. FOXL2 gene detection was carried out via RT-PCR, while cytotoxicity and apoptosis of LACS2/FOXL2 complexes were assessed using MTT assay and caspase-3 activity, respectively. In vivo gene delivery was evaluated in Wistar rats. Phytochemical analysis revealed the presence of many metabolites such as alkaloids, flavonoids, tannins, phenols, saponins, phytosteroids, and triterpenes. Among the nanoparticle formulations, LACS2 demonstrated superior physicochemical properties: 68.22 % yield, 120.0 ± 2.11 nm particle size, 88.04 % entrapment efficiency, 98.88 % cumulative drug release, 74.01 % swelling index, 25.22 ± 0.24 mV zeta potential, and a PDI of 0.50. Gel retardation assay showed efficient complexation between LACS2 and FOXL2 gene, with maximum complex formation (86.22 ± 2.04 %) observed at 32 mg chitosan and 8 mg FOXL2. Molecular characterization (FTIR, ¹H NMR, ¹³C NMR, MS) confirmed the presence of LA in the ethyl acetate fraction. Transfection with LACS2/FOXL2 reversed the TGC→TGG mutation, restoring the wild-type FOXL2 gene. In vivo, LACS2/FOXL2 significantly reduced tumor cell volume (24.01 ± 0.01 mL), viable cell counts (8.44 × 10⁴/mL), and increased non-viable cell counts (15.57 × 10⁴/mL). Hematological indices improved remarkably: RBC (52.11 ± 2.01 × 10¹²/L), Hb (38.02 ± 1.01 g/dL), and WBC (36.56 ± 1.01 × 10⁹/L). Furthermore, LACS2/FOXL2 increased mean survival time to 28.14 days and ILS to 74.16 %, while reducing tumor biomarkers: CEA (0.0012 ± 0.010 µg/L) and α-fetoprotein (0.11 ± 0.010 µg/L). This study showed that LACS2/FOXL2 is a promising gene-targeted nanotherapeutic candidate against FOXL2-mutated ovarian cancer.