**Background:** The liver performs essential functions including detoxification, but is susceptible to injury from chemicals like carbon tetrachloride (CCL4). Melatonin (Mel) has antioxidant and anti-inflammatory properties but has a short half-life and can cause side effects such as drowsiness and hormonal disturbances. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles offer controlled drug release and improved bioavailability. This study aimed to synthesize Mel-loaded PLGA nanoparticles (Mel-PLGA NPs) and evaluate their hepatoprotective effects against CCL4-induced liver injury in rats.
**Methods:** Mel-PLGA NPs were prepared by nanoprecipitation-solvent volatilization. Nanoparticles were characterized by TEM, zeta potential, and size analysis. Encapsulation efficiency (EE%) and drug loading (DL%) were measured by HPLC. In vitro assays assessed antioxidant (DPPH scavenging), cytotoxicity (MTT on Caco2 cells), anticoagulant (PT/PTT), and anti-inflammatory (hemolysis inhibition) effects. In vivo, 50 male Sprague Dawley rats were divided into healthy and CCL4-injured groups, each with five subgroups (n=5): control, free Mel (5 or 10 mg/kg), or Mel-PLGA NPs (5 or 10 mg/kg). CCL4 (0.5 mg/kg i.p. twice/week for 4 weeks) induced liver injury, followed by 4 weeks of daily treatment while continuing CCL4. Serum liver function (ALT, AST, albumin, total bilirubin), liver homogenate oxidative stress markers (MDA, GPx, SOD, CAT), cytokines (IL-1β, TNF-α, IL-6, IL-10), matrix metalloproteinases (MMP9, TIMP1), and flow cytometry for apoptosis (Bax, p53, caspase 3/8, Bcl2) were measured. Histopathology and immunohistochemistry for NF-κB and CRP were performed.
**Key Results:** Mel-PLGA NPs were spherical (87–96 nm by TEM, hydrodynamic diameter 41 nm, zeta potential −6 mV) with EE% 59.9% and DL% 12.5%. In vitro, NPs showed dose-dependent antioxidant activity, low cytotoxicity (IC50 175.9 µg/ml), anticoagulant effects, and hemolysis inhibition comparable to indomethacin. In vivo, CCL4 caused significant liver injury: ALT 80.2 vs 26.4 U/l, AST 64.8 vs 19.9 U/l, total bilirubin 1.9 vs 0.4 mg/dl, albumin 1.2 vs 3.8 g/dl (healthy control). Free Mel (5 or 10 mg/kg) failed to normalize these parameters. Mel-PLGA NPs (5 or 10 mg/kg) restored all liver function markers to healthy control levels. Oxidative stress: CCL4 increased MDA to 16.2 nmol/g (healthy 10.4) and decreased GPx, SOD, CAT. Mel-PLGA NPs (5 mg/kg) reduced MDA to 11.2 nmol/g and normalized antioxidant enzymes. Inflammation: CCL4 elevated IL-1β (156.2 vs 50.9 pg/g), TNF-α (750.4 vs 229.4), IL-6 (210.1 vs 69.4), and reduced IL-10 (55.5 vs 115.9 pg/g). Mel-PLGA NPs (10 mg/kg) returned cytokines to normal. MMP9 and TIMP1 were elevated in CCL4 (11.4 ng/g and 220.1 pg/g vs healthy 4.2 and 165.2); Mel-PLGA NPs significantly reduced both. Flow cytometry showed Mel-PLGA NPs reduced apoptosis and normalized pro-apoptotic proteins (Bax, p53, caspase 3/8) and increased Bcl2. Histopathology confirmed that Mel-PLGA NPs prevented CCL4-induced necrosis, inflammation, and fibrosis, with negative NF-κB and CRP immunostaining.
**Clinical Implications:** Mel-PLGA NPs at 5 mg/kg effectively protected rat livers from CCL4 toxicity, outperforming free melatonin at 10 mg/kg. The sustained release formulation reduced the required melatonin dose, potentially minimizing side effects. These findings suggest that Mel-PLGA NPs could be developed as a hepatoprotective therapy for liver injury. However, further studies in higher animals and humans are needed to confirm efficacy and safety.