Assessment of the hepatoprotective effect of developed lipid-polymer hybrid nanoparticles (LPHNPs) encapsulating naturally extracted β-Sitosterol against CCl4 induced hepatotoxicity in rats

Abstract

The hepatoprotective effect of β-Sitosterol (BSS), a natural phytosterol, after being formulated into a suitable pharmaceutical drug delivery system has not been widely explored. BSS was isolated from Centaurea pumilio L., identified and formulated as lipid-polymer hybrid nanoparticles (LPHNPs) using the poly(D,L-lactide-co-glycolide) polymer and DSPE-PEG-2000 lipid in different ratios. The selected formulation, prepared with a lipid: polymer: drug ratio of 2:2:2, had an entrapment efficiency (EE%) of 94.42 ± 3.8, particle size of 181.5 ± 11.3 nm, poly dispersity index (PDI) of 0.223 ± 0.06, zeta potential of -37.34 ± 3.21 and the highest drug release after 24 h. The hepatoprotective effect of the formulation at two different doses against CCl₄ induced hepatotoxicity was evaluated in rats. The results showed that the BSS-LPHNPs (400 mg/kg) have the ability to restore the liver enzymes (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)), liver lipid peroxidation markers (malondialdehyde (MDA) and catalase (CAT)), total bilirubin and albumin to their normal levels without inhibitory effect on the CYP2E1 activity. Also, the formulation could maintain the normal histological structure of liver tissue and decrease the cleaved caspase-3 expression. LPHNPs formulation encapsulating natural BSS is a promising hepatoprotective drug delivery system.

Figure 1

In-vitro BSS release from the prepared BSS-LPHNPs formulations.

Figure 2

TEM micro-photo of the prepared BSS-LPHNPs (F5).

Figure 3

Effect of BSS-LPHNPs on hepatic functional enzymes and hepatic lipid profile after CCl₄-induced hepatotoxicity.

Figure 4

Liver of rat (a) from normal group showing the normal histological structure of hepatic lobule. Note normal central vein (CV) and normal hepatocytes (H). (b) Treated with CCl4 showing massive centrilobular hepatocellular necrosis associated with haemorrhage and mononuclear inflammatory cells infiltration (arrow), insert image showing ballooning degeneration of hepatocytes with pyknosis of their nuclei (black arrow) and apoptosis of hepatocytes (red arrow). (c) Co-treated with CCl4 + BSS suspension showing ballooning degeneration of hepatocytes with pyknosis of their nuclei (black arrow), apoptosis of hepatocytes (red arrow) and mononuclear inflammatory cells infiltration. (d) Co-treated with CCl4 + plain LPHNPs formula, showing ballooning degeneration of hepatocytes (black arrow) and apoptosis (red arrow). (e) Co-treated with CCl4 + BSS-LPHNPs (200 mg/kg), showing ballooning degeneration of some hepatocytes (black arrow) and cytoplasmic vacuolization and microvesicular steatosis of other hepatocytes (red arrow), (f) co-treated with CCl4 + BSS-LPHNPs (400 mg/kg) showing necrosis of sporadic hepatocytes (long arrow). (H&E, scale bar 50um, X200) (inserted images X 400).

Figure 5

Histopathological lesions score in different groups. Data shown as mean ± SE; error bars show the variations of determinations in terms of standard error.

Figure 6

Immunostaining for cleaved caspase-3 protein in liver sections of rats, (a) normal control group showing no cleaved caspase-3 immune-reactive cells. (b) Treated with CCl₄ showing strong positive immune expression (arrow). (c) Co-treated with CCl₄ + BSS suspension showing strong positive immune reactive hepatocytes (arrow). (d) Co-treated with CCl₄ + plain LPHNPs formula, showing moderate positive immune expression (arrow). (e) Co-treated with CCl₄ + BSS-LPHNPs (200 mg/kg), (f) co-treated with CCl₄ + BSS-LPHNPs (400 mg/kg), (e,f) showing weak positive cleaved caspase-3 reaction (arrow). (g) Immunostaining area (%) of cleaved caspase-3 expression. Data shown as mean ± SE; error bars show the variations of determinations in terms of standard errors, mean values with unlike superscript letters were significantly different(p ≤ 0.05).