Ganoderic acid loaded nano-lipidic carriers improvise treatment of hepatocellular carcinoma

Abstract

This work evaluates nano-lipid carrier of ganoderic acid (GA) and molecular docking on various cancer signaling pathways, an attempt to improve the hepatic condition associated with hepatic carcinoma (HCC) induced by diethyl-nitrosamine (DEN) in Wistar rats. Molecular docking mechanism of GA was performed through binding simulation analysis for various cancer signaling pathway, viz., Bcl-2, Pl3K, NF-κB, Akt/PKB, and Stat-3. Double emulsion solvent displacement method was implied for preparation of GA-loaded nano-lipid carrier. GA-NLCs were evaluated for drug loading capacity, entrapment efficiency, particle size, gastric stability, in vitro drug release, cytotoxicity, cellular uptake, and in vivo studies including macroscopical, hepatic injury markers, non-hepatic, biochemical, antioxidant parameters, and histopathological evaluation. HCC was induced by intraperitoneal injection of DEN (200 mg/kg). Both in vivo and molecular docking results were compatible in establishing the alteration in hepatic nodules, hepatic, non-hepatic, and antioxidant parameters, in a significant manner (p < .001) by GA and GA-NLC along with signal alteration of Bcl-2, Pl3K, NF-κB Akt/PKB, and Stat-3 pathway. Histopathological observation confirmed and supported the above result by GA and GA-NLC. On the basis of our results, we can advocate that, GA interferes with various cancer signaling proteins involved in pathogenesis of cancer and was able to cease the progression of disease. Additionally, GA-NLCs proved its chemoprotective effect against the DEN-induced HCC by modulation of hepatic and non-hepatic parameters through various mechanisms.

Keywords: Akt/PKB; Ganoderic acid; HepG2 cell; antioxidant; antioxidants enzymes; entrapment efficiency; hepatoprotective; nano-lipid carrier.

Figure 1.

Uptake of (A) free coumarin-6 (C-6) and (B) C-6 loaded GA-nano-lipid carrier, upon incubation at 1 µg/mL for 2 h. In all the images, (a) images under the green fluorescence channel; (b) corresponding differential interface contrast images of HepG2 cells. (c) Superimposition of figure (a) and figure (b). (d, e) In all, the images show horizontal line series analysis of fluorescence along the white line.

Figure 2.

Macroscopical observation of DEN-induced HCC group rats. (A) DEN control group showed the expansion of hepatic nodules (white in color) and decolorization of tissue, (B) DEN control group treated with GA (25 mg/kg) illustrated the expansion of pre-cancerous nodules (white in color) and decolorization of tissue color, which was less as compared to DEN group, (C) DEN control group treated with GA (50 mg/kg) demonstrated the less pre-cancerous nodules (white in color) and decolorization of tissue color, (D) DEN control group treated with GA (100 mg/kg) illustrated the expansion of pre-cancerous nodules and decolorization of tissue color, which was less as compared to other group, and (E) DEN control group treated with GA-NLC (25 mg/kg) illustrated the decolorization of tissue color, which was less as compared to other group rats. Note: Normal control and normal control rats treated with GA (100 mg/kg) did not show any sign of precancerous cells and decolorization of skin (data not shown).

Figure 3.

The histopathological evaluation of DEN-induced HCC group rats. (A) DEN control group showed the expansion of hepatic nodules (white in color) and decolorization of tissue, (B) DEN control group treated with GA (25 mg/kg) illustrated the expansion of pre-cancerous nodules (white in color) and decolorization of tissue color, which was less as compared to DEN group, (C) DEN control group treated with GA (50 mg/kg) demonstrated the less pre-cancerous nodules (white in color) and decolorization of tissue color, (D) DEN control group treated with GA (100 mg/kg) illustrated the expansion of pre-cancerous nodules and decolorization of tissue color, which was less as compared to other group, and (E) DEN control group treated with GA-NLC (25 mg/kg) illustrated the decolorization of tissue color, which was less as compared to other group rats. Note: Normal control and normal control rats treated with GA (100 mg/kg) did not show any sign of alteration in the histopathology (data not shown).