Antiviral Activity of Chitosan Nanoparticles Encapsulating Curcumin Against Hepatitis C Virus Genotype 4a in Human Hepatoma Cell Lines

Abstract

Purpose: Current direct-acting antiviral agents for treatment of hepatitis C virus genotype 4a (HCV-4a) have been reported to cause adverse effects, and therefore less toxic antivirals are needed. This study investigated the role of curcumin chitosan (CuCs) nanocomposite as a potential anti-HCV-4a agent in human hepatoma cells Huh7.

Methods: Docking of curcumin and CuCs nanocomposite and binding energy calculations were carried out. Chitosan nanoparticles (CsNPs) and CuCs nanocomposite were prepared with an ionic gelation method and characterized with TEM, zeta size and potential, and HPLC to calculate encapsulation efficiency. Cytotoxicity studies were performed on Huh7 cells using MTT assay and confirmed with cellular and molecular assays. Anti-HCV-4a activity was determined using real-time PCR and Western blot.

Results: The strength of binding interactions between protein ligand complexes gave scores with NS3 protease, NS5A polymerase, and NS5B polymerase of -124.91, -159.02, and -129.16, for curcumin respectively, and -68.51, -54.52, and -157.63 for CuCs nanocomposite, respectively. CuCs nanocomposite was prepared at sizes 29-39.5 nm and charges of 33 mV. HPLC detected 4% of curcumin encapsulated into CsNPs. IC50 was 8 µg/mL for curcumin and 25 µg/mL for the nanocomposite on Huh7 but was 25.8 µg/mL and 34 µg/mL on WISH cells. CsNPs had no cytotoxic effect on tested cell lines. Apoptotic genes' expression revealed the caspase-dependent pathway mechanism. CsNPs and CuCs nanocomposite demonstrated 100% inhibition of viral entry and replication, which was confirmed with HCV core protein expression.

Conclusion: CuCs nanocomposite inhibited HCV-4a entry and replication compared to curcumin alone, suggesting its potential role as an effective therapeutic agent.

Keywords: Huh7; caspase-dependent pathway; chitosan curcumin nanocomposite; docking; hepatitis C virus genotype 4a.

Figure 1

In silico docking study of interactions using Molegro Virtual Docker software, showing 2- and 3-dimensional representations for: Ligand interaction of curcumin (A) and chitosan curcumin nanoparticles (B) with NS3 protease; binding interaction of curcumin (C) and chitosan curcumin nanoparticles (D) with NS5A polymerase; ligand interaction of curcumin (E) and chitosan curcumin nanoparticles (F) with NS5B polymerase.

Scheme 1

An illustration of the workflow of methods performed in the current research. Abbreviation: CuCs, curcumin chitosan.

Figure 2

(A). Size of curcumin chitosan nanocomposite ranged from 29.6 to 39.5 nm as detected with TEM, (B). Zeta potential of curcumin chitosan nanocomposite (CuCsNPs) equal to +33 mV, (C). Infrared spectrum of: Curcumin, Chitosan nanoparticles, Curcumin-loaded chitosan-TPP nanoparticles, (D). HPLC of curcumin as a standard, (E). HPLC of curcumin chitosan nanocomposite, (F). Curcumin release percentage profile from chitosan nanoparticles.

Figure 3

(A). IC50 of curcumin on Huh7, (B). IC50 of curcumin chitosan nanocomposite on Huh7, (C). IC50 of curcumin on WISH cells, (D). IC50 of curcumin chitosan nanocomposite on WISH cells.

Figure 4

Morphological examination of Huh7 and WISH cells treated with an IC50 concentration of the tested materials (A). Untreated Huh 7, (B). Huh7 treated with IC50 of curcumin, (C). Huh7 treated with curcumin chitosan nanocomposite, (D). Huh7 treated with chitosan nanoparticles, (E). Untreated WISH cells, (F). WISH cells treated with IC50 of curcumin, (G). WISH cells with IC50 of curcumin chitosan nanocomposite, (H). WISH cells treated with chitosan nanoparticles at 100 µg/mL.

Figure 5

TEM images showing (A). Untreated Huh7 using STEM techniques, scale bar 1 micron, (B). Localization of curcumin chitosan nanocomposite (CuCsNPs; red circles) into Huh7 cells, scale bar 2 micron, (C). Localization of CuCsNPs in Huh7 cells, scale bar 500 nanometer.

Figure 6

(A). Cell cycle analysis of untreated Huh7 cells, (B). Cell cycle analysis post-treatment of Huh7 with IC50 concentration of nanocomposite, showing preG1 arrest by 6.7% compared to control cells and therefore inhibiting cells from entering S phase and G2/M phase.

Figure 7

(A). Effect of curcumin chitosan nanocomposite treated Huh7 on protein levels of Caspase 3, Bcl-2, caspase 8, and p53, (B). Effect of paclitaxel treatment on the expression levels of the same proteins. Error bars represent standard deviation.

Figure 8

(A). The scanned densitometry Western blot of viral replication in Huh7 (a) versus β-actin (b). Lane 1: protein levels of infected cells treated, Lane 2: infected cells treated with curcumin, Lane 3: infected cells treated with chitosan nanoparticles, Lane 4: infected cells treated with curcumin chitosan nanocomposite, (B). The scanned densitometry Western blot of viral entry (a) versus β-actin (b) protein levels in positive Lane 1: untreated infected cells, Lane 2: cells treated with CsNPs, Lane 3: cells treated with nanocomposite, Lane 4: cells treated with nanocomposite, Lane 5: cells treated with curcumin, Lane 6: cells treated with sovaldi. HCV core protein at size of 22 KD.