Biocompatibility, Cytotoxicity, Antimicrobial and Epigenetic Effects of Novel Chitosan-Based Quercetin Nanohydrogel in Human Cancer Cells

Abstract

Background: Previous studies have reported that quercetin (Q) has a potential antibacterial and anticancer activity. However, its application is limited by many important factors including high hydrophobicity and low absorption.

Methodology: In the current study, we synthesized and characterized (Patent) a novel chitosan-based quercetin nanohydrogel (ChiNH/Q). Encapsulation efficiency was confirmed by UV/VIS spectrophotometer. Physicochemical characterization of ChiNH/Q was assessed by PDI, DLS, SEM, FTIR, and XRD. The toxicity of the ChiNH/Q against five strains of the pathogen and HepG2 cells was examined. Moreover, the quantification of ChiNH/Q on genomic global DNA methylation and expression of DNMTs (DNMT1/3A/3B) in HepG2 cancer cells were evaluated by ELISA and real-time PCR, respectively.

Results: Under the SEM-based images, the hydrodynamic size of the ChiNH/Q was 743.6 nm. The changes in the PDI were 0.507, and zeta potential was obtained as 12.1 mV for ChiNH/Q. The FTIR peak of ChiNH/Q showed the peak at 627 cm^-1 corresponded to tensile vibrational of NH₂-groups related to Q, and it is the indication of Q loading in the formulation. Moreover, XRD data have detected the encapsulation of ChiNH/Q. The ChiNH/Q showed a potent antimicrobial inhibitory effect and exerted cytotoxic effects against HepG2 cancer cells with IC₅₀ values of 100 µg/mL. Moreover, our data have shown that ChiNH/Q effectively reduced (65%) the average expression level of all the three DNMTs (p<0.05) and significantly increased (1.01%) the 5-methylated cytosine (5-mC) levels in HepG2 cells.

Conclusion: Our results showed for the first time the bioavailability and potentiality of ChiNH/Q as a potent antimicrobial and anticancer agent against cancer cells. Our result provided evidence that ChiNH/Q could effectively reduce cellular DNMT expression levels and increase genomic global DNA methylation in HepG2 cancer cells. Our results suggest a potential clinical application of nanoparticles as antimicrobial and anticancer agents in combination cancer therapy.

Keywords: DNA methylation; antimicrobial activity; chitosan nanohydrogel; cytotoxic activity; gene expression; quercetin.

Figure 1

(A) The scanning electron microscopy (SEM) micrographs at 15 kV of unloaded ChiNH and ChiNH/Q. (B) The FTIR spectrum analysis of ChiNH/Q, ChiNH, Q, TPP and chitosan. (C) The XRD peaks of ChiNH and ChiNH/Q formulation.

Figure 2

The kinetic release of Q from ChiNH in PBS. (A) The cumulative release of Q in a 300-min time course was calculated as a percentage of total Q loaded into chitosan nanohyrogel. (B) the Korsmeyer–Peppas mathematical model was used to calculate n.

Figure 3

The pH of ChiNH and ChiNH/Q formulation throughout 90 days.

Figure 4

The optical microscopy images of the morphological changes of HepG2 cells after treatment with 50 µg/mL of ChiNH, Q and ChiNH/Q for 48 h.

Figure 5

The cytotoxicity of ChiNH (nano-blank), Q and ChiNH/Q in HepG2 cancer cells. ChiNH/Q (combination of ChiNH and Q) synergistically improved cytotoxicity in HepG2 cells line in a dose-dependent manner at 48 h. All data repeated in triplicate shown as means ± SD.

Figure 6

(A) The relative expression of DNMT1, DNMT3A and DNMT3B in HepG2 cells treated with ChiNH, Q and ChiNH/Q, measured by real time-PCR. The expression of each gene was normalized to GAPDH. Cells treated with only RPMI 1640 were considered as controls and the rate of expression of the genes of interest in other groups was calculated. The data were presented as mean ± SD and each experiment was conducted in triplicate. The results were analyzed by one-way ANOVA and Tukey’s post hoc test. The significance level is considered as p <0.05. The bars marked with different letters (a, b, c) are significantly different from other samples. (B) A summary of the change in the average expression of all 3 DNMTs (DNMT1, DNMT3A and DNMT3B) in HepG2 cells treated with ChiNH, Q and ChiNH/Q for 48 h.

Figure 7

The Effects of 50 µg/mL concentration of ChiNH, Q and ChiNH/Q with concentrations on global DNA methylation in HepG2 cells. Values represent mean ± SD of three experiments. The bars marked with asterisk are significantly different as verified by Tukey’s honestly significant difference multiple comparison test (p < 0.05).