Antimicrobial activity and cytotoxic and epigenetic effects of tannic acid-loaded chitosan-based nanoparticles

These data are associated with the physicochemical evaluation of tannic acid-loaded chitosan-based nanoparticles. In addition, their cytotoxic effects were investigated. The following sections describe the methodologies used to obtain data.Encapsulation efficiencyThe encapsulation efficiency was evaluated by pouring 2M hydrochloric acid (4 mL) into a test tube containing lyophilized hydrogel (0.02 g). The obtained solution was ultrasonicated (UP200H, Germany) for 30 minutes. Subsequently, the solution was filtered using filter paper, and the total volume was increased to 10 mL using ddH2O. The amount of TA incorporated into the nanohydrogel was determined by monitoring the absorbance of the sample at 254 nm (Shimadzu UV-1900i, Japan)31,33. The following equation was used to calculate the encapsulation efficiency31:Physicochemical evaluation of the nanoparticles Size, polydispersity index (PDI), and zeta potentialThe size distribution and zeta potential of Chi-NPs and Chi-TA-NPs were evaluated using dynamic light scattering (Malvern Instruments Ltd., Malvern, UK). Colloidal samples were dispersed in ddH2O at a ratio of 1:1000 (v/v) to prepare the samples. The samples were evaluated in triplicate (at 25 °C) at the same angle using this apparatus33.Field emission scanning electron microscopy (FE-SEM)Briefly, freeze-dried chitosan-based tannic acid nanohydrogel and chitosan-based nanohydrogel (stored at −50 °C for 24 h) were placed on a stub, sputter-coated with gold, and analyzed at 15 kV using a TESCAN MIRA3 field-emission scanning electron microscope (TESCAN, Czech Republic)34. Fourier-transform infrared (FTIR) spectroscopyFTIR spectroscopy was used to characterize the chemical groups and bonds in Chi-TA-NPs. FTIR spectroscopy was conducted to measure the infrared absorbance spectra of different compounds, such as Chi, TPP, Chi-NPs, and Chi-TA-NPs (BRUKER, TENSOR 27. Germany)34.X-ray diffraction (XRD) analysisThe XRD profiles of the formulations were determined using a diffractometer (Dmax 2100; USA). UV-filtered with a current of 20 mA and potential of 40 kV was used. The scan rate was 4°/min over a range of 0º–90º of diffraction angle (2θ)35.       Release kinetics test A dialysis bag was used to determine the amount of TA that was released. First, it was kept in ddH2O at 25 °C for 24 h. Then, the Chi-TA-NPs solution (5 mL) containing 1.5 mg of TA as the donor was placed in a dialysis bag (molecular weight cut-off = 12000 Da). The filled bag was then immersed in a solution including PBS (60 mL) and ethanol (30 mL), as recipients of TA (pH = 7.4). The recipient solution was then stirred at 600 rpm. The release kinetics of TA was analyzed by examining the recipient solution (1 mL) after 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, and 300 min and by replacing it with a fresh solution (1 mL; pH = 7.4). TA concentrations were quantified spectrophotometrically at 254 nm (Shimadzu UV-1900i, Japan)36. The cumulative release of TA from the prepared nanoparticles was determined using the following equation:After determining the cumulative release, a graph representing released TA against time was plotted.The mechanism of TA release from the nanoparticles was assessed using the Korsmeyer-Peppas equation:Mt is the level of released TA at each time, M∞ is the level of TA at the start of the experiment, t shows the release time, K indicates the release kinetic constant, and n represents the characteristic power required for release. The mechanism of release is explained according to the n value and mimics the Fickian and non-Fickian patterns. n ≤ 0.43 shows case I transport (Fickian release); however, n = 0.85 exhibits case II transport (non-Fickian release). 0.43 < n < 0.85 represents a non-Fickian pattern, and the release mechanism includes diffusion and inflation33. A graph was drawn in a linear form using Ln Mt/M∞ against Ln (t) to calculate the K and n values. pH testTo assess pH changes, unloaded and TA-loaded nanoparticles (0.5 g) were dispersed in 10 mL ddH2O with stirring and sonication. Then, pH was measured for 90 days (0, 1, 2, 7, 14, 30, 45, 60, and 90 days).HepG2 cell cultureHuman hepatocellular HepG2 cancer cells were obtained from the National Cell Bank of Iran (Pasteur Institute, Tehran, Iran). HepG2 cells were grown in a prepared culture medium (RPMI 1640) containing 10% FBS, 2 mM glutamine, penicillin (100 U/mL), and streptomycin (100 μg/mL) under suitable conditions in an incubator with CO2 (5%) at 37 °C38.MTT assayTo evaluate the cell toxicity of Chi-NPs, TA, and Chi-TA-NPs, cells at a density of 3 × 104 were cultured in six-well plates after the addition of the desired amount of complete culture medium and allowed to attach for 48 h to prepare for treatment with Chi-NPs, TA, and Chi-TA-NPs. Cell viability was assessed using the MTT assay38. Briefly, cells were exposed to five different concentrations (10, 50, 100, 500, and 1000 μg/mL) of each compound for 48 h, as described in our recent study28. After treatment, the cells were incubated with MTT solution (0.5 mg/mL), followed by exposure to 100 μL of DMSO. Finally, absorbance was measured at 570 nm using an ELISA reader (Stat Fax 4700, USA). Each experiment was conducted three times. The Ethics Committee of the Lorestan University of Medical Sciences reviewed and accepted the methodology of this study (IR.LUMS.REC.1397.085).RNA isolation and reverse transcription-quantitative real-time PCR (RT-qPCR) Total RNA was isolated from the cells using an RNA Isolation Reagent (Roche Applied Science, Germany) according to the manufacturer’s protocol. Electrophoresis on a 2% formaldehyde containing 1.5% agarose gel was used to evaluate the integrity of the separated RNA. The extracted RNA was stored at -80 °C until further analysis. Complementary DNA (cDNA) synthesis was performed with isolated RNA (2.0 µg) and oligo dT primers using an M-MuLV RT kit (MBI Fermentas, Lithuania). DNase I (Yekta Tajhiz Azma, Iran) was used to extract DNA from the RNA samples prior to cDNA synthesis. Gene expression levels of DNMT1, DNMT3A, DNMT3B, and GAPDH were analyzed in triplicate using the Corrbet sequence detection system (Rotor Gene 6000). Primer sequences used for RT-qPCR are available in Table 1. Relative expression was determined using the 2–ΔΔCt standard method39,40. Genomic DNA extraction and global DNA methylation analysisGlobal DNA methylation was analyzed in DNA extracted from the cells using an ELISA kit (Zymo Research, Germany) following the manufacturer’s  protocol, as previously explained41. Briefly, 100 ng of isolated genomic DNA was denatured (98 °C for 5 min) and coated onto the wells of the plate by incubation with 5-mC coating buffer (37 °C for 1 h). After incubation, the wells were washed three times with 200 μL of 5-mC ELISA buffer, and then 100 μL of an antibody mix comprising Anti-5-Methylcytosine and Secondary Antibody conjugated with horseradish peroxidase (HRP) was added to each well. The plate was covered with foil and incubated at 37 °C for 1 h. Next, the antibody mix was removed from the wells using 5-mC ELISA buffer, and HRP developer (100 μL) was added to each well and incubated at room temperature for 1 h. Finally, optical density (OD) was measured at 450 nm using an ELISA plate reader (Stat Fax 4700, USA).