DOI: 10.57760/sciencedb.15081
title: Chitosan-based tannic acid nanoparticles: Antimicrobial activity and cytotoxic and epigenetic effects on HepG2 cancer cell line
title: Chitosan-based tannic acid nanoparticles: Antimicrobial activity and cytotoxic and epigenetic effects on HepG2 cancer cell line

description: These data are related to our study, which investigated the antimicrobial activity and cytotoxic and epigenetic effects of tannic acid-loaded chitosan nanoparticles. In the following sections, we describe the methodologies used to obtain data.Formulation of chitosan-based nanoparticles (Chi-NPs) and tannic acid-loaded Chi-NPs (Chi-TA-NPs)Chitosan-based nanoparticles (Chi-NPs) were formulated using the ionic gelling assay28,31. First, 0.2% (v/v) acetic acid was used to prepare the Chi solutions (0.125, 0.25, 0.5, and 1% Chi). The solutions were vigorously stirred using a magnetic stirrer (HMS8805, Germany) for 12 h at pH 4.7. Next, TPP solutions (0.125, 0.25, 0.5, and 1% TPP) were prepared as the aqueous solutions. After preparing the solutions, a filter paper (0.45 μ pore size) was used to eliminate unsolvable particles. Subsequently, TPP solution (5 mL) was mixed dropwise with Chi solution (20 mL), and the mixture was agitated for 10 min. To prepare the Chi-TA-NPs, 5 mg of TA (0.625, 1.25, 2.5, and 5 mg TA) was poured into the Chi solution before adding the TPP solution. Subsequently, uninterrupted stirring was applied for 12 h. The produced nanoparticles were freeze-dried using a freeze-drying instrument (Alpha1-2 LD plus, Germany) and maintained at 4 °C for future analysis. Optimized levels of the constituents (1% Chi, 0.5% TPP, and 5 mg TA) were used to prepare nanoparticles28.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 &lt; n &lt; 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).Evaluation of antimicrobial activityThe antimicrobial effects of Chi-TA-NPs were examined using several strains of pathogens, such as Staphylococcus aureus (S. aureus) (ATCC 12600), Listeria monocytogenes (L. monocytogenes) (PTCC 1297), Escherichia coli (E. coli) (PTCC 1395), Klebsiella pneumoniae (K. pneumoniae) (PTCC 1290), and Candida albicans (C. albicans) (PTCC 5072), which were purchased from the National Cell Bank of Iran (Pasteur Institute, Tehran, Iran). Antimicrobial properties were assessed by identifying the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) using the microdilution method in line with the CLSI standard method37. Briefly, different concentrations of Chi-NPs, Chi-TA-NPs, TA, ceftriaxone, and fluconazole were prepared using Mueller-Hinton Broth medium (MHB, Merck, Germany) for bacterial pathogens and Sabouraud Dextrose Broth (SDB, Merck, Germany) for fungal pathogens. Serial dilutions were performed to obtain a concentration range of 5000–3.125 μg/mL. Each subsequent well contained half the concentration in the previous well. The prepared concentrations were loaded into a microplate (96-well), and a suspension comprising microbial strain (1.5 × 108 CFU/mL) was immediately added. The plates were then incubated at 37°C for 24 h. After incubation, the microbial viability was examined using 2,3,5-Triphenyltetrazolium chloride. The results were obtained by observing the color of the wells as follows: pink wells exhibited live microbes, and non-colored wells showed microbial inhibition. To determine MBC and MFC, wells with no color formation were seeded (5 μL) onto Muller-Hinton Agar and Sabouraud Dextrose Agar plates for bacterial and fungal pathogens, respectively. The cultured plates were maintained at 37 °C for 24 h, and the plates with the minimum concentrations of Chi-NPs, Chi-TA-NPs, TA, ceftriaxone, and fluconazole that exhibited no colony growth were reported as MBC or MFC37.  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 explained.  
description: These data are related to our study, which investigated the antimicrobial activity and cytotoxic and epigenetic effects of tannic acid-loaded chitosan nanoparticles. In the following sections, we describe the methodologies used to obtain data.Formulation of chitosan-based nanoparticles (Chi-NPs) and tannic acid-loaded Chi-NPs (Chi-TA-NPs)Chitosan-based nanoparticles (Chi-NPs) were formulated using the ionic gelling assay28,31. First, 0.2% (v/v) acetic acid was used to prepare the Chi solutions (0.125, 0.25, 0.5, and 1% Chi). The solutions were vigorously stirred using a magnetic stirrer (HMS8805, Germany) for 12 h at pH 4.7. Next, TPP solutions (0.125, 0.25, 0.5, and 1% TPP) were prepared as the aqueous solutions. After preparing the solutions, a filter paper (0.45 μ pore size) was used to eliminate unsolvable particles. Subsequently, TPP solution (5 mL) was mixed dropwise with Chi solution (20 mL), and the mixture was agitated for 10 min. To prepare the Chi-TA-NPs, 5 mg of TA (0.625, 1.25, 2.5, and 5 mg TA) was poured into the Chi solution before adding the TPP solution. Subsequently, uninterrupted stirring was applied for 12 h. The produced nanoparticles were freeze-dried using a freeze-drying instrument (Alpha1-2 LD plus, Germany) and maintained at 4 °C for future analysis. Optimized levels of the constituents (1% Chi, 0.5% TPP, and 5 mg TA) were used to prepare nanoparticles28.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 &lt; n &lt; 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).Evaluation of antimicrobial activityThe antimicrobial effects of Chi-TA-NPs were examined using several strains of pathogens, such as Staphylococcus aureus (S. aureus) (ATCC 12600), Listeria monocytogenes (L. monocytogenes) (PTCC 1297), Escherichia coli (E. coli) (PTCC 1395), Klebsiella pneumoniae (K. pneumoniae) (PTCC 1290), and Candida albicans (C. albicans) (PTCC 5072), which were purchased from the National Cell Bank of Iran (Pasteur Institute, Tehran, Iran). Antimicrobial properties were assessed by identifying the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) using the microdilution method in line with the CLSI standard method37. Briefly, different concentrations of Chi-NPs, Chi-TA-NPs, TA, ceftriaxone, and fluconazole were prepared using Mueller-Hinton Broth medium (MHB, Merck, Germany) for bacterial pathogens and Sabouraud Dextrose Broth (SDB, Merck, Germany) for fungal pathogens. Serial dilutions were performed to obtain a concentration range of 5000–3.125 μg/mL. Each subsequent well contained half the concentration in the previous well. The prepared concentrations were loaded into a microplate (96-well), and a suspension comprising microbial strain (1.5 × 108 CFU/mL) was immediately added. The plates were then incubated at 37°C for 24 h. After incubation, the microbial viability was examined using 2,3,5-Triphenyltetrazolium chloride. The results were obtained by observing the color of the wells as follows: pink wells exhibited live microbes, and non-colored wells showed microbial inhibition. To determine MBC and MFC, wells with no color formation were seeded (5 μL) onto Muller-Hinton Agar and Sabouraud Dextrose Agar plates for bacterial and fungal pathogens, respectively. The cultured plates were maintained at 37 °C for 24 h, and the plates with the minimum concentrations of Chi-NPs, Chi-TA-NPs, TA, ceftriaxone, and fluconazole that exhibited no colony growth were reported as MBC or MFC37.  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 explained.