Preparation of QI extract and TA

First, the oak gall was collected from the oak forest in Zagros Mountains, Lorestan, Iran, with the permission of Lorestan Natural Resources and Watershed Management Organization. Identification and verification of the collected samples were done in the herbarium department of the Faculty of Pharmacy, University of Tehran (Voucher number: PMP-1852). Air-dried QI galls were powdered, and then 100 g were extracted in 500 ml of 70% methanol for seven days. QI extraction was done at 25 °C without agitation. The extraction efficiency was 12.7%.

TA (Sigma-Aldrich, St. Louis, MO, USA) was also purchased for the chemical analysis of QI extract and nano hydrogel formulation.

HPLC quantification of TA in QI extract

Quantitation of TA in QI extract was done by reversed-phase High-performance liquid chromatography (HPLC) method that was conducted on a Shimadzu (model 10Avp) instrument. Isocratic HPLC method with 0.05 mol/L acetate buffer pH 5.0 in water (solvent A) and acetonitrile (solvent B) was used (Acetate buffer: acetonitrile ratio 70:30) with a flow rate of 1 ml/min. This process was performed on a 25 cm × 4.6 mm i.d. RP-8 column with the temperature maintained at 30 °C. The peaks of the QI sample were detected compared to the TA solution as a standard at 280 nm. 1 mg of QI extract was dissolved in 10 ml of distilled water and after filtration with 0.45 μm nylon filter membrane, it was injected into the device. Finally, the percentage of TA in the QI extract was calculated based on the calibration curve of standard samples (5–200 µg/ml TA).

Preparation of nano hydrogels

To synthesize QI-NH, TA-NH, and unloaded-NH, 20 ml of 0.5% w/v chitosan solution containing 1% acetic acid (Merck, Darmstadt, Germany) was used. Low molecular weight chitosan (50,000-190,000 Da) was used for the formulations. After stirring for 24 h, 100 mg QI extract and 20 mg TA were added separately to 2 chitosan solutions gradually. No bioactive agent (QI or TA) was used for unloaded-NH formulation. After 4 h and complete dissolution of QI extract and TA, 5 ml of 0.3% w/v tripolyphosphate (TPP) solution was dropped to all three samples within 30 min. To separate the insoluble particles, the solutions were filtered using filter paper with 0.45 µ pore size in the synthesis steps. Prepared nano hydrogels were kept at 4 °C until analytical, antibacterial, and cytotoxic assays.

Encapsulation efficiency (EE)

5 ml of 2 M acetic acid was added to 0.3 mg of freeze-dried nano hydrogels (QI-NH and TA-NH) in test tubes. These two solutions were placed in a water bath (Memert, Germany) for 30 min at 90 ℃ to release the encapsulated natural products by breaking the bonds between chitosan and TPP. After the paper filtration of the solutions, the volume of each solution was doubled (10 ml) by adding distilled water, and finally, the absorbance of the samples was measured at 280 nm with a UV/Vis spectrophotometer (Shimadzuo, Japan). EE was calculated based on the following formula:

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Physicochemical analysis of the nanohydrogelsScanning electron microscopy (SEM)

SEM was used to examine the morphology of nano hydrogels. For this purpose, freeze-dried nano hydrogels (at -50 °C for 24 h) were coated with gold layers with a thickness of 5 nm. Then, SEM images were taken with a 6300 field emission scanning electron microscope (Hitachi, S-4160) at 15 kV.

Fourier-transform infrared (FT-IR) spectroscopy

In this method, the main functional groups around the samples including QI extract, TA, chitosan, TPP, QI-NH, and TA-NH were found based on infrared wavelength absorption between 4000 and 400 cm− 1.

Dynamic light scattering (DLS) and zeta potential

The emulsions were diluted 1:1000 in distilled water, and the samples were filtered through 0.45 µ pores. The size distribution of QI-NH and TA-NH was measure using the DLS method. Polydispersity index and zeta potential to evaluate the stability of nanohydrogels were also determined with Malvern Instruments Ltd., Malvern, UK.

Release kinetics

To evaluate the release rate of QI extract and TA at different times, the dialysis bag was immersed in phosphate-buffered saline (PBS) (PH = 7.4). After 24 h, 5 ml PBS solution containing 0.2 g QI-NH or TA-NH was completely sealed in two separate bags. These bags were kept in 100 ml of PBS while being stirred, and 1 ml of PBS was sampled in 5, 10, 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240 min and immediately replaced with 1 ml of fresh PBS. The absorbance of 12 samples of each nano hydrogel was measured by UV/Vis spectrophotometer (Shimadzu, Japan) at 280 nm. Finally, the percentage of QI extract and TA released from nano hydrogels was calculated separately based on the following formula [11]:

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Bacterial strain and culture condition

The standard strain of S. mutans (ATCC 35668) was acquired from the Iranian Biological Resource Center (IBRC, Tehran, Iran). S. mutans was cultured in Brucella broth (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C under anaerobic conditions (5% CO2, 80% N2) for 24 h.

Antibacterial assay

The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined by microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines [24]. Briefly, different concentrations of QI extract (29.29 to 15000 µg/ml), QI-NH (0.93 to 480 µg/ml), TA—NH(0.62 to 320 µg/ml), and unloaded-NH (117.18 to 60000 µg/ml) were prepared by serial dilution method in 96-well polystyrene plates. Then, 1.5 × 105 (CFU/mL) bacteria were added to the wells and incubated for 24 h at 37 °C and under anaerobic culture conditions. Finally, MIC was determined using 2,3,5-Triphenyltetrazolium chloride to indicate bacterial viability.

To determine MBC, 10 µl of supra-MIC concentrations were subcultured in Brucella agar (Sigma-Aldrich, St. Louis, MO, USA) for 24 h under the same conditions. The minimum concentration of extract or nano hydrogels that inhibited visible growth in the culture medium was equal to MBC.

Anti-biofilm assayQuantitative anti-biofilm assay

The minimum biofilm inhibition concentration (MBIC50): MBIC50 was determined by the microtiter method, by investigating the inhibition of biofilm formation at MIC and sub-MIC concentrations. MBIC50 is the minimum concentration that inhibits biofilm formation by 50% or more. Briefly, 1.5 × 106 CFU/mL bacteria were added to the wells containing extract or nano hydrogels in the mentioned concentrations in the flat-bottom 96-well plate. In this method, Brucella broth contained 1% sucrose. After 48 h of cultivation in anaerobic conditions and 37 °C. Free cells were discarded by gently washing the wells twice with PBS. Then, by adding 200 µl of 10% formaldehyde, the attached bacteria were fixed. The biofilms formed on the surface of the wells were stained with crystal violet for 30 min. After its dissolving with ethanol, absorbance was measured at 595 nm [25].

Qualitative anti-biofilm assay on the dental surface

Evaluation of biofilm formation on the dental surface was done according to Ashrafi et al.‘s study with a slight change [11]. Seventeen healthy premolar teeth extracted in the orthodontic department of dental school, Shahed University, were first washed with normal saline. In the next step, 25.5% sodium hypochlorite and then 20% H2O2 were used to remove the smear layer. After they were sterilized by autoclaving, the teeth were immersed in 4 mL of culture medium containing 1.5 × 106 CFU/mL of bacteria and extracts or nano hydrogels at concentrations of MIC, 1/2 MIC, and 1/4 MIC. Finally, they were cultured for 48 h in a shaker incubator under anaerobic conditions at 37 °C.

RNA extraction and real-time PCR

Total RNA was extracted from untreated S. mutans and S. mutans treated with sub-MIC concentrations of extract and nano hydrogels for 48 h with DNAbiotech kit (DNAbiotech, Iran). RNA extraction steps proceeded according to the manufacturer’s instructions. The quantity and integrity of purified RNA samples were analyzed by spectrophotometry (Nanodrop, USA) and agarose gel electrophoresis (1.5% agarose gel), respectively. Purified RNA was stored at -80 °C until use. cDNA synthesis kit (Parstous, Iran) was used for reverse transcription of 2 µg of total RNA of each sample to cDNA.

The expression level of 4 essential genes in biofilm formation and 16SrRNA as a reference gene was determined in triplicate through quantitative Real-Time PCR. Samples were analyzed using a SYBR Green master mix (Amplicon, Denmark) with an ABI 7500 detector (Applied Biosystems, USA). Informations of primers and PCR conditions are described in Table 1. The conditions of the real-time PCR amplification reactions were as follows:

95 °C for 10 min for initial pre-cycling heat activation, then 40 cycles of 95 °C for 20s, annealing at 60 °C for 45 s for gtfB, gtfD and gbpB and at 58 °C for 40 s for gtfC and 16SrRNA.

The 2-ΔΔCT standard method was used to determine relative expression levels [26].

Table 1 Characteristics of primers for genes related to S.mutans biofilm formationCytotoxicity assay

In this study, for the first time, the cytotoxicity of QI extract, QI-NH and TA-NH against KB cells was assessed by MTT method. The toxicity of CHX as one of the most common antimicrobial agents in dentistry was investigated too. In this regard, KB cell line was purchased from National cell bank of Iran (NCBI, Pasteur Institute, Tehran). RPMI 1640 medium containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin was employed. Cells (3 × 104 cells/well) were cultured at 37 °C with 5% CO2 in 96-well plates. To evaluate cell viability, five different concentrations of the studied substances were mixed in culture medium in triplicate. After 24 h, the culture medium was removed and equal (20 ml) MTT or 2,5-diphenyl-2 H-tetrazolium bromide (Sigma-Aldrich, St. Louis, MO, USA) was added to the wells and incubated again for 1 h at 37 °C in the dark. The supernatants were removed and again by adding DMSO to the wells and gently shaking for 10 min, the remaining sediments were removed from the wells. Finally, the absorption of samples in different concentrations was measured with a microplate reader and, cell viability was calculated based on the following formula:

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Statistical analysis

In this study, Graph Pad Prism (Version 9.5.1) was used for data analysis. One-way ANOVA test was used for statistical analysis of data and, Tukey’s test was used to compare means. Differences were considered statistically significant at p < 0.05.