Thymol-based Chitosan Nanogels Have Strong Antibacterial and Anti-biolm Effects on Multidrug-resistant Pathogens

In the present study, the antibacterial and antibiolm effects of Thymol-based chitosan nanogels were investigated. After clinical screening of MDR strains from the hospital environment, the morphological characteristics of the synthesized nanoparticles were identied using SEM, DLS, XRD and FTIR and the eciency of encapsulation, stability and drug release were evaluated. The expression of OmpA and PgaB biolm genes were determined by q-Real-Time PCR and the antibacterial and cytotoxic properties of the nanoparticles were determined by well diffusion and MTT methods, respectively. Nanoparticles with a size of 82.71 nm, encapsulation eciency of 76.54% and stability up to 60 days at 4 °C were prepared. The results of the biological study showed strong antibacterial properties of Thymol-based chitosan nanoparticles by reducing the expression of OmpA and PgaB biolm genes at a signicant level of P ≤ 0.05 and reducing antibiotic resistance compared to the free drug thymol and chitosan nanogels. Thymol-based chitosan nanogels at concentrations of 0.125 to 256μg/mL showed the lowest cytotoxicity against HEK-293 compared to chitosan and free Thymol nanogels. The results showed very strong antibacterial properties of Thymol-based chitosan nanogels against MDR strains such as Staphylococcus, Acinetobacter and Pseudomonas as the challenging bacteria of the century. as fever and chills, dizziness and drowsiness, burning when urinating, the strange smell of urine, respiratory problems, muscle and pain, cough with yellow, green or bloody mucus were selected and informed consent and ethical approval was received from these patients. The initial diagnosis of the disease in these people was made fellow This study was conducted ethical principles and expression of PgaB gene in Pseudomonas, Acinetobacter, and Staphylococcus bacteria was 0.148, 208, 0.412 and the expression of OmpA gene was 0.212, 0.256, and 0.324, respectively. The results showed that different bacterial strains with different concentrations of MIC, under the inuence of chitosan nanogels and Thymol-based chitosan nanoparticles had different expression changes and showed a statistically signicant difference compared to the expression of the al. (2018) reported the antibacterial activity of Thymol nanoemulsion [51]. Abdelhamid et al. (2017) showed the combined effect of Thymol and silver nanoparticles against Staphylococcus aureus [52]. The last aim of this study was to investigate the non-toxicity of Thymol-based chitosan nanogels on human cells. For this purpose, MTT test was performed and the results showed low or no toxicity of this nanoparticle on HEK-293 cells. These results can be attributed to the amazing properties of chitosan and its non-toxicity. Chitosan can also reduce the toxicity of drug agents [53]. Many studies have reported the toxicity-modifying properties of chitosan nanoparticles [54]. Shukla et al. (2015) showed that the cellular damage of iron oxide nanoparticles was signicantly reduced after encapsulation in chitosan [55]. The present study also showed that the coating of chitosan nanogels on Thymol reduced cell damage and, as a result, the cytotoxic effect of free Thymol was signicantly reduced.

According to the fellow doctor, the history of smoking, weakened immune system and history of chronic disease was recorded in individuals. For screening, blood, urine, respiratory samples (bronchial lavage, secretions suction), and chest x-ray were performed according to the fellow doctor's instructions.

Prescribing antibiotics and clinical examination of patients
After diagnosing the infection, the decision about the type of antibiotic to be used to treat the infection was made separately by the fellow doctor based on each individual's symptoms. Prescribed antibiotics include; Cipro oxacin, erythromycin, chloramphenicol, amikacin, tetracycline, imipenem, tazobactam, ampicillin-sulbactam, kanamycin, ceftazidime, and clistin. After 15 days, the results of controlling or not controlling the disease were recorded by a fellow doctor. A sampling of patients was also performed.

Recovery of bacterial isolates and biochemical tests
From patients who did not recover after antibiotic administration, blood, urine, and respiratory samples were collected in the hospital laboratory, and biochemical tests, including growth on blood agar, McCangi agar, and oxidase, catalase, urease, lysine-decarboxylase, MR-VP, SIM, TSA, Simon citrate and arginine tests were performed.

Bio lm detection phenotypic test
According to the method of Mariana et al. [22], Congo Red Agar (CRA) medium was prepared by combining BHI (brain heart infusion broth) (37 g/L), sucrose (50 g/L), agar (10 g/L) and Congo Red indicator (0.8 g/L) and clinical strains of bacteria were cultured on plates containing Congo Red culture medium for 24 hours at 37 °C. Under such conditions, bio lm-producing bacteria form black colonies and other bacteria form red colonies.

Bio lm detection genotype test
In this test, after culturing the bacteria, 200 μL of them was added to 96-well sterile polystyrene wells and incubated for 24 hours at 37 °C. 200 μL of 1% violet crystal dye was added to it for staining for 15 minutes and then washed with PBS. After adding 200 μL of 33% acetic acid, absorbance was measured at 570 nm by ELISA Reader Stat Fax2100 (Awareness Technology, Ukraine). In order to investigate by Optical density cut-off (ODc) method, rst the standard deviation and mean OD of negative control wells and ODc were calculated according to Formula 1 and OD of the studied wells was classi ed according to Table 1. Initially, DNA extraction from screened resistant bacteria was performed according to the protocol of the DNA extraction kit (Sinaclon, Iran). Also, to detect bacterial densities in clinical specimens, polymerase chain reaction (PCR) method using universal primer and sequence (F: 5'-AGAGTTTGATYMTGGCTCAG-3') and (R: 5'-AGAAAGGAGGTGATCCAGCC-3') used. After determining the microbial density by polymerase chain reaction, the presence of Acinetobacter, Pseudomonas and Staphylococcus as well as the presence of OmpA and PgaB genes were con rmed. The primer sequences used in this study are listed in the Table 2.
The nal volume of the reaction ( 1 gram of chitosan powder (Sigma-Aldrich, USA) was added to a container containing 1 mL of distilled water and 1 mL of hydrogen chloride (pH=4.5) and mixed for 1 hour using a magnetic stirrer with 800 rpm. It was then centrifuged at 1000 rpm for 5 minutes. Dissolved 50 mg of myristic acid and 100 mg of EDC and NHS in 1 ml of ethanol and then the resulting solution was added dropwise to chitosan for 2 hours. The pH of the reaction was then increased with dilute sodium (0.1 M) to precipitate chitosan nanogels (pH=8.5). Finally, the chitosan nanogel precipitate was separated by centrifugation and washed with ethanol and then distilled water.

Thymol encapsulation in chitosan nanogels
By adding dilute hydrochloric acid, the chitosan nanogel was dissolved and the resulting mixture was sonicated for 30 minutes using an ultrasonic homogenizer. 0.5 mL of 99.9% Thymol essential oil (Plant Therapy, USA) was dissolved in 2 mL of ethanol, and myristate was added dropwise at the same time as chitosan nanogel sonication. The sonication was continued for 15 minutes to encapsulate Thymol essential oil in nanogels.

2.3.3.
Characterization and investigation of drug loading in nanoparticles with FESEM, XRD, and FTIR electron microscopy Determination of particle size and the charge was performed using Dynamic light scattering devices and palladium electrodes of ZetaPals device (Brookhaven Instruments Corp., USA) at a light scattering angle of 90° and at a temperature of 25 °C. The morphology of nanoparticles was investigated using eld scanning electron microscopy (FESEM) model MIRA3 (TESCAN, Czech Republic) and XRD model X' Pert Pro (Panalytical, Netherlands). 20 μL of dilute nanoparticle solution was dried on aluminum foil at room temperature and then coated with a layer of gold to conduct electrical conductivity. For FTIR spectroscopy, one percent of each sample was converted to a tablet by potassium bromide and then spectroscopy was performed in the frequency range of 400-4000 cm -1 .

Evaluation of encapsulation e ciency and release of encapsulated thymol in chitosan
After the encapsulation of thymol in chitosan nanogels, encapsulation e ciency (EE) was evaluated. Then 1 mL of thymol-based chitosan was centrifuged for 1 hour at 14000 g at 4 °C and then washed with PBS. After determining the amount of encapsulated thymol in each sample by measuring the maximum adsorption of the supernatant at a wavelength of 653 nm, the percentage of encapsulation e ciency was calculated using Formula 2.
Formula 2: EE%= Total amount of initial drug entrapped into the Thymol formulations -amount of free drug in supernatant/ total amount of drug × 100 The process of examining drug release in vitro was performed in a 12 kDa dialysis bag (MWCO). For this purpose, after adding 2 mL of free thymol and 2 mL of thymol encapsulated in chitosan nanogels to the dialysis bag, the whole fraction was placed in 50 mL of PBS solution at different pHs of 3, 5, and 7.4 and gently mixed at 37 °C and 50 rpm. It was then aliquoted at speci ed intervals and a new environment was added to it. Different kinetic diffusion models were analyzed to evaluate drug release characteristics. To determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), the serial dilution method with three replications in a culture medium was used. Dilutions ranging from 256 to 1 μL/mL of chitosan nanoparticles, Thymol essential oil, and Thymol-based chitosan nanoparticles were added to the 96-well plate well. Then 100 μL of microbial suspension was added to each well and the plates were incubated overnight at 37 °C. One well was selected as blank (empty culture medium), and one well was selected as the positive control (culture medium + standard bacteria). Finally, the turbidity of all wells was measured using the ELISA Reader Stat Fax2100 (Awareness Technology, Ukraine) apparatus at 630 nm. The values of MIC, MBC (the lowest concentration of drugs that inhibit bacterial growth or death, respectively), and subMIC (the highest concentrations of drugs that have no effect on bacterial growth) were determined.

Evaluation of OmpA, and PgaB bio lm gene expression
Genomic RNA was extracted according to the protocol of the RNX-Plus kit (Sinagen, Iran) from bacteria isolated from clinical specimens after treatment with subMIC concentrations of chitosan nanoparticles, Thymol essential oil, and Thymol-based chitosan nanoparticles. Concentrations of 1 to 2 μg of RNA were determined by the Nanodrop Model One C (Thermo, USA) with a light absorption reading at 260 nm. Also, light absorption was measured at 260/230 nm to ensure the absence of contamination and the light absorption ratio for RNA was considered equal to 2. CDNA was constructed from RNA extracted from treated and untreated bacteria according to the YTA Kit Protocol (Yekta Tajhiz, Iran). Quantitative Real-Time PCR test was performed with Mastermix Cybergreen YTA (Yekta Tajhiz, Iran) and 16SrRNA gene as a reference gene. 15 μL of reaction volume consisted of 0.5 μL of cDNA, 0.5 μL of the forward primer, 0.5 μL of back primer, 10 μL of mastermix, and 3.5 μL of double sterile distillation water. The temperature cycle program also included initial denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 20 sec and 60 °C for 40 sec. The nal step to draw the Melting Curve was selected from 55 to 95 °C.

Investigation of antibacterial properties of synthesized nanoparticles
After preparing the Müller Hinton agar culture medium, using a sterile pipette No. 5, made 4 mm deep wells on a plate containing the culture medium and then the suspension of resistant bacteria was cultured on the culture medium surface with a sterile swab. 30 μL of subMIC concentration of chitosan nanoparticles, Thymol essential oil and Thymol-based chitosan nanoparticles were poured into wells and incubated for 24 hours. DMSO and gentamicin were used as negative control and positive control, respectively, and the diameter of the growth inhibition zone was measured.
2.6. Investigation of cytotoxicity of synthesized nanoparticles HEK 293 cells are widely used in biocellular research due to their reliable growth. Therefore, to evaluate the cytotoxicity and survival rate of synthesized nanoparticles, the MTT tetrazolium salt-based colorimetric method was used on HEK 293 cells. According to the instructions of the DMA500 kalazist kit, cells were cultured in 96-well plates with 2-5 ×104 cells per 100 μL of culture medium and incubated overnight in a CO 2 incubator. Then the fresh culture medium was replaced and incubation was performed at 37 °C for 24 hours. Dilutions of 256 to 0.125 μL/mL of chitosan nanogels, thymol essential oil, and thymolbased chitosan nanogels were rst dissolved in DMSO and then diluted in a culture medium and added to the cells. After incubation of CO 2 at 37 °C for 48 hours, 20 mL of MTT solution (5 mg/mL in the colorless buffer PBS) (Kalazist, Iran) was added to the wells and after 4 hours of incubation, the contents of the wells were drained and 100 μL of DMSO solution was added. The plates were placed on a shaker at 400 rpm for 6 minutes to completely dissolve the formed formazan crystals in DMSO. The color intensity was then read by the ELISA Reader Stat Fax2100 (Awareness Technology, Ukraine) at 570 nm. The cell-free culture medium was used as the blank of the ELISA reader apparatus and the culture medium with the cell without drug was used as the control of the living cell. The percentage of viable cells was obtained from Formula 3.

Statistical analysis
Statistical analysis in this study was calculated using SPSS software version 16 and the results were subjected to One-way Analysis Of Variance (ANOVA). Also, the expression of target genes between the control and treated samples was calculated by Tukey's HSD post statistical method.

Results of clinical studies
The clinical part of the study was a descriptive-analytical study.  ) did not respond well to antibiotic therapy. Thus, the rates of resistant bacteria in pneumonia and sepsis were higher than wound infection and urinary tract infection, respectively, which indicates the failure of antibiotic treatment in these infections. These patients were selected as a source of MDR bacterial isolation and sampling was performed for laboratory evaluation.

Laboratory diagnosis results
Of the samples collected from 112 unhealed patients, after biochemical tests, the most common bacterial densities were related to gram-negative organisms and Escherichia coli, Acinetobacter, Pseudomonas aeruginosa, and other Enterobacter species. Then, antibiotic susceptibility testing was performed and the highest resistance to broad-spectrum antibiotics was related to Acinetobacter, Staphylococcus, and Pseudomonas, respectively. Phenotypic bio lm detection test was performed in isolated strains according to Table 3. Resistant bacteria were then selected for isolation and species identi cation and PCR was performed with 16SrRNA primer and 27 pathogens with 110 bp length were identi ed as Ab, Pseudomonas aeruginosa, and Staphylococcus. According to the aim of the study to evaluate the strength of bio lm in isolated strains, a microtiter test was performed and the results showed strong and moderate bio lms in 18 and 9 samples of each bacterium, respectively (Graph 1).
To evaluate the presence of bio lm genes in bacteria, PCR test was performed using speci c primers and con rmed the presence of OmpA and pgaB genes in the screened bacteria. Standard strains were used as positive control in this test.

Characterization and investigation of drug loading in nanoparticles
chitosan nanogels and Thymol-based chitosan nanoparticles were synthesized and optimized in terms of size, charge and shape. The average size of chitosan nanogels and Thymol-based chitosan nanoparticles were 72.35 and 82.71 nm with spherical structure, respectively ( Figure 1).
Suitable uniformity of the produced nanoparticles was determined using the DLS (Dynamic Light Scattering) technique. The Poly Dispersity Index (PDI) and mean diameter of chitosan nanogels were 0.178 and 258.2 nm, respectively, and for Thymol-based chitosan nanoparticles were 0.115 and 179.3 nm, respectively. The zeta potential of drug-free chitosan nanogels and thymol-based chitosan showed 64.19 and 66.35 mV, respectively. Also according to the XRD diagram, in the range of 2θ, the intensity of the peak increased with increasing irradiation time and peaks with higher intensities have more re ection, which indicates the presence of nanoparticles. Accordingly, the highest re ections related to peak intensities at 29.470 angles in Figure 2C and angles 32.470, and 38.940 in Figure 2D were related to chitosan nanogels and Thymol-based chitosan nanoparticles, respectively; Therefore, the amount of nanoparticles formed in the range of these peaks is higher. FTIR test was performed and in the spectrum of chitosan nanoparticles, peaks in the range of 2858.54 cm -1 and 2928.83 cm -1 were related to C-H bond and peak 3329.24 cm -1 belonged to the amino group of chitosan and peaks of 1082.15, 1158.47 and 1739.11 cm -1 belonged to the carboxyl group. In the FTIR spectrum of the thymol, the peak of 2919.19 cm -1 is associated with the tensile vibration of C-H in the benzene ring. The absorption peaks at 1742.34, 1630.90, 1460.77, and 1378.23 were four unequal stretches corresponded to a speci c peak of Thymol. The peaks at 1247.83 and 1098.26 cm -1 were related to the -OH and C-O exural vibrations. The peak of 3370.00 cm -1 belongs to the O-H group. The presence of these peaks in Thymol-based chitosan nanoparticles indicates the loading of Thymol in the chitosan nanogel ( Figure 2).
According to Table 4, encapsulation e ciency (EE), PDI dispersion index, hydrodynamic diameter size and surface charge of nanoparticles indicate proper synthesis and high encapsulation e ciency of Thymol in chitosan nanogels. A dialysis bag containing Thymol-free drug and Thymol-based chitosan nanogel was used to evaluate the controlled release pro le of the drug. The resulting curve is similar to the bacterial growth curve in that in the rst 9 hours; the Thymol-free drug is released rapidly, followed by a slow release of the drug from the dialysis bag. This reduction in emissions lasted up to 48 hours. According to the results, the release of Thymol-free drug and Thymol-based chitosan nanogel in the rst 9 hours was 88% and 69%, respectively, and then the drug release rate was proven so that after 48 hours, the release rate of the two drugs increased to 97 and 76.54%, respectively. Morphological properties including; mean size, Poly Dispersity Index (PDI), and encapsulation e ciency (EE) were measured to evaluate the stability of Thymol-based nanogels at different temperatures and time intervals. The results showed that with increasing temperature, it changed the morphological properties of chitosan-based nanogels that as a result of EE drug reduction was reduced. However, increasing the shelf life of the drug has little effect on the morphological characteristics and EE of the drug (Figure 3).

Investigation of inhibitory ability of synthesized nanoparticles
The susceptibility of 27 bio lm-producing isolates in 3 bacterial genera was determined based on the minimum growth inhibitory concentration (MIC) for chitosan nanogels, Thymol essential oil, and Thymol-based chitosan nanoparticles. Bacterial positive bio lm strains were exposed to 100% to 0.39% of essential oils and nanoparticles for 24 hours. According to Table 5, each strain had a range of MICs with concentrations between 256 and 1 µg/mL. All isolates were treated with Sub-MIC concentrations of chitosan nanoparticles, Thymol essential oil, and Thymol-based chitosan nanoparticles. According to the results, Thymol-based chitosan nanoparticles had the highest inhibition and then chitosan nanoparticles and Thymol essential oil had the highest inhibition in all 3 bacterial genera, respectively. Pseudomonas was also less resistant than the other two genera. The highest resistance to synthesized compounds belonged to the genus Staphylococcus. After determining the MIC of nanoparticles in 3 bacterial genera, samples S4, S12, and S27 were identi ed as the most resistant pathogens, respectively, and were selected to evaluate the e ciency of the synthesized nanoparticles. For this purpose, fresh cultures of bacteria were rst prepared and a colony of each bacterium was cultured in a tube containing the Müller-Hinton broth medium. Then S4, S12, and S27 bacteria were cultured at concentrations of 256 to 1 μg/mL chitosan nanogels and thymol-based chitosan nanoparticles and during 24 hours of incubation at 37 °C every 4 hours light absorption of the samples was read.  Figure 3, Staphylococcus bacteria show a higher growth curve at concentrations of 1 to 256 μg/mL against synthesized nanoparticles, indicating that Staphylococcus is more resistant to nanoparticles. Acinetobacter also has a growth curve close to Staphylococcus, although it lacks a cell wall. This indicates the high e ciency of Acinetobacter resistance mechanisms compared to other gram-negative bacteria such as Pseudomonas. Pseudomonas was the most sensitive bacterial strain to the synthesized nanoparticles. On the other hand, Thymol-based chitosan nanoparticles showed the highest inhibitory effect in all three bacterial genera. Concentrations between 256 and 8 μg/mL of Thymol-based chitosan nanoparticles showed the best inhibitory effects in all 3 bacterial genera.
Then, in order to evaluate the anti-bio lm activity, the subMIC concentration of nanoparticles is used and the expression of PgaB and OmpA genes in all 3 bacterial genera is examined by Quantitative Real-Time PCR and the results of PgaB and OmpA gene expression relative to 16SrRNA reference gene in positive bio lm strains are shown in Graph 2. According to the results, the mean of the highest decrease in PgaB and OmpA gene expression was related to Pseudomonas strain.
Thymol-based chitosan nanoparticles also showed the greatest inhibitory effect on the expression of bio lm genes in all three bacterial genera; the expression of PgaB gene in Pseudomonas, Acinetobacter, and Staphylococcus bacteria was 0.148, 208, 0.412 and the expression of OmpA gene was 0.212, 0.256, and 0.324, respectively. The results showed that different bacterial strains with different concentrations of MIC, under the in uence of chitosan nanogels and 16SrRNA gene (P≤ 0.05). Finally, the antibacterial activity of the synthesized nanoparticles was investigated by the standard method of well diffusion. After culturing Pseudomonas, Acinetobacter, and Staphylococcus separately, four wells were created in the plate containing the culture medium and chitosan nanogels, Thymol-based chitosan nanoparticles, Thymol-free drug, and gentamicin antibiotic were added as positive controls to wells 1 to 4, respectively. According to Table 6, the highest growth inhibition zone is related to Thymol-based chitosan nanoparticles, which in Pseudomonas, Acinetobacter, and Staphylococcus bacteria are 30, 25, and 18 mm, respectively. Also, all three bacterial genera were resistant to the antibiotic gentamicin. These results indicate the appropriate antibacterial properties of the synthesized nanoparticles. Finally, the cytotoxicity of chitosan nanogels, free Thymol, and Thymol-based chitosan nanogels were measured at concentrations of 0.125 to 256 μg/mL for HEK-293 cells. According to Figure 4, the results showed that Thymol-based chitosan nanogels had the least inhibitory effect on HEK-293 compared to chitosan nanogels and Thymol-free drug in all samples.

Discussion
The discovery of antibiotics to treat bacterial infections was one of the most important advances in medical history. Unfortunately, bacteria are very adaptable, and overuse of antibiotics has made many bacteria resistant to antibiotics [23]. With the increase in the range of antibiotic resistance, the density of MDR bacteria in hospitals has increased and has created a new challenge called nosocomial infections [24]. According to the clinical results of this study, the main spectrum of bacteria resistant to various nosocomial infections belonged to the three genera Staphylococcus, Pseudomonas and Acinetobacter. Previous studies have reported an increase in the prevalence of antibiotic resistance in these three genera [25]. On the other hand, the results of the present study showed that 83.3% of the bacteria are resistant to MDR and have the ability to form bio lms. These results are consistent with the results of previous studies indicates an increase in resistance to various antibiotics due to increased expression of bio lm genes and consequently increased bio lm formation capacity [26]. 66% of the bacteria studied in this study formed strong bio lms; while other bacteria were Intermediate in terms of bio lm potency, indicating a direct effect of bio lm on antibiotic resistance and high activity of bio lm genes in multidrug-resistant bacteria. According to previous studies, bacteria use various physical, physiological and genetic factors to develop resistance mechanisms, that the highest resistance being related to bio lm and expression of bio lm genes [27]. In this study, it was shown that OmpA and pgaB genes are actively present in MDR bacteria, which increase the ability of bio lms to form in bacteria and contribute to bacterial resistance to various antibiotics. Past studies indicate an important role for the pgaB protein in activating the bio lm response under stress conditions [28]. Many studies have identi ed OmpA as a factor in antibiotic resistance and bio lm formation [29]. Today, due to the increase in nosocomial infections, the prevalence of MDR bacteria and the slow production of new antibiotics, the need to use natural antibacterial agents is felt more than ever [30]. Therefore, most research is on various strategies to introduce new antibacterial agents or even overcoming bacterial resistance.
Therefore, in this study, after preparing chitosan nanogels, encapsulation of Thymol in the structure of chitosan nanogels was performed with the aim of introducing a new antibacterial compound. In the formulation used, Thymol, as the main extract of thyme, is a known antibacterial agent (31). While chitosan polymer, due to its positive charge, has a high ability to react with biological membranes (negative charge) and increases the transport capacity of drugs [32].
Chitosan also has different and special properties such as; non-toxic, biodegradable and emulsifying, which is used to convert oily and water-insoluble drugs into soluble form [33]. According to previous studies, chitosan amino groups are responsible for properties such as; Controlled drug release, mucosal adhesion, increased penetration, and bacterial growth inhibitory and inhibitory properties of the e ux pump [34]. By converting chitosan polymer to nanoparticles, its chemical properties can be improved and the transport e ciency of drugs can be increased [35]. Therefore, encapsulation of drugs such as thymol can be promising for new antibacterial compounds. Encapsulation e ciency is an important factor in the synthesis of nanomaterials and various factors such as; Nanoparticle diameters affect capsulation e ciency and consequently drug release ability [36]. The results of our study showed that the capsulation of thymol by chitosan nanogels increases its antibacterial properties. The results of this study are consistent with previous studies that show that the antibacterial properties of thymol are increased at the nano level [37]. In this study, the uniformity of nanoparticles was con rmed by DLS and the size of nanoparticles for chitosan nanogels and Thymol encapsulated in chitosan nanogels were determined to be 258.2 and 179.3, respectively, while the size of nanoparticles obtained by SEM indicates the appropriate size for chitosan nanogels (72.35) and Thymol encapsulated in chitosan nanogels (82.71). Since DLS determines the hydrodynamic diameter, it consists of nanoparticles and ions or molecules attached to it.
But SEM is the exact method that determines the dry size of nanoparticles. These results, in line with previous studies, indicate the effect of scattering on particle measurement error by DLS [38]. Also, the study of drug release pro le showed that drug diffusion at PH = 7.4 in 48 hours is 76%. While in the rst 9 hours, the highest rate of drug diffusion is seen. The initial diffusion rate can be attributed to the release of surface drugs. As time goes on, the surfaceencapsulated drugs are released more until after 9 hours the Thymol concentration at the surface is minimized and the encapsulated drugs in the underlying layers begin to release. This slows down the release of the drug. Previous studies have introduced drug release control agents in polymer nanoparticles diffusion and biodegradation and reported the rapid release of the drug in the early hours as an "explosion diffusion" [39]. The results of this study in line with previous studies indicate the explosive diffusion of encapsulated Thymol release [40]. Protein stability study showed that temperature as a destructive factor plays an important role in drug preservation [41]. So that with increasing temperature in 60 days interval, the morphological properties of nanoparticles changed and as a result, the e ciency of nanoparticles was decreased. However, according to the results, nanoparticles have a storage capacity of 15 to 30 days with good e ciency. However, time has little effect on the morphology and e ciency of nanoparticles, and thus by storing Thymol-based chitosan