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Composition and characterization of chlorhexidine hydrochloride nanoemulsion as a promising antibacterial root canal irrigant: in vitro and ex vivo studies
作者 Abdelmonem R., Younis M.K., Hassan D.H., El-Sayed Ahmed MAEG, Hassanien E., El-Batuti K., Elfaham A.
Rehab Abdelmonem, 1 Mona K. Younis, 1 Doaa H. Hassan, 1 Mohamed Abd El-Gawad El-Sayed Ahmed, 2 Ehab Hassanein, 3 Kariem El-Batuti, 3 Alaa Elfaham 31 Science and Technology, Faculty of Pharmacy and Industrial Pharmacy, Misr University, 6 October City, Egypt; 2 Department of Microbiology and Immunology, Faculty of Pharmacy, Misr University of Science and Technology, October 6, Egypt; 3 Department of Endodontics, Ain Shams University, Cairo, Egypt Introduction and Purpose: Chlorine hexidine hydrochloride [Chx.HCl] has a broad spectrum antibacterial activity, prolonged action and low toxicity, therefore it is recommended as a potential root canal irrigant. The aim of this study was to use a new composition Chx.HCl nanoemulsion to increase the penetrating power, cleansing and antibacterial action of Chx.HCl and use it as a root canal irrigant. Methods: Chx.HCl nanoemulsions were prepared using two different oils: oleic acid and Labrafil M1944CS, two surfactants, Tween 20 and Tween 80, and co-surfactant, propylene glycol. Plot a pseudo-ternary phase diagram to indicate the optimal system. The prepared nanoemulsion formulations were evaluated for drug content, emulsification time, dispersibility, droplet size, in vitro drug release, thermodynamic stability, in vitro antibacterial activity, and in vitro studies of selected formulations. The penetrating, cleansing and antibacterial action of Chx.HCl 0.75% and 1.6% nanoemulsion was compared with normal particle size as a root canal irrigant. Results. The formulation chosen was F6 with 2% Labrafil, 12% Tween 80 and 6% propylene glycol. Small particle size (12.18 nm), short emulsification time (1.67 seconds) and fast dissolution after 2 minutes. It has been found to be a thermodynamically/physically stable system. Compared to the conventional Chx.HCl particle size, the higher concentration of Chx.HCl 1.6% nanoemulsion showed better penetration due to the smaller particle size. Compared to a normal particle size material (2609.56 µm2), the 1.6% Chx.HCl nanoemulsion has the smallest average surface area of ​​residual debris (2001.47 µm2). Conclusion: Nanoemulsion composition Chx.HCl has better cleaning ability and antibacterial action. It has a highly effective bactericidal action against Enterococcus faecalis, and the bacterial cell contraction rate is high or completely destroyed. Key words: chlorhexidine hydrochloride, nanoemulsion, root canal irrigant, penetration, cleansing effect, antibacterial irrigant.
Nanoemulsions, a class of emulsions with droplet sizes in the range of 50–500 nm, have received much attention in recent years due to their unique properties. Good cleaning properties, they are not affected by water hardness, in most cases they have low toxicity and the absence of electrostatic interactions. 2 Nanotechnology has an ultra-small particle size, a large surface area to mass ratio and unique physical and chemical properties compared to similar bulk products, and also opens up new perspectives in the treatment and prevention of dental infections. 3 Chlorhexidine hydrochloride (Chx.HCl) is slightly soluble in water, very slightly soluble in alcohol and gradually stains in the light. 4.5 SH. HCl has a broad spectrum antibacterial action, prolonged action and low toxicity. Because of these properties, it is also recommended as a potential root canal irrigant. The main advantages of Chx.HCl are low cytotoxicity, no odor and no unpleasant taste. 6-9 Several types of lasers have been used to improve root canal disinfection. The bactericidal effect of lasers depends on the wavelength and energy, as well as on thermal exposure, which causes changes in the bacterial cell wall, which leads to a change in the osmotic gradient up to cell death. The interaction between lasers and root canal irrigators opens up new horizons in pulp disinfection. 10 Ultrasonic energy produces high frequencies but low amplitudes. The files are designed to oscillate at ultrasonic frequencies of 25–30 kHz, which are beyond the limit of human auditory perception (>20 kHz). The files are designed to oscillate at ultrasonic frequencies of 25–30 kHz, which are beyond the limit of human auditory perception (>20 kHz). Файлы предназначены для колебаний на ультразвуковых частотах 25–30 кГц, которые находятся за пределами слухового восприятия человека (> 20 кГц). The files are designed to vibrate at ultrasonic frequencies of 25-30 kHz, which are beyond the range of human hearing (> 20 kHz).这些文件被设计成在25–30 kHz 的超声波频率下振荡，这超出了人类听觉感知的极限(>20 kHz)。这些文件被设计成在 25–30 kHz Файлы рассчитаны на колебания на ультразвуковых частотах 25–30 кГц, что выходит за пределы слухового восприятия человека (>20 кГц). The files are designed for vibrations at ultrasonic frequencies of 25-30 kHz, which is beyond the limits of human hearing (>20 kHz). They operate in transverse oscillation, setting the characteristic modes of knots and antinodes along their length. The term “passive ultrasonic irrigation” (PUI) is an irrigation protocol in which no instruments or walls come into contact with endodontic files or instruments. During PUI, ultrasound energy is transferred from the vibrating file to the irrigation solution in the root canal. The latter can cause sonic flow and cavitation of the flushing agent. 11 Based on the above data, it is considered appropriate to use nanotechnology to evaluate the improved penetrating and cleaning action of Chx.HCl.
Chlorhexidine hydrochloride Chx.HCl was kindly provided by Arab Drug Company for Pharmaceuticals (Cairo, Egypt). Labrafil M 1944 CS (oleoylpolyoxy-6-glyceride) was generously provided by Gattefosse (Saint Priest, France). Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 80 (polyoxyethylene (80) sorbitan monooleate), oleic acid, propylene glycol from Gomhorya Company (Cairo, Egypt)). Extraction of non-carious single-rooted teeth for periodontal or orthodontic treatment, Department of Maxillofacial Sciences, Faculty of Dentistry, Ain Shams University, Cairo, Egypt. Pure culture of Enterococcus faecalis (strain ATCC 29212) grown in brain heart extract (BHI) broth (RC CLEANER, IIchung Dental Ltd., Seoul, Korea).
The solubility of Chx.HCl in various media (oleic acid, Labrafil M 1944CS, Tween 20, Tween 80, propylene glycol, and water) was studied. A large excess of Chx.HCl (50 mg) is placed in a centrifuge tube and 5.0 g of the medium phase is added. The mixture was shaken in a vortex mixer for 15 minutes and then stored at room temperature. After 24 hours, the insoluble drug pellet in the tube was centrifuged at 3000 rpm for 5 minutes to obtain a clear supernatant. Collect enough sample solution and dilute it with n-butanol. The diluted samples were filtered through Whatman 102 filter paper and then diluted appropriately with n-butanol to determine the drug concentration in the saturated solution. Samples were analyzed with a UV spectrophotometer at 260 nm with n-butanol as a control. 12.13
A pseudo-triple phase diagram was built to determine the exact ratio of each component required in the formulation to obtain the optimal parameters of an ideal nanoemulsion. 14 The formulation was formulated using oils (i.e. oleic acid and Labrafil M1944CS), surfactants (i.e. Tween 20 and Tween 80) and an additional surfactant, i.e. propylene glycol. First, separate mixtures of surfactants (without cosurfactants) and oils were prepared in different volume ratios (from 1:9 to 9:1). When the mixture is titrated with water (adding water dropwise), closely monitor the mixture from clear to cloudy as the end point. These end points are then marked on a pseudo-triple phase diagram. The entire process was repeated for mixtures of surfactant and secondary surfactant (Smix) prepared in 2:1 and 3:1 ratios and mixed with selected oils15,16 one.
Nanoemulsion systems containing Chx.HCl were prepared using Labrafil M 1944 CS as oil phase and Tween 80 or 20 surfactant and propylene glycol as additional surfactant and finally water, Table 1. The drug was dissolved in Labrafil M 1944 CS and the combined water of surfactant and secondary surfactant was added at a slow rate with gradual mixing. The amount of surfactant and co-surfactant added, as well as the percentage of oil phase that can be added, is determined using a pseudo-ternary phase diagram. An ultrasonic generator (Ultrasonic LC 60 H, Elma, Germany) was used to achieve the desired size range for dispersing the granules. Then balance it. 17
Dispersibility testing was carried out using a dissolution apparatus (Dr. Schleuniger Pharmaton, Model Diss 6000, Thun, Switzerland) in which 1 ml of each preparation was added to 500 ml of water at 37±0.5°C. Gentle agitation is ensured by standard stainless steel dissolving paddles rotating at 50 rpm. The resulting emulsion was determined visually and classified as clear, translucent with a bluish tinge, milky or hazy. Choose a clear formula for further research. 18.19
Extraction of Chx.HCl from optimized nanoemulsion compositions based on pseudo-triple phase diagram leads to the production of n-butanol using ultrasonic technology. After appropriate dilution, the extracts were analyzed spectrophotometrically at a wavelength of 260 nm for the content of Chx.HCl. twenty
To test the self-emulsification time, 1 ml of each composition was added to a beaker filled with 250 ml of distilled water and maintained at 37 ± 1°C with constant stirring at 50 rpm. The self-emulsification time is taken as the time during which the preconcentrate forms a homogeneous mixture after dilution. twenty one
For droplet size analysis, dilute 50 mg of the optimized formulation to 1000 ml with water in a flask and mix gently by hand. Droplet size distribution was determined using a Malvern Zetasizer 2000 instrument (Malvern Instruments Ltd., Malvern, UK) under backscatter detection conditions of 173º, temperature of 25ºC, and refractive index of 1.330. twenty two
In vitro dissolution studies were performed using a USP Type II apparatus (paddle) (Dr. Schleuniger Pharmaton, Diss Model 6000) at 50 rpm. Distilled water (500 ml) maintained at a temperature of 37±0.5°C was used as the dissolution medium, and 5 ml of the prepared composition was added dropwise to the dissolution medium. Then, at various intervals, 5 ml of the dissolution medium was taken and the amount of released drug was determined spectrophotometrically at 254 nm. The experiments were carried out in triplicate. twenty three
Then, the kinetic parameters of Chx.HCl release in vitro from nanoemulsions prepared on its basis were measured. Zero, first and second order kinetics and Higuchi diffusion models were tested to select the kinetic sequence that is best suited for the release of Chx.HCl.
2 ml of each formulation was stored at ambient temperature for 48 hours before phase separation was observed. 1 ml samples of each Chx.HCl nanoemulsion formulation was then diluted to 10 ml and 100 ml with distilled water at 25° C. and stored for 24 hours. Then phase separation was observed. twenty one
Then samples of 2 ml of each composition were transferred separately into transparent bottles with a screw cap and stored in a refrigerator at 2°C for 24 hours. Then they were removed and stored at 25°C and 40°C. A single cooling-thawing cycle was carried out. The samples were then observed for phase separation and drug precipitation. twenty one
A 5 ml sample of each Chx.HCl nanoemulsion formulation was transferred into a glass tube and placed in a laboratory centrifuge (Shanghai Surgical Instrument Factory Microcentrifuge Model 800, Shanghai, People’s Republic of China) and centrifuged at 4000 rpm for 5 minutes. The samples were then observed for phase separation and drug precipitation. twenty one
All experiments were approved by the Institutional Ethics Committee of Ain Shams University, Egypt. 50 non-carious single-rooted human teeth with a formed apex were selected. Extracted teeth were used after obtaining written informed consent signed by the patient. Teeth include maxillary and mandibular incisors and mandibular premolars. The outer surfaces of the roots were treated with a curette and all teeth were subjected to surface sterilization in 0.5% NaOCl for 24 hours and then stored in sterile saline until use. The crown was removed with a safe side diamond disc and the length of the tooth was normalized to 16 mm from apex to coronal margin. 24,25 According to the rinse solution, the teeth are divided into the following groups:
(A) Group (n=24) samples were washed with Chx.HCl nanoemulsion. Subgroup (I) (n = 12) rinsed samples with 5 ml Chx.HCl nanoemulsion 0.75% concentration. Subgroup (II) (n=12) rinsed the samples with 5 ml of 1.6% Chx.HCl nanoemulsion. (B) A group (n=24) of samples will be washed with 5 ml 2% Chx.HCl normal particle size. Control group: (n=2) washed with 5 ml saline without activation.
Were selected 44 non-carious single-rooted human teeth with a formed tip. Teeth include maxillary and mandibular incisors and mandibular premolars. The outer surfaces of the roots were treated with a curette and all teeth were subjected to surface sterilization in 0.5% NaOCl for 24 hours and then stored in sterile saline until use. The crowns were removed with a safety diamond disc and the length of the tooth was normalized to 16 mm from apex to coronal margin. 24,25,29
Mechanical preparation of the main apical file size 50 using standard methods. Use sterile saline as an irrigant during surgery. Finally, the root canal was flushed with 2 ml of 17% EDTA for 1 minute to remove the smear layer. The entire root surface, including the apical foramen of each specimen, was covered with two coats of nail polish (cyanoacrylate glue) to prevent leakage. The teeth are then set vertically in a block of tartar for ease of handling and identification. 29-33 Samples were then autoclaved at 121ºC and 15 psi for 20 minutes. After sterilization, all samples were transported and processed under sterile conditions using sterile instruments. The root canals were contaminated with a pure culture of Enterococcus faecalis (strain ATCC 29212) grown in brain heart extract (BHI) broth for 24 hours at 37°C. Using a sterile micropipette, inject a clear suspension of E. faecalis inoculum into the prepared root canals of all teeth. The blocks were then placed in sterile beakers and incubated at 37°C for 24 hours. 31, 34, 35
(A) Group (n=24) samples were washed with Chx.HCl nanoemulsion. Samples of subgroup (I) (n=12) were rinsed with 5 ml of Chx.HCl nanoemulsion 0.75% concentration. Subgroup (II) (n = 12) rinsed the samples with 5 ml of Chx.HCl nanoemulsion 1.6% concentration.
Control group: positive control, (n=4) the contaminated root canal was flushed with 5 ml saline and kept as a positive control. Negative Control: (n=4) Specimens were not injected with suspension, i.e. the root canal was not contaminated with E. faecalis, and was kept sterile as a negative control to confirm sterilization and reliability of the procedure. Use 5 ml test wash solution in each sample. Each sample was then subjected to a final wash with 1 ml of sterile saline.
A size 35 sterile paper tip is used to collect samples from root canals. The paper tip was inserted into the tube to working length, left for 10 seconds, and then transferred to agar plates to determine the number of colony forming units (CFU) per plate. The plates were incubated at 37ºC for 24 hours and then visually assessed for bacterial growth. The transparent plate shows complete sterilization. Blurred plates are considered to show positive growth. The average number of CFUs in the bacterial growth zone per dish was determined and the number of CFUs was calculated. Survivors are primarily measured with viable counts on drip plates. In addition, a pouring cup was used to count low CFUs, and a dilution to 106 was used to count high CFUs. 36.37
Prepare tubes containing 15 ml of thawed agar medium pre-sterilized in an autoclave on the same day as for the experiment. Enterococcus faecalis is a facultative Gram-positive anaerobic coccus that can survive at very high pH, ​​acidity and high temperatures. 39 Bacterial samples (Enterococcus faecalis ATCC 29212) were prepared by mixing cells from colonies with sterile saline. The bacterial samples were then diluted with saline to match McFarland 0.5, equivalent to 108 CFU/mL. The sample volume added was 10 µl. 39 A turbidity standard (McFarland 0.5)40 was prepared by pouring 0.6 ml of 1% (10 g/l) barium chloride dihydrate solution into a 100 ml graduated cylinder and filling to 100 ml with 1% (10 g/l) sulfuric acid. Turbidity standards were placed in the same tubes as the broth samples and stored at room temperature for 6 months in the dark and sealed to prevent evaporation. Open the lid of the empty Petri dish and pour the sample into the middle of the dish. If the agar is completely solidified, invert the plate and incubate at 37°C for 24 hours.
All data were collected, tabulated and subjected to statistical analysis. Statistical analysis was performed using IBM® SPSS® Statistical Version 17 for Windows (SPSS Inc., IBM Corporation, Armonk, NY, USA).
The solubility of Chx.HCl in various oil phases, surfactant solutions, co-surfactant solutions, and water was studied. Chx.Hcl has the highest solubility in Labrafil M and the lowest solubility in oleic acid. Higher drug solubility in the oil phase is important for nanoemulsions because nanoemulsions are able to keep the drug in dissolved form, meaning that higher drug solubility in oil results in less oil in the formulation and therefore less drug. loading A certain amount of surfactant and co-surfactant is required to emulsify the oil droplets.
A pseudo-triple phase diagram was constructed to define nanoemulsion regions and optimize the concentrations of selected oils, surfactants, and additional surfactants (Labrafil M, Tween 80, Tween 20, and propylene glycol, respectively). Chx.Hcl shows very low solubility in oleic acid, resulting in cloudiness when the oleic acid is titrated with the first drop of water. Therefore, the oleic acid system was excluded from this study. Other formulations have been prepared using a 1:9 mixture of oil and surfactant. range of pH and ionic strength, so these surfactants were chosen.
All formulations prepared were clear except for System F2, which appeared cloudy and was therefore excluded from further evaluation studies.
The ideal nanoemulsion formulation should be able to disperse completely and quickly when diluted with gentle agitation. Chx.HCl nanoemulsion formulations showed short emulsification times, from 1.67 to 12.33 seconds. Tween 80 has the shortest emulsification time. This can be explained by the higher solubilizing capacity of Tween 80. The self-emulsification time increases with increasing surfactant concentration, which may be due to the increase in the viscosity of the system under the action of the surfactant.
The droplet size of the emulsion determines the rate and extent of drug release. Smaller emulsion droplet size results in shorter emulsification time and more surface area for drug absorption. The average droplet sizes of the selected compositions of the Chx.HCl nanoemulsion were 711±0.44, 587±15.3, 10.97±0.11, 16.43±4.55, and 12.18±2.48, and the PDI was 0 .76, 0.19, 0.61, 0.47 and 0.76 for F1, F2. , F3 and 0.16 respectively F4, F5 and F6. Formulations containing Tween 80 as a surfactant showed smaller spherulites. This may be due to its higher emulsifying power. A lower PDI value indicates a narrower system size distribution. These formulations have a clean appearance because their droplet radii are smaller than the optical wavelength of visible light (390-750 nm) at which minimal light scattering occurs. 41
On fig. 2 shows the percentage of Chx.HCl released from the formulated formulation. The complete release of the drug from the prepared formulations of the Chx.HCl nanoemulsion ranged from 2 to 7 minutes. It was observed that the highest drug release rate was obtained in the case of the Chx.HCl F6 nanoemulsion formulation (2 min), which may be due to the presence of Tween 80, which showed a higher degree of emulsification, and the resulting nanoemulsion. provides a large surface area for drug release, allowing for increased drug release rates. At the same time, the solubility properties of propylene glycol allow a large amount of hydrophilic surfactants to be dissolved in the oil. 40
Chx.HCl release in vitro has been found to follow a different kinetic order, and no clear kinetic order can reflect drug release from differently prepared nanoemulsion formulations. The kinetic release of F4 drugs is first order kinetics, which means that they are released in proportion to the amount of drug remaining inside them. 42 The kinetic release of other drugs was consistent with the Higuasha diffusion model, which indicated that the amount of drug released was proportional to the square root of the total drug and drug solubility in the nanoemulsion. 42
Selected formulations were subjected to varying thermodynamic stability by stress testing using heat-cool cycles, centrifugation and freeze-thaw cycles. It was observed that formulations F3 and F4 showed precipitation of the drug after thawing cycles, while F1 showed thickening (gelling). F5 and F6 passed the continuous centrifugation cycle, heating-cooling test and freeze-thaw test. Nanoemulsions are thermodynamically stable systems formed at certain concentrations of oil, surfactant and water without phase separation, emulsification or cracking. It is thermal stability that distinguishes nanoemulsions from emulsions, which are kinetically stable and will eventually separate into phases. 19 F3 showed larger particle size (587 nm) than other formulations, which may explain phase separation and drug precipitation in thermodynamic stability tests. F4 containing Tween 80 and no co-surfactant showed drug precipitation, this may indicate on the need to use propylene glycol and Tween 80 to improve the stability of the nanoemulsion formulations. F1 containing Tween 20 without additional surfactant exhibited thickening (gelling), which is an increase in gel viscosity or strength due to droplet aggregation.
The stability results demonstrate the importance of the presence of an additional propylene glycol surfactant to increase particle dispersion and prevent drug precipitation. 43 F6 was the best formulation due to the small particle size (12.18 nm), short emulsification time (1.67 seconds) and fast dissolution rate after 2 minutes. It was found to be a thermodynamically/physically stable system and was therefore selected for further study.
Failures after root canal treatment are becoming more frequent, meaning that patients are at an increased risk of developing more complex infections. 44,45 Biofilm must be removed during disinfection and filling of root canals. 46,47 Because of the complexity of the root canal system, it becomes difficult to completely remove bacterial root canals using only instruments and irrigation. 48 The effectiveness of root canal rinsing solutions depends on the penetration of the irrigant into the DT and the duration of exposure to bacteria. 49 Therefore, new methods of thorough root canal sterilization have been tried and tested. Conventional rinses do not completely eliminate E. faecalis due to less penetration of DT.50.
The average cleaning power of the nanoemulsion rinse was 2001.47 µm2, and the average particle size of the rinse aid was 2609.56 µm. The average difference between the nanoemulsion wash and the normal particle size wash was 608.09 µm2. There was a statistically highly significant (P<0.001) difference between nanoemulsion irrigants and normal particle size irrigants with (P-value 0.00052). There was a statistically highly significant (P<0.001) difference between nanoemulsion irrigants and normal particle size irrigants with (P-value 0.00052). Между ирригационными растворами наноэмульсии и ирригационными растворами с нормальным размером частиц наблюдалась статистически высокозначимая (P<0,001) разница (значение P 0,00052). There was a statistically highly significant (P<0.001) difference (P value 0.00052) between nanoemulsion irrigants and normal particle irrigants.纳米乳液冲洗剂和正常粒径冲洗剂之间存在统计学上高度显着的差异（P<0.001）（P 值0.00052）。纳米乳液冲洗剂和正常粒径冲洗剂之间存在统计学上高度显着的差异（P<0.001）（P 值0.00052）。 Между ополаскивателем с наноэмульсией и ополаскивателем с нормальным размером частиц была статистически очень значимая разница (P<0,0001) (значение P 0,00052). There was a statistically very significant difference (P<0.0001) between the nanoemulsion rinse and the normal particle size rinse (P value 0.00052). The nanoemulsion showed a statistically very significant difference compared to the normal particle size material, showing a lower mean residual debris surface area, i.e. the nanoemulsion material had the best cleaning ability, as shown in figure 3.
Figure 3. Comparison of cleaning performance of rinse aids: (A) with Nano CHX laser activated, (B) with CHX laser activated, (C) with PUI Nano CHX, (D) without Nano CHX activation, (E) without CHX activation, and (F) ) CHX PUI activation.
The average surface area of ​​the remaining Chx.HCl 1.6% fragments was 2320.36 µm2, and the average surface area of ​​Chx.HCl 2% was 2949.85 µm2. There was a statistically highly significant (P<0.001) difference between the higher concentration of nanoemulsion irrigants and the normal particle size irrigants (P-value 0.00000). There was a statistically highly significant (P<0.001) difference between the higher concentration of nanoemulsion irrigants and the normal particle size irrigants (P-value 0.00000). Наблюдалась статистически высокозначимая (P<0,001) разница между более высокой концентрацией наноэмульсионных ирригационных растворов и ирригационными растворами с нормальным размером частиц (значение P 0,00000). There was a statistically highly significant (P<0.001) difference between the higher concentration of nanoemulsion irrigants and normal particle size irrigants (P value 0.00000).较高浓度的纳米乳液冲洗剂与正常粒径冲洗剂之间存在统计学上高度显着的差异（P<0.001）（P 值0.00000）。较高浓度的纳米乳液冲洗剂与正常粒径冲洗剂之间存在统计学显着的差异（P<0.001）（P 0 0.0 Наблюдалась статистически очень значимая разница (P<0,001) между более высокими концентрациями ополаскивателя с наноэмульсией и ополаскивателя с нормальным размером частиц (значение P 0,00000). There was a statistically very significant difference (P<0.001) between higher concentrations of nanoemulsion rinse and normal particle size rinse (P value 0.00000). Although the concentration of the nanoemulsion irrigant was lower than that of the normal particle size irrigant, this lower concentration was significantly more effective in removing debris and more effective in cleaning root canals.
PUI had a statistically highly significant difference (p<0.001) when compared to other activation methods. PUI had a statistically highly significant difference (p<0.001) when compared to other activation methods. PUI имел статистически высокозначимую разницу (p<0,001) по сравнению с другими методами активации. PUI had a statistically highly significant difference (p<0.001) compared to other activation methods.与其他激活方法相比，PUI 具有统计学上非常显着的差异(p<0.001)。与其他激活方法相比，PUI 具有统计学上非常显着的差异(p<0.001)。 По сравнению с другими методами активации PUI имел статистически очень значимую разницу (p<0,001). Compared to other activation methods, PUI had a statistically very significant difference (p<0.001). With the activation of the ISP, the average area of ​​the residual surface of the debris was 1695.31 µm2. The mean difference between PUI and Laser was 987.89929 showing highly statistically significant (P<0.001) difference with (p-value 0.00000). The mean difference between PUI and Laser was 987.89929 showing highly statistically significant (P<0.001) difference with (p-value 0.00000). Средняя разница между PUI и Laser составила 987,89929, демонстрируя высокостатистически значимую (P<0,001) разницу с (p-значение 0,00000). The mean difference between PUI and Laser was 987.89929, showing a highly statistically significant (P<0.001) difference from (p-value 0.00000). PUI 和Laser 之间的平均差异为987.89929，显示出高度统计学显着性(P<0.001) 差异(p 值0.00000)。 PUI 和Laser Средняя разница между PUI и Laser составила 987,89929, что свидетельствует о высокой статистической значимости (P<0,001) разницы (p-значение 0,00000). The mean difference between PUI and Laser was 987.89929, indicating a high statistical significance (P<0.001) difference (p-value 0.00000). The mean difference between PUI and no activation was 712.40643 showing a highly statistically significant (P<0.001) difference with a p-value of 0.00098).The use of either Laser activation or no activation was not significantly statistically (P>0.05) different with a P-value of 0.451211. The mean difference between PUI and no activation was 712.40643 showing a highly statistically significant (P<0.001) difference with a p-value of 0.00098). a P-value of 0.451211. Средняя разница между PUI и отсутствием активации составила 712,40643, демонстрируя высокостатистически значимую (P<0,001) разницу с p-значением 0,00098). The mean difference between PUI and no activation was 712.40643, showing a highly statistically significant (P<0.001) difference with a p-value of 0.00098). P-value 0.451211. PUI 和未激活之间的平均差异为712.40643，显示高度统计学显着性差异(P<0.001)，p 值为0.00098)。 PUI Средняя разница между PUI и инактивацией составила 712,40643, что свидетельствует о высокой статистической значимости разницы (P<0,001, p-значение 0,00098). The mean difference between PUI and inactivation was 712.40643, indicating a high statistical significance of the difference (P<0.001, p-value 0.00098).使用激光激活或不激活在统计学上没有显着差异(P>0.05) P 值为0.451211。使用激光激活或不激活在统计学上没有显着差异(P>0.05) P 值为0.451211。 Статистически значимой разницы (P>0,05) с лазерной активацией или без нее не было со значением P 0,451211. There was no statistically significant difference (P>0.05) with or without laser activation with a P value of 0.451211. The average surface area of ​​the remaining fragments upon laser activation was 2683.21 µm2. The average surface area of ​​the remaining fragments without activation was 2407.72 µm2. Compared with laser activation or no activation, PUI had a statistically smaller average chip surface area, i.e. the best cleaning power.
The average cleaning power of the nanoemulsion rinse was 2001.47 µm2, and the average particle size of the rinse aid was 2609.56 µm. The average difference between the nanoemulsion wash and the normal particle size wash was 608.09 µm2. There was statistically highly significant (P<0.001) difference between nanoemulsion irrigants and normal particle size irrigants with (P-value 0.00052). There was statistically highly significant (P<0.001) difference between nanoemulsion irrigants and normal particle size irrigants with (P-value 0.00052). Между ирригационными растворами наноэмульсии и ирригационными растворами с нормальным размером частиц была статистически высокозначимая (P<0,001) разница (значение P 0,00052). There was a statistically highly significant (P<0.001) difference (P value 0.00052) between nanoemulsion irrigants and normal particle irrigants.纳米乳液冲洗剂与正常粒径冲洗剂之间存在统计学上高度显着的差异（P<0.001）（P值0.00052）。 P<0.001）（P值0.00052）。 Между ополаскивателем с наноэмульсией и ополаскивателем с нормальным размером частиц была статистически очень значимая разница (P<0,0001) (значение P 0,00052). There was a statistically very significant difference (P<0.0001) between the nanoemulsion rinse and the normal particle size rinse (P value 0.00052). Compared to a normal particle size material, the nanoemulsion has a statistically very significant difference, showing a lower mean residual debris surface area, i.e. Nanoemulsion material has better cleaning ability as shown in Figure 3.
The average surface area of ​​the remaining Chx.HCl 1.6% fragments was 2320.36 µm2, and the average surface area of ​​Chx.HCl 2% was 2949.85 µm2. There was statistically highly significant (P<0.001) difference between the higher concentration of nanoemulsion irrigants and the normal particle size irrigants (P-value 0.00000). There was a statistically highly significant (P<0.001) difference between the higher concentration of nanoemulsion irrigants and the normal particle size irrigants (P-value 0.00000). Имелась статистически высокодостоверная (P<0,001) разница между более высокой концентрацией наноэмульсионных ирригационных средств и ирригационными растворами с нормальным размером частиц (значение P 0,00000). There was a statistically significant (P<0.001) difference between the higher concentration of nanoemulsion irrigants and normal particle size irrigants (P value 0.00000).较高浓度的纳米乳液冲洗剂与正常粒径冲洗剂之间存在统计学上高度显着的差异（P<0.001）（P值0.00000）。较高浓度的纳米乳液冲洗剂与正常粒径冲洗剂之间存在统计学上高度显着的差异（P<0.001）（P000 Наблюдалась статистически высокозначимая разница (P <0,001) между более высокими концентрациями ополаскивателя с наноэмульсией и ополаскивателем с нормальным размером частиц (значение P 0,00000). There was a statistically highly significant difference (P < 0.001) between higher concentrations of nanoemulsion rinse and normal particle size rinse (P value 0.00000). Although the concentration of the nanoemulsion irrigant was lower than that of the normal particle size irrigant, this lower concentration was significantly more effective in removing debris and more effective in cleaning root canals.
PUI had statistically high significant difference (p<0.001) when compared to other activation methods. PUI had statistically high significant difference (p<0.001) when compared to other activation methods. PUI имел статистически высокую значимую разницу (p<0,001) по сравнению с другими методами активации. PUI had a statistically significant difference (p<0.001) compared to other methods of activation.与其他激活方法相比，PUI 具有统计学上的显着差异(p<0.001)。 Compared with other activation methods, PUI has a statistically significant difference (p<0.001). PUI статистически значимо отличался (p<0,001) по сравнению с другими методами активации. PUI was statistically significantly different (p<0.001) compared to other activation methods. During the activation of PUI, the average area of ​​residual surface debris was 1695.31 μm2. The mean difference between PUI and Laser was 987.89929 showing highly statistically significant (P<0.001) difference with (p-value 0.00000).The mean difference between PUI and no activation was 712.40643 showing highly statistically significant (P<0.001) difference with (p-value 0.00098).The use of either Laser activation or no activation was none significantly statistical (P>0.05) different with (P-value 0.451211). The mean difference between PUI and Laser was 987.89929 showing highly statistically significant (P<0.001) difference with (p-value 0.00000). The mean difference between PUI and no activation was 712.40643 showing highly statistically significant (P<0.001) difference with (p -value 0.00098). The use of either Laser activation or no activation was none significantly statistically (P>0.05) different with (P-value 0.451211). Средняя разница между PUI и лазером составила 987,89929, демонстрируя высокостатистически значимую (P<0,001) разницу с (p-значение 0,00000). The mean difference between PUI and laser was 987.89929, demonstrating a highly statistically significant (P<0.001) difference with (p-value 0.00000). - значение 0,00098).Использование лазерной активации или отсутствие активации не имело статистически значимой разницы (P>0,05) с (P-значение 0,451211). - value 0.00098). The use of laser activation or no activation had a statistically significant difference (P>0.05) with (P-value 0.451211). PUI 和激光之间的平均差异为987.89929，与(p 值0.00000) 差异具有高度统计学意义(P<0.001)。 The average difference between PUI and laser is 987.89929, and the difference (p 值0.00000) has a high statistical significance (P<0.001). Средняя разница между PUI и лазером составила 987,89929, что было высоко статистически значимым (P<0,001) с (значение p 0,00000). The mean difference between PUI and laser was 987.89929, which was highly statistically significant (P<0.001) with (p value 0.00000). PUI 与未激活之间的平均差异为712.40643，与(p) 差异具有高度统计学意义(P<0.001) -值0.00098)。 The average difference between PUI and inactive is 712.40643, and the difference (p) has a high statistical significance (P<0.001) – value 0.00098. Средняя разница между PUI и инактивацией составила 712,40643, что было высоко статистически значимым с разницей (p) (P<0,001 — значение 0,00098). The mean difference between PUI and inactivation was 712.40643, which was highly statistically significant with difference (p) (P<0.001 – value 0.00098).使用激光激活或不激活没有显着统计学差异(P>0.05) 与(P 值0.451211)。 There was no significant statistical difference between laser activation and non-activation (P>0.05) and (P 值0.451211). Не было статистически значимой разницы (P>0,05) по сравнению с (значение P 0,451211) с лазерной активацией или без нее. There was no statistically significant difference (P>0.05) compared to (P value 0.451211) with or without laser activation. The average surface area of ​​the remaining fragments during laser activation was 2683.21 μm2. The average surface area of ​​the remaining fragments without activation was 2407.72 μm2. Compared with laser activation or no activation, PUI has a statistically smaller average surface area of ​​the chip, i.e. better cleaning ability.
The mean effect of the nanoemulsion rinse on debris removal was statistically significantly higher than that of the normal particle size rinse. Chx.HCl 1.6%, PUI 1938.77 µm2, 2510.96 µm2 with laser. Without activation, the average value is 2511.34 µm2. When 2% Chx.HCl was used and activated with a laser, the results were the worst and the amount of debris was maximum. The same results were obtained when 0.75% Chx.HCl was not activated. Obviously, the best results were obtained using higher concentrations of rinse aid in the nanoemulsion. PUI was most effective in irrigant activation and debris flushing, as shown in Figure 3A-F)).
As shown in Table 2, the Chx.HCl nanoemulsion performed better than normal size particles in terms of viable microorganism count and had a good correlation with formulation penetration and cleansing effect according to the following parameters: size, flushing agent concentration and activation method.
Bacteria can be completely destroyed by using a higher concentration of rinse aid. Even with PUI activation, 0.75% Chx.HCl had the worst antibacterial effect. Laser activation has a negative effect on nano-emulsion rinses. As can be seen from all previous results, the use of a laser reduces the efficiency of the Chx.HCl 0.75% nanoemulsion, where the CFU of nanoChx.HCl 0.75% is 195, which is a very high value, indicating that the reagents at this concentration are comparable to laser activation. Diode lasers are photothermal, so either light or heat can cause the nanoemulsion to lose its antibacterial effect. The result of high concentrations is the complete destruction of bacteria. Nano Chx.HCl 1.6% showed negative bacterial growth in the presence of laser activation, which means that the laser did not affect the antibacterial ability of nano Chx.HCl 1.6%. It can be concluded that the nanoemulsion material with a higher concentration has a better antibacterial effect.
In this work, Chx.HCl nanoemulsions were prepared using two different oils, two surfactants and a co-surfactant, the optimal formulation (F6) with small particle size, short emulsification time and high dissolution rate) was chosen. In addition, (F6) was tested for thermodynamic/physical stability. In the Chx.HCl nanoemulsion at a concentration of 1.6%, the Chx.HCl nanoemulsion showed the best permeability in the dentinal tubules compared to the traditional Chx.HCl as a rinsing fluid, and PUI as an activation method had a cleansing ability. In addition, antibacterial studies of the Chx.HCl nanoemulsion showed complete elimination of bacteria. The results confirmed this. The Chx.HCl nanoemulsion can be considered as a promising washing liquid.
We are very grateful to the staff of the research laboratory of the Misr University of Science and Technology for their great support.