A novel root canal irrigation system based on photodynamic / NO combined therapy and its construction method

Abstract

The present invention belongs to the field of biomedicine, and particularly relates to a novel root canal irrigation system based on photodynamic / nitric oxide (NO) combined therapy and its construction method. The system comprises a polyethylene glycol-polycaprolactone (PEG-PCL) carrier surface-modified with guanidine-CN3H4, and a photosensitizer (Ce6) carried on the carrier. The present invention has the following beneficial effects: the root canal irrigation system constructed in accordance with the present invention has good biocompatibility and can safely and effectively treat refractory apical periodontitis by penetrating biomembranes, synergistically inhibiting bacteria with NO and ROS, and promoting bone defect repair, exerting a strong therapeutic effect on refractory apical periodontitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a novel root canal flushing system based on photodynamic / NO combined therapy and a construction method thereof. Background Art

[0002] Apical periodontitis is an inflammatory disease caused by bacterial infection that damages periapical tissues. It can lead to destruction of the dental pulp, periapical ligament, and alveolar bone. If left untreated, it can even cause serious complications such as cellulitis, mandibular osteomyelitis, and sepsis. The global prevalence of apical periodontitis has reached 52%, while the incidence of apical periodontitis after conventional root canal treatment remains as high as 39%. Refractory apical periodontitis is defined as persistent apical periodontitis that persists despite repeated conventional root canal treatment. Root canal therapy (RCT) is currently one of the most commonly used clinical treatments for apical periodontitis. This technique primarily involves mechanical preparation and chemical irrigation to eliminate bacteria and microorganisms within the root canal and control infection. Sodium hypochlorite (NaClO) is the most commonly used chemical irrigant in clinical practice; however, it is associated with numerous serious complications, such as facial hematoma, mucosal necrosis, bone necrosis, and even life-threatening airway obstruction. Therefore, the efficient and safe management of persistent apical periodontitis is crucial.

[0003] Numerous studies have shown that the primary cause of root canal treatment failure is microbial persistence, with Enterococcus faecalis (E. faecalis) being the most prominent species associated with refractory periapical periodontitis. E. faecalis is resistant to disinfectants and antiseptics and can survive harsh environmental conditions of high alkalinity (pH up to 11.5) and prolonged starvation. Furthermore, without the support of other bacteria, E. faecalis can grow as a single infection in treated root canals and form biofilms. Biofilms protect bacteria by hindering the penetration and subsequent effects of exogenous drugs. The drug resistance of bacteria in biofilms is approximately 100–1000 times higher than that of free bacteria. NaClO, the preferred root canal irrigant in clinical practice, possesses highly effective antimicrobial properties and tissue-dissolving abilities. However, its harmful effects, such as cytotoxicity, are well known. Leakage outside the root canal can cause severe pain and acute damage. Upon contact with tissue, NaClO rapidly oxidizes surrounding tissue, leading to rapid hemolysis and ulceration, and damaging the endothelium and fibroblasts. In refractory apical periodontitis, the root apex is exposed to pathogenic stimuli within the root canal, leading to resorption and destruction of the adjacent alveolar bone and cementum. In addition to controlling infection, promoting the healing of periapical bone defects to shorten the course of the disease and avoid secondary infection is of great significance. Therefore, the development of a new root canal disinfection system that is safe, controllable, and can efficiently penetrate and eliminate Enterococcus faecalis biofilm while promoting the repair of periapical bone defects is of great significance for the effective treatment of refractory apical periodontitis. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a new root canal irrigation system based on photodynamic / NO combined therapy and a construction method thereof to solve the above problems.

[0005] The technical solution of the present invention is achieved as follows: a new root canal irrigation system based on photodynamic / NO combined therapy, including a polyethylene glycol-polycaprolactone PEG-PCL carrier with a surface modified with guanidine-CN3H4, and a photosensitizer Ce6 carried on the carrier.

[0006] Furthermore, a method for constructing a new root canal irrigation system based on photodynamic / NO combined therapy includes the following steps:

[0007] a. Weigh a certain amount of amino-polyethylene glycol-polycaprolactone NH2-PEG-PCL and 1H-pyrazole-1-carboxamidine hydrochloride and add them to a branched pear-shaped flask;

[0008] b. Add anhydrous dimethyl sulfoxide (DMSO) under nitrogen protection;

[0009] c. Slowly add N,N-diisopropylethylamine (DIPEA) under stirring, and then react at room temperature;

[0010] d. After the reaction in step c is completed, the solution is placed in a dialysis bag and slowly stirred in pure water. After stirring, it is freeze-dried to obtain the product guanidine-polyethylene glycol-polycaprolactone G-PEG-PCL, and stored at -20°C for use.

[0011] Furthermore, a method for constructing a new root canal irrigation system based on photodynamic / NO combined therapy further includes the following steps:

[0012] Step 1: Weigh a certain amount of Ce6 and G-PEG-PCL and dissolve them in DMSO;

[0013] Step 2: After the mixture in step 1 is evenly mixed, the mixture is slowly added dropwise into pure water to form a micellar suspension, and then stirred at room temperature in the dark;

[0014] Step 3: After the stirring in step 2 is completed, the solution is placed in a dialysis bag, and slowly stirred in pure water, and then freeze-dried to obtain the final product, Ce6@GP nanomicelles.

[0015] Furthermore, in step a, amino-polyethylene glycol-polycaprolactone NH2-PEG-PCL is 4.5 g, 1 mmol, and 1H-pyrazole-1-carboxamidine hydrochloride is 146.58 mg, 1 mmol.

[0016] Furthermore, in step b, the amount of anhydrous dimethyl sulfoxide (DMSO) is 20 ml.

[0017] Furthermore, in step c, the amount of N,N-diisopropylethylamine DIPEA was 174.2 μL.

[0018] Furthermore, in step d, the pore size of the dialysis bag is 1000 Da, the stirring time is 48 h, and the freeze-drying time is 48 h.

[0019] Furthermore, in step 1, Ce6 is 1 mg, G-PEG-PCL is 20 mg, and DMSO is 4 ml.

[0020] Furthermore, in step 2, the amount of pure water was 24 ml and the stirring time was 24 h.

[0021] Furthermore, in step three, the pore size of the dialysis bag is 3500 Da, the stirring time is 48 h, and the freeze-drying time is 48 h.

[0022] The beneficial effects of the present invention are:

[0023] 1. Successfully developed a treatment system that can safely and effectively treat refractory periapical periodontitis;

[0024] 2. It has good biocompatibility and can treat refractory apical periodontitis by penetrating biofilms, synergistically inhibiting NO and ROS, and promoting bone defect repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 This is a comparison chart of the penetration of Ce6@GP, PBS and Ce6 in biofilm according to a specific embodiment of the present invention;

[0027] Figure 2 This is the ROS production rate of Ce6@GP under light in a specific embodiment of the present invention;

[0028] Figure 3 The NO production rate of Ce6@GP under light in a specific embodiment of the present invention;

[0029] Figure 4 This is a comparison chart of bacterial survival rates of PBS, Ce6 and Ce6@GP according to a specific embodiment of the present invention. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0031] In the description of this application, it should be noted that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments according to this application. For ease of description, the dimensions of the various parts shown in the drawings are not drawn according to the actual proportional relationship. Technologies, methods and equipment known to ordinary technicians in the relevant fields may not be discussed in detail, but where appropriate, the technologies, methods and equipment should be considered as part of the authorization specification. In all examples shown and discussed here, any specific values should be interpreted as merely exemplary and not as limitations. Therefore, other examples of the exemplary embodiments may have different values. It should be noted that similar numbers and letters represent similar items in the following figures, so once an item is defined in one figure, it does not need to be further discussed in subsequent figures.

[0032] Example 1:

[0033] First, accurately weighed NH2-PEG-PCL (4.5 g, 1 mmol) and 1H-pyrazole-1-carboxamidine hydrochloride (146.58 mg, 1 mmol) were added to a 50 mL branched pear-shaped flask. Then, under nitrogen protection, 20 mL of anhydrous DMSO was added, and 174.2 μL of DIPEA was slowly added under stirring, followed by reaction at room temperature for 24 hours. After the reaction, the solution was placed in a dialysis bag (1000 Da) and slowly stirred in pure water for 48 hours, followed by freeze-drying for 48 hours to obtain the product guanidine-PEG-PCL, which was stored at -20°C for future use.

[0034] Ce6@GP nanomicelles were then prepared using a nanoprecipitation method. First, 1 mg of Ce6 and 20 mg of G-PEG-PCL were dissolved in 4 mL of DMSO. After mixing thoroughly, the mixture was slowly added dropwise to 24 mL of pure water to form a micellar suspension. Stirring was continued at room temperature in the dark for 24 hours. The solution was then placed in a dialysis bag (3500 Da), slowly stirred in pure water for 48 hours, and then freeze-dried for 48 hours to obtain the final product, Ce6@GP.

[0035] NH2-PEG-PCL was purchased from Shanghai Pengshuo Biotechnology Co., Ltd., and 1H-pyrazole-1-carboxamidine hydrochloride, dimethyl sulfoxide (DMSO), and N,N-diisopropylethylamine (DIPEA) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0036] This embodiment constructs a photodynamically activated NO multifunctional nanomedicine that can treat refractory periapical periodontitis by penetrating biofilms, synergistic antibacterial effects of NO and ROS, and promoting bone defect repair. Using guanidine-modified PEG-PCL as a carrier and Ce6 molecules as photosensitizers, a new root canal irrigation system (Ce6@GP) based on PDT / NO combined therapy was constructed. Ce6@GP can penetrate the biofilm in the root canal during root canal irrigation, and then produce high levels of ROS through PDT activation. The guanidine groups on the surface can quickly release NO under the action of ROS, avoiding accidental damage to the oral mucosa and apical tissue through precise spatiotemporal control; efficient penetration of complex root canal systems and biofilms on their surfaces; excellent synergistic antibacterial and biofilm ablation effects mediated by ROS and NO; NO can further promote periapical angiogenesis and bone defect repair. Due to the above-mentioned outstanding biofilm penetration, synergistic antibacterial and bone tissue healing effects, Ce6@GP exerts a powerful therapeutic effect on refractory periapical periodontitis and has good biocompatibility. It can safely and effectively treat refractory periapical periodontitis.

[0037] Experimental Example 1:

[0038] Biofilm permeability:

[0039] First, the round cell slide was placed in a 24-well plate, and then 450 μL BHI medium and 50 μL Enterococcus faecalis suspension (10 8 CFU / mL), and static culture was carried out at 37°C under anaerobic conditions for 7 days to form a biofilm, and the medium was changed every 2 days. Ce6 was used as a fluorescent probe to evaluate the penetration performance of Ce6@GP. The supernatant was removed, and 500μL of Ce6@GP (36μg / mL) was added to a 24-well plate and incubated at 37°C in the dark for 5 minutes. The laser group used a laser emitter (665nm, 43mW / cm 2 ) for 5 minutes. The supernatant was then removed and the cells were stained with SYTO-9 dye for 15 minutes. The dye was then removed by aspiration, and the cells were washed twice with PBS and dried naturally in the dark. Finally, the slides were removed, placed on glass slides, and mounted. Laser confocal microscopy was used to observe the penetration of each material into the biofilm. PBS and Ce6 (2 μg / mL) were used as control samples.

[0040] result:

[0041] See also Figure 1,Three-dimensional laser confocal scanning microscopy showed that only a very weak fluorescence signal was observed for free Ce6, indicating that the biofilm can prevent the entry of foreign drugs, while a large amount of red fluorescence signal can be observed in the biofilm of the Ce6@GP group, indicating that the prepared Ce6@GP does have the effect of penetrating the biofilm.

[0042] Experimental Example 2:

[0043] Active oxygen release capacity:

[0044] The ROS generation of Ce6@GP under light was measured using a ROS fluorescent probe, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). The experimental steps were as follows: First, 0.5 mL of DCFH-DA (0.487 mg / mL) in ethanol was mixed with 2 mL of sodium hydroxide solution (0.4 mg / mL). The mixture was stirred in the dark for 30 minutes to hydrolyze DCFH. Then, 7.5 mL of PBS was added to dilute the DCFH concentration to 100 μM. The resulting solution was stored in the dark at -20°C until ready for use.

[0045] 50 μL of Ce6@GP (54 μg / mL) and 100 μL of DCFH (6 μM) were mixed and added to a 96-well plate. The final concentration of Ce6@GP in the mixture was 18 μg / mL. Then, a 665 nm laser (output power 43 mW / cm 2 The mixture was irradiated for 0, 1, 3, 5, and 10 min. Finally, the fluorescence intensity was measured at each time point using a multifunctional microplate reader (excitation wavelength 488 nm, emission wavelength 525 nm).

[0046] result:

[0047] See also Figure 2 , Ce6@GP produced a large amount of ROS under photodynamic drive, and the concentration gradually increased with the increase of illumination time.

[0048] Experimental Example 3:

[0049] Nitric oxide releasing capacity:

[0050] The modified Griess assay was used to measure the NO production of Ce6@GP under light irradiation. The experimental steps were as follows: First, 50 μL of Ce6@GP (54 μg / mL) was mixed with 100 μL of Griess reagent and added to a 96-well plate. The final concentration of Ce6@GP in the mixture was 18 μg / mL, and the mixture was left in the dark for 30 minutes. Then, a 665 nm laser (output power of 43 mW / cm 2The mixture was irradiated for 0, 1, 3, 5, and 10 minutes. Finally, the absorbance at 540 nm was measured using a microplate reader at each time point, and the amount of NO produced was determined based on the standard curve.

[0051] result:

[0052] See also Figure 3 After 1 minute of irradiation, Ce6@GP can rapidly produce NO under laser driving, and the cumulative amount of NO produced at 10 minutes is about 4.2μM. The experimental results clearly show that Ce6@GP has good NO production ability under the driving force of photodynamics.

[0053] Experimental Example 4:

[0054] Antimicrobial properties:

[0055] The antibacterial properties of Ce6@GP were evaluated using the colony counting method. The specific experimental steps were as follows: First, 950 μL of Ef bacterial suspension (10 8 CFU / mL) was mixed with 50 μL of Ce6@GP (360 μg / mL), then added to a centrifuge tube and placed in a water-insulated constant temperature incubator (37°C, dark, anaerobic) for 5 min. Then, a laser with a wavelength of 660 nm (light intensity of 43 mW / cm 2 ) irradiated for 5 min, incubated for 30 min, and then 100 μL of bacterial suspension was collected and serially diluted in a 96-well plate (from 10 8 Dilute to 10 4 CFU / mL), 5 μL was dripped onto solid culture medium. Each concentration was repeated three times, and the cells were incubated under anaerobic conditions at 37°C for 24 hours. Finally, the bacterial count and survival rate were calculated. PBS, Ce6 (20 μg / mL), and Ce6@GP without laser irradiation were used as controls.

[0056] result:

[0057] See also Figure 4 The bacterial survival rate in the Ce6@GP group without laser irradiation was consistent with that in the PBS group, exceeding 8 log units. However, after laser irradiation, the bacterial survival rate in the Ce6+Laser group was 3.84 log, while that in the Ce6@GP+Laser group dropped to 1.53 log.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A new root canal irrigation system based on photodynamic / NO combined therapy, characterized in that: The root canal irrigation system is a Ce6@GP nanomicelle, which includes a polyethylene glycol-polycaprolactone PEG-PCL carrier (GP) with a surface modified with guanidine-CN3H4, and a photosensitizer Ce6 carried on the carrier.

2. The method for constructing a new root canal irrigation system based on photodynamic / NO combined therapy according to claim 1, characterized in that: The steps include: a. Weigh a certain amount of amino-polyethylene glycol-polycaprolactone NH2-PEG-PCL and 1H-pyrazole-1-carboxamidine hydrochloride and add them to a branched pear-shaped flask; b. Add anhydrous dimethyl sulfoxide (DMSO) under nitrogen protection; c. Slowly add N,N-diisopropylethylamine (DIPEA) under stirring, and then react at room temperature; d. After the reaction in step c is completed, the solution is placed in a dialysis bag and slowly stirred in pure water. After stirring, it is freeze-dried to obtain the product guanidine-polyethylene glycol-polycaprolactone G-PEG-PCL, and stored at -20°C for use.

3. The method for constructing a new root canal irrigation system based on photodynamic / NO combined therapy according to claim 2, characterized in that: The following steps are also included: Step 1: Weigh a certain amount of Ce6 and G-PEG-PCL and dissolve them in DMSO; Step 2: After the mixture in step 1 is evenly mixed, the mixture is slowly added dropwise into pure water to form a micellar suspension, and then stirred at room temperature in the dark; Step 3: After the stirring in step 2 is completed, the solution is placed in a dialysis bag and slowly stirred in pure water. After the stirring is completed, freeze-drying is performed to obtain the final product, Ce6@GP nanomicelles.

4. The method for constructing a new root canal irrigation system based on photodynamic / NO combined therapy according to claim 2, characterized in that: In step a, amino-polyethylene glycol-polycaprolactone NH2-PEG-PCL is 4.5 g, 1 mmol, and 1H-pyrazole-1-carboxamidine hydrochloride is 146.58 mg, 1 mmol.

5. The method for constructing a new root canal irrigation system based on photodynamic / NO combined therapy according to claim 2, characterized in that: In step b, the amount of anhydrous dimethyl sulfoxide (DMSO) is 20 ml.

6. The method for constructing a new root canal irrigation system based on photodynamic / NO combined therapy according to claim 2, characterized in that: In step c, N,N-diisopropylethylamine DIPEA was 174.2 μL.

7. The method for constructing a new root canal irrigation system based on photodynamic / NO combined therapy according to claim 2, characterized in that: In step d, the pore size of the dialysis bag was 1000 Da, the stirring time was 48 h, and the freeze-drying time was 48 h.

8. The method for constructing a new root canal irrigation system based on photodynamic / NO combined therapy according to claim 3, characterized in that: In step 1, Ce6 is 1 mg, G-PEG-PCL is 20 mg, and DMSO is 4 ml.

9. The method for constructing a new root canal irrigation system based on photodynamic / NO combined therapy according to claim 3, characterized in that: In step 2, the amount of pure water is 24 ml and the stirring time is 24 h.

10. The method for constructing a new root canal irrigation system based on photodynamic / NO combined therapy according to claim 3, characterized in that: In step three, the pore size of the dialysis bag is 3500 Da, the stirring time is 48 h, and the freeze-drying time is 48 h.

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