A dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel and its preparation method and application

Abstract

The present invention discloses a dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel and its preparation method and application. The present invention first uses microemulsion-lipid cascade drug delivery technology to co-load quercetin and caffeic acid phenethyl ester into liposomes, and then distributes the liposomes in a dual-responsive gel composed of chitosan, poloxamer 407 and poly-N-isopropylacrylamide to construct a dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel. The composite gel has a temperature-sensitive and pH-sensitive dual-responsive performance, and after administration, quercetin can be preferentially released to achieve rapid anti-inflammatory effects, and then caffeic acid phenethyl ester is released to promote osteogenic differentiation in the periodontal part, which can synergistically play the therapeutic effects of eliminating periodontal tissue inflammation and promoting alveolar bone repair, and provides a new preparation for the treatment of periodontitis.

Description

Technical Field

[0001] The invention belongs to the field of drug-carrying hydrogels, and particularly relates to a dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel and a preparation method and application thereof. Background Art

[0002] Periodontitis is a chronic inflammatory disease that has been rated by the World Health Organization as the third most threatening disease to human health after malignant tumors and cardiovascular and cerebrovascular diseases. Epidemiological studies have shown that the incidence of periodontitis is over 30% worldwide, which can have adverse effects on the diet, work, social life, etc. of patients, seriously affecting their physical and mental health.

[0003] Periodontitis is a chronic inflammatory reaction induced by abnormal proliferation of oral pathogenic microorganisms. The proportion of proinflammatory cells, led by M1 macrophages, increases in periodontitis tissues, and the large amount of inflammatory factors secreted leads to symptoms such as gingival swelling, bleeding, alveolar bone destruction, loose and falling teeth. In order to relieve the symptoms of gingival pain, swelling, bleeding, etc. in patients with periodontitis, the treatment of periodontitis needs to give priority to quickly controlling the inflammation of patients and reducing the expression of inflammatory cells and inflammatory factors. In addition, in order to achieve the ultimate treatment goal of repairing damaged periodontal tissues, continuous alveolar bone repair treatment is required after controlling inflammation. Therefore, the treatment of periodontitis needs to give priority to quickly controlling inflammation, followed by continuous repair of alveolar bone tissue. At present, most of the clinical periodontitis treatment drugs are single ingredients with single therapeutic effects, and cannot achieve the synergistic therapeutic effects of anti-inflammatory and osteogenesis at the same time. The development of a periodontal local drug delivery system that can programmatically release anti-inflammatory and osteogenic drugs is urgently needed in the current field of periodontitis treatment.

[0004] Studies have shown that quercetin can regulate the conversion of M1 macrophages to M2 macrophages, reduce the secretion of inflammatory factors such as IL-6 and TNF-α, and has excellent anti-inflammatory therapeutic effects. Caffeic acid phenethyl ester can promote the expression of osteogenic proteins such as OPN and OPG, promote the osteogenic differentiation of periodontal ligament stem cells, and also has a good effect on periodontal bone tissue repair. However, quercetin and caffeic acid phenethyl ester have low water solubility. It is difficult to dissolve and release quercetin and caffeic acid phenethyl ester locally in periodontal tissues when they are directly formulated into solutions for administration. In addition, due to physiological activities such as oral chewing and swallowing, the solution is difficult to stay in the periodontal mucosa for a long time, and it is difficult to achieve an ideal therapeutic effect. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel, which has dual-responsive properties of temperature and pH sensitivity. After injection, it can form a gel in situ in the periodontal tissue and retain it for a long time, preferentially releasing quercetin, which exerts an anti-inflammatory effect, and then continuously releases caffeic acid phenethyl ester, which promotes alveolar bone repair.

[0006] The present invention is achieved through the following technical solutions:

[0007] A dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel comprises quercetin-caffeic acid phenethyl ester liposomes and a dual-responsive gel matrix; the quercetin-caffeic acid phenethyl ester liposomes are prepared by using quercetin and caffeic acid phenethyl ester as raw materials, lipid compounds, surfactants, co-surfactants, cholesterol, and phospholipid materials as auxiliary materials through microemulsion-lipid cascade drug delivery technology, and the weight proportions of each component are as follows:

[0008] 1-20 parts of quercetin, 1-10 parts of caffeic acid phenethyl ester, 100-250 parts of lipid compounds, 180-280 parts of surfactants, 50-100 parts of co-surfactants, 10-50 parts of phospholipid materials, and 5-15 parts of cholesterol;

[0009] The dual-responsive gel matrix comprises: 5-20 parts of poloxamer 407, 10-100 parts of chitosan, and 50-150 parts of poly N-isopropylacrylamide.

[0010] The present invention co-loads water-insoluble drugs quercetin and caffeic acid phenethyl ester into liposomes, combines the pharmacological effects of the two, and preferentially releases quercetin quickly to exert anti-inflammatory effects through programmed release technology, and then releases caffeic acid phenethyl ester to exert sustained osteogenic effects. The dual-responsive gel prepared by cross-linking poly N-isopropylacrylamide, poloxamer, and chitosan can cause the gel to undergo phase change at body temperature after being injected into periodontal tissues, thereby achieving local periodontal retention, and chitosan can cause the gel to be responsively released under the low pH environment of periodontal tissues. The dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel prepared by the present invention has dual-responsive performance that is sensitive to temperature and pH.

[0011] Preferably, the lipid compound is selected from one or more of triacetin, tributyrin, tripropionin, tricaprylin or tricaprin.

[0012] Preferably, the surfactant is selected from one or more of polyethylene glycol 15 hydroxystearate HS-15, polyoxyethylene 40 hydrogenated castor oil RH-40, castor oil polyoxyethylene ether, Tween 80, Tween 20, poloxamer 188, carbitol, and lecithin.

[0013] Preferably, the co-surfactant is selected from one or more of polyethylene glycol 400, glycerol, 1,2-propylene glycol, n-butanol, isopropanol, and anhydrous ethanol.

[0014] Preferably, the phospholipid material is selected from one or more of dipalmitoylcholine (DPPC), distearoylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin phosphatidic acid, phosphatidylglycerol, dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylserine (DOPS), lysophosphatidylethanolamine (LPE), palmitoyl lysophosphatidylcholine (P-LysoPC), dimyristoylphosphatidylethanolamine (DMPE), dioleoylphosphatidylglycerol (DOPG), DOP-DEDA, DOTMA, Dlin-MC3-DMA, DODMA, and DOTAP.

[0015] The present invention also provides a method for preparing the dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel, comprising the following steps:

[0016] (1) Microemulsion-lipid cascade drug delivery technology: quercetin, lipid compounds, surfactants, and co-surfactants are fully stirred, and then deionized water is added to obtain quercetin microemulsion by emulsification; caffeic acid phenethyl ester, cholesterol, and phospholipid materials are used to prepare lipid films, and then quercetin microemulsion is added, deionized water is used for hydration, and extrusion is performed to obtain quercetin-caffeic acid phenethyl ester liposomes;

[0017] (2) chitosan and poloxamer 407 are cross-linked to prepare a chitosan-poloxamer 407 solution, quercetin-caffeic acid phenethyl ester liposomes and poly (N-isopropylacrylamide) are added to the chitosan-poloxamer 407 solution to obtain a dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel.

[0018] Preferably, in step (1), the mass ratio of the lipid compound to the surfactant is 1:1-5:1, the mass ratio of the lipid compound to the co-surfactant is 1:5-1:1, and the mass ratio of the lipid compound to the phospholipid material is 1:5-1:1.

[0019] Preferably, in step (2), the mass ratio of chitosan to poloxamer 407 is 2:1-10:1, and the mass ratio of chitosan to poly (N-isopropylacrylamide) is 1:5-5:1, preferably 2:1-3:1.

[0020] The present invention also provides the use of the dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel in the preparation of a drug for treating periodontitis.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention first uses microemulsion-lipid cascade drug loading technology to co-load quercetin and caffeic acid phenethyl ester into liposomes, and then distributes the liposomes in a dual-responsive gel composed of chitosan, poloxamer 407 and poly N-isopropylacrylamide to construct a dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel. The microemulsion-lipid cascade drug loading technology can efficiently encapsulate quercetin and caffeic acid phenethyl ester at the same time, and realize the preferential release of quercetin, quercetin plays an anti-inflammatory effect, and then releases caffeic acid phenethyl ester, which promotes periodontal bone formation. The gel has dual response characteristics of temperature and pH, and the temperature-sensitive characteristic contributes to the periodontal in situ injection of the gel, and the pH response characteristic contributes to the controllable release of the gel, and the intelligent response release. The evaluation results show that the preparation can play a therapeutic role in subsiding periodontal tissue inflammation and promoting alveolar bone repair, and provides a new preparation for the treatment of periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure shows the appearance of the quercetin-caffeic acid phenethyl ester liposome (CQ-ML) solution of the present invention;

[0024] Figure 2 The particle size and potential characterization results of the quercetin-caffeic acid phenethyl ester liposome (CQ-ML) and the blank control liposome (Blank-Lip) of the present invention;

[0025] Figure 3 The transmission electron microscopy (TEM) characterization results of the quercetin microemulsion (Qu-MEs), caffeic acid phenethyl ester liposomes (CAPE-L), and quercetin-caffeic acid phenethyl ester liposomes (CQ-ML) of the present invention;

[0026] Figure 4 The scanning electron microscope (SEM) characterization results of the dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel (CQ-ML@gel) of the present invention;

[0027] Figure 5 It is the temperature response phase transition diagram of the dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel (CQ-ML@gel) of the present invention;

[0028] Figure 6 It is the pH response dissolution diagram of the dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel (CQ-ML@gel) of the present invention;

[0029] Figure 7 This is a result diagram of the programmed release of quercetin and caffeic acid phenethyl ester from the dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel (CQ-ML@gel) of the present invention;

[0030] Figure 8H&E staining and immunohistochemical staining of tissue sections of rat periodontitis treated with the dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel (CQ-ML@gel) of the present invention;

[0031] Fig. 9 These are micro-CT images of alveolar bones and immunohistochemical staining images of tissue sections of rats with periodontitis treated with the dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel (CQ-ML@gel) of the present invention. DETAILED DESCRIPTION

[0032] The present invention is further described below by means of specific implementation methods. The following examples are preferred implementation methods of the present invention, but the implementation methods of the present invention are not limited to the following examples.

[0033] The raw materials used in the embodiments of the present invention are all commercially available.

[0034] Example 1: Screening of quercetin-caffeic acid phenethyl ester liposome formulations

[0035] (1) Preparation of quercetin-caffeic acid phenethyl ester liposomes (CQ-ML)

[0036] Weigh 5 parts of Quercetin Qu, mix with 150 parts of tributyrin, 225 parts of HS15, and 75 parts of PEG400 in a cillin bottle, stir overnight at room temperature until completely homogenized, add pure water to 5 mL under magnetic stirring to obtain quercetin microemulsion (Qu-MEs) for later use. Weigh 30 parts of soybean lecithin, mix with 7.5 parts of cholesterol and 3 parts of caffeic acid phenethyl ester (CAPE) in an eggplant-shaped bottle, dissolve in 10 mL of chloroform, rotate at 100 rpm in a 37°C constant temperature water bath to reduce pressure to form a uniform film. 0.5mL, 1.0mL, 1.5mL Qu-MEs and 9.5mL, 9.0mL, 8.5mL pure water were added to the above films, respectively, and the phospholipid membrane was incubated in a constant temperature water bath at 37°C and 100rpm; the solution was then placed in an ultrasonic cell crusher for 5 minutes and extruded through an extruder to prepare CQ-ML with a mass ratio of tributyrin to soybean lecithin of 1:2, 1:1, and 3:2, respectively.

[0037] (2) Preparation of Blank Liposomes

[0038] Weigh 30 parts of soybean lecithin, mix with 7.5 parts of cholesterol in an eggplant-shaped bottle, dissolve in 10 mL of chloroform, and rotate at 100 rpm in a constant temperature water bath at 37°C to form a uniform film under reduced pressure; add 10 mL of pure water to hydrate the phospholipid film; then place the solution in an ultrasonic cell crusher for 5 minutes and extrude it through an extruder to prepare Blank-Lip.

[0039] (3) CQ-ML particle size, potential value and encapsulation efficiency

[0040] The particle size and potential of Blank-lip and CQ-ML were measured by Malvern particle size analyzer. As shown in Table 1, when the mass ratio of tributyrin to soy lecithin in the CQ-ML formula was 1:2, the particle size and potential values ​​of CQ-ML were not significantly different from those of Blank-lip, indicating that the outer layer of the quercetin microemulsion inside it was wrapped with a lipid membrane, showing similar particle size and potential properties to Blank-lip. And when the mass ratio of tributyrin to soy lecithin in the CQ-ML formula was 1:2, the encapsulation rate of quercetin and caffeic acid phenethyl ester was the highest, indicating that quercetin and caffeic acid phenethyl ester can be efficiently encapsulated, so the optimal mass ratio of tributyrin to soy lecithin in CQ-ML is 1:2.

[0041] Table 1 Particle size, potential value and encapsulation efficiency of CQ-ML with different mass ratios of tributyrin and soybean lecithin

[0042]

[0043] Note: *P<0.05.

[0044] Example 2: Preparation and characterization of quercetin-caffeic acid phenethyl ester liposomes (CQ-ML)

[0045] (1) Preparation of CQ-ML

[0046] Weigh 5 parts of Qu, mix with 150 parts of tributyrin, 225 parts of HS15, and 75 parts of PEG400 in a vial, and stir overnight at room temperature until completely homogeneous; add pure water dropwise under magnetic stirring to form Qu-MEs.

[0047] Weigh 30 parts of soybean lecithin, mix with 7.5 parts of cholesterol and 3 parts of CAPE in an eggplant-shaped bottle, dissolve in 10 mL of chloroform, and evaporate under reduced pressure at 100 rpm in a constant temperature water bath at 37°C to form a uniform film; add 0.5 mL of Qu-MEs and 9.5 mL of pure water to hydrate the phospholipid film; then place the solution in an ultrasonic cell crusher for 5 minutes, and extrude it into CQ-ML using an extruder.

[0048] (2) Preparation of Caffeic Acid Phenethyl Ester Liposomes (CAPE-L)

[0049] Weigh 30 parts of soybean lecithin, mix with 7.5 parts of cholesterol and 3 parts of CAPE in an eggplant-shaped bottle, dissolve in 10 mL of chloroform, and rotate at 100 rpm in a constant temperature water bath at 37°C to form a uniform film under reduced pressure; add 10 mL of pure water to hydrate the phospholipid film; then place the solution in an ultrasonic cell crusher for 5 minutes and extrude it through an extruder to prepare CAPE-L.

[0050] (3) Particle size, potential, and TEM images

[0051] The particle size, PDI and potential of CQ-ML were measured using a Malvern particle size analyzer. Figure 2 As shown in Figure 2, the particle size of CQ-ML is 113.33±1.11nm and the potential is -39.9±0.70mV. The morphology of Qu-MEs, CAPE-L and CQ-ML was investigated using transmission electron microscopy (TEM). Figure 3 As shown, CQ-ML is spherical and forms an encapsulation structure for Qu-MEs.

[0052] Example 3: Formulation screening of dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel (CQ-ML@gel)

[0053] (1) Preparation of CQ-ML@gel

[0054] Weigh 5 parts of Qu, mix with 150 parts of tributyrin, 225 parts of HS15, and 75 parts of PEG400 in a vial, and stir overnight at room temperature until completely homogeneous; add pure water dropwise under magnetic stirring to form Qu-MEs.

[0055] Weigh 30 parts of soybean lecithin, mix with 7.5 parts of cholesterol and 3 parts of CAPE in an eggplant-shaped bottle, dissolve in 10 mL of chloroform, and evaporate under reduced pressure at 100 rpm in a constant temperature water bath at 37°C to form a uniform film; add 0.5 mL of Qu-MEs and 9.5 mL of pure water to hydrate the phospholipid film; then place the solution in an ultrasonic cell crusher for 5 minutes and extrude it through an extruder to prepare CQ-ML for use.

[0056] Weigh 12 parts of poloxamer 407 (P407) into a vial, add 1.5 mL of deionized water, 40 μL of glycerol, and 20 μL of glacial acetic acid and stir evenly, add 60 parts of chitosan (CS) and stir evenly at 50°C, add 0.2 mL of NaHCO3 (1 M), add deionized water to 2 mL and stir evenly to obtain chitosan-poloxamer

[0057] 407(CS-P407) is for spare use.

[0058] CQ-ML was used as the aqueous phase, and CS-P407 and poly (N-isopropylacrylamide) (PNIPAM) were added to prepare dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel (CQ-ML@gel), in which the mass ratios of CS:PNIPAM were 15:30, 30:30, 60:30, and 90:30.

[0059] (2) Investigation of gelation time of CQ-ML@gel

[0060] The gelation time of CQ-ML@gel at 30°C was used as the evaluation index, and the test tube inversion method was used to select the best formula of CQ-ML@gel with different CS and PNIPAM mass ratios per unit volume. The specific operation method is to measure 30 portions of 1mL of CQ-ML@gel, place them in 5mL test tubes, fix them with floating plates, and place them in a 30°C water bath. Starting from the 21st second, pick up a test tube every 1s to observe whether it turns into a gel state, record the gelation time, and repeat the experiment 3 times. The results are shown in Table 2. When the CS:PNIPAM mass ratio is 60:30, CQ-ML@gel can undergo a phase change in 30s, which meets the needs of periodontal drug delivery.

[0061] Table 2 Gel state and gel time of different prescriptions

[0062]

[0063] Example 4: Characterization of dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel

[0064] (1) Preparation of CQ-ML@gel

[0065] Weigh 5 parts of Qu, mix with 150 parts of tributyrin, 225 parts of HS15, and 75 parts of PEG400 in a vial, and stir overnight at room temperature until completely homogeneous; add pure water dropwise under magnetic stirring to form Qu-MEs.

[0066] Weigh 30 parts of soybean lecithin, mix with 7.5 parts of cholesterol and 3 parts of CAPE in an eggplant-shaped bottle, dissolve in 10 mL of chloroform, and evaporate under reduced pressure at 100 rpm in a constant temperature water bath at 37°C to form a uniform film; add 0.5 mL of Qu-MEs and 9.5 mL of pure water to hydrate the phospholipid film; then place the solution in an ultrasonic cell crusher for 5 minutes and extrude it through an extruder to prepare CQ-ML for use.

[0067] Weigh 12 parts of poloxamer 407 (P407) into a vial, add 1.5 mL of deionized water, 40 μL of glycerol, and 20 μL of glacial acetic acid and stir evenly, add 60 parts of chitosan (CS) and stir evenly at 50°C, add 0.2 mL of NaHCO3 (1 M), add deionized water to 2 mL and stir evenly to obtain chitosan-poloxamer 407 (CS-P407) for use.

[0068] Take 1 mL of CQ-ML as the aqueous phase, add 90 parts of poly (N-isopropylacrylamide) (PNIPAM) and 2 mL of CS-P407, stir evenly and prepare CQ-ML@gel for use.

[0069] (2) Scanning electron microscopy characterization of CQ-ML@gel

[0070] After freeze-drying, CQ-ML@gel was sprayed with gold to prepare a scanning electron microscope sample. The gel morphology was observed and photographed under an electron microscope. The results are as follows: Figure 4 As shown, the gel has a porous network structure, which is conducive to loading drug delivery systems.

[0071] (3) Temperature-responsive phase transition results of CQ-ML@gel

[0072] like Figure 5 As shown, it can be observed from the macroscopic state that at 30°C, CQ-ML@gel transforms into a gel state, indicating that it has temperature-responsive properties.

[0073] (4) pH-responsive dissolution results of CQ-ML@gel

[0074] like Figure 6 As shown in the figure, the dissolution rate of CQ-ML@gel in a 37°C pH 3.6 environment was significantly faster than that in a pH 7.4 environment, indicating that it has pH-responsive properties.

[0075] (5) CQ-ML@gel programmed release of quercetin (Qu) and caffeic acid phenethyl ester (CAPE)

[0076] like Figure 7 As shown in the figure, in the environment of pH 7.4 and 37°C, CQ-ML@gel preferentially releases Qu and then slowly releases CAPE. This is because the microemulsion-lipid cascade preparation method improves the solubility of Qu and enhances the dissolution performance, so Qu can be released preferentially; since CAPE is distributed in the lipid layer, it dissolves slowly and shows slow release. The above results show that CQ-ML@gel can programmatically release Qu and CAPE.

[0077] Example 5: Preparation of dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel

[0078] Weigh 10 parts of Qu, mix with 200 parts of glycerol tripropionate, 200 parts of RH-40, and 50 parts of glycerol in a vial, and stir overnight at room temperature until completely homogeneous; add pure water dropwise under magnetic stirring to form Qu-MEs.

[0079] Weigh 20 parts of hydrogenated soybean lecithin, mix with 5 parts of cholesterol and 2 parts of CAPE in an eggplant-shaped bottle, dissolve in 10 mL of chloroform, and rotate at 100 rpm in a constant temperature water bath at 37°C to form a uniform film under reduced pressure; add 0.5 mL of Qu-MEs and 9.5 mL of pure water to hydrate the phospholipid film; then place the solution in an ultrasonic cell crusher for 5 minutes and extrude it through an extruder to prepare CQ-ML for use.

[0080] Weigh 15 parts of poloxamer 407 (P407) into a vial, add 1.5 mL of deionized water, 40 μL of glycerol, and 20 μL of glacial acetic acid and stir evenly, add 60 parts of chitosan (CS) and stir evenly at 50°C, add 0.2 mL of NaHCO3 (1 M), add deionized water to 2 mL and stir evenly to obtain chitosan-poloxamer 407 (CS-P407) for use.

[0081] Take 1 mL of CQ-ML as the aqueous phase, add 30 parts of poly (N-isopropylacrylamide) (PNIPAM) and 2 mL of CS-P407, stir evenly and prepare CQ-ML@gel for use.

[0082] Example 6: Preparation of dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel

[0083] Weigh 15 parts of Qu, mix with 250 parts of tricaprylic acid glycerol, 250 parts of Tween 80, and 100 parts of 1,2-propylene glycol in a vial, and stir overnight at room temperature until completely homogeneous; add pure water dropwise under magnetic stirring to form Qu-MEs.

[0084] Weigh 40 parts of soybean lecithin, mix with 10 parts of cholesterol and 5 parts of CAPE in an eggplant-shaped bottle, dissolve in 10 mL of chloroform, and evaporate under reduced pressure at 100 rpm in a constant temperature water bath at 37°C to form a uniform film; add 0.5 mL of Qu-MEs and 9.5 mL of pure water to hydrate the phospholipid film; then place the solution in an ultrasonic cell crusher for 5 minutes and extrude it through an extruder to prepare CQ-ML for use.

[0085] Weigh 20 parts of poloxamer 407 (P407) into a vial, add 1.5 mL of deionized water, 40 μL of glycerol, and 20 μL of glacial acetic acid and stir evenly, add 80 parts of chitosan (CS) and stir evenly at 50°C, add 0.2 mL of NaHCO3 (1 M), add deionized water to 2 mL and stir evenly to obtain chitosan-poloxamer 407 (CS-P407) for use.

[0086] Use 1 mL of CQ-ML as the aqueous phase, add 35 parts of poly (N-isopropylacrylamide) (PNIPAM) and 2 mL of CS-P407, stir evenly to prepare CQ-ML@gel for use.

[0087] Example 7: Evaluation of the efficacy of dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel in the treatment of periodontitis in rats

[0088] After 100 healthy male SPF rats were adaptively fed for 2 weeks, 3% sodium pentobarbital (30 mg / kg) was used for intraperitoneal anesthesia in all groups except the normal group. The rats were placed supine on the experimental table, the head and limbs of the rats were fixed, the upper and lower jaws were pulled to fully expose the left maxillary first molar, and a 0.2 mm ligature was passed through the gap between the first and second molars of the rats, ligated at the neck of the first molar, and tied on the palate side. The excess length of the ligature was cut off, and the ligature was buried under the gums. After 4 weeks, the periodontal condition of the rats was observed, and the rats with successful modeling were randomly divided into normal group (Normal), model group (Model), positive drug (Perio) group, quercetin microemulsion gel group (Qu-MEs@gel), caffeic acid phenethyl ester liposome gel group (CAPE-L@gel), and dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel group (CQ-ML@gel). Every 7 days, 20 μL of the corresponding preparation was injected subperiosteally into the central alveolar ridge of the buccal and palatal sides of the maxillary first molar, and the normal group and the model group were injected with the same volume of normal saline.

[0089] Preparation method of quercetin microemulsion gel (Qu-MEs@gel): Qu, tributyrin, HS15, and PEG400 were mixed in a vial, stirred until completely homogeneous, and pure water was added dropwise under magnetic stirring to form Qu-MEs. CS-P407 and PNIPAM were added to Qu-MEs and stirred evenly to obtain Qu-MEs@gel.

[0090] Preparation method of caffeic acid phenethyl ester liposome gel (CAPE-L@gel): Soybean lecithin, cholesterol, and CAPE were placed in an eggplant-shaped bottle, 10 mL of chloroform was added to dissolve, and the mixture was rotated in a constant temperature water bath at 37°C at 100 rpm to form a uniform film under reduced pressure, and pure water was added, and the mixture was hydrated in a constant temperature water bath at 37°C at 100 rpm, and ultrasonicated and extruded to obtain caffeic acid phenethyl ester liposome (CAPE-L). CS-P407 and PNIPAM were added to CAPE-L, and CAPE-L@gel was obtained after uniform stirring.

[0091] Preparation method of dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel group (CQ-ML@gel): Qu, tributyrin, HS15, and PEG400 were mixed in a vial, stirred until completely homogenized, and pure water was added under magnetic stirring to form Qu-MEs. Soy lecithin, cholesterol, and CAPE were placed in an eggplant-shaped bottle, 10 mL of chloroform was added to dissolve, and a uniform film was formed by rotating at 100 rpm in a 37°C constant temperature water bath under reduced pressure; quercetin microemulsion and pure water were added, hydrated, ultrasonicated, and extruded to prepare CQ-ML. CS-P407 and PNIPAM were added to CQ-ML and stirred evenly to obtain CQ-L@gel.

[0092] Four weeks after administration, SD rats were euthanized, the left maxilla was separated, blood stains were cleaned with saline, tissues near the first molar were collected, rinsed, and stored at -80°C. Excess soft tissue was removed with fine scissors, and the specimens were fixed in 4% paraformaldehyde for 48 hours. Standard procedures such as decalcification, dehydration, embedding, and sectioning were performed. The hematoxylin-eosin staining (H&E) method was used to observe the histological changes of periodontal tissues after 4 weeks.

[0093] The results of H&E staining are as follows Figure 8 As shown in A, the periodontal tissue structure of the Normal group was clear, the periodontal fibers were intact and tightly arranged, there was no alveolar bone absorption, and almost no inflammatory cell infiltration was observed. The interdental papilla of the Model group disappeared, the junctional epithelium migrated to the root of the tooth, the periodontal fiber structure was disordered, and periodontal pockets were formed, with a large number of inflammatory cell infiltrations. The inflammatory cell infiltration was reduced in the Qu-MEs@gel group, the CAPE-L@gel group, and the CQ-ML@gel group. It can be seen that the epithelial structure of the CQ-ML@gel group was clear, and its anti-inflammatory effect was better than that of the Qu-MEs@gel and CAPE-L@gel groups.

[0094] Immunohistochemical staining was performed on each group using IL-6 and IL-10 as inflammatory indicators. Figure 8 As shown in Figures B and C, compared with the Model, the expression of the pro-inflammatory factor IL-6 in the CQ-ML@gel group and the positive drug group (Perio) was reduced, and the expression of the anti-inflammatory factor IL-10 was increased. The reason is that Qu in CQ-ML@gel reduces the secretion of inflammatory factors by regulating the conversion of M1 macrophages to M2 macrophages. In addition, CAPE in CQ-ML@gel is expected to play an auxiliary anti-inflammatory role by promoting the reconstruction of periodontal bone tissue and inhibiting the infiltration of inflammatory cells into the deep layer of bone tissue. Therefore, under the combined action of Qu and CAPE, the anti-inflammatory ability of CQ-ML@gel is further improved.

[0095] Micro-CT technology was used to reconstruct the three-dimensional structure of rat alveolar bones. Fig. 9 As shown in A, the gaps between teeth in the Normal group were small and the alveolar bone was intact, while the gaps between teeth in the Model group were obvious and the alveolar bone was severely lost. The gaps between teeth in the CQ-ML@gel group were significantly reduced and the alveolar bone was significantly repaired. Its treatment effect was better than that of the CAPE-L@gel and Qu-MEs@gel groups. Fig. 9As shown in Figures B and C, the expression of periodontal osteogenesis-related proteins OPG and OPN in the CQ-ML@gel group was also significantly improved. On the one hand, this is because CAPE in CQ-ML@gel can promote the expression of osteogenic proteins such as OPN and OPG, and promote the osteogenic differentiation of periodontal ligament stem cells; on the other hand, Qu in CQ-ML@gel is expected to inhibit the differentiation of M1 macrophages into osteoclast precursors by inhibiting the secretion of inflammatory factors, thereby assisting in promoting periodontal bone tissue regeneration.

[0096] The above results indicate that, compared with a single active ingredient drug delivery system, the CQ-ML@gel prepared in the present invention, through co-loading of Qu and CAPE technology, as well as programmed drug release design, can exert excellent anti-inflammatory and alveolar bone regeneration therapeutic effects, and is a new drug delivery system and treatment strategy for periodontitis.

Claims

1. A dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel, characterized in that: It includes quercetin-caffeic acid phenethyl ester liposomes and a dual-responsive gel matrix; the quercetin-caffeic acid phenethyl ester liposomes are prepared by microemulsion-lipid cascade drug delivery technology using quercetin and caffeic acid phenethyl ester as raw materials, lipid compounds, surfactants, co-surfactants, cholesterol, and phospholipid materials as auxiliary materials, and the weight proportions of each component are as follows: 1-20 parts of quercetin, 1-10 parts of caffeic acid phenethyl ester, 100-250 parts of lipid compounds, 180-280 parts of surfactants, 50-100 parts of co-surfactants, 10-50 parts of phospholipid materials, and 5-15 parts of cholesterol; The dual-responsive gel matrix comprises: 5-20 parts of poloxamer 407, 10-100 parts of chitosan, and 50-150 parts of poly-N-isopropylacrylamide; The preparation method of the dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel comprises the following steps: (1) Microemulsion-lipid cascade drug delivery technology: Quercetin, lipid compounds, surfactants, and co-surfactants are fully stirred, and then deionized water is added to obtain quercetin microemulsion by emulsification. Caffeic acid phenethyl ester, cholesterol, and phospholipid materials are used to prepare lipid films, and then quercetin microemulsion is added. Deionized water is used for hydration and extrusion to obtain quercetin-caffeic acid phenethyl ester liposomes. (2) Chitosan and poloxamer 407 were cross-linked to prepare chitosan-poloxamer 407 solution, and quercetin-caffeic acid phenethyl ester liposomes and poly (N-isopropylacrylamide) were added to the chitosan-poloxamer 407 solution for further cross-linking to obtain a dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel.

2. The dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel according to claim 1, characterized in that: The lipid compound is selected from one or more of triacetin, tributyrin, tripropionin, tricaprylin or tricaprin.

3. The dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel according to claim 1, characterized in that: The surfactant is selected from one or more of polyethylene glycol 15 hydroxystearate HS-15, polyoxyethylene 40 hydrogenated castor oil RH-40, castor oil polyoxyethylene ether, Tween 80, Tween 20, poloxamer 188, carbitol, and lecithin; the co-surfactant is selected from one or more of polyethylene glycol 400, glycerol, 1,2-propylene glycol, n-butanol, isopropanol, and anhydrous ethanol.

4. The dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel according to claim 1, characterized in that: The phospholipid material is selected from one or more of dipalmitoylcholine (DPPC), distearoylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin phosphatidic acid, phosphatidylglycerol, dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylserine (DOPS), lysophosphatidylethanolamine (LPE), palmitoyl lysophosphatidylcholine (PLysoPC), dimyristoylphosphatidylethanolamine (DMPE), dioleoylphosphatidylglycerol (DOPG), DOP-DEDA, DOTMA, Dlin-MC3-DMA, DODMA, and DOTAP.

5. The method for preparing the dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel according to any one of claims 1 to 4, characterized in that: The steps include: (1) Microemulsion-lipid cascade drug delivery technology: Quercetin, lipid compounds, surfactants, and co-surfactants are fully stirred, and then deionized water is added to obtain quercetin microemulsion by emulsification. Caffeic acid phenethyl ester, cholesterol, and phospholipid materials are used to prepare lipid films, and then quercetin microemulsion is added. Deionized water is used for hydration and extrusion to obtain quercetin-caffeic acid phenethyl ester liposomes. (2) Chitosan and poloxamer 407 were cross-linked to prepare chitosan-poloxamer 407 solution, and quercetin-caffeic acid phenethyl ester liposomes and poly (N-isopropylacrylamide) were added to the chitosan-poloxamer 407 solution for further cross-linking to obtain a dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel.

6. The method for preparing the dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel according to claim 5, characterized in that: In step (1), the mass ratio of the lipid compound to the surfactant is 1:1-5:1, the mass ratio of the lipid compound to the co-surfactant is 1:5-1:1, and the mass ratio of the lipid compound to the phospholipid material is 1:5-1:

1.

7. The method for preparing the dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel according to claim 5, characterized in that: In step (2), the mass ratio of chitosan to poloxamer 407 is 2:1-10:1, and the mass ratio of chitosan to poly (N-isopropylacrylamide) is 1:5-5:

1.

8. The method for preparing the dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel according to claim 7, characterized in that: In step (2), the mass ratio of chitosan to poly (N-isopropylacrylamide) is 2:1-3:

1.

9. Use of the dual-responsive quercetin-caffeic acid phenethyl ester lipid composite gel according to any one of claims 1 to 4 in the preparation of a drug for treating periodontitis.

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