A photosensitive gel of eucalyptus oil microemulsion wrapped by a biomimetic membrane, and a preparation method and application thereof

Abstract

The application discloses a photosensitive gel of eucalyptus oil microemulsion wrapped by a biomimetic membrane and a preparation method and application thereof. The photosensitive gel of eucalyptus oil microemulsion wrapped by a biomimetic membrane is prepared by taking eucalyptus oil and a lipid compound as a mixed oil phase, wrapping the eucalyptus oil microemulsion with a biomimetic membrane, and distributing the eucalyptus oil microemulsion wrapped by the biomimetic membrane in a photosensitive gel matrix of dopamine-modified methyl methacrylate gelatin. The photosensitive gel has high adhesion and the ability of actively targeting periodontitis pathogenic bacteria, and significantly improves the treatment effect on periodontitis.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of drug-loaded hydrogels, and particularly relates to a photosensitive hydrogel of eucalyptus oil microemulsion wrapped by a biomimetic membrane as well as a preparation method and application thereof. BACKGROUND

[0002] Periodontitis is an infectious disease characterized by bacterial infection, gum inflammation, tooth loosening and migration, and eventually tooth loss. Chronic periodontitis is clinically manifested as gum swelling, attachment loss, alveolar bone resorption, and periodontal pocket formation. If not intervened in time, it will lead to tooth loosening and even tooth loss, which seriously affects the appearance, pronunciation and even mental health of patients.

[0003] Studies have confirmed that periodontitis is induced by periodontitis pathogenic bacteria such as Porphyromonas gingivalis, Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans. Periodontitis pathogenic bacteria can destroy the normal function of the host immune system and aggravate the inflammatory symptoms of periodontal tissue. For example, Porphyromonas gingivalis can selectively down-regulate the expression of macrophage CD14, hindering macrophage-mediated phagocytosis. At the same time, the virulence factors of bacteria will activate macrophages, leading to massive secretion of inflammatory factors (TNF-alpha, IL-1 beta, IL-6), causing periodontal tissue destruction. Although antibiotics can play a role in bacterial clearance, long-term use of antibiotics will induce bacterial drug resistance, kill beneficial bacteria in the oral cavity, and destroy the balance of the normal human microbiota. Therefore, the development of a new type of preparation that can effectively target and eliminate periodontitis pathogenic bacteria is of great significance for clinical periodontitis.

[0004] Eucalyptus oil, also known as eucalyptus oil or eucalyptus oil, is a volatile oil extracted by steam distillation from Eucalyptus globulus Labill. of Myrtaceae, Cinnamomum camphora (L.) Presl of Lauraceae or other plants of the same genus. Eucalyptus oil is rich in active ingredients such as eucalyptus oil, 1,8-cineole, etc., and has various effects such as antibacterial, anti-inflammatory, etc. Studies have shown that eucalyptus oil can effectively inhibit various pathogenic bacteria such as Proteus, Streptococcus and anaerobic bacteria, etc. In addition, eucalyptus oil can also reduce oral inflammation such as periodontitis and promote tissue healing. However, due to the poor water solubility, low bioavailability, lack of bacterial targeting, low tissue adhesion and other biopharmaceutical property defects of eucalyptus oil, its practical application in the treatment of periodontitis is limited. SUMMARY

[0005] In view of this, the purpose of the present application is to provide a photosensitive hydrogel of eucalyptus oil microemulsion wrapped by a biomimetic membrane, which has high adhesion and the ability to actively target periodontitis pathogenic bacteria, significantly improving the treatment effect on periodontitis.

[0006] The present application is realized by the following technical solutions:

[0007] The photosensitive gel of the eucalyptus oil microemulsion wrapped by a biomimetic membrane comprises a biomimetic membrane wrapped eucalyptus oil microemulsion and a gel matrix; the biomimetic membrane wrapped eucalyptus oil microemulsion is composed of a eucalyptus oil microemulsion and a biomimetic membrane modified on the surface of the eucalyptus oil microemulsion; the biomimetic membrane is selected from one or more of a macrophage membrane, a neutrophil membrane, an NK cell membrane, an erythrocyte membrane, and a monocyte membrane; and the gel matrix is dopamine modified methyl methacrylate gelatin (GelMA-DA).

[0008] The biomimetic membrane wrapped eucalyptus oil microemulsion of the present application is composed of a eucalyptus oil microemulsion and a biomimetic membrane modified on the surface of the eucalyptus oil microemulsion. The eucalyptus oil microemulsion uses eucalyptus oil and a lipid compound as a mixed oil phase. A large number of compatibility experiments show that the compatibility of eucalyptus oil and a lipid compound can significantly improve the solubility of a poorly soluble active ingredient, improve the overall drug loading performance of the microemulsion, and reduce the amount of inactive excipients. In addition, the biomimetic membrane retains the membrane structure and function during extraction and separation, and has the characteristics of actively targeting homologous cells or pathogenic bacteria. The present application uses distearoyl phosphatidyl acetylamine-polyethylene glycol-amino (DSPE-PEG-NH2) to provide -NH2 and -COOH on the surface of the macrophage membrane, and modifies the biomembrane protein on the outer layer of the microemulsion through chemical bonding reaction to construct a biomimetic cell membrane wrapped eucalyptus oil microemulsion, so that the eucalyptus oil microemulsion has the ability to actively target homologous cells or pathogenic bacteria of periodontitis, and improves the therapeutic efficacy of the drug.

[0009] The eucalyptus oil microemulsion of the present application is made of eucalyptus oil and a lipid compound as a mixed oil phase, supplemented with a surfactant, a co-surfactant, an active ingredient and the like.

[0010] As a preferred embodiment of the present application, the eucalyptus oil microemulsion is made of the following components by weight:

[0011] Eucalyptus oil 20-80 parts, lipid compound 10-160 parts, surfactant 20-240 parts, co-surfactant 20-440 parts, active ingredient 1-5 parts, DSPE-PEG-NH2 1-5 parts.

[0012] Preferably, the lipid compound is one or more of glycerol triacetate, glycerol tributyrate, glycerol tripropionate, glycerol trioctanoate, and glycerol tricaprate.

[0013] Preferably, the surfactant is 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, carbopol, and lecithin.

[0014] Preferably, the co-surfactant is one or more of polyethylene glycol 400, glycerol, 1,2-propanediol, n-butanol, isopropyl alcohol, and anhydrous ethanol.

[0015] Preferably, the active ingredient is one or more of quercetin, caffeic acid phenethyl ester, baicalin, gallic acid, catechin.

[0016] Preferably, the mass ratio of the eucalyptus oil to the lipid compound is 1:2-1:1; the mass ratio of the surfactant to the co-surfactant is 1:2-2:1; the mass ratio of the mixed oil phase to the mixed surfactant is 1:3-1:1.3; and the mass ratio of the active ingredient to the lipid compound is 1:10-1:20.

[0017] The active hydroxyl group in the photoinitiator can react with the unsaturated group of the methacrylated gelatin (GelMA), causing the methacrylamide and methacrylate side groups in the structure of the methacrylated gelatin to be polymerized to form a gel through a free radical addition reaction. However, the bioadhesion performance of the methacrylated gelatin is weak, and the large amount of saliva in the oral cavity makes it difficult for the GelMA to stably reside. The ortho-phenolic group of dopamine can form hydrogen bonds with polymers such as proteins and polysaccharides in the oral tissue, forming a firm adhesion connection. Therefore, the GelMA is first modified by dopamine hydrochloride (DA) to endow the GelMA with self-adhesion performance, and dopamine-modified methacrylated gelatin (GelMA-DA) is prepared. Then, the GelMA-DA is used to load the eucalyptus oil microemulsion wrapped with a biomimetic membrane, and a photosensitive gel of the biomimetic membrane-wrapped eucalyptus oil microemulsion is formed in situ in the oral cavity through ultraviolet light irradiation, so that the biomimetic membrane-wrapped eucalyptus oil microemulsion stably resides in the oral cavity periodontal pocket, and the treatment effect of periodontitis is improved.

[0018] The application also provides a preparation method of the photosensitive gel of the biomimetic membrane-wrapped eucalyptus oil microemulsion, comprising the following steps:

[0019] (1) uniformly stir eucalyptus oil, a surfactant, a co-surfactant, an active ingredient and DSPE-PEG-NH2, and then add deionized water to obtain eucalyptus oil microemulsion by emulsification;

[0020] (2) uniformly stir the prepared eucalyptus oil microemulsion, a cell membrane solution, 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide (EDC) and N-hydroxy succinimide (NHS) to obtain biomimetic membrane-wrapped eucalyptus oil microemulsion by chemical bonding;

[0021] (3) disperse dopamine-modified methacrylated gelatin into the biomimetic membrane-wrapped eucalyptus oil microemulsion to obtain the photosensitive gel of the biomimetic membrane-wrapped eucalyptus oil microemulsion.

[0022] Preferably, in step (2), the protein concentration of the cell membrane is 0.002-0.060 mg / mL.

[0023] Preferably, in step (2), the molar ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide to N-hydroxysuccinimide is 1:1-3:1.

[0024] Preferably, in step (3), the mass ratio of dopamine-modified methyl acrylate gelatin to biomimetic membrane-coated eucalyptus oil microemulsion is 40:227-100:81.

[0025] The application also provides a use of the above-mentioned photosensitive gel of the biomimetic membrane-coated eucalyptus oil microemulsion in the preparation of a drug for treating periodontitis.

[0026] Compared with the prior art, the application has the following beneficial effects:

[0027] 1. The application selects eucalyptus oil and lipid compounds as a mixed oil phase, which on the one hand reduces the use amount of traditional inactive oil phase, and significantly improves the solubility of a poorly soluble small molecule active ingredient; and on the other hand, fully plays the inhibitory activity of eucalyptus oil on a variety of pathogenic bacteria, and is conducive to reducing the number of pathogenic bacteria and tissue damage in the periodontal lesion area.

[0028] 2. The application prepares a biomimetic membrane-coated eucalyptus oil microemulsion by chemically bonding the cell membrane to the surface of the eucalyptus oil microemulsion. Compared with other microemulsion preparations, the biomimetic membrane-coated eucalyptus oil microemulsion avoids drug leakage, actively targets pathogenic bacteria or homologous cells, and greatly improves the antibacterial, anti-inflammatory and other therapeutic properties of eucalyptus oil and active ingredients.

[0029] 3. In addition, the application further disperses the biomimetic membrane-coated eucalyptus oil microemulsion in a dopamine-modified methyl acrylate gelatin matrix to construct a photosensitive gel of the biomimetic membrane-coated eucalyptus oil microemulsion. The gel is a liquid at room temperature, and automatically gels when irradiated with ultraviolet light. Compared with other gels, it is more convenient for oral injection and can fully fill the periodontal pocket. By utilizing the characteristics of the dopamine groups in the gel matrix structure, high adhesion performance is achieved, which can realize efficient adhesion and residence in periodontal tissue, effectively prevent drug loss, and further enhance the performance of the biomimetic membrane-coated eucalyptus oil microemulsion in eliminating periodontitis pathogenic bacteria and tissue inflammation. The evaluation results show that the preparation can kill periodontitis pathogenic bacteria and relieve periodontal tissue inflammation, providing a new preparation for the treatment of periodontitis. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Figure is the rheological characterization diagram of GelMA-DA of the application;

[0031] Figure 2 Figure is the particle size diagram of the biomimetic membrane-coated eucalyptus oil microemulsion;

[0032] Figure 3 Figure is the appearance and scanning electron microscope diagram of the photosensitive gel of the biomimetic membrane-coated eucalyptus oil microemulsion of the application;

[0033] Figure 4 This invention demonstrates the photosensitive gel adhesion properties of the biomimetic membrane encapsulating eucalyptus oil microemulsion.

[0034] Figure 5 To prepare pseudo-ternary phase diagrams for microemulsions using different surfactants EL, HS-15, and Tween 80;

[0035] Figure 6 To obtain pseudo-ternary phase diagrams of microemulsions using different Km values ​​(1:1, 1:2, 2:1);

[0036] Figure 7 For response surface contour plots (A, B, C, G, H, I) and 3D model plots (D, E, F, J, K, L);

[0037] Figure 8 This is a graph showing the evaluation of the antibacterial properties of the biomimetic membrane-encapsulated eucalyptus oil microemulsion of this invention.

[0038] Figure 9 This is an evaluation diagram of the biomimetic membrane-encapsulated eucalyptus oil microemulsion targeting homologous cells of the present invention;

[0039] Figure 10 Evaluation of the antibacterial effect of the photosensitive gel containing eucalyptus oil microemulsion encapsulated in the biomimetic membrane of this invention on rats with periodontitis; Figure 11 This image shows the therapeutic effect of the photosensitive gel containing eucalyptus oil microemulsion encapsulated in the biomimetic membrane of this invention on the bone tissue of rats with periodontitis. Detailed Implementation

[0040] The present invention will be further illustrated below through specific embodiments. The following embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the following embodiments.

[0041] Example 1: Synthesis and Rheological Characterization of Dopamine-Modified Methacrylic Anhydride Gelatin (GelMA-DA)

[0042] The GelMA-DA described in this invention uses GelMA and DA as raw materials, and the specific preparation method is as follows: GelMA prepolymer is dissolved in PBS and stirred at 50°C to obtain a homogeneous solution. Triethylamine, succinic anhydride, and dimethyl sulfoxide are added to the solution, and the mixture is stirred for 12 hours. The solution is then diluted with PBS. The solution is dialyzed against deionized water for one week at room temperature using a dialysis tube to remove impurities. The dialyzed solution is placed in a centrifuge tube and frozen at -80°C for two days, then freeze-dried for four days to obtain the GelMA-COOH prepolymer. The prepolymer is dissolved in MES buffer, degassed with nitrogen, and EDC, NHS, and DA are added. The mixture is stirred for 12 hours under nitrogen. Finally, the solution is placed in a centrifuge tube and refrigerated at -80°C for two days, then freeze-dried for four days to obtain GelMA-DA.

[0043] The viscosity and injectability of hydrogels are very important for practical applications, especially for the filling of irregular periodontal pockets. Rheological studies were performed using a DHR-2 rheometer equipped with a 20 mm parallel plate fixture. As Figure 1 The rheological properties of GelMA and GelMA-DA were shown. When the shear rate increased from 10 to 100 s -1 The viscosity of GelMA-DA was always higher than that of GelMA due to the modification of DA, and the results showed that GelMA-DA belonged to a typical pseudoplastic fluid with high viscosity and injectability.

[0044] Example 2:

[0045] Quercetin 5 parts, eucalyptus oil 40 parts, glycerol tributyrate 40 parts, HS-158 80 parts, anhydrous ethanol 20 parts, glycerol 20 parts, DSPE-PEG-NH2 5 parts;

[0046] The above components were placed in a container and stirred at 37℃ until completely homogeneous. Ultra-pure water was slowly added dropwise, and the volume was adjusted to 5mL to obtain eucalyptus oil microemulsion (QE-MEs);

[0047] Take 1 part of eucalyptus oil microemulsion in a vial, accurately weigh EDC, NHS, and macrophage membrane aqueous solution (protein concentration is 0.031mg / mL) in a vial, adjust the volume to 10mL, and incubate at 37℃ for 6h to obtain biomimetic membrane-coated eucalyptus oil microemulsion (M-QE-MEs); the molar ratio of EDC to NHS is 1:1;

[0048] Disperse GelMA-DA into the biomimetic membrane-coated eucalyptus oil microemulsion to obtain biomimetic membrane-coated eucalyptus oil microemulsion photosensitive gel (M-QE-MEs@Gel).

[0049] Performance characterization:

[0050] (1) Particle size characterization of biomimetic membrane-coated eucalyptus oil microemulsion

[0051] Figure 2 The particle size of the biomimetic membrane-coated eucalyptus oil microemulsion obtained in Example 2 is shown, with a particle size of 46.65±0.45nm.

[0052] (2) Appearance and morphology characterization of biomimetic membrane-coated eucalyptus oil microemulsion photosensitive gel

[0053] From the macroscopic state, it can be observed that Figure 3The transformation of the eucalyptus oil microemulsion photosensitive gel solution into a gel is illustrated in Example 2 (Figure A). After freeze-drying the photosensitive gel of the eucalyptus oil microemulsion encapsulated in the biomimetic membrane, the sample was adhered to conductive tape on a sample holder. The sample holder was then placed in a vacuum coating machine, the lid closed, and the vent valve opened to evacuate the gold vapor deposition chamber. Gold was then sprayed onto the sample surface. The sample was removed and the gel morphology was observed under a scanning electron microscope, and photographs of the gel morphology were taken. The results are as follows: Figure 3 As shown in Figure B, the photosensitive gel solution scaffold of eucalyptus oil microemulsion encapsulated in Example 2 has a porous network structure, which is beneficial for drug loading.

[0054] (3) Adhesion performance characterization of biomimetic membrane-encapsulated eucalyptus oil microemulsion photosensitive gel

[0055] As shown in the figure, the biomimetic membrane-encapsulated eucalyptus oil microemulsion photosensitive gel of Example 2 can firmly adhere to corks and rubber materials. Figure 4 (A, B). The oral cavity is a moist and dynamic environment, and the persistent adhesion of the gel in the oral cavity is crucial for drug retention and sustained delivery. The adhesion strength of the gel to various moist tissues was tested, including the oral mucosa, stomach, large intestine, small intestine, and myocardial tissue. Figure 4 (C) The result is as follows Figure 4 As shown in Figure D, the biomimetic membrane-encapsulated eucalyptus oil microemulsion photosensitive gel adheres immediately upon contact with wet gingival tissue and can withstand vigorous water rinsing without peeling off, further demonstrating the excellent adhesion of the biomimetic membrane-encapsulated eucalyptus oil microemulsion photosensitive gel to the oral mucosa.

[0056] Example 3:

[0057] 5 parts of phenethyl caffeate, 20 parts of eucalyptus oil, 40 parts of glyceryl tripropionate, 50 parts of poloxamer 188, 50 parts of polyethylene glycol 400, 50 parts of glycerol, and 3 parts of DSPE-PEG-NH2.

[0058] The above components were placed in a container and stirred at 37°C until completely homogeneous. Ultrapure water was slowly added dropwise and the volume was adjusted to 5 mL to obtain eucalyptus oil microemulsion.

[0059] Take one part of eucalyptus oil microemulsion into a vial, accurately weigh EDC, NHS, and macrophage membrane aqueous solution (protein concentration of 0.002 mg / mL) into the vial, make up to 10 mL, and incubate at 37℃ for 6 h to obtain eucalyptus oil microemulsion encapsulated by biomimetic membrane; wherein the molar ratio of EDC to NHS is 2:1.

[0060] GelMA-DA was dispersed into eucalyptus oil microemulsions encapsulated in a biomimetic membrane to obtain a photosensitive gel of eucalyptus oil microemulsions encapsulated in a biomimetic membrane.

[0061] Example 4:

[0062] Baicalin 5 parts, eucalyptus oil 80 parts, glyceryl trioctanate 40 parts, RH-406 60 parts, absolute ethanol 40 parts, 1,2-propanediol 40 parts, DSPE-PEG-NH2 2 parts;

[0063] Put the above components in a container, stir at 37°C until completely homogeneous, slowly drop into ultrapure water, dilute to 5 mL, and obtain eucalyptus oil microemulsion;

[0064] Take 1 part of eucalyptus oil microemulsion in a vial, weigh EDC, NHS, and macrophage membrane aqueous solution (protein concentration 0.004 mg / mL) in the vial, dilute to 10 mL, and incubate at 37°C for 6 h to obtain a biomimetic membrane-coated eucalyptus oil microemulsion; the molar ratio of EDC to NHS is 3:1;

[0065] Disperse GelMA-DA into the biomimetic membrane-coated eucalyptus oil microemulsion to obtain a biomimetic membrane-coated eucalyptus oil microemulsion photosensitive gel.

[0066] Example 5: Solubility of quercetin in mixed oil phase

[0067] Excess quercetin was weighed and dissolved in equal volumes of eucalyptus oil, glyceryl tributyrate, and mixed oil EG11 (eucalyptus oil: glyceryl tributyrate, 1:1, w:w), EG2 (eucalyptus oil: glyceryl tributyrate, 2:1, w:w), and EG3 (eucalyptus oil: glyceryl tributyrate, 3:1, w:w), respectively, and placed in a stoppered glass test tube. Ultrasonic treatment was performed for 30 min, and the sample was placed in a 37°C constant temperature oscillator for 24 h to reach solubility equilibrium. Centrifugation was performed at 13000 r / min for 10 min, 20 μL of supernatant was taken, diluted with methanol, and the solubility was detected by HPLC method. The results are shown in Table 1. The solubility of quercetin in EG1 was as high as 263.70 ± 9.04 μg / mL 1 , indicating that when the mass ratio of eucalyptus oil to lipid compound is 1:1, not only can the use amount of traditional inactive oil phase be reduced, but also the solubility of quercetin can be significantly improved. Moreover, eucalyptus oil itself has inhibitory effect on a variety of pathogenic bacteria, and can play a therapeutic performance of antibacterial and anti-inflammatory together with active ingredients. -1

[0068] Table 1 Solubility of quercetin in different oil phases (n = 3,

[0069]

[0070] Example 6: Formulation screening of microemulsion surfactant, surfactant / surfactant (Km) value

[0071] ​​The surface active agent of the microemulsion is screened: fixed eucalyptus oil-glycerol tributyrate as a mixed oil phase (1:1, w:w), anhydrous ethanol-glycerol as a co-surfactant, and the surface active agent of the microemulsion is preferably selected. Different types of surfactants (HS-15, Tween 80, castor oil polyoxyethylene ether) are mixed with the co-surfactant at a mass ratio of 1:1 to form a mixed surfactant. Titrate with ultrapure water, record the critical change of the system, draw a pseudo-ternary phase diagram with Origin 2021 software, determine the microemulsion area, and determine the optimal surfactant according to the microemulsion area.

[0072] Fixed polyethylene glycol 15 hydroxystearate (HS-15) as a surface active agent, anhydrous ethanol-glycerol (1:1, w:w) as a co-surfactant, and Km values of 1:1, 1:2, and 2:1 are selected. Mix evenly. Titrate with ultrapure water, record the critical change of the system, draw a pseudo-ternary phase diagram with Origin 2021 software, determine the microemulsion area, and determine the optimal Km value according to the microemulsion area.

[0073] The results are shown in Figure 5 and Figure 6 The microemulsion area prepared with HS-15 as the surface active agent and a Km value of 1:1 has the largest area, so HS-15 is preferably selected as the surface active agent and the Km value is 1:1.

[0074] Example 7: Prescription optimization of eucalyptus oil microemulsion wrapped with biomimetic membrane

[0075] Plackett-Burman design method is used to screen the key influencing factors of eucalyptus oil microemulsion wrapped with biomimetic membrane. The experimental results are shown in Tables 2 and 3. The results show that the mass ratio of eucalyptus oil-glycerol tributyrate (X1), the mass ratio of quercetin-excipient (X3), the concentration of macrophage membrane protein (X4), and the molar ratio of EDC-NHS (X7) have a significant effect on the microemulsion.

[0076] Based on the Plackett-Burman experiment, the Box-Behnken experiment is used to optimize the prescription of eucalyptus oil microemulsion wrapped with biomimetic membrane. The mass ratio of eucalyptus oil-glycerol tributyrate (X1), the mass ratio of quercetin-polyethylene glycol 15 hydroxystearate (HS15) (X2), the concentration of macrophage membrane protein (X3), and the molar ratio of EDC-NHS (X4) are used as independent variables, and the encapsulation efficiency of the core drug component eucalyptus oil (Y3) is used as the response value. The prescription ratio of the eucalyptus oil wrapped with biomimetic membrane is optimized. The experimental scheme is generated by Design Expert software, and the experimental arrangement and results are shown in Tables 4 and 5.

[0077] The Box-Behnken experiment data were fitted with Design Expert software to obtain a multiple quadratic regression model for the encapsulation rate Y = 1.74A - 0.0988B + 0.1317C - 0.7042D - 0.8310AB - 0.5950AC + 0.7950AD - 0.9750BC - 1.13BD + 1.24CD - 4.27A2 - 2.76B2 - 3.21C2 - 4.40D2 + 51.00. The model was subjected to F test for variance analysis and model coefficient significance check, and the results are shown in Table 6.

[0078] Table 2 Factors and levels of Plackett-Burman design

[0079]

[0080]

[0081] Table 3 Key factors of eucalyptus oil wrapped by biomimetic membrane Plackett-Burman design experiment and results (n = 3, )

[0082]

[0083] Table 4 BBD design factor and level table

[0084]

[0085] Table 5 BBD experimental scheme and results (n = 3, )

[0086]

[0087]

[0088] Table 6 Variance analysis of encapsulation rate

[0089]

[0090] As shown in the table, the variance analysis model of the encapsulation rate has significant difference; the lack of fit term P > 0.05 proves that the model has good fitting degree; the multiple correlation coefficient R2 is 0.8701, proving that the model has similarity with the actual results; AdeqPrecision > 5, CV < 10%, which again verifies the credibility and precision of the model. Through the binomial fitting equation, with the help of Design Expert software, the response surface contour line graph and 3D model graph are drawn Figure 7), the key prescription and process parameters are obtained as follows: mass ratio of eucalyptus oil-glycerol tributyrate (1:1, w:w), mass ratio of quercetin-HS15 (1:14, w:w), concentration of macrophage membrane protein is 0.031 mg / mL, and molar ratio of EDC-NHS (1:1, M:M).

[0091] Preparation of quercetin gel

[0092] Quercetin was dissolved in PBS to obtain a quercetin solution (Qu); GelMA-DA was dispersed into the quercetin naked drug solution to obtain Qu@Gel.

[0093] Preparation of eucalyptus oil gel

[0094] GelMA-DA was dispersed into the eucalyptus oil solution (Eu) to obtain Eu@Gel.

[0095] Preparation of quercetin eucalyptus oil mixed gel

[0096] Quercetin was dissolved in 40 parts of eucalyptus oil solution to obtain a quercetin eucalyptus oil solution (Qu+EuMix); GelMA-DA was dispersed into the quercetin eucalyptus oil solution to obtain Qu+Eu Mix@Gel.

[0097] Preparation of common eucalyptus oil microemulsion gel

[0098] Quercetin 5 parts, eucalyptus oil 40 parts, glycerol tributyrate 40 parts, HS-15 80 parts, anhydrous ethanol 20 parts, glycerol 20 parts, and DSPE-PEG-NH2 5 parts; the above components were placed in a container, stirred at 37°C until completely homogeneous, and then ultrapure water was slowly added dropwise, and the volume was adjusted to 5 mL to obtain a eucalyptus oil microemulsion (QE-MEs); GelMA-DA was dispersed into the QE-MEs solution to obtain QE-MEs@Gel.

[0099] Experimental Example 1: Evaluation of antibacterial performance

[0100] Pg was taken as a suspension, diluted 1:100 in BHI medium, and then inoculated into a 6-well plate; 100 μL of bacterial solution was added to each well, and then 100 μL of quercetin solution (Qu), eucalyptus oil solution (Eu), physical mixture of quercetin and eucalyptus oil (Qu+EuMix), common eucalyptus oil microemulsion (QE-MEs), and biomimetic membrane-wrapped eucalyptus oil microemulsion (M-QE-MEs) were added, respectively; an equal amount of PBS solution was added as a Control group for comparison; after sealing, the plate was placed in an anaerobic workstation and incubated at 37°C for 24 h; the optical density (OD) value of the bacterial solution at 600 nm was detected by an enzyme marker instrument, and the antibacterial rate was calculated. As shown in Table A, the results show that the antibacterial performance of M-QE-MEs is the best and is better than that of QE-MEs and Eu. Figure 8 Table A

[0101] 200 μL Pg bacteria suspension and cell climbing sheet were placed in a 24-well plate, 500 μL Qu, Eu, Qu+Eu Mix, QE-MEs, M-QE-MEs were added to each well, and an equal amount of aqueous solution was added to the control group as a control, and incubated for 24 hours. 500 μL of 2.5% glutaraldehyde was added to each well, and the plate was placed in a dark environment at 4°C for 24 hours. Then the plate was rinsed with PBS buffer (pH = 7) for 4 times, 5 minutes each time, to completely remove the glutaraldehyde on the surface of the mycelium. Sequentially dehydrated with 30%, 50%, 70%, 90% ethanol, 20 minutes each time, and finally placed in anhydrous ethanol for 15 minutes. The anhydrous ethanol was naturally evaporated in air overnight, coated, and the morphology was observed by SEM. As shown in Figure 8 As shown in B, the bacterial membrane is ruptured after the action of M-QE-MEs, and the integrity is seriously damaged, further proving that M-QE-MEs have good antibacterial performance.

[0102] Experimental Example 2: Evaluation of homophilic performance

[0103] Macrophage RAW 264.7 was inoculated in a 6-well plate at a density of 7.8 x 10 6 / well, and the culture solution was removed after incubation at 37°C, 5% CO2 for 24 hours. Coumarin 6 fluorescein (C6) was used to label ordinary eucalyptus oil microemulsion (C6-QE-MEs) and biomimetic membrane wrapped eucalyptus oil microemulsion (C6-M-QE-MEs). C6 solution, C6-QE-MEs, and C6-M-QE-MEs were diluted with culture solution to 2 μM and added to the 6-well plate. After incubation at 37°C for 2 hours, the cells were washed with PBS for 3 times. Fixed with paraformaldehyde for 10 minutes (500 μL / well), stained with DAPI for 5 minutes (500 μL / well), and then washed with PBS for 3 times. Finally, 500 μL of PBS was added to infiltrate the cells. The uptake of different groups of cells was observed by inverted fluorescence microscope. The results showed that Figure 9 ), the green fluorescence intensity of the C6-M-QE-MEs group was significantly higher than that of the C6-QE-MEs. The results showed that C6-M-QE-MEs can target homophilic cells, and because the surface of C6-M-QE-MEs is wrapped with macrophage membrane, the modification of macrophage membrane makes C6-M-QE-MEs more easily recognized and taken up by macrophages.

[0104] Experimental Example 3: Treatment effect of gels prepared in the examples and comparative examples on periodontitis rats

[0105] After 2 weeks of adaptive feeding, male healthy SPF rats were anesthetized with 3% sodium pentobarbital (30 mg / kg) except for the normal group (Normal), and the rats were placed on the experimental table in a supine position. The head and limbs of the rats were fixed, the upper and lower jaws were pulled to fully expose the left first maxillary molar, a 0.2 mm ligature wire was passed through the gap between the first and second molars of the rats, and was ligated at the neck of the first molar and knotted on the palate side. The excess length of the ligature wire was cut off, and the ligature knot was buried under the gum and given the bacterial suspension. After 4 weeks, the periodontal condition of the rats was observed, and the successfully modeled rats were randomly divided into groups: model group (Model), quercetin gel group (Qu@Gel), eucalyptus oil gel group (Eu@Gel), quercetin and eucalyptus oil mixed gel group (Qu+Eu Mix@Gel), ordinary eucalyptus oil microemulsion gel group (QE-MEs@Gel), and the biomimetic membrane-coated eucalyptus oil microemulsion photosensitive gel group (M-QE-MEs@Gel) prepared in Example 2. Every 3 days, 20 μL of each corresponding preparation was injected submucosally on the buccal and palatal sides of the central alveolar ridge of the first maxillary molar. The normal group (Normal) and the model group (Model) were injected with the same volume of normal saline.

[0106] After four weeks of administration, the gingival crevicular fluid was absorbed with a hygroscopic paper tip, and was immersed in PBS for 24 h. 100 μL of the bacterial suspension was coated, sealed, and placed in an anaerobic workstation at 37°C for 3-4 d. Photographs were taken Figure 10 A). 100 μL of the bacterial suspension after administration was sealed and placed in an anaerobic workstation at 37°C for 24 h. The absorbance was measured on a microplate reader. As shown in Figure 10 A and Figure 10 B, the number of pathogenic bacteria colonies in the Model group was the largest, and a large number of pathogenic bacteria colonies were visible in the Qu@Gel, Eu@Gel, and Qu+Eu Mix@Gel groups. In the ordinary microemulsion gel group, the solubility of eucalyptus oil and quercetin was improved by the microemulsion, and the number of pathogenic bacteria colonies in the QE-MEs@Gel group was reduced. After the preparation of the biomimetic membrane-coated eucalyptus oil microemulsion gel, the number of bacteria colonies in the M-QE-MEs@Gel group was the lowest, and the antibacterial effect was the best.

[0107] SD rats were euthanized, and the left maxillary bone was isolated. The blood stains were washed with normal saline, and the tissue around the first molar was collected. After washing, the samples were stored at -80℃. Excess soft tissue was removed with a sharp knife, and the specimens were fixed in 4% paraformaldehyde for 48 h. The standard procedures of decalcification, dehydration, embedding, and sectioning were performed. After the fixed and stored maxillary bone samples were removed, a German Bruker SkyScan 1176 small animal Micro-CT scanning system was used for scanning, with the scanning parameters set as follows: 65 kV, current 385 μL, exposure time 340 ms, and layer distance 20 μm. After scanning, the reconstructed images were obtained by CTvox software. The results are shown in Figure 11 As shown in Fig. 6, the alveolar bone of the Model group rats was severely damaged due to the action of pathogenic bacteria, and obvious bone defects were observed in the circle. Qu@Gel had no obvious effect on repairing the alveolar bone. Eu@Gel, Qu+Eu Mix@Gel, and QE-MEs@Gel could repair the alveolar bone, but the alveolar bone gap of the M-QE-MEs@Gel group was the smallest, indicating that the biomimetic membrane coated with eucalyptus oil microemulsion photosensitive gel had the strongest ability to promote bone tissue regeneration.

Claims

1. A photosensitive gel with a biomimetic membrane encapsulating eucalyptus oil microemulsion, characterized in that, The invention comprises a eucalyptus oil microemulsion encapsulated by a biomimetic membrane and a gel matrix; the eucalyptus oil microemulsion encapsulated by the biomimetic membrane is composed of a eucalyptus oil microemulsion and a biomimetic membrane modified on the surface of the eucalyptus oil microemulsion; the biomimetic membrane is selected from one or more of macrophage membranes, neutrophil membranes, and monocyte membranes; the gel matrix is ​​dopamine-modified methacrylic anhydride gelatin. The eucalyptus oil microemulsion is prepared by using eucalyptus oil and lipid compounds as a mixed oil phase, and surfactants and co-surfactants as a mixed surfactant. The components by weight are as follows: Eucalyptus oil 20-80 parts, lipid compounds 10-160 parts, surfactants 20-240 parts, co-surfactants 20-440 parts, active ingredients 1-5 parts, distearylphosphatidylacetamide-polyethylene glycol-amino 1-5 parts; The lipid compounds are selected from one or more of glyceryl triacetate, glyceryl tributyrate, glyceryl tripropionate, glyceryl tricaprylate, and glyceryl tridecanoate; The surfactant is polyethylene glycol 15-hydroxystearate; the co-surfactant is selected from one or more of polyethylene glycol 400, glycerin, 1,2-propanediol, n-butanol, isopropanol, and anhydrous ethanol. The active ingredient is selected from one or more of quercetin, phenethyl caffeate, baicalin, gallic acid, and catechin; The mass ratio of eucalyptus oil to lipid compounds is 1:2-1:1; Eucalyptus oil microemulsion, cell membrane solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were thoroughly mixed and chemically bonded to obtain a biomimetic membrane-encapsulated eucalyptus oil microemulsion.

2. The photosensitive gel with biomimetic membrane-encapsulated eucalyptus oil microemulsion according to claim 1, characterized in that, The mass ratio of the surfactant to the co-surfactant is 1:2 to 2:1; the mass ratio of the mixed oil phase to the mixed surfactant is 1:3 to 1:1.

3.

3. The method for preparing a photosensitive gel encapsulated in a biomimetic membrane with eucalyptus oil microemulsion according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Take eucalyptus oil, surfactant, co-surfactant, active ingredient, lipid compound, distearate phosphatidyl acetamide-polyethylene glycol-amino and stir thoroughly until homogeneous. Then add deionized water and emulsify to obtain eucalyptus oil microemulsion. (2) Take the prepared eucalyptus oil microemulsion, cell membrane solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide and stir them thoroughly. The biomimetic membrane-encapsulated eucalyptus oil microemulsion is obtained by chemical bonding. (3) Dopamine-modified methacrylic anhydride gelatin was dispersed into eucalyptus oil microemulsion wrapped in a biomimetic membrane to obtain a photosensitive gel of eucalyptus oil microemulsion wrapped in a biomimetic membrane.

4. The preparation method according to claim 3, characterized in that, In step (2), the protein concentration of the cell membrane is 0.002-0.060 mg / mL; the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:1-3:

1.

5. The preparation method according to claim 3, characterized in that, In step (3), the mass ratio of the dopamine-modified methacrylic anhydride gelatin to the eucalyptus oil microemulsion wrapped in the biomimetic membrane is 40:227-100:81.

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