Photosensitive gel with eucalyptus oil microemulsion wrapped by biomimetic membrane as well as preparation method and application of photosensitive gel

Abstract

The invention discloses photosensitive gel with eucalyptus oil microemulsion wrapped by a biomimetic membrane as well as a preparation method and application of the photosensitive gel. The preparation method comprises the following steps: taking eucalyptus oil and a lipid compound as a mixed oil phase, wrapping the outer layer of eucalyptus oil microemulsion with a biomimetic membrane, and then distributing the eucalyptus oil microemulsion wrapped with the biomimetic membrane in a photosensitive gel matrix of dopamine modified methacrylic anhydride gelatin to prepare the photosensitive gel with the eucalyptus oil microemulsion wrapped with the biomimetic membrane. The photosensitive gel has high adhesion and capability of actively targeting periodontitis pathogenic bacteria, and the treatment effect on periodontitis is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the field of drug-carrying hydrogels, and in particular relates to a photosensitive gel of eucalyptus oil microemulsion wrapped in a bionic membrane, and a preparation method and application thereof. Background Art

[0002] Periodontitis is an infectious disease characterized by bacterial infection, gum inflammation, loose and displaced teeth, and eventually tooth loss. Chronic periodontitis is clinically manifested by gingival swelling, loss of attachment, alveolar bone resorption, and periodontal pocket formation. If not intervened in time, it will lead to loose teeth or even tooth loss, seriously affecting the patient's face, pronunciation, and even mental health.

[0003] Studies have confirmed that periodontitis is caused by periodontitis pathogens, such as Porphyromonas gingivalis, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans. Periodontitis pathogens can destroy the normal function of the host immune system and aggravate the inflammatory symptoms of periodontal tissues. For example, Porphyromonas gingivalis can selectively downregulate the expression of macrophage CD14 and hinder macrophage-mediated phagocytosis. At the same time, bacterial virulence factors activate macrophages, leading to the massive secretion of inflammatory factors (TNF-α, IL-1β, IL-6), causing periodontal tissue damage. Although antibiotics can play a bacterial clearance role, long-term use of antibiotics can induce bacterial resistance, kill beneficial oral bacteria, and destroy the balance of normal microbial communities in the human body. Therefore, the development of a new preparation that can effectively target and eliminate periodontitis pathogens is of great significance for clinical periodontitis.

[0004] Eucalyptus oil, also known as eucalyptus oil and eucalyptus oil, is a volatile oil extracted by steam distillation from Eucalyptus globulus Labill., a plant of the Myrtaceae family, Cinnamomum camphora (L.) Presl, a plant of the Lauraceae family, or other plants of the same genus in the above two families. Eucalyptus oil is rich in a variety of active ingredients, such as eucalyptol, 1,8-cineole, etc., and has multiple effects such as antibacterial and anti-inflammatory. Studies have shown that eucalyptus oil can effectively inhibit a variety of pathogenic bacteria, such as Proteus, Streptococcus and anaerobic bacteria. In addition, eucalyptus oil can also reduce oral inflammation such as periodontitis and promote tissue healing. However, due to the biopharmaceutical properties of eucalyptus oil, such as poor water solubility, low bioavailability, no bacterial targeting, and low tissue adhesion, its practical application in the treatment of periodontitis is limited. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a photosensitive gel of eucalyptus oil microemulsion wrapped in a bionic membrane, which has the ability of high adhesion and active targeting of periodontitis pathogens, and significantly improves the therapeutic effect on periodontitis.

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

[0007] A photosensitive gel with an eucalyptus oil microemulsion encapsulated by a biomimetic membrane, comprising an eucalyptus oil microemulsion encapsulated by a biomimetic membrane and a gel matrix; the eucalyptus oil microemulsion encapsulated by the biomimetic membrane is composed of an 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 cell membrane, neutrophil cell membrane, NK cell membrane, red blood cell membrane, and monocyte cell membrane; the gel matrix is dopamine-modified gelatin methacrylate (GelMA-DA).

[0008] The eucalyptus oil microemulsion encapsulated by the biomimetic membrane of the present invention is composed of an eucalyptus oil microemulsion and a biomimetic membrane modified on the surface of the eucalyptus oil microemulsion. Among them, the eucalyptus oil microemulsion uses eucalyptus oil and lipid compounds as a mixed oil phase. Through a large number of compatibility experiments in the early stage, it was found that the compatibility of eucalyptus oil and lipid compounds can significantly improve the solubility of poorly soluble active ingredients, improve the overall drug-loading performance of the microemulsion, and reduce the dosage of inactive excipients. In addition, the biomimetic membrane completely retains the membrane structure and function during the extraction and separation process, and has the characteristics of actively targeting homologous cells or pathogenic bacteria. The present invention uses distearoyl phosphatidylethanolamine-polyethylene glycol-amine (DSPE-PEG-NH2) to provide -NH2 and -COOH on the surface of the macrophage cell membrane, and modifies the biomembrane protein on the outer layer of the microemulsion through a chemical bonding reaction to construct a biomimetic cell membrane-encapsulated eucalyptus oil microemulsion, enabling the eucalyptus oil microemulsion to have the ability to actively target homologous cells or periodontitis pathogenic bacteria, and improving the therapeutic efficacy of the drug.

[0009] The eucalyptus oil microemulsion of the present invention is prepared using eucalyptus oil and lipid compounds as a mixed oil phase, supplemented with substances such as surfactants, co-surfactants, and active ingredients.

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

[0011] 20 - 80 parts of eucalyptus oil, 10 - 160 parts of lipid compounds, 20 - 240 parts of surfactants, 20 - 440 parts of co-surfactants, 1 - 5 parts of active ingredients, and 1 - 5 parts of DSPE-PEG-NH2.

[0012] Preferably, the lipid compound is one or more of triacetin, tributyrin, tripropionin, trioctanoin, and tridecanoin.

[0013] Preferably, the surfactant is one or more of polyethylene glycol 15 hydroxystearate (HS-15), polyoxyl 40 hydrogenated castor oil (RH-40), polyoxyethylene castor oil, Tween 80, Tween 20, poloxamer 188, carbitol, and lecithin.

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

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

[0016] Preferably, the mass ratio of eucalyptus oil to lipid compounds is 1:2 - 1:1; the mass ratio of surfactant to 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; the mass ratio of the active ingredient to lipid compounds is 1:10 - 1:20.

[0017] The active hydroxyl groups in the photoinitiator can react with the unsaturated groups of gelatin methacrylate (GelMA), causing the methacrylamide and methacrylate side groups in the structure of gelatin methacrylate to polymerize through free radical addition reaction to form a gel. However, the bioadhesive property of gelatin methacrylate is weak, and a large amount of saliva flushing in the oral cavity makes it difficult for GelMA to stably stay. The catechol group of dopamine can form hydrogen bonds with polymers such as proteins and polysaccharides in oral tissues to form a firm adhesive connection. Therefore, in the present invention, gelatin methacrylate (GelMA) is first functionalized and modified with dopamine hydrochloride (DA) to endow GelMA with self-adhesive properties, and dopamine-modified gelatin methacrylate (GelMA-DA) is prepared. Then, GelMA-DA is used to load the eucalyptus oil microemulsion wrapped with a biomimetic membrane, and a photosensitive gel of the eucalyptus oil microemulsion wrapped with a biomimetic membrane is formed in situ in the oral periodontium by ultraviolet light irradiation, so that the eucalyptus oil microemulsion wrapped with a biomimetic membrane can stably stay in the oral periodontal pocket, improving the treatment effect of periodontitis.

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

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

[0020] (2) Then take the prepared eucalyptus oil microemulsion, cell membrane solution, 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), stir well, and obtain the eucalyptus oil microemulsion wrapped with a biomimetic membrane by chemical bonding method;

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

[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 methacrylated gelatin to eucalyptus oil microemulsion encapsulated by the biomimetic membrane is 40:227 - 100:81.

[0025] The present invention also provides the application of the photosensitive gel of the above-mentioned eucalyptus oil microemulsion encapsulated by the biomimetic membrane in the preparation of drugs for treating periodontitis.

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

[0027] 1. The present invention selects eucalyptus oil and lipid compounds as the mixed oil phase. On the one hand, it reduces the usage amount of traditional inactive oil phase and significantly improves the solubility of poorly soluble small molecule active ingredients; on the other hand, it fully exerts the inhibitory activity of eucalyptus oil against various pathogenic bacteria, which is beneficial to reducing the number of pathogenic bacteria in the periodontal lesion area and tissue damage.

[0028] 2. The present invention prepares the eucalyptus oil microemulsion encapsulated by the biomimetic membrane by chemically bonding the cell membrane to the surface of the eucalyptus oil microemulsion. Compared with other microemulsion preparations, the eucalyptus oil microemulsion encapsulated by the biomimetic membrane 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 present invention further disperses the eucalyptus oil microemulsion encapsulated by the biomimetic membrane in the dopamine-modified methacrylated gelatin matrix to construct the photosensitive gel of the eucalyptus oil microemulsion encapsulated by the biomimetic membrane. This gel is liquid at room temperature and automatically gels upon ultraviolet light irradiation. Compared with other gels, it is more convenient for oral injection and can fully fill the periodontal pocket. Utilizing the characteristics of dopamine groups in the gel matrix structure, it exhibits high adhesion performance, can achieve efficient adhesion and retention in the periodontal tissue, effectively prevent drug loss, and further enhance the performance of the eucalyptus oil microemulsion encapsulated by the biomimetic membrane in clearing periodontitis pathogenic bacteria and tissue inflammation. The evaluation results show that this preparation can kill periodontitis pathogenic bacteria and relieve periodontal tissue inflammation, providing a new preparation for the treatment of periodontitis. Description of the Drawings

[0030] Figure 1 It is the rheological characterization diagram of GelMA-DA of the present invention;

[0031] Figure 2 It is the particle size diagram of the eucalyptus oil microemulsion encapsulated by the biomimetic membrane;

[0032] Figure 3 The appearance and scanning electron microscope diagram of the photosensitive gel of the eucalyptus oil microemulsion encapsulated by the biomimetic membrane of the present invention;

[0033] Figure 4 The photosensitive gel adhesion performance of the eucalyptus oil microemulsion encapsulated by the biomimetic membrane of the present invention;

[0034] Figure 5 Pseudo-ternary phase diagrams of microemulsions prepared with different surfactants EL, HS-15, and Tween 80;

[0035] Figure 6 Pseudo-ternary phase diagrams of microemulsions prepared with different Km values (1:1, 1:2, 2:1);

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

[0037] Figure 8 Antibacterial performance evaluation diagram of the eucalyptus oil microemulsion encapsulated by the biomimetic membrane of the present invention;

[0038] Figure 9 Targeted homologous cell evaluation diagram of the eucalyptus oil microemulsion encapsulated by the biomimetic membrane of the present invention;

[0039] Figure 10 Antibacterial effect evaluation diagram of the photosensitive gel of the eucalyptus oil microemulsion encapsulated by the biomimetic membrane of the present invention on periodontitis rats;

[0040] Figure 11 Bone tissue treatment effect diagram of the photosensitive gel of the eucalyptus oil microemulsion encapsulated by the biomimetic membrane of the present invention on periodontitis rats. Specific embodiments

[0041] The present invention will be further described below through specific embodiments. The following examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the following examples.

[0042] Example 1: Synthesis and rheological characterization of dopamine-modified methacrylic anhydride gelatin (GelMA-DA)

[0043] The GelMA-DA described in the present invention uses GelMA and DA as raw materials, and the specific preparation method is as follows: Dissolve the GelMA prepolymer in PBS and stir at 50°C to obtain a homogeneous solution. Add triethylamine, succinic anhydride, and dimethyl sulfoxide to the solution and stir for 12 h, then dilute with PBS solution. Dialyze the solution with deionized water at room temperature for 1 week using a dialysis tube to remove impurities in the solution. Place the dialyzed solution in a centrifuge tube, freeze it in a -80°C refrigerator for 2 days, and then freeze-dry it for 4 days to obtain the GelMA-COOH prepolymer. Take the prepolymer and dissolve it in MES buffer, degas it with nitrogen, add EDC, NHS, and DA, and stir in a nitrogen environment for 12 h. Finally, place the solution in a centrifuge tube and refrigerate it at -80°C for two days, and then freeze-dry it for four days to obtain GelMA-DA.

[0044] The viscosity and injectability of the hydrogel are very important for practical applications, especially for the filling of irregular periodontal pockets. Rheological studies were carried out using a DHR-2 rheometer equipped with a 20 mm parallel plate fixture. As Figure 1 shows the rheological properties of GelMA and GelMA-DA. When the shear rate increased from 10 to 100 s -1 , due to the modification of DA, the viscosity of GelMA-DA was always higher than that of GelMA, indicating that GelMA-DA belongs to a typical pseudoplastic fluid with high viscosity and injectability.

[0045] Example 2:

[0046] 5 parts of quercetin, 40 parts of eucalyptus oil, 40 parts of tributyrin, 80 parts of HS-15, 20 parts of absolute ethanol, 20 parts of glycerol, 5 parts of DSPE-PEG-NH2;

[0047] Put the above components in a container, stir at 37 °C until completely homogeneous, slowly drop in ultrapure water, and make up to 5 mL to obtain eucalyptus oil microemulsion (QE-MEs);

[0048] Take 1 part of eucalyptus oil microemulsion in a vial, accurately weigh EDC, NHS, and macrophage cell membrane aqueous solution (protein concentration is 0.031 mg / mL) into the vial, make up to 10 mL, and incubate at 37 °C for 6 h to obtain eucalyptus oil microemulsion encapsulated by biomimetic membrane (M-QE-MEs); the molar ratio of EDC to NHS is 1:1;

[0049] Disperse GelMA-DA into the eucalyptus oil microemulsion encapsulated by the biomimetic membrane to obtain a photosensitive gel of eucalyptus oil microemulsion encapsulated by the biomimetic membrane (M-QE-MEs@Gel).

[0050] Performance characterization:

[0051] (1) Particle size characterization of eucalyptus oil microemulsion encapsulated by biomimetic membrane

[0052] Figure 2 shows the particle size diagram of the eucalyptus oil microemulsion encapsulated by the biomimetic membrane obtained in Example 2, and the particle size is 46.65 ± 0.45 nm.

[0053] (2) Appearance and morphology characterization of photosensitive gel of eucalyptus oil microemulsion encapsulated by biomimetic membrane

[0054] It can be observed from the macroscopic state that Figure 3The transformation of the bionic membrane - encapsulated eucalyptus oil micro - emulsion photosensitive gel solution into gel in Example 2 as shown in A. After freeze - drying the bionic membrane - encapsulated eucalyptus oil micro - emulsion photosensitive gel, the sample was pasted on the conductive tape on the sample holder. Then the sample holder was placed in a vacuum coating machine, the lid was closed, and the vent valve was opened to evacuate the gold evaporation chamber. After sputtering gold onto the sample surface, the sample was taken out and observed in a scanning electron microscope to photograph the gel morphology. The results are as Figure 2 shown in B in Figure 2. The scaffold of the bionic membrane - encapsulated eucalyptus oil micro - emulsion photosensitive gel in Example 2 has a porous network structure, which is beneficial for drug loading.

[0055] (3) Characterization of the adhesion performance of the bionic membrane - encapsulated eucalyptus oil micro - emulsion photosensitive gel

[0056] As shown in the figure, the bionic membrane - encapsulated eucalyptus oil micro - emulsion photosensitive gel in Example 2 can firmly adhere to the cork and rubber materials ( Figure 3 shown in A and B in Figure 2). 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 wet tissues was tested, including oral mucosa, stomach, large intestine, small intestine, and myocardial tissue ( Figure 4 shown in C in Figure 2). The results are as Figure 4 shown in D in Figure 2. The bionic membrane - encapsulated eucalyptus oil micro - emulsion photosensitive gel adheres immediately once it contacts the wet gum tissue and can withstand severe water rinsing without peeling, further demonstrating the excellent adhesion of the bionic membrane - encapsulated eucalyptus oil micro - emulsion photosensitive gel to the oral mucosa.

[0057] Example 3:

[0058] 5 parts of phenethyl caffeate, 20 parts of eucalyptus oil, 40 parts of tripropionin, 50 parts of poloxamer 188, 50 parts of polyethylene glycol 400, 50 parts of glycerol, 3 parts of DSPE - PEG - NH2;

[0059] Put the above components in a container, stir at 37 °C until completely homogeneous, and slowly add ultrapure water to make the volume up to 5 mL to obtain the eucalyptus oil micro - emulsion;

[0060] Take 1 part of the eucalyptus oil micro - emulsion in a vial, accurately weigh EDC, NHS, and macrophage cell membrane aqueous solution (protein concentration is 0.002 mg / mL) into the vial, make the volume up to 10 mL, and incubate at 37 °C for 6 h to obtain the bionic membrane - encapsulated eucalyptus oil micro - emulsion; where the molar ratio of EDC to NHS is 2:1;

[0061] Disperse GelMA - DA into the bionic membrane - encapsulated eucalyptus oil micro - emulsion to obtain the bionic membrane - encapsulated eucalyptus oil micro - emulsion photosensitive gel.

[0062] Example 4:

[0063] 5 parts of baicalin, 80 parts of eucalyptus oil, 40 parts of glyceryl trioctanoate, 60 parts of RH-40, 40 parts of absolute ethanol, 40 parts of 1,2-propanediol, 2 parts of DSPE-PEG-NH2;

[0064] Put the above components in a container, stir at 37 °C until completely homogeneous, slowly drop in ultrapure water, and make up the volume to 5 mL to obtain eucalyptus oil microemulsion;

[0065] Take 1 part of eucalyptus oil microemulsion in a vial, weigh EDC, NHS, and macrophage cell membrane aqueous solution (protein concentration 0.004 mg / mL) in the vial, make up the volume to 10 mL, and incubate at 37 °C for 6 h to obtain eucalyptus oil microemulsion wrapped with biomimetic membrane; the molar ratio of EDC to NHS is 3:1;

[0066] Disperse GelMA-DA into the eucalyptus oil microemulsion wrapped with biomimetic membrane to obtain a photosensitive gel of eucalyptus oil microemulsion wrapped with biomimetic membrane.

[0067] Example 5: Solubility of quercetin in mixed oil phase

[0068] Weigh an excess of quercetin and dissolve it in equal volumes of eucalyptus oil, glyceryl tributyrate, and mixed oil EG1 (eucalyptus oil:glyceryl tributyrate, 1:1, w:w), EG2 (eucalyptus oil:glyceryl tributyrate, 2:1, w:w), EG3 (eucalyptus oil:glyceryl tributyrate, 3:1, w:w), place in a stoppered glass test tube, ultrasonicate for 30 min, place in a 37 °C constant temperature oscillator and shake for 24 h to reach dissolution equilibrium, 13000 r / min -1 Centrifuge for 10 min, take 20 μL of the supernatant, dilute with methanol, and use HPLC method to detect its solubility. The results are shown in Table 1. The solubility of quercetin in EG1 is 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 usage amount of traditional inactive oil phase be reduced, the solubility of quercetin can be significantly improved, but also eucalyptus oil itself has an inhibitory effect on a variety of pathogenic bacteria and can play antibacterial, anti-inflammatory and other therapeutic properties together with the active ingredient.

[0069] Table 1 Solubility of quercetin in different oil phases (n = 3, )

[0070]

[0071] Example 6: Prescription screening of microemulsion surfactant and surfactant / cosurfactant (Km) value

[0072] Screening of surfactants for microemulsion: Fix eucalyptus oil - tributyrin as the mixed oil phase (1:1, w:w), anhydrous ethanol - glycerol as the co - surfactant, and optimize the surfactant for the microemulsion. Mix different types of surfactants (HS - 15, Tween 80, polyoxyethylene castor oil ether) with the co - surfactant at a mass ratio of 1:1 to form a mixed surfactant. Titrate with ultrapure water, record the critical changes of the system, draw a pseudo - ternary phase diagram with the help of Origin2021 software, determine the microemulsion region, and determine the optimal surfactant according to the area of the microemulsion region.

[0073] Fix polyethylene glycol 15 - hydroxystearate (HS - 15) as the surfactant and anhydrous ethanol - glycerol (1:1, w:w) as the co - surfactant. Select Km values of 1:1, 1:2, and 2:1 respectively, and mix evenly. Titrate with ultrapure water, record the critical changes of the system, draw a pseudo - ternary phase diagram with the help of Origin 2021 software, determine the microemulsion region, and determine the optimal Km value according to the area of the microemulsion region.

[0074] The results are shown in Figure 5 and Figure 6 As shown, when HS - 15 is used as the surfactant and the Km value is 1:1, the area of the microemulsion region prepared is the largest. Therefore, HS - 15 is preferably selected as the surfactant and the Km value is 1:1.

[0075] Example 7: Optimization of the prescription of eucalyptus oil microemulsion encapsulated by biomimetic membrane

[0076] Use the Plackett - Burman design method to screen the key influencing factors of the prescription of eucalyptus oil microemulsion encapsulated by biomimetic membrane. Conduct experiments according to the prescription ratios and process conditions in the table, and the experimental results are shown in Table 2 and Table 3. It is found that the mass ratio of eucalyptus oil - tributyrin (X1), the mass ratio of quercetin - excipients (X3), the concentration of macrophage cell membrane protein (X4), and the molar ratio of EDC - NHS (X7) have significant effects on the microemulsion.

[0077] On the basis of the Plackett - Burman experiment, select the Box - Behnken experiment to optimize the prescription of eucalyptus oil microemulsion encapsulated by biomimetic membrane. Take the mass ratio of eucalyptus oil - tributyrin (X1), the mass ratio of quercetin - polyethylene glycol 15 - hydroxystearate (HS15) (X2), the concentration of macrophage cell membrane protein (X3), and the molar ratio of EDC - NHS (X4) as independent variables, and take the encapsulation efficiency (Y3) of the core drug ingredient eucalyptus oil as the response value to optimize the prescription ratio of eucalyptus oil encapsulated by biomimetic membrane. The experimental scheme is generated by Design Expert software, and the experimental arrangements and results are shown in Table 4 and Table 5.

[0078] Using Design Expert software, the Box-Behnken test data was subjected to model fitting, and the multiple quadratic regression model for the encapsulation efficiency was obtained as 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 analyzed for variance and the significance of the model coefficients was examined using the F-test. The results are shown in Table 6 below.

[0079] Table 2 Factors and Levels of Plackett-Burman Design

[0080]

[0081]

[0082] Table 3 Plackett-Burman Design Experiment and Results of Key Influencing Factors of Eucalyptus Oil Encapsulated in Bionic Membrane (n = 3, )

[0083]

[0084] Table 4 Factors and Levels of BBD Design

[0085]

[0086] Table 5 BBD Experimental Scheme and Results (n = 3, )

[0087]

[0088]

[0089] Table 6 Variance Analysis of Encapsulation Efficiency

[0090]

[0091] As can be seen from the table, the variance analysis model of the encapsulation efficiency has significant differences; the lack-of-fit term P > 0.05 proves that the model has a good fitting degree; the multiple correlation coefficient R2 is 0.8701, proving that the model is similar to the actual results; AdeqPrecision > 5 and CV < 10%, verifying the credibility and precision of the model again. Through the binomial fitting equation, with the help of Design Expert software, the response surface contour map and 3D model map were drawn ( Figure 7), the key prescription and process parameters obtained were as follows: the mass ratio of eucalyptus oil to tributyrin was (1:1, w:w), the mass ratio of quercetin to HS15 was (1:14, w:w), the concentration of macrophage cell membrane protein was 0.031 mg / mL, and the molar ratio of EDC-NHS was (1:1, M:M).

[0092] Comparative Example 1: Preparation of quercetin gel

[0093] 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.

[0094] Comparative Example 2: Preparation of eucalyptus oil gel

[0095] GelMA-DA was dispersed into the eucalyptus oil solution (Eu) to obtain Eu@Gel.

[0096] Comparative Example 3: Preparation of quercetin and eucalyptus oil mixed gel

[0097] 5 parts of quercetin were dissolved in 40 parts of eucalyptus oil solution to obtain a quercetin and eucalyptus oil solution (Qu+Eu Mix); GelMA-DA was dispersed into the quercetin and eucalyptus oil solution to obtain Qu+Eu Mix@Gel.

[0098] Comparative Example 4: Preparation of ordinary eucalyptus oil microemulsion gel

[0099] 5 parts of quercetin, 40 parts of eucalyptus oil, 40 parts of tributyrin, 80 parts of HS-15, 20 parts of absolute ethanol, 20 parts of glycerol, and 5 parts of DSPE-PEG-NH2; the above components were placed in a container, stirred at 37°C until completely homogeneous, and slowly added dropwise with ultrapure water, and the volume was fixed to 5 mL to obtain eucalyptus oil microemulsion (QE-MEs); GelMA-DA was dispersed into the QE-MEs solution to obtain QE-MEs@Gel.

[0100] Experimental Example 1: Antibacterial performance evaluation

[0101] Porphyromonas gingivalis suspension (Pg) was taken, diluted 1:100 with BHI medium and inoculated into a 6-well plate. After adding 100 μL of bacterial solution to each well, 100 μL of quercetin solution (Qu), eucalyptus oil solution (Eu), physical mixture of quercetin and eucalyptus oil (Qu+EuMix), ordinary eucalyptus oil microemulsion (QE-MEs), and biomimetic membrane-coated eucalyptus oil microemulsion (M-QE-MEs) were added respectively. An equal amount of PBS solution was added as the Control group for comparison. After sealing, it was placed in an anaerobic workstation and incubated at 37°C for 24 h. The enzyme-linked immunosorbent assay (ELISA) reader was used to detect the optical density (OD) value of the bacterial solution at 600 nm, and the antibacterial rate was calculated. As Figure 8As shown in A, the results indicate that M-QE-MEs has the best antibacterial performance, which is superior to QE-MEs and the Eu group.

[0102] Put 200 μL of Pg bacterial suspension and cell slides together in a 24-well plate. Add 500 μL of Qu, Eu, Qu+EuMix, QE-MEs, and M-QE-MEs to each well, and use the addition of an equal amount of aqueous solution as the Control group for comparison. Incubate for 24 hours. Add 500 μL of 2.5% glutaraldehyde to each well, place it in the dark at 4°C for 24 h, and then rinse 4 times with PBS buffer (pH = 7), 5 min each time, to thoroughly remove the glutaraldehyde on the surface of the mycelium. Dehydrate successively with 30%, 50%, 70%, and 90% ethanol, 20 min for each dehydration, and finally place it in absolute ethanol for 15 min, and dehydrate with absolute ethanol 3 times. Let the absolute ethanol evaporate naturally in the air overnight, coat with a film, and observe the morphology with SEM. As Figure 8 As shown in B, the bacterial membrane after the action of M-QE-MEs ruptures, and its integrity is severely damaged, further proving that M-QE-MEs has good antibacterial performance.

[0103] Experimental Example 2: Evaluation of the performance of targeting homologous cells

[0104] Macrophage RAW 264.7 was seeded in a 6-well plate at a density of 7.8×10 6 / well, incubated at 37°C and 5% CO2 for 24 h, and then the culture medium was removed. Coumarin 6 fluorescein (C6) was used to label common eucalyptus oil microemulsion (C6-QE-MEs) and eucalyptus oil microemulsion wrapped with biomimetic membrane (C6-M-QE-MEs). C6 solution, C6-QE-MEs, and C6-M-QE-MEs were diluted to 2 μM with the culture medium and added to the 6-well plate. After incubation at 37°C for 2 hours, the cells were washed three times with PBS. Add paraformaldehyde to fix for 10 min (500 μL / well), stain with DAPI for 5 min (500 μL / well), then wash 3 times with PBS, and finally add 500 μL of PBS to infiltrate the cells. An inverted fluorescence microscope was used to observe the uptake of the preparations in cells of different groups. The results showed ( Figure 9 ), the green fluorescence intensity of the C6-M-QE-MEs group was significantly higher than that of C6-QE-MEs. The results indicate that C6-M-QE-MEs can target homologous cells. Because the surface of C6-M-QE-MEs is wrapped with macrophage membrane, the macrophage membrane modification makes C6-M-QE-MEs more easily recognized and taken up by macrophages.

[0105] Experimental Example 3: Therapeutic effect of the gels prepared in the examples and comparative examples on periodontitis rats

[0106] After 2 weeks of adaptive feeding of male healthy SPF rats, except for the normal group (Normal), the remaining groups were anesthetized intraperitoneally with 3% sodium pentobarbital (30 mg / kg). The rats were placed supine on the experimental table, and their heads and limbs were fixed. The upper and lower jaws were pulled to fully expose the left maxillary first molar. A ligature wire with a diameter of 0.2 mm was passed through the gap between the first and second molars of the rat, ligated around the neck of the first molar, and knotted on the palatal side. The excess length of the ligature wire was cut off. At the same time, the ligature knot was buried under the gingiva and the bacterial suspension was given. After 4 weeks, the periodontal condition of the rats was observed. The rats with successful modeling 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+EuMix@Gel), common eucalyptus oil microemulsion gel group (QE-MEs@Gel), and photosensitive gel group of eucalyptus oil microemulsion wrapped with the biomimetic membrane prepared in Example 2 (M-QE-MEs@Gel). Every 3 days, 20 μL of the corresponding preparation of each group was injected subperiosteally into the central alveolar ridge apex of the buccal and palatal sides of the maxillary first molar. The normal group (Normal) and the model group (Model) were injected with the same volume of normal saline.

[0107] Four weeks after administration, gingival crevicular fluid was aspirated with a moisture-absorbing paper tip, soaked in PBS for 24 h, 100 μL of the bacterial suspension was taken for coating, sealed, and placed in an anaerobic workbench for incubation at 37 °C for 3-4 d, and photographed ( Figure 10 A). 100 μL of the bacterial suspension after administration was taken, sealed, placed in an anaerobic workbench for incubation at 37 °C for 24 h, and the absorbance was measured on an enzyme-linked immunosorbent assay (ELISA) reader. As Figure 10 A and Figure 10 B show, the number of periodontitis 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 common microemulsion gel group, due to the improvement of the solubility of eucalyptus oil and quercetin by the microemulsion, the number of pathogenic bacteria colonies in the QE-MEs@Gel group decreased. After the eucalyptus oil microemulsion gel was wrapped with the biomimetic membrane, due to the targeting effect of the biomimetic membrane on bacteria, the number of colonies in the M-QE-MEs@Gel group was the lowest and the antibacterial effect was the best.

[0108] SD rats were euthanized, and the left maxilla was isolated. The bloodstains were washed with normal saline, and the tissues near the first molar were collected. After rinsing, they were stored at -80°C. Excess soft tissues were removed with fine scissors, and the specimens were fixed in 4% paraformaldehyde for 48 h. Standard procedures such as decalcification, dehydration, embedding, and sectioning were carried out. After taking out the fixed and preserved maxilla specimens, they were scanned using the SkyScan 1176 small animal Micro-CT scanning imaging system of Bruker Company in Germany. The scanning parameters were set as: 65 kV, current 385 μL, exposure time 340 ms, layer spacing 20 μm. After scanning, the reconstructed images were obtained through CTvox software. The results are as Figure 11 shown. Due to the action of pathogenic bacteria, the alveolar bone of the rats in the Model group was severely damaged, and obvious bone defects could be seen in the round frame. Qu@Gel had no obvious effect on repairing the alveolar bone. Alveolar bone repair was visible in the Eu@Gel, Qu+Eu Mix@Gel, and QE-MEs@Gel groups, but the alveolar bone gap in the M-QE-MEs@Gel group was the smallest, indicating that the biomimetic membrane-coated eucalyptus oil microemulsion photosensitizing gel had the strongest ability to promote bone tissue regeneration.

Claims

1. A photosensitive gel with eucalyptus oil microemulsion wrapped by a bionic membrane, characterized in that, Including eucalyptus oil microemulsion wrapped by biomimetic membrane and a gel matrix; the eucalyptus oil microemulsion wrapped by the biomimetic membrane is composed of the eucalyptus oil microemulsion and the biomimetic membrane modified on the surface of the eucalyptus oil microemulsion; the biomimetic membrane is selected from one or more of macrophage membrane, neutrophil membrane, NK cell membrane, red blood cell membrane, and monocyte membrane; the gel matrix is dopamine-modified methacrylated gelatin.

2. The photosensitive gel of eucalyptus oil microemulsion wrapped by the bionic membrane according to claim 1, characterized in that, 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 in parts by weight are as follows: 20 - 80 parts of eucalyptus oil, 10 - 160 parts of lipid compounds, 20 - 240 parts of surfactants, 20 - 440 parts of co-surfactants, 1 - 5 parts of active ingredients, and 1 - 5 parts of distearoyl phosphatidylethanolamine-polyethylene glycol-amine.

3. The photosensitive gel with eucalyptus oil microemulsion wrapped by the bionic membrane according to claim 2, characterized in that, The lipid compounds are selected from one or more of triacetin, tributyrin, tripropionin, trioctanoin, and tridecanoin.

4. The photosensitive gel with eucalyptus oil microemulsion wrapped by the bionic membrane according to claim 2, characterized in that, The surfactants are selected from one or more of polyethylene glycol 15 hydroxystearate, polyoxyethylene 40 hydrogenated castor oil, castor oil polyoxyethylene ether, Tween 80, Tween 20, poloxamer 188, carbitol, and lecithin; the co-surfactants are selected from one or more of polyethylene glycol 400, glycerol, 1,2-propanediol, n-butanol, isopropanol, and absolute ethanol.

5. The photosensitive gel with eucalyptus oil microemulsion wrapped by the bionic membrane according to claim 2, characterized in that, The active ingredients are selected from one or more of quercetin, caffeic acid phenethyl ester, baicalin, gallic acid, and catechin.

6. The photosensitive gel with eucalyptus oil microemulsion wrapped by the bionic membrane according to claim 2, characterized in that, The mass ratio of the eucalyptus oil to the lipid compounds is 1:2 - 1:1; the mass ratio of the surfactants to the co-surfactants is 1:2 - 2:1; the mass ratio of the mixed oil phase to the mixed surfactant is 1:3 - 1:1.3; the mass ratio of the active ingredients to the lipid compounds is 1:10 - 1:

20.

7. The preparation method of the photosensitive gel encapsulated with eucalyptus oil microemulsion by the bionic membrane according to any one of claims 1-6, characterized in that, Including the following steps: (1) Take eucalyptus oil, surfactants, co-surfactants, active ingredients, and distearoyl phosphatidylethanolamine-polyethylene glycol-amine, stir well, and then add deionized water to obtain eucalyptus oil microemulsion by emulsification method; (2) Then take the prepared eucalyptus oil microemulsion, cell membrane solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide, stir well, and obtain the eucalyptus oil microemulsion wrapped by the biomimetic membrane by chemical bonding method; (3) Disperse dopamine-modified methacrylated gelatin into the eucalyptus oil microemulsion wrapped by the biomimetic membrane to obtain a photosensitive gel of the eucalyptus oil microemulsion wrapped by the biomimetic membrane.

8. The preparation method according to claim 7, 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.

9. The preparation method according to claim 7, characterized in that, In step (3), the mass ratio of dopamine-modified methacrylated gelatin to the eucalyptus oil microemulsion wrapped by the biomimetic membrane is 40:227 - 100:

81.

10. Use of the photosensitive gel of the eucalyptus oil microemulsion wrapped by the biomimetic membrane according to any one of claims 1 - 6 in the preparation of a drug for treating periodontitis.

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