Ionic liquid transmucosal delivery system based on polyphenol modification

Abstract

The invention discloses an ionic liquid transmucosal delivery system based on polyphenol modification. A preparation method comprises the following steps: S1, preparing polyphenol modified ionic liquid by adopting aromatic folic acid, polyphenol A and choline bicarbonate; the polyphenol A is gallic acid or caffeic acid; s2, polyphenol B is dissolved in ultrapure water, a biomacromolecule medicine is added, freeze drying is performed after ultrasonic treatment, and polyphenol-biomacromolecule nanoparticles are obtained; the polyphenol A is epigallocatechin-3-gallate, and the polyphenol B is epigallocatechin-3-gallate or tannic acid; s3, adding the polyphenol-biomacromolecule nanoparticles into the ionic liquid, and performing ultrasonic treatment to obtain the ionic liquid transmucosal delivery system based on polyphenol modification. According to the drug delivery system, polyphenol participates in ionic liquid construction, more intermolecular interaction force sites are provided for the ionic liquid, the charge property of the ionic liquid is reduced, and meanwhile, polyphenol-biomacromolecule nanoparticles are constructed to prolong the in-vivo action time of the drug.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polyphenol-modified ionic liquid transmucosal delivery system. Background Technology

[0002] Biological macromolecules such as proteins, peptides, and nucleic acids have the advantages of high specificity and good efficacy, making them an important area of ​​drug development. Injection is currently the main route of administration for biological macromolecules, but patient compliance is poor due to pain, needle phobia, and infection risks. In contrast, non-invasive percutaneous / mucosal administration is an attractive route worth exploring, and its advantage lies in avoiding the first-pass effect of the liver, allowing for convenient patient self-administration. However, biological macromolecules struggle to penetrate biological barriers (such as skin or mucous membranes) to reach their targets. Compared to the skin system, the mucosal system has a thinner and more sensitive barrier structure. The stratum corneum of the skin has a dense lipid bilayer structure and high hydrophobicity, making it a major barrier that drug delivery needs to overcome, often requiring enhancers such as ionic liquids to improve permeability. However, the mucosal system lacks a strong barrier similar to the stratum corneum; its tissues are rich in water and directly exposed to the external environment, making drug delivery easier but also more susceptible to stimulation by chemical enhancers. The high charge density in ionic liquids may disrupt the charge balance of mucosal cell membranes, leading to inflammation or cell damage. In particular, strongly charged or hydrophobic ionic liquids may cause irritation and damage to the mucosal system, which places higher design requirements on the safe delivery of mucosal liquids.

[0003] Direct delivery of macromolecular drugs such as proteins and peptides using ionic liquids faces other challenges, such as the tendency of proteins to denature or aggregate, leading to loss of biological activity. This is mainly due to the interfacial incompatibility between ionic liquids and protein molecules, limiting their application in protein delivery. The interfacial incompatibility between ionic liquids and proteins primarily stems from the conflict between their strong polarity (strong charge) and the sensitivity of protein structure. First, the cations and anions of ionic liquids may interfere with charged groups (such as carboxyl and amino groups) on the protein surface, disrupting the salt bridges and hydrogen bond networks that maintain its higher-order structure, leading to conformational relaxation or denaturation. Second, hydrophobic ionic liquids may bind to nonpolar regions of proteins through hydrophobic interactions, exposing their internal hydrophobic core and causing aggregation or inactivation; while the high ionic strength of hydrophilic ionic liquids may shield functional sites of proteins, affecting their biological activity. Furthermore, the acidity or basicity of some ionic liquids may catalyze protein hydrolysis or side-chain modification, while redox-active anions may oxidize thiol groups or aromatic amino acid residues, further compromising protein stability. This incompatibility limits the application of ionic liquids in protein delivery. Therefore, it is necessary to introduce interface modifiers to reduce the strong charge characteristics of ionic liquids and instead form functional coatings by creating more hydrogen bonds, hydrophobic interactions, and other intermolecular forces. This enhances the interaction between ionic liquids and protein drugs, thereby improving their stability and delivery efficiency.

[0004] Furthermore, the direct delivery of biomolecules in vivo is prone to degradation, thus failing to achieve the desired therapeutic effect. Existing methods for protecting proteins include covalently linking polyethylene glycol molecules, glycosylation or fatty acid modification, constructing lipid nanoparticles, and condensate delivery, but these methods suffer from drawbacks such as high research and development costs and complex and difficult synthesis processes. Summary of the Invention

[0005] To address the problems of mucosal irritation, reduced biomolecular activity, and easy degradation of biomolecular drugs in current ionic liquid delivery processes, this invention provides a polyphenol-modified ionic liquid transmucosal delivery system.

[0006] The polyphenol-modified ionic liquid transmucosal delivery system of the present invention utilizes polyphenol A to participate in the construction of ionic liquid, providing more intermolecular interaction sites for ionic liquid, and at the same time constructs a polyphenol B-biomacromolecule complex system to protect biomacromolecule drugs, enhance the interaction between biomacromolecule drugs and ionic liquid, and further form a transmucosal delivery system in ionic liquid, which can effectively penetrate gingival mucosa.

[0007] The transmucosal delivery system of the present invention is prepared by the following method:

[0008] S1. Preparation of polyphenol-modified ionic liquids;

[0009] First, geranic acid is recrystallized to improve its purity. The method is as follows: Geranic acid is liquid at room temperature. It is cooled to -70℃ to solidify it, and then acetone is added. The acetone carries away impurities, thus purifying the geranic acid. This recrystallization process is repeated several times to improve its purity. Polyphenol A is dissolved in geranic acid, and then choline bicarbonate is added. The reaction is stirred at room temperature until CO2 production stops. The resulting solution is then rotary evaporated at 50-70℃ for 20-30 minutes, and further dried under vacuum to obtain a polyphenol-modified ionic liquid.

[0010] The polyphenol A is a plant polyphenol containing a carboxyl group in its molecular structure; preferably gallic acid (GA) or caffeic acid.

[0011] S2. Polyphenol B is dissolved in ultrapure water, then a biological macromolecular drug is added. The mixed solution is sonicated for several minutes and then freeze-dried to obtain polyphenol-biological macromolecular nanoparticles.

[0012] The polyphenol B is a plant polyphenol that contains at least two catechol or pyrogallol structures in its molecular structure and does not contain a carboxyl group, preferably epigallocatechin-3-gallate (EGCG) or tannic acid.

[0013] The biological macromolecular drug is any one of polypeptide, protein, DNA or RNA.

[0014] S3. Add polyphenol-biomacromolecule nanoparticles to the ionic liquid obtained in step S1, and sonicate for several minutes to obtain a polyphenol-modified ionic liquid transmucosal delivery system for the target material, named [PM]. PIL system.

[0015] Preferably, in step S1, the molar ratio of geranilic acid, polyphenol A, and choline bicarbonate is 2:2:1.

[0016] Preferably, in step S1, the vacuum drying temperature is 50-70℃ and the drying time is 40-50 hours.

[0017] Preferably, in step S2, when the biomolecule is a polypeptide or protein, the mass ratio of polyphenol B to the biomolecule drug is 1:1. When the biomolecule is DNA or RNA, the mass ratio of polyphenol B to the biomolecule drug is 100:8.

[0018] Preferably, in step S3, the mass ratio of polyphenol-biomacromolecule nanoparticles to ionic liquid is 1:4.

[0019] Compared with the prior art, the advantages of the present invention are:

[0020] (1) The polyphenol A used in this invention can be blended with choline and geraniol to form a composite ionic liquid system. The phenolic hydroxyl group of polyphenol A provides hydrogen bond and hydrophobic bond sites for the choline-geraniol system. While avoiding the destruction of biological macromolecular drugs by the ionic properties of the ionic liquid, it also enhances the interaction between the ionic liquid and the biological macromolecular drugs. At the same time, it reduces the charge of the ionic liquid and avoids the strong charge of the ionic liquid from causing damage to the mucosal system.

[0021] (2) Polyphenol B can generate non-covalent interactions with a variety of biomacromolecules and small molecule drugs, exhibiting strong intermolecular adhesion, and thus assembles with biomacromolecule drugs into nanoparticles. Through ionic liquid media, it can be effectively delivered to the periodontal tissues through the gums. Polyphenol B can not only prevent the degradation of biomacromolecule drugs, but also enhance drug adhesion, preventing them from being washed away by saliva and gingival crevicular fluid, thus prolonging the duration of drug action in vivo and significantly improving the efficacy of clinical medication.

[0022] (3) By introducing polyphenol-biomacromolecule nanoparticles into ionic liquids, this invention can efficiently and safely deliver various types of biomacromolecules into the deep periodontal tissues without trauma, achieving bactericidal and osteogenic effects, without the need for chemical modification of the biomacromolecules, thus maintaining their activity; a novel transmucosal system with high efficiency, high biocompatibility, low toxicity and side effects and universal drug loading is obtained.

[0023] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0024] Figure 1 For representative [PM] PIL Transmission electron microscope image of the system.

[0025] Figure 2 This is a snapshot of a representative molecular dynamics simulation.

[0026] Figure 3 For [EGCG-TPTD] CAGE-GA and [TPTD] CAGE-GA Release curve of TPTD in solution.

[0027] Figure 4 For [EGCG-TPTD] CAGE-GA The biocompatibility results are shown in the figure.

[0028] Figure 5 Confocal fluorescence micrograph of ionic liquid through the gingiva.

[0029] Figure 6This is a SEM image of bacteria.

[0030] Figure 7 Image showing the results of ALP staining. Detailed Implementation

[0031] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0032] Example 1

[0033] A polyphenol-modified ionic liquid transmucosal delivery system is prepared as follows:

[0034] (1) Synthesis of polyphenol-modified ionic liquid (CAGE-GA):

[0035] First, geranilic acid was recrystallized in acetone. The method involved cooling geranilic acid liquid (9.88 g, 0.059 mol) to -70°C to solidify it, then adding acetone to dissolve and remove impurities. This recrystallization process was repeated five times to ensure the purity of geranilic acid. Next, gallic acid (12.51 g, 0.059 mol) was added to the purified geranilic acid liquid until it dissolved, with a molar ratio of geranilic acid to gallic acid of 1:1. Choline bicarbonate (6.06 g, 0.029 mol) was then added, resulting in a molar ratio of geranilic acid, gallic acid, and choline bicarbonate of 2:2:1. The reaction was stirred at room temperature until CO2 production ceased. The solution was then rotary evaporated at 60°C for 20 minutes and further dried in a vacuum oven at 60°C for 48 hours to obtain CAGE-GA ionic liquid.

[0036] (2) Biomolecular selection of peptides, with teriparatide (TPTD) as the representative. 1 mg of EGCG was dissolved in 1 mL of Milli-Q ultrapure water, and then 1 mg of TPTD was dissolved in the EGCG solution. The mixed solution was sonicated for 2 minutes and then freeze-dried to obtain EGCG-TPTD nanoparticles.

[0037] Compared with existing methods for constructing peptide carriers, including desolvation, self-assembly, emulsification, biomineralization, and covalent modification, the method of this invention does not require complicated preparation processes and large amounts of crosslinking agents or organic reagents. It can construct carriers and achieve drug loading in a short time, and the prepared EGCG-TPTD nanoparticles have high biocompatibility.

[0038] (3) Take 2 mg of EGCG-TPTD nanoparticles and add them to 8 mg of CAGE-GA ionic liquid, and sonicate for 2 minutes to obtain [EGCG-TPTD]. CAGE-GAComplex, also known as transmucosal delivery system.

[0039] Example 2

[0040] A polyphenol-modified ionic liquid transmucosal delivery system is prepared as follows:

[0041] (1) Synthesis of polyphenol-modified ionic liquid (CAGE-GA) was carried out in the same manner as in Example 1.

[0042] (2) Biomolecular selection of proteins, with human serum albumin (HSA) as the representative. 1 mg of EGCG was dissolved in 1 mL of Milli-Q ultrapure water, and then 1 mg of HSA was dissolved in the EGCG solution. The mixed solution was sonicated for 2 minutes and then freeze-dried to obtain EGCG-HSA nanoparticles.

[0043] (3) Take 2 mg of EGCG-HSA nanoparticles and add them to 8 mg of CAGE-GA ionic liquid, and sonicate for 2 minutes to obtain [EGCG-HSA]. CAGE-GA Complex, also known as transmucosal delivery system.

[0044] Example 3

[0045] A polyphenol-modified ionic liquid transmucosal delivery system is prepared as follows:

[0046] (1) Synthesis of polyphenol-modified ionic liquid (CAGE-GA) was carried out in the same manner as in Example 1.

[0047] (2) Selection of DNA by biomacromolecules, represented by pUC19 plasmid DNA. 2 mg of EGCG was dissolved in 1 mL of Milli-Q ultrapure water, and then 80 μg of pUC19 plasmid DNA was dissolved in the EGCG solution. The mixed solution was sonicated for 2 minutes and then freeze-dried to obtain EGCG-plasmid DNA nanoparticles.

[0048] (3) Add 2 mg of EGCG-plasmid DNA nanoparticles to 8 mg of CAGE-GA ionic liquid and sonicate for 2 minutes to obtain [EGCG-pUC-19]. CAGE-GA Complex, also known as transmucosal delivery system.

[0049] Example 4

[0050] A polyphenol-modified ionic liquid transmucosal delivery system is prepared as follows:

[0051] (1) Synthesis of polyphenol-modified ionic liquid (CAGE-GA) was carried out in the same manner as in Example 1.

[0052] (2) Selective RNA from biomacromolecules, represented by siGAPDH. 2 mg of EGCG was dissolved in 1 mL of Milli-Q ultrapure water, and 80 μg of siGAPDH was dissolved in the EGCG solution. The mixed solution was sonicated for 2 minutes and then freeze-dried to obtain EGCG-siGAPDH nanoparticles.

[0053] (3) Add 2 mg of EGCG-siGAPDH nanoparticles to 8 mg of CAGE-GA ionic liquid and sonicate for 2 minutes to obtain [EGCG-siGAPDH]. CAGE-GA Complex, also known as transmucosal delivery system.

[0054] The performance of the transmucosal delivery systems prepared in Examples 1-4 was tested, as follows:

[0055] (1) TEM detection;

[0056] Take [EGCG-TPTD] respectively CAGE-GA [EGCG-HSA] CAGE-GA [EGCG-pUC-19] CAGE-GA [EGCG-siGAPDH] CAGE-GA The composite was dropped onto a copper grid supported by an ultrathin carbon film and observed using TEM at an accelerating voltage of 100 kV. The test results are shown in [Figure number missing]. Figure 1 As can be seen, under non-covalent modification by EGCG, peptides, proteins, DNA, and RNA can all successfully form [PM]. PIL The system. The figure shows the morphology of the nanoparticles in CAGE-GA, which still exhibit a uniform spherical structure with an average particle size of about 200 nm and no obvious aggregation, indicating that the polyphenol-modified ionic liquid CAGE-GA does not change the morphological characteristics of the nanoparticles.

[0057] (2) Molecular dynamics simulation evaluation [EGCG-TPTD] CAGE-GA Stability;

[0058] Using TPTD as a representative of biopharmaceutical macromolecules, a model of [TPTD] was constructed through molecular dynamics (MD) simulation experiments. CAGE-GA and [EGCG-TPTD] CAGE-GA Two complex systems were analyzed for simulation convergence parameters, conformational changes, intermolecular interaction forces, and intermolecular interaction energies. Figure 2 It is [TPTD] CAGE-GA and [EGCG-TPTD] CAGE-GA A snapshot of molecular dynamics simulations. It can be observed that in [TPTD] without the addition of EGCG molecules... CAGE-GIn the system, during the interaction between TPTD peptide molecules and the ionic liquid, some peptide molecules did not completely enter the ionic liquid, with one end of each of the two peptides floating outside the ionic liquid. In contrast, when EGCG molecules were added, [EGCG-TPTD]... CAGE-GA In the system, the TPTD peptide molecule was almost completely encapsulated within the ionic liquid. This result indicates that the addition of EGCG molecules strengthens the interaction between the ionic liquid, the peptide molecule, and EGCG, leading to improved overall system stability. The number of hydrogen bonds formed between molecules in both complex systems was also analyzed, and the results are shown in Table 1. [TPTD] CAGE-GA In the system, the average number of hydrogen bonds between TPTD molecules and choline bicarbonate molecules was 14.99 throughout the simulation, while the average number of hydrogen bonds between TPTD molecules and geranic acid molecules was 172.23. (In [EGCG-TPTD]) CAGE-GA In this system, TPTD and EGCG molecules were considered as a whole, and the changes in the number of hydrogen bonds between them and the two types of ionic liquid molecules were calculated. The results showed that after the addition of EGCG molecules, the number of hydrogen bonds between TPTD and EGCG molecules and choline bicarbonate molecules increased to 36.22 groups, and the number of hydrogen bonds with geranic acid molecules increased to 488.86 groups. Comparatively, the total number of hydrogen bonds between the ionic liquid and peptide molecules increased by approximately 2.8 times after the addition of EGCG molecules, indicating that the introduction of EGCG significantly enhanced the hydrogen bonding interactions in the complex system.

[0059] Table 1. [TPTD] CAGE-GA and [EGCG-TPTD] CAGE-GA Analysis results of the average number of intermolecular hydrogen bonds in the system

[0060]

[0061] (3) Determination of [TPTD] by high performance liquid chromatography (HPLC) CAGE-GA and [EGCG-TPTD] CAGE-GA The release rate of TPTD under simulated in vivo enzymatic hydrolysis conditions was measured. Proteinase K was introduced into the experiment, and high-performance liquid chromatography (HPLC) was used to evaluate [EGCG-TPTD]. CAGE-GA and [TPTD] CAGE-GA The release behavior of TPTD under the same concentration of proteinase K. Among them, [EGCG-TPTD]... CAGE-GA and [TPTD] CAGE-GAThe concentration of TPTD used in the experiments was 1 mg / mL, and the concentration of proteinase K was 100 ng / mL. The experiments were conducted in phosphate buffer solution at pH 7.4, and the release of TPTD was measured at 0, 5, 10, 20, 30, 60, 120, and 180 minutes. The mobile phase consisted of 0.05 mol / L potassium chloride solution (adjusted to pH 4.5 with phosphoric acid) and acetonitrile in a 75:25 volume ratio, with a flow rate of 1.0 mL / min. The detection wavelength was set to 210 nm, and the column temperature was maintained at room temperature. The release of TPTD at different time points was quantitatively determined by HPLC analysis to evaluate its release kinetics. The test results are shown below. Figure 3 Experiments have shown that [EGCG-TPTD] CAGE-GA Approximately 80% of the TPTD remained after 3 hours of release. In contrast, TPTD without EGCG protection was rapidly degraded by proteases. This demonstrates that EGCG can protect TPTD from protease degradation.

[0062] (4) Detection of [EGCG-TPTD] CAGE-GA Biocompatibility;

[0063] MC3T3 cells were loaded at 5×10 3 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured overnight with 100 μL of medium. Then, [EGCG-TPTD] was administered at concentrations of 500 μg / mL, 250 μg / mL, 125 μg / mL, and 62.5 μg / mL, respectively. CAGE-GA Co-cultured with MC3T3 cells for 24 hours. Cytotoxicity was detected using the CCK-8 assay; results are shown below. Figure 4 The results showed that at 500 μg / mL [EGCG-TPTD]... CAGE-GA At the specified concentration, cells still maintained a survival rate of approximately 90%, demonstrating that [EGCG-TPTD] CAGE-GA It has good biocompatibility and will not affect biosafety.

[0064] (5) Detection of [EGCG-TPTD] CAGE-GA Its ability to penetrate mucous membranes;

[0065] This experiment compared the permeability of Mulli-Q ultrapure water (Aqua) and ionic liquids to the gingival mucosa. Rhodamine B was dissolved in ultrapure water and ionic liquids, respectively. First, fluorescently labeled TPTD (FITC-TPTD) was synthesized using a solid-phase synthesis method. The synthesis method of FITC-TPTD was as follows: Step 1, Resin selection based on the target peptide chain sequence and terminal properties: The C-terminus of this sequence is a carboxylic acid terminus, so 1g of king resin was used. Step 2, Resin pretreatment and Fmoc group deprotection: The resin was placed in a reaction column, soaked in DMF for 10 minutes, and dried; 20% piperidine / DMF solution was added, shaken at room temperature for 5 minutes, and dried; another 20% piperidine / DMF solution was added, shaken at room temperature for 15 minutes, and dried; the resin was washed with DMF 5–9 times and dried for later use. Step 3: Add the Fmoc (9-fluorenylmethoxycarbonyl) protected amino acid, condensing agent (HOBt), base reagent (Collidine), and DMF to the reaction column. Shake at room temperature for 20-60 minutes, then dry under vacuum. Wash 3 times with DMF, 3 times with anhydrous methanol, and 3 times with dichloromethane, then dry under vacuum for later use. Use Kaiser's reagent to check if the coupling reaction is complete. If incomplete, repeat the coupling steps. Step 4: Repeat the Fmoc deprotection and washing steps: After each coupling, deprotect with 20% piperidine / DMF solution (5 minutes first, then 15 minutes), then wash the resin 3-6 times with DMF, 3 times with anhydrous methanol, and 3 times with dichloromethane, then dry under vacuum. Step 5: Repeat steps 3 and 4 sequentially according to the sequence to synthesize the polypeptide sequence SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ ID No: 1), coupling each amino acid one by one. Step 6: FITC Labeling: After sequence synthesis, remove the Fmoc protecting group from the N-terminus and label with FITC. Step 7: Add FITC and DIPEA to the resin in DMF solution and shake overnight at room temperature. After the reaction, wash the resin 5 times with DMF, 3 times with anhydrous methanol, and 3 times with dichloromethane, then dry under vacuum. Step 8: Resin Washing and Drying: After completing all sequence synthesis and FITC labeling, wash the resin 3-9 times with diethyl ether, thoroughly dry under vacuum, and set aside. Step 9: Peptide Cleavage: Prepare the cleavage reagent: use TFA:H2O:p-cresol:ethylenedithiol = 92.5:2.5:2.5:2.5 (volume ratio). Place the resin in the cleavage solution and shake at room temperature for 1-4 hours. Filter and wash the resin with a small amount of TFA. Add cold anhydrous diethyl ether to the filtrate to precipitate the crude product. Wash the crude product several times with diethyl ether and thoroughly dry under vacuum to obtain the crude product. Step 10, Crude Product Identification: The purity and accuracy of the peptide were identified by mass spectrometry and chromatography using an LCMS-8040 and an Agilent 1260 high-performance liquid chromatography system, respectively, and the target pure product FITC-TPTD was finally obtained.

[0066] Then, EGCG-TPTD nanoparticles were prepared using FITC-TPTD as the raw material according to step (2) of this invention. The EGCG-TPTD nanoparticles were then added to ultrapure water and polyphenol-modified ionic liquid, respectively, to obtain [EGCG-TPTD]. Aqua and [EGCG-TPTD] CAGE-GA 10 μL of [EGCG-TPTD] was added separately. CAGE-GA and [EGCG-TPTD] Aqua The solution was applied to the area around the maxillary second molar of Sprague-Dawley (SD) rats and left for 1 minute. After sacrifice, the maxillary bone specimens were decalcified in 10% EDTA solution at 4°C for four weeks, and then embedded in FSC 22 frozen sections. 10 μm sections were cut along the long axis of the teeth, and images were obtained using a confocal laser scanning microscope. Fluorescence intensity and colocalization analysis were performed using ImageJ software. The results are shown in [Figure number missing]. Figure 5 Experiments have shown that water cannot carry [EGCG-TPTD] nanoparticles into the deep gingival tissue, while polyphenol-modified ionic liquids can carry large molecular nanoparticles into the deep tissue.

[0067] (6) Detection of [EGCG-TPTD] CAGE-GA bactericidal activity;

[0068] Bactericidal activity was assessed using *Escherichia coli*, *Staphylococcus aureus*, and *Porphyromonas gingivalis*. *E. coli* colonies were transferred from solid medium to 10 mL of Luria-Bertani (LB) broth. *Staphylococcus aureus* colonies were transferred from solid medium to 10 mL of trypsin-enriched soybean broth. The *E. coli* and *Staphylococcus aureus* cultures were then incubated at 37°C for 12 h with shaking at 200 rpm to ensure optimal growth and aeration. Separately, *Porphyromonas gingivalis* colonies were transferred from solid medium to 10 mL of brain-heart perfusion (BHI) broth supplemented with heme chloride (5 μg / mL) and vitamin K1 (0.5 μg / mL). This was then cultured anaerobically at 37°C for 12 h. After 12 h, the bacteria were harvested by centrifugation at 8000 rpm for 3 minutes. Subsequently, it was washed three times with PBS, resuspended in PBS, and diluted to an optical density of approximately 10. 7 CFU / mL. Add bacterial solution (10...) 7 CFU / mL) and 100μg / mL[EGCG-TPTD] CAGE-GAAfter co-culturing for 30 min, the bacteria were centrifuged at 10,000 rpm for 8 min, mixed thoroughly with 2.5% glutaraldehyde, and fixed for 6 h. They were then dehydrated using a gradient of 20%, 40%, 60%, 80%, 95%, and 100% ethanol, each step lasting 10 min. The 2.5% glutaraldehyde was prepared by adding 50 ml of 0.2 M phosphate buffer to 10 ml of 10 ml of 25% glutaraldehyde stock solution, and finally bringing the volume to 100 ml with distilled water, resulting in a final concentration of 2.5%. The bacterial suspension was then dropped onto a silicon wafer for SEM imaging analysis. A blank control group (Control) was set up using the same method, without the addition of [EGCG-TPTD]. CAGE-GA , bacterial solution (10 7 After being cultured alone for 30 min with CFU / mL, the bacteria were centrifuged at 10,000 rpm for 8 min, mixed thoroughly with 2.5% glutaraldehyde, and fixed for 6 h. The bacteria were then dehydrated using a gradient of 20%, 40%, 60%, 80%, 95%, and 100% ethanol, each step lasting 10 min. The bacterial suspension was then dropped onto a silicon wafer for SEM imaging analysis. The experimental results are shown below. Figure 6 The experiment found that, compared to the control group, [EGCG-TPTD] CAGE-GA It has highly efficient bactericidal activity.

[0069] (7) Detection of [EGCG-TPTD] CAGE-GA Osteogenic activity;

[0070] The control group, osteogenic induction group (OM), and [TPTD] were set up separately. CAGE-GA Group and [EGCG-TPTD] CAGE-GA Groups. Among them, the osteogenic induction group (OM) and [TPTD] CAGE-GA Group and [EGCG-TPTD] CAGE-GA All groups were supplemented with osteogenic induction medium, including 50 μg / mL L-ascorbic acid 2-phosphate and 5 mM β-glycerophosphate. All three groups were also supplemented with 0.1 μg / mL proteinase K. After 12 days of MC3T3 cell culture, ALP staining was performed using the BCIP / NBT ALP staining kit, following the kit's instructions. Experimental results are shown below. Figure 7 As can be seen, compared to the other three groups, [EGCG-TPTD] CAGE-GA The group exhibited the highest osteogenic activity.

[0071] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A polyphenol-modified ionic liquid transmucosal delivery system, characterized in that, The preparation method is as follows: S1. Preparation of polyphenol-modified ionic liquids; Polyphenol A was dissolved in geranium acid, and then choline bicarbonate was added. The mixture was stirred at room temperature until CO2 production stopped. The resulting solution was then rotary evaporated at 50-70°C for 20-30 minutes and further dried under vacuum to obtain a polyphenol-modified ionic liquid. The polyphenol A was a plant polyphenol containing a carboxyl group in its molecular structure. S2. Polyphenol B is dissolved in ultrapure water, then a biomolecular drug is added. The mixed solution is sonicated for several minutes and then freeze-dried to obtain polyphenol-biomolecular nanoparticles. The polyphenol B is a plant polyphenol whose molecular structure contains at least two catechol or pyrogallol structures and does not contain a carboxyl group. The biomolecular drug is any one of polypeptide, protein, DNA or RNA. S3. Add polyphenol-biomacromolecule nanoparticles to the ionic liquid obtained in step S1, and sonicate for several minutes to obtain a polyphenol-modified ionic liquid transmucosal delivery system for the target substance.

2. The polyphenol-modified ionic liquid transmucosa delivery system as described in claim 1, characterized in that, In step S1, the molar ratio of geranilic acid, polyphenol A, and choline bicarbonate is 2:2:

1.

3. The polyphenol-modified ionic liquid transmucosa delivery system as described in claim 1, characterized in that, Geranilic acid needs to be recrystallized to improve its purity before use. The method is as follows: the geranilic acid is cooled to -70℃ to solidify it, and then acetone is added. The acetone dissolves and removes impurities, thereby purifying the geranilic acid. The recrystallization process is repeated multiple times.

4. The polyphenol-modified ionic liquid transmucosa delivery system as described in claim 1, characterized in that, The polyphenol A is gallic acid or caffeic acid.

5. The polyphenol-modified ionic liquid transmucosa delivery system as described in claim 1, characterized in that, In step S1, the vacuum drying temperature is 50-70℃ and the drying time is 40-50 hours.

6. The polyphenol-modified ionic liquid transmucosal delivery system as described in claim 1, characterized in that, The polyphenol B is epigallocatechin-3-gallate or tannic acid.

7. The polyphenol-modified ionic liquid transmucosal delivery system as described in claim 6, characterized in that, When the biomolecule is a polypeptide or protein, the mass ratio of polyphenol B to the biomolecule drug is 1:

1.

8. The polyphenol-modified ionic liquid transmucosa delivery system as described in claim 6, characterized in that, When the biomolecule is DNA or RNA, the mass ratio of polyphenol B to the biomolecule drug is 100:

8.

9. The polyphenol-modified ionic liquid transmucosa delivery system as described in claim 1, characterized in that, In step S3, the mass ratio of polyphenol-biomacromolecule nanoparticles to ionic liquid is 1:4.

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