Mussel mucin-hyaluronic acid nanogel as well as preparation method, application and pharmaceutical composition thereof

Abstract

The invention relates to mussel mucin-hyaluronic acid nanogel and a preparation method and application thereof and a pharmaceutical composition, and belongs to the field of biomedical materials, the structural formula of the mussel mucin-hyaluronic acid nanogel is # imgabs0 #, Mussel mucin is a mussel mucin molecule, and n is the number of repetitive units. The mussel mucin-hyaluronic acid nanogel can be swelled in a water-based medium to form a three-dimensional space net-shaped structure, pharmaceutical active molecules are better entrapped, lysine and dopa groups in the structure can be tightly combined with the surface or mucous membrane of an organism through electrostatic interaction, hydrogen-bond interaction and oxidative crosslinking, so that the bioavailability of the mussel mucin-hyaluronic acid nanogel is improved, and the bioavailability of the mussel mucin-hyaluronic acid nanogel is improved. The carrier has good biological adhesion, can prolong the residence time of the carrier on an organism or mucous membrane surface, and promotes drug absorption. The mussel mucin-hyaluronic acid nanogel can also be used as a drug storage cavern, so that the slow release effect of drugs is improved, the action time of the drugs is prolonged, and the administration frequency is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and specifically relates to a mussel mucin-hyaluronic acid nanogel and a preparation method, application and pharmaceutical composition thereof. Background Art

[0002] Mussel Adhesive Protein (MAP) is a macromolecular protein secreted and purified from the foot gland of marine mussels. It has strong adhesion and anti-oxidation, anti-inflammatory, and repair properties. It has good affinity with the human body and is widely used in the biomedical field. The structure of mussel mucin contains a large amount of lysine, which has a high load of positive charge under normal physiological conditions and can be quickly adsorbed to various negatively charged cell surfaces (epidermal cells, fibroblasts, vascular endothelial cells, nerve cells, etc.). The high content of DOPA in the structure further enhances the adhesion of mussel mucin through hydrogen bonding and oxidative cross-linking. It is suitable for surfaces such as human skin and mucous membranes, and has broad application prospects in the field of biomedical materials.

[0003] Nanogel is a hydrogel particle with a three-dimensional polymer network structure and a nanometer particle size. It has good biocompatibility, large specific surface area, good stability and high drug loading capacity. The three-dimensional network structure of nanogel can encapsulate drugs or biological molecules and protect them from damage by the external environment. Its nanometer size can promote the penetration of drugs through biological membranes, prolong the circulation time in the body, and can passively target penetration and be retained in tumor tissues. It is a nano drug carrier with excellent performance.

[0004] Hyaluronic acid is a glycosaminoglycan widely found in the body. It is widely used as a nano drug delivery carrier due to its good hydrophilicity, permeability, biocompatibility and biodegradability. There are a large number of negatively charged carboxyl groups in the hyaluronic acid structure, which can reduce the uptake of macrophages. Therefore, the drug delivery system based on hyaluronic acid can significantly prolong the blood circulation time of drugs; hyaluronic acid can specifically bind to the CD44 receptor overexpressed on the surface of various tumor cells, enter the cells through endocytosis, and increase the drug concentration at the target cell site to achieve targeted treatment; hyaluronic acid in skin topical preparations can not only delay the release rate of drugs, but also promote the transdermal absorption of drugs. However, the bioadhesion ability of hyaluronic acid is weak and has certain limitations. Therefore, it needs to be modified with materials with bioadhesion to further enhance its bioadhesion and applicability.

[0005] At present, there is no report on the research on preparing a mussel mucin-hyaluronic acid nanogel using mussel mucin and hyaluronic acid and applying it to the preparation of drug carriers. Summary of the invention

[0006] One of the purposes of the present invention is to provide a mussel mucin-hyaluronic acid nanogel.

[0007] The second object of the present invention is to provide a method for preparing mussel mucin-hyaluronic acid nanogel.

[0008] The third object of the present invention is to provide an application of mussel mucin-hyaluronic acid nanogel in the preparation of drug carriers.

[0009] A fourth object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned mussel mucin-hyaluronic acid nanogel.

[0010] A fifth object of the present invention is to provide a method for preparing a pharmaceutical composition.

[0011] The technical solution adopted by the present invention to solve the technical problem is as follows:

[0012] The present invention provides a mussel mucin-hyaluronic acid nanogel, and the structural formula of the mussel mucin-hyaluronic acid nanogel is:

[0013]

[0014] Wherein, Mussel adhesive protein represents mussel adhesive protein molecule, n represents the number of repeating units, and n is a natural number, 10≤n≤130.

[0015] The present invention provides a method for preparing a mussel mucin-hyaluronic acid nanogel, comprising the following steps:

[0016] Step S1: dissolving hyaluronic acid in a reaction solvent, and adding EDC and NHS to activate the carboxyl group;

[0017] Step S2: adding mussel mucin to the activated carboxyl groups in the hyaluronic acid structure to undergo amide reaction;

[0018] Step S3: adjusting the pH value with a pH regulator, dialyzing and drying to obtain the mussel mucin-hyaluronic acid nanogel.

[0019] Preferably, the weight ratio of the mussel mucin to hyaluronic acid is 1:0.5-5.

[0020] Preferably, in step S1, the molecular weight of the hyaluronic acid is 0.5×10 4 ~5×10 4; The weight ratio of the hyaluronic acid to the reaction solvent is 1:5-60; the weight ratio of the hyaluronic acid, EDC and NHS is 5-50:1-5:1; the reaction solvent is selected from at least one of MES buffer, acetate buffer and phosphate buffer; the reaction temperature of the activated carboxyl group is 25-45°C, the reaction time is 30min-4h, and the reaction conditions are magnetic stirring and light-proof reaction.

[0021] Preferably, in step S2, the reaction temperature of the amide reaction is 25-45° C., the reaction time is 4-48 h, and the reaction conditions are magnetic stirring and light-proof reaction.

[0022] Preferably, in step S3, the pH regulator is selected from one of sodium hydroxide, potassium hydroxide, sodium bicarbonate, and ammonia water; the pH value is adjusted to 7-9; the dialysis and drying method is: placing the product after the pH value is adjusted in a dialysis bag with a molecular weight cutoff of 3500-14000Da, dialyzing in deionized water at 4-30°C for 1-4 days, and then freeze-drying to obtain the mussel mucin-hyaluronic acid nanogel.

[0023] The invention provides an application of a mussel mucin-hyaluronic acid nanogel in the preparation of a drug carrier.

[0024] Preferably, the drug carrier is a drug carrier for transdermal administration, mucosal administration, oral administration, ocular administration, rectal administration or vaginal administration.

[0025] The present invention provides a pharmaceutical composition, which comprises a drug and the mussel mucin-hyaluronic acid nanogel according to claim 1; the drug is selected from at least one of anti-inflammatory drugs, antibacterial drugs, anti-tumor drugs, analgesics, chemotherapeutic drugs, central nervous system drugs, digestive system drugs, circulatory system drugs, immune system drugs, and hormone drugs; the anti-inflammatory drug is selected from at least one of hormones, non-steroidal drugs, and steroid drugs.

[0026] The present invention provides a method for preparing a pharmaceutical composition, comprising the following steps:

[0027] The drug and mussel mucin-hyaluronic acid nanogel are dissolved in an organic solvent at a weight ratio of 1:1-40, and stirred for 1-4 hours at room temperature in the dark; 10% PBS is slowly added dropwise to the above solution, stirred for 4-12 hours at room temperature in the dark, and then transferred to a dialysis bag with a molecular weight cutoff of 3500-7000Da, dialyzed in deionized water for 12-24 hours, and the dialyzed product is centrifuged and the supernatant is collected to obtain a pharmaceutical composition.

[0028] The beneficial effects of the present invention are:

[0029] The mussel mucin-hyaluronic acid nanogel prepared by the present invention is a functional derivative carrier prepared by introducing mussel mucin into the structure of hyaluronic acid. The mussel mucin-hyaluronic acid nanogel can swell in an aqueous medium to form a three-dimensional spatial network structure, and is used as a delivery carrier for pharmaceutically active substances. It has good encapsulation of pharmaceutically active molecules and encapsulates hydrophilic and hydrophobic drugs. The lysine and dopa groups in the mussel mucin-hyaluronic acid nanogel structure can be tightly combined with the surface of the organism or mucosa through electrostatic action, hydrogen bonding and oxidative cross-linking, have good bioadhesion, can prolong the retention time of the carrier on the surface of the organism or mucosa, and promote the absorption of the drug. The mussel mucin-hyaluronic acid nanogel can also be used as a drug reservoir to increase the sustained release effect of the drug, prolong the action time of the drug, and reduce the number of administrations.

[0030] The mussel mucin-hyaluronic acid nanogel prepared by the present invention can be used as a drug carrier for transdermal administration, mucosal administration, oral administration, eye administration, rectal or vaginal cavity administration, etc., can increase local drug concentration, and has local targeting. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 These are the nuclear magnetic resonance hydrogen spectra of mussel mucin, hyaluronic acid and mussel mucin-hyaluronic acid.

[0032] Figure 2 This is a scanning electron micrograph of curcumin-loaded mussel mucin-hyaluronic acid nanogel.

[0033] Figure 3 Particle size distribution of curcumin-loaded mussel mucin-hyaluronic acid nanogel.

[0034] Figure 4 This is the potential distribution diagram of mussel mucin-hyaluronic acid nanogel loaded with curcumin.

[0035] Figure 5 In vitro release profile of curcumin-loaded mussel mucin-hyaluronic acid nanogel.

[0036] Figure 6 This is the particle size distribution diagram of the mussel mucin-hyaluronic acid nanogel loaded with doxorubicin in Example 8.

[0037] Figure 7 This is the potential distribution diagram of the mussel mucin-hyaluronic acid nanogel loaded with doxorubicin in Example 8.

[0038] Figure 8 Fluorescence micrographs of skin treated with curcumin solution and curcumin-loaded mussel mucin-hyaluronic acid nanogel. DETAILED DESCRIPTION

[0039] The present invention provides a mussel mucin-hyaluronic acid nanogel, the structural formula of which is as follows:

[0040]

[0041] Wherein, Mussel adhesive protein represents mussel adhesive protein molecule, n represents the number of repeating units, and n is a natural number, 10≤n≤130.

[0042] The present invention also provides a method for preparing mussel mucin-hyaluronic acid nanogel, and the specific implementation steps are as follows:

[0043] Step S1: hyaluronic acid (molecular weight 0.5×10 4 ~5×10 4 ) is dissolved in a reaction solvent (selected from at least one of MES buffer, acetate buffer, and phosphate buffer), EDC and NHS are added to activate the carboxyl group according to a weight ratio of hyaluronic acid, EDC, and NHS of 5 to 50:1 to 5:1, the reaction temperature is 25 to 45° C., the reaction time is 30 min to 4 h, and the reaction conditions are magnetic stirring and light-proof reaction;

[0044] Step S2: adding mussel mucin to hyaluronic acid at a weight ratio of 1:0.5-5 to react the mussel mucin with the activated carboxyl group in the hyaluronic acid structure by amide reaction, the reaction temperature is 25-45° C., the reaction time is 4-48 hours, and the reaction conditions are magnetic stirring and light-proof reaction;

[0045] Step S3: After adjusting the pH value to 7-9 with a pH regulator (selected from sodium hydroxide, potassium hydroxide, sodium bicarbonate, and ammonia water), the obtained product is placed in a dialysis bag with a molecular weight cutoff of 3500-14000Da, dialyzed in deionized water at 4-30°C for 1-4 days, and then freeze-dried to obtain the mussel mucin-hyaluronic acid nanogel.

[0046] The chemical reaction equation involved in the preparation method of a mussel mucin-hyaluronic acid nanogel of the present invention is as follows:

[0047]

[0048] Among them, Hyaluronic acid means hyaluronic acid, MAP means mussel mucin, and MAP-HA means mussel mucin-hyaluronic acid nanogel.

[0049] The present invention also provides an application of mussel mucin-hyaluronic acid nanogel in the preparation of a drug carrier, wherein the drug carrier is a drug carrier for transdermal administration, mucosal administration, oral administration, ocular administration, rectal administration or vaginal administration, but is not limited thereto.

[0050] The invention also provides a pharmaceutical composition, which comprises a drug and the mussel mucin-hyaluronic acid nanogel.

[0051] Furthermore, the drug is selected from at least one of anti-inflammatory drugs, antibacterial drugs, anti-tumor drugs, analgesics, chemotherapeutic drugs, central nervous system drugs, digestive system drugs, circulatory system drugs, immune system drugs, and hormone drugs, but is not limited thereto.

[0052] Furthermore, the anti-inflammatory drug is selected from at least one of hormones, non-steroidal drugs, and steroidal drugs, but is not limited thereto.

[0053] The present invention also provides a method for preparing the pharmaceutical composition, and the specific implementation steps are as follows:

[0054] The drug and mussel mucin-hyaluronic acid nanogel are dissolved in an organic solvent at a weight ratio of 1:1-40, and stirred for 1-4 hours at room temperature in the dark; 10% PBS is slowly added dropwise to the above solution, stirred for 4-12 hours at room temperature in the dark, and then transferred to a dialysis bag with a molecular weight cutoff of 3500-7000Da, dialyzed in deionized water for 12-24 hours, and the dialyzed product is centrifuged and the supernatant is collected to obtain a pharmaceutical composition.

[0055] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this description, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0056] Example 1 Preparation of mussel mucin-hyaluronic acid nanogel

[0057] (1) Weigh 200 mg of hyaluronic acid and dissolve it in 4 mL of MES buffer with a pH of 5.6. Ultrasonicate until completely dissolved. Add 11.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 7.0 mg of N-hydroxysuccinimide (NHS) respectively. Activate the carboxyl groups by magnetic stirring at 37°C in the dark for 60 min to obtain a carboxyl-activated sodium hyaluronate solution.

[0058] (2) Weigh 200 mg of mussel mucin and add it to the carboxyl-activated sodium hyaluronate solution obtained above, and stir it with a magnetic stirrer at 37° C. in the dark for 6 h.

[0059] (3) The pH value was adjusted to 7.4 dropwise with 0.1 mol / L sodium hydroxide solution, and the resulting product was then placed in a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed with deionized water at room temperature for 72 h, and then freeze-dried to obtain mussel mucin-hyaluronic acid nanogel.

[0060] Example 2 Nuclear magnetic resonance hydrogen spectrum of mussel mucin-hyaluronic acid nanogel ( 1 HNMR) characterization

[0061] 10 mg of mussel mucin (MAP), hyaluronic acid (HA) and mussel mucin-hyaluronic acid nanogel (MAP-HA) were weighed and dissolved in 0.5 mL of deuterated water (D2O), and the hydrogen nuclear magnetic resonance spectra were measured respectively.

[0062] Results Figure 1 , comparing MAP, HA and MAP-HA 1 It can be seen from the H NMR spectrum that MAP-HA has characteristic peaks at chemical shifts of 6.9ppm and 6.6ppm, and the peak at chemical shift of 1.85ppm belongs to the characteristic peak of N-acetylmethyl of HA (-NHCOCH3). The H NMR spectrum of MAP-HA contains characteristic peaks of MAP and HA, indicating that MAP-HA was successfully synthesized.

[0063] Example 3 Preparation of curcumin-loaded mussel mucin-hyaluronic acid nanogel

[0064] Curcumin-loaded mussel mucin-hyaluronic acid nanogels were prepared by nanoprecipitation method. Weigh 20 mg of mussel mucin-hyaluronic acid nanogel, dissolve it in 2 mL of N,N-dimethylformamide (DMF), and ultrasonically disperse it until it is completely dissolved; weigh 10 mg of curcumin, dissolve it in 2.5 mL of DMF, and ultrasonically disperse it until it is completely dissolved; mix the curcumin DMF solution and the mussel mucin-hyaluronic acid DMF solution according to the mass ratio of curcumin to mussel mucin-hyaluronic acid of 1:5, and stir it at room temperature in the dark for 2 hours; slowly add 3 mL of 10% PBS (0.3 mL PBS 7.4 and 2.7 mL pure water) dropwise to the DMF mixed solution, and stir it magnetically at room temperature in the dark for 8 hours; the stirred solution is transferred to a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed with deionized water for 12 hours, collect the dialyzed product and centrifuge it at 3500 rpm for 10 minutes to remove the unencapsulated curcumin, and take out the supernatant to obtain the mussel mucin-hyaluronic acid nanogel loaded with curcumin.

[0065] Example 4 Scanning electron microscopic observation of mussel mucin-hyaluronic acid nanogel loaded with curcumin

[0066] The curcumin-loaded mussel mucin-hyaluronic acid nanogel was diluted with deionized water to 0.5 mg / mL, and the diluted sample was dropped on the surface of a silicon wafer and stored at room temperature until completely dry; then gold was sprayed under vacuum conditions, and scanning electron micrographs of the sample were obtained by applying an accelerating voltage of 4.0 kV and a working distance of 6.7 mm.

[0067] Results Figure 2 ,from Figure 2 It can be seen that the mussel mucin-hyaluronic acid nanogels loaded with curcumin are mostly irregular spherical in shape, with good dispersion and no agglomeration, indicating that a drug carrier preparation with good dispersion has been formed.

[0068] Example 5 Determination of particle size and potential of curcumin-loaded mussel mucin-hyaluronic acid nanogel

[0069] The particle size and potential of the mussel mucin-hyaluronic acid nanogel loaded with curcumin were measured by dynamic light scattering. The mussel mucin-hyaluronic acid nanogel loaded with curcumin was diluted to 1.0 mg / mL with deionized water, placed in a measuring cell, and the particle size and potential were measured at 25°C.

[0070] Particle size distribution diagram Figure 3 As shown, from Figure 3 It can be seen that the particle size of the mussel mucin-hyaluronic acid nanogel loaded with curcumin is 293.77±7.41nm, the PDI is 0.064±0.04, the particle size distribution is unimodal, and the distribution is relatively uniform.

[0071] Potential distribution diagram Figure 4 As shown, from Figure 4 It can be seen that the potential of the mussel mucin-hyaluronic acid nanogel loaded with curcumin is a single peak with a potential of -28.0±0.06mV, indicating that there is a high charge repulsion between the mussel mucin-hyaluronic acid nanogel particles loaded with curcumin and good stability.

[0072] Example 6 Determination of drug loading and encapsulation efficiency of curcumin-loaded mussel mucin-hyaluronic acid nanogel

[0073] The drug loading and encapsulation efficiency of mussel mucin-hyaluronic acid nanogel loaded with curcumin were determined by organic solvent extraction method. 0.5 mL of mussel mucin-hyaluronic acid nanogel aqueous solution loaded with curcumin was taken, and the volume was made up to 5 mL with anhydrous ethanol, and the solution was ultrasonicated in a water bath for 20 min. The absorbance at 425 nm was measured by UV-visible spectrophotometer, and the concentration of curcumin in the mussel mucin-hyaluronic acid nanogel solution loaded with curcumin was calculated. The encapsulation efficiency and drug loading were calculated according to the formula.

[0074] Encapsulation efficiency = mass of curcumin in drug-loaded nanogel / mass of curcumin × 100%

[0075] Drug loading = mass of curcumin in drug-loaded nanogel / (mass of curcumin in drug-loaded nanogel + mass of nanogel) × 100%

[0076] The calculated drug loading of curcumin-loaded mussel mucin-hyaluronic acid nanogel was 5.6%, and the encapsulation efficiency was 58.7%, indicating that mussel mucin-hyaluronic acid nanogel had a good drug loading capacity for curcumin.

[0077] Example 7 In vitro release study of curcumin-loaded mussel mucin-hyaluronic acid nanogel

[0078] The in vitro release of curcumin from mussel mucin-hyaluronic acid nanogels was investigated using the dialysis bag method. 2mL of curcumin ethanol solution (1 part of curcumin is dissolved in 5 parts of ethanol to prepare the curcumin ethanol solution) and a mussel mucin-hyaluronic acid nanogel solution (embodiment 3) loaded with curcumin are placed in a dialysis bag, and the two ends are sealed and immersed in a centrifuge tube equipped with 40mL of a release medium (PBS 7.4 with 0.5% Tween-80); The sealed centrifuge tube is placed in a 37°C constant temperature shock box with a rotation speed of 100rpm, and 2mL of samples are taken successively at 0.5h, 1h, 2h, 4h, 6h, 8h, 12h and 24h, and 2mL of isothermal fresh release medium is immediately supplemented to maintain a sink condition; A UV-visible spectrophotometer is used to quantitatively measure the drug in the sample at 425nm, and the content of curcumin in the release medium at different time points is calculated by a standard curve, and the cumulative release percentage of curcumin is calculated according to a formula to draw a release curve.

[0079] Results Figure 5 ,from Figure 5 It can be seen that the cumulative release percentage of curcumin ethanol solution in 24 hours exceeded 98%, indicating that the curcumin solution was basically released completely within 24 hours and had no sustained release effect. The mussel mucin-hyaluronic acid nanogel loaded with curcumin released 25.86% in 24 hours, indicating that the mussel mucin-hyaluronic acid nanogel loaded with curcumin well controlled the burst release behavior and showed a good sustained release effect.

[0080] Example 8 Preparation of mussel mucin-hyaluronic acid nanogel loaded with doxorubicin

[0081] The mussel mucin-hyaluronic acid nanogel loaded with doxorubicin was prepared by nanoprecipitation method. The mussel mucin-hyaluronic acid nanogel and doxorubicin were weighed and dissolved in DMF respectively, and ultrasonic dispersion was performed until completely dissolved; the doxorubicin DMF solution and the mussel mucin-hyaluronic acid DMF solution were mixed according to the mass ratio of doxorubicin to mussel mucin-hyaluronic acid of 1:10, and stirred at room temperature in the dark for 2 hours; an equal volume of ultrapure water containing 10% PBS was slowly added dropwise to the DMF mixed solution, and magnetic stirring was performed at room temperature in the dark for 12 hours; the stirred solution was transferred to a dialysis bag with a molecular weight cutoff of 3500Da, dialyzed with deionized water for 12 hours, and the dialyzed product was collected and centrifuged at 3500rpm for 10 minutes to remove the unencapsulated doxorubicin, and the supernatant was taken out to obtain the mussel mucin-hyaluronic acid nanogel loaded with doxorubicin.

[0082] According to calculation, the encapsulation efficiency and drug loading of the prepared mussel mucin-hyaluronic acid nanogel loaded with doxorubicin were 63.1% and 7.7%, respectively, indicating that the mussel mucin-hyaluronic acid nanogel has a good drug loading capacity for doxorubicin.

[0083] The results showed that the particle size of the mussel mucin-hyaluronic acid nanogel loaded with doxorubicin was 275.35±9.12nm ( Figure 6 ), PDI was 0.165±0.14, indicating that the particle size distribution was relatively uniform. The potential of the mussel mucin-hyaluronic acid nanogel loaded with doxorubicin was -30.1±0.15mV ( Figure 7 ), indicating that there is a high charge repulsion between the mussel mucin-hyaluronic acid nanogel particles loaded with doxorubicin and good stability.

[0084] Example 9 Fluorescence penetration study of curcumin-loaded mussel mucin-hyaluronic acid nanogel on ex vivo skin

[0085] The back skin of Bama miniature pigs was taken, and the back hair was shaved with an animal shaver, and the subcutaneous fat and adhesion tissue were carefully peeled off; the prepared ex vivo skin stratum corneum was fixed upward between the supply pool and the receiving pool of the Franz diffusion cell using a Franz test diffusion device, and 0.1 mL of curcumin solution and mussel mucin-hyaluronic acid solution loaded with curcumin were added to the supply pool respectively; the skin was removed at 2h and 8h, and repeatedly washed with physiological saline, and then the drug-dosing area was cut off, and the skin sample was wrapped with tin foil and stored at -20°C. Under light-proof conditions, the frozen skin was sliced, stained with the nuclear dye DAPI, and sealed with an anti-fluorescence quenching sealing agent, and placed under a fluorescence microscope for observation.

[0086] Results Figure 8 ,from Figure 8 It can be seen that the curcumin solution showed weak green fluorescence at 2h and 8h, indicating that the amount of curcumin penetrating into the skin was small. The mussel mucin-hyaluronic acid nanogel loaded with curcumin had strong green fluorescence in the epidermis and dermis at 2h and 8h, and could accumulate in skin appendages such as hair follicles, indicating that the mussel mucin-hyaluronic acid nanogel loaded with curcumin could effectively promote the amount and depth of drug transdermal penetration, form a drug reservoir on the biomembrane, slowly release the drug, and keep the drug at a high retention level in the skin for a long time, which is beneficial to ensure the effectiveness of local drug treatment.

[0087] Example 10 Study on the transdermal penetration and retention of curcumin-loaded mussel mucin-hyaluronic acid nanogel

[0088] The back skin of Bama miniature pigs was taken, and the back hair was shaved with an animal shaver, and the subcutaneous fat and adhesion tissue were carefully peeled off; the Franz test diffusion device was used to conduct an in vitro percutaneous permeation and retention experiment. The prepared ex vivo skin was fixed between the supply pool and the receiving pool of the Franz diffusion cell, with the stratum corneum facing the drug preparation and the dermis facing the receiving liquid. The effective permeation area was 2.8 cm 2The volume of the receiving pool is 6.5 mL, the receiving solution is a PBS (pH 7.4) buffer solution containing 0.5% Tween-80, the water bath temperature of the diffusion pool is 37°C, the in vitro skin temperature is controlled at 32±1°C, and the stirring speed is set to 300 r / min; 1.0 mL of curcumin solution and mussel mucin-hyaluronic acid nanogel solution containing curcumin are added to the dosing chamber respectively, 1.0 mL of receiving solution is taken out from the receiving pool at a predetermined time point, and immediately supplemented with an equal volume of fresh receiving solution at the same temperature; the concentration of curcumin in the sample is determined by high performance liquid chromatography, and the cumulative transdermal amount of curcumin is calculated; after the transdermal experiment, the skin is taken out from the diffusion pool, washed with deionized water, cut into pieces, homogenized with methanol for 2 minutes, and ultrasonicated at room temperature for 30 minutes; the obtained sample is filtered through a 0.22 μm microporous filter membrane, and the content of curcumin is determined by high performance liquid chromatography, and the intradermal retention of curcumin is calculated.

[0089] The results showed that after 12 hours of percutaneous transdermal transdermal administration of curcumin-loaded mussel mucin-hyaluronic acid nanogel, the cumulative transdermal amount was 0.088 μg / cm 2 , while the cumulative transdermal amount of curcumin solution was 0.037 μg / cm 2 , indicating that mussel mucin-hyaluronic acid nanogel can promote the transdermal absorption of curcumin. After 12 hours of transdermal permeation, the retention of curcumin in the ex vivo skin was 1.828 μg / cm 2 , while the retention of curcumin solution was only 0.118 μg / cm 2 Compared with the curcumin solution, the skin retention of mussel mucin-hyaluronic acid nanogel loaded with curcumin was significantly increased (p<0.01), indicating that mussel mucin-hyaluronic acid nanogel as a carrier of curcumin can form a drug reservoir in the skin and has a certain sustained-release effect. While increasing the skin retention of the drug, it also increases the transdermal absorption of the drug. Therefore, this drug carrier helps the drug to exert its efficacy locally on the skin.

[0090] The mucin-hyaluronic acid nanogel provided by the present invention not only has good drug loading capacity and sustained release effect, but also has excellent bioadhesion, can adhere tightly to human skin and mucous membranes, prolong the local retention of drugs, and promote drug absorption. It is a drug carrier with good performance.

[0091] The present invention discloses a mussel mucin-hyaluronic acid nanogel and a preparation method, application and pharmaceutical composition thereof. Those skilled in the art can refer to the content of the present invention and appropriately improve the process parameters to achieve the present invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The product of the present invention has been described through a preferred embodiment, and relevant personnel can obviously modify or appropriately change and combine the product described in the present invention without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

Claims

1. A mussel mucin-hyaluronic acid nanogel, characterized in that: The structural formula of the mussel mucin-hyaluronic acid nanogel is: Wherein, Mussel adhesive protein represents mussel adhesive protein molecule, n represents the number of repeating units, and n is a natural number, 10≤n≤130.

2. A method for preparing a mussel mucin-hyaluronic acid nanogel as claimed in claim 1, characterized in that: The following steps are involved: Step S1: dissolving hyaluronic acid in a reaction solvent, and adding EDC and NHS to activate the carboxyl group; Step S2: adding mussel mucin to the activated carboxyl groups in the hyaluronic acid structure to undergo amide reaction; Step S3: adjusting the pH value with a pH regulator, dialyzing and drying to obtain the mussel mucin-hyaluronic acid nanogel.

3. The method for preparing a mussel mucin-hyaluronic acid nanogel according to claim 2, characterized in that: The weight ratio of the mussel mucin to the hyaluronic acid is 1:0.5-5.

4. The method for preparing a mussel mucin-hyaluronic acid nanogel according to claim 2, characterized in that: In step S1, the molecular weight of the hyaluronic acid is 0.5×10 4 ~5×10 4 ; The weight ratio of the hyaluronic acid to the reaction solvent is 1:5-60; the weight ratio of the hyaluronic acid, EDC and NHS is 5-50:1-5:1; the reaction solvent is selected from at least one of MES buffer, acetate buffer and phosphate buffer; the reaction temperature of the activated carboxyl group is 25-45°C, the reaction time is 30min-4h, and the reaction conditions are magnetic stirring and light-proof reaction.

5. The method for preparing a mussel mucin-hyaluronic acid nanogel according to claim 2, characterized in that: In step S2, the reaction temperature of the amide reaction is 25-45° C., the reaction time is 4-48 h, and the reaction conditions are magnetic stirring and light-proof reaction.

6. The method for preparing a mussel mucin-hyaluronic acid nanogel according to claim 2, characterized in that: In step S3, the pH regulator is selected from one of sodium hydroxide, potassium hydroxide, sodium bicarbonate, and ammonia water; the pH value is adjusted to 7-9; the dialysis and drying method is: placing the product after the pH value is adjusted in a dialysis bag with a molecular weight cutoff of 3500-14000Da, dialyzing in deionized water at 4-30°C for 1-4 days, and then freeze-drying to obtain the mussel mucin-hyaluronic acid nanogel.

7. Use of the mussel mucin-hyaluronic acid nanogel as claimed in claim 1 in preparing a drug carrier.

8. The use according to claim 7, characterized in that: The drug carrier is a drug carrier for transdermal administration, mucosal administration, oral administration, ocular administration, rectal administration or vaginal administration.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a drug and a mussel mucin-hyaluronic acid nanogel as described in claim 1; the drug is selected from at least one of anti-inflammatory drugs, antibacterial drugs, anti-tumor drugs, analgesics, chemotherapeutic drugs, central nervous system drugs, digestive system drugs, circulatory system drugs, immune system drugs, and hormone drugs; the anti-inflammatory drug is selected from at least one of hormones, non-steroidal drugs, and steroid drugs.

10. The method for preparing a pharmaceutical composition according to claim 9, characterized in that: The following steps are involved: The drug and mussel mucin-hyaluronic acid nanogel are dissolved in an organic solvent at a weight ratio of 1:1-40, and stirred for 1-4 hours at room temperature in the dark; 10% PBS is slowly added dropwise to the obtained solution, and after stirring for 4-12 hours at room temperature in the dark, the solution is transferred to a dialysis bag with a molecular weight cutoff of 3500-7000Da, and dialyzed in deionized water for 12-24 hours, the dialyzed product is centrifuged and the supernatant is collected to obtain a pharmaceutical composition.

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