Double-drug-one-peptide drug delivery system as well as preparation method and application thereof

Abstract

The invention discloses a double-drug-one-peptide drug delivery system as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The drug loading system provided by the invention comprises Ce (at) MPBMPs, DAP, RosA, MM and E7 homing peptide. The drug loading system can be attached to an intervertebral disc segment corresponding to lumbago in situ, CeO2 is triggered in an inflammatory acid environment by means of a microneedle transdermal drug delivery technology to generate oxygen vortex, and then response release of double-loaded drugs is achieved to synergistically inhibit pathological microenvironment deterioration; meanwhile, endogenous mesenchymal stem cells can be collected specifically through homing peptide E7 modified on the outer layer, the endogenous mesenchymal stem cells are activated in a space-time specific mode to secrete anti-inflammatory external vesicles carrying regulatory factors, and the anti-inflammatory effect is achieved; therefore, the drug loading system can effectively inhibit the deterioration of the key pathological factor inflammation microenvironment of the intervertebral disc degeneration, and realizes the delayed treatment effect on the intervertebral disc degeneration.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a double-drug one-peptide drug delivery system, a preparation method and application thereof. BACKGROUND

[0002] Lumbar pain caused by intervertebral disc degeneration (IDD) accounts for about 40% of the world, which seriously affects the physical and mental health of patients. Pathological inflammatory microenvironment may be a key factor affecting IDD, and the main features include the production of inflammatory mediators, the gradual loss of extracellular matrix, the increase of cell aging and apoptosis, and the phenotype changes of nucleus pulposus cells. Studies have shown that long-term inflammatory microenvironment will recruit more inflammatory cells, further exacerbating the situation. Therefore, inhibiting the deterioration of pathological microenvironment caused by intervertebral disc degeneration is one of the main strategies for treating and relieving intervertebral disc degeneration, and apoptosis of nucleus pulposus cells is one of the important indicators of deterioration of inflammatory microenvironment, so delaying apoptosis of nucleus pulposus cells is of great significance for prevention and treatment of IDD. Mitophagy homeostasis imbalance and M1 macrophage recruitment are two key factors leading to apoptosis of nucleus pulposus cells, and a double-pathway drug regulation strategy designed for this purpose is expected to improve the treatment efficiency of IDD. In addition, studies have shown that bone marrow mesenchymal stem cells (BMSCs) are deeply involved in the repair process of IDD, and interact with the surrounding environment by secreting various factors, including growth factors, cytokines and chemotactic factors, to regulate the high expression of type II collagen and slow down the apoptosis of nucleus pulposus cells in the intervertebral disc. At the same time, BMSCs play an immunoregulatory role in the inflammatory microenvironment of IDD. BMSCs have the potential to induce differentiation, but are difficult to induce aggregation due to their small amount of distribution, so how to construct a strategy to induce endogenous BMSCs to aggregate in the inflammatory area of degenerative intervertebral disc to secrete beneficial cytokines to regulate the microenvironment and resist inflammatory response, and to synergistically treat or delay IDD is a technical problem to be solved in the field. SUMMARY

[0003] To solve the above problems in the prior art, the application provides a double-drug one-peptide drug delivery system, a preparation method and application thereof. The drug delivery system provided by the application designs a target site on-demand drug release strategy based on the transdermal drug delivery route, delivers the drug combination to the lesion site in a convenient and efficient manner, and synergistically regulates the endogenous stem cell microenvironment (a "three synergistic" strategy of double-drug one-peptide), inhibits the inflammatory microenvironment, and delays the occurrence and development of IDD.

[0004] To achieve the above purpose, the application provides the following technical solutions.

[0005] One of the technical solutions of the present application: a double-drug one-peptide drug delivery system, comprising mesoporous prussian blue nanoparticles with built-in cerium Ce@MPBMPs, daphne alpine DAP, rosmarinic acid RosA, M1 macrophage membrane MM and E7 homing peptide.

[0006] As a preferred solution of the present application, the Ce@MPBMPs comprise cerium dioxide and mesoporous prussian blue nanoparticles; the mass ratio of the Ce@MPBMPs, DAP, RosA, MM and E7 homing peptide is (5-10):(1-5):(1-5):(0.2-1):(0.1-0.5); the built-in cerium dioxide is a nanoparticle structure with a particle size of 2-10 nm; the particle size of the mesoporous prussian blue nanoparticles is 70-120 nm; the MM is obtained from RAW264.7 cells by ultrasonic crushing method.

[0007] The E7 homing peptide has high affinity with bone marrow mesenchymal stem cells (BMSCs) and can effectively induce BMSCs homing.

[0008] The second technical solution of the present application: a preparation method of the double-drug one-peptide drug delivery system described above, comprising the following steps:

[0009] S1, preparing a Ce@MPBMPs drug carrier: forming micelles of CeO2 and cationic surfactant in an alkaline solvent, adding K3[Fe(CN)6] and polyvinylpyrrolidone (PVP), heating to 75-85 DEG C, stirring for 18-22 h, cooling to room temperature, adding hydrochloric acid to the obtained mixture, continuing to stir for 3-5 h, then putting into a stainless steel autoclave and heating to 135-145 DEG C, 0.1-3 MPa, keeping for 2-3 h, cooling, centrifuging, collecting the precipitate, washing, and obtaining the Ce@MPBMPs drug carrier;

[0010] S2, preparing a MM / DR-Ce@MPBNPs double-drug unit: adding DAP, RosA and Ce@MPBNPs in a benign solvent, stirring, centrifuging, collecting the precipitate, dissolving in water again, then adding MM, stirring and mixing, to obtain a MM / DR-Ce@MPBNPs double-drug unit solution;

[0011] S3, preparing an E7-MM / DR-Ce@MPBNPs double-drug one-peptide drug delivery system: dissolving E7 homing peptide in a solvent, then adding the MM / DR-Ce@MPBNPs double-drug unit solution obtained in step S2, stirring, to obtain the E7-MM / DR-Ce@MPBNPs double-drug one-peptide drug delivery system.

[0012] As a preferred scheme of the present application, in S1, the use amount ratio of the CeO2, the alkaline solvent, K3[Fe(CN)6], polyvinylpyrrolidone and hydrochloric acid is (1-5) mg:(50-80) mL:(100-250) mg:(1-3) g:(2-10) mL; the use amount ratio of the CeO2 and the cationic surfactant is 1 mg:(20-200) μL, the cationic surfactant comprises one or more of cetyltrimethylammonium chloride, octadecyltrimethylammonium chloride, dodecyl dimethyl benzyl ammonium chloride and dodecyl dimethyl amine oxide; the alkaline solvent comprises a sodium hydroxide or potassium hydroxide solution with a concentration of 0.5 mol / L; the hydrochloric acid concentration is 0.1-1 M, heating and stirring is 80-140 ℃ stirring for 5-20 h; cooling to 20-30 ℃; centrifugation is 10000-12000 rpm speed centrifugation for 15-30 min; washing is using ethanol for 3-6 times, the ethanol concentration is 50-100 wt%.

[0013] The hydrochloric acid functions in adjusting PH, catalyzing reaction and promoting precipitation formation.

[0014] After heating and stirring, the sealed container needs to be placed in an autoclave for further heating to 140 ℃ for 2.5-10 h; after ending, it needs to be cooled to room temperature. The present application utilizes an improved hydrothermal synthesis method to prepare the mesoporous Prussian blue nanoparticles with built-in cerium dioxide, through controlling conditions such as raw material ratio, reagent type and heat treatment time, the Prussian blue nanoparticles with built-in cerium dioxide which are good in monodispersity, uniform in particle size and present mesoporous structure are synthesized. Subsequently, the Ce@MPBNPs which are uniform in structure and stable in performance are obtained through centrifugation, precipitation and washing, and are used as a drug carrier of daphnetin and rosmarinic acid.

[0015] As a preferred scheme of the present application, in S2, the benign solvent is 20-50 mL of ethanol or methanol, the use amount ratio of the benign solvent and the Ce@MPBNPs is (1.5-2.5) mL:1 mg; the mass ratio of DAP, RosA, Ce@MPBNPs and MM is (1-5):(1-5):(5-10):(0.2-1); the stirring is all 20-25 ℃ stirring for 30-60 min; the centrifugation is 10000-12000 rpm speed centrifugation for 15-30 min.

[0016] The present application loads the drug through a self-assembly method before loading the drug of the drug carrier Ce@MPBNPs and DAP, RosA, and then carries out the M1 macrophage membrane loading, which can increase the drug loading rate and the stability of the membrane structure.

[0017] As a preferred scheme of the present application, in S3, the solvent comprises water, DMSO or isopropyl alcohol, the concentration of the DMSO is 50-70 wt%, and the concentration of the isopropyl alcohol is 30-70 wt%; the stirring is stirring at 20-25 DEG C for 60-300 min; and the dosage ratio of the E7 homing peptide, the solvent and the MM / DR-Ce@MPBNPs double-drug unit solution is 1 mg:10 mL:(40-60) mL.

[0018] The E7 homing peptide is grafted to the surface of the MM by a non-covalent bond; the non-covalent bond is one or more of a hydrogen bond, an ionic bond and intermolecular force.

[0019] The sixth technical scheme of the present application is the use of the "double-drug one-peptide type" drug delivery system in the preparation of anti-inflammatory drugs and drugs for treating intervertebral disc degeneration.

[0020] The fourth technical scheme of the present application is a "double-drug one-peptide type" drug delivery system, comprising the double-drug one-peptide drug delivery system and the soluble microneedle DMNs.

[0021] The fifth technical scheme of the present application is a preparation method of the "double-drug one-peptide type" drug delivery system, comprising the following steps: dissolving the E7-MM / DR-Ce@MPBNPs double-drug one-peptide drug delivery system in a microneedle matrix, placing the microneedle matrix in a PDMS template, centrifuging, and drying to obtain the E7-MM / DR-Ce@MPBNPs-DMNs "double-drug one-peptide type" drug delivery system.

[0022] As a preferred technical scheme of the present application, the microneedle matrix comprises one or more of hyaluronic acid, polyvinyl alcohol and polyvinylpyrrolidone at a concentration of 10-30 wt%; the dosage ratio of the E7-MM / DR-Ce@MPBNPs double-drug one-peptide drug delivery system to the microneedle matrix is 1 mg:(1.5-2.5) mL; the centrifuging is centrifuging at a speed of 10000-12000 rpm at 4 DEG C for 15-30 min; and the drying is drying in a vacuum drying box at 37-50 DEG C and under a pressure of 0.01-0.05 MPa for 2-5 h.

[0023] The sixth technical scheme of the present application is the use of the "double-drug one-peptide type" drug delivery system in the preparation of anti-inflammatory drugs and drugs for treating intervertebral disc degeneration.

[0024] The present application uses the micro-molding method to prepare the DMNs, and optimizes the best microneedle matrix of the DMNs through the control of hardness, morphology, drug loading amount and dissolution rate.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] The drug delivery system provided by the present invention can be in situ fitted to the intervertebral disc segment corresponding to low back pain, and with the help of microneedle transdermal drug delivery technology, CeO2 can be triggered to generate oxygen vortex in the inflammatory acidic environment, thereby realizing the responsive release of dual-loaded drugs. The two drugs synergistically inhibit the deterioration of the pathological microenvironment by regulating the mitochondrial autophagy homeostasis of nucleus pulposus cells and inducing macrophage M2 polarization respectively; at the same time, the outer modified homing peptide E7 can specifically recruit endogenous mesenchymal stem cells, and activate them to secrete anti-inflammatory extracellular vesicles carrying regulatory factors in a spatiotemporal specific manner, thereby realizing the reconstruction of the inflammatory microenvironment gradient and exerting an anti-inflammatory effect; therefore, by grafting the drug to the homing peptide E7, the drug is released into the inflammatory area of ​​the lumbar intervertebral disc. Because E7 has a high affinity with BMSCs, it can induce BMSCs to aggregate and participate in the regulation through proliferation, differentiation, etc. This drug delivery system can deliver the drug combination to the lesion site in a convenient and efficient manner, and synergistically recruit endogenous stem cells to their home follicles, forming a "dual-drug, one-peptide" treatment strategy, inhibiting the inflammatory microenvironment, effectively inhibiting the deterioration of the inflammatory microenvironment, a key pathological factor of intervertebral disc degeneration, and achieving a delayed therapeutic effect on intervertebral disc degeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 In the figure, A is the TEM image of the E7-MM / DR-Ce@MPBNPs dual-drug and one-peptide drug delivery system prepared in step (3) of Example 1, and B is the SEM image of DMNs;

[0029] Figure 2 In the figure, A is an immunofluorescence image of the effect of co-culture of daphnetin (DAP) and nucleus pulposus cells on mitochondrial autophagy at different concentrations in Example 2; B is a quantitative statistical analysis of the immunofluorescence image results of Figure A;

[0030] Figure 3 In the figure, a is a FACS detection diagram of the effect of co-culturing macrophages with different concentrations of rosmarinic acid (RosA) on phenotypic polarization in Example 3; b is a quantitative statistical analysis diagram of the FACS detection results of A;

[0031] Figure 4 In the figure, A is a FACS detection diagram of the effect of co-culture of nucleus pulposus cells with different mass ratios of daphnetin and rosmarinic acid (DR) in Example 4 on cell apoptosis; B is a quantitative statistical analysis diagram of the FACS detection results of A;

[0032] Figure 5 In the figure, A is a scratch test graph for observing the effect of E7 homing peptide co-cultured with bone marrow mesenchymal stem cells (BMSCs) on cell migration; B is a quantitative statistical analysis graph of the results of the scratch test graph of A;

[0033] Figure 6 In the figure, A is an immunofluorescence graph for observing the effect of different drug groups on mitochondrial autophagy when co-cultured with nucleus pulposus cells; B is a quantitative statistical analysis graph of the M2 phenotype of the FACS detection results of A;

[0034] Figure 7 In the figure, A is a FACS graph for observing the effect of different drug groups on phenotype polarization when co-cultured with macrophages; B is a quantitative statistical analysis graph of the M2 phenotype of the FACS detection results of A;

[0035] Figure 8 In the figure, A is a FACS detection graph for observing the effect of different drug groups on cell apoptosis when co-cultured with nucleus pulposus cells; B is a quantitative statistical analysis graph of the FACS detection results of A. DETAILED DESCRIPTION

[0036] A number of exemplary embodiments of the present application are now described in detail. The following description of certain aspects of the application is not intended to be limiting of the scope of the application, but rather is intended to be illustrative of certain aspects of the application. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments of the application only and is not intended to limit the scope of the application.

[0037] In addition, for numerical ranges that are expressly recited herein, it is to be understood that every intervening value between the upper and lower limits of the range is also specifically contemplated. In lay terms, every value between the upper and lower limits of a range recited is specifically contemplated. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In the case of conflict between the present specification and any document incorporated by reference, the present specification controls.

[0039] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0040] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" and the like are open-ended terms that are intended to permit but not limit the inclusion of elements or the number of elements, as well as the possibility that an element or a number of elements can be added later.

[0041] The raw materials used in the following examples are all commercially available conventional raw materials, which are not particularly limited; room temperature is 20-25°C. The preparation method of M1 macrophage membrane is as follows: first, RAW264.7 cells are cultured and collected, and the cells are resuspended with a hypotonic buffer, and protease inhibitors and phosphatase inhibitors are added to prevent membrane protein degradation. Next, the cell suspension is placed in an ultrasonic disrupter, and when using a probe disrupter, it is recommended to use a 3mm (1 / 8 inch) probe to process 1-5mL samples, set the power to 35%-40%, and perform ultrasonic disruption with 10 seconds of pulse and 10 seconds of interval. The sample is kept on ice during disruption to prevent overheating. After disruption is complete, the cell suspension is transferred to a centrifuge tube, centrifuged at 12000r / min for 10min at 4°C, and the supernatant contains cell membrane fragments, and the precipitate is unbroken cells and contents. If membrane proteins are needed, the supernatant is subjected to ultracentrifugation (4°C) for 1h, the precipitate is discarded, and after washing once with PBS, it is resuspended, the protein concentration is determined using a kit, and it is stored at -80°C for later use. The following will not be repeated.

[0042] Example 1

[0043] The preparation of the dual-drug one-peptide drug delivery system is as follows:

[0044] (1) Preparation of Ce@MPBNPs drug carrier: 5mg of CeO2 was added to 50mL of sodium hydroxide solution (concentration: 0.5mol / L), 500μL of cetyltrimethylammonium chloride cationic surfactant was added dropwise, and the micelles were formed by stirring, then PVP (3g) and K3[Fe(CN)6] (150mg) were added to the micelle solution, and the mixture was stirred vigorously and heated to 80°C for 20h, further 10mL of HCl (0.2M) was added, and the mixture was stirred at room temperature for 4h, then it was placed in a stainless steel autoclave, heated to 140°C for 2.5h at 2MPa, cooled to room temperature, then centrifuged at 12000rpm for 20min, the sample was collected, and further washed with 75wt% ethanol for 3 times, and the Ce@MPBNPs drug carrier was obtained;

[0045] (2) Preparation of MM / DR-Ce@MPBNPs dual drug delivery unit: 5 mg of Ce@MPBNPs obtained in (1) was added to 10 mL of methanol, followed by further addition of 1 mg of daphnetin (DAP) and 1 mg of rosmarinic acid (RosA), and stirred at room temperature for 30 min, and then centrifuged at 12000 rpm for 20 min, and the precipitate was collected and transferred to 10 mL of deionized water solution, and 0.5 mg of extracted M1 macrophage membrane (MM) was added, and stirred at room temperature for 15 min to obtain the MM / DR-Ce@MPBNPs dual drug delivery unit;

[0046] (3) E7-MM / DR-Ce@MPBNPs dual drug and peptide delivery unit: 0.2 mg of E7 homing peptide was dissolved in 2 mL of DMSO solution (50 wt%), and then added to the MM / DR-Ce@MPBNPs solution (10 mL) prepared in step (2), and stirred at room temperature for 120 min to obtain the E7-MM / DR-Ce@MPBNPs dual drug and peptide delivery system;

[0047] (4) Preparation of E7-MM / DR-Ce@MPBNPs-DMNs dual drug and peptide drug delivery system: 5 mg of E7-MM / DR-Ce@MPBNPs powder prepared in (3) was dissolved in 10 mL of 15 wt% hyaluronic acid solution, and then transferred to a PDMS mold, and centrifuged at 15000 rpm at 4°C for 30 min, and then the mold was removed and transferred to a 37°C (0.01 MPa) oven for drying for 3 h, and then the microneedle patch DMNs was taken out with tweezers, which was the E7-MM / DR-Ce@MPBNPs-DMNs dual drug and peptide drug delivery system.

[0048] The dual drug and peptide delivery system obtained in step (3) of Example 1 and the microneedle patch DMNs obtained in step (4) were subjected to transmission electron microscopy and scanning electron microscopy scanning, respectively, and the scanning results are shown in Figure 1 Fig. A is a TEM image of the E7-MM / DR-Ce@MPBNPs dual drug and peptide delivery unit prepared in step (3) of Example 1, and Fig. B is a SEM image of the DMNs. As can be seen from Fig. A, the dual drug and peptide delivery unit structure was successfully prepared; and as can be seen from Fig. B, the soluble microneedle was successfully prepared.

[0049] Example 2

[0050] Effect of DAP on mitochondrial autophagy of nucleus pulposus cells at a set concentration

[0051] DAP used in Example 1 was prepared under sterile conditions to a concentration of 10 μg / mL and stored at 4°C until use. Two 12-well culture plates were prepared and placed in a biosafety cabinet for UV sterilization for 1 h. Rat nucleus pulposus cells (NPCs) were prepared into a cell suspension and inoculated into the sterilized 6-well cell culture plates at a seeding density of 1 x 10 5 cells / well, and 2 mL of complete culture medium (90% DMEM / F12 + 10% FBS + 1% PS) was added. The cell culture plates were transferred to a 37°C, 5% CO2 incubator and incubated for 4 h until the cells were completely adherent. The cells were divided into 4 groups, each with 3 parallel samples, and the 4 groups were: Control (blank control), 90 ng / mL rapamycin (Rapa), 10 μg / mL DAP, and 90 ng / mL Rapa + 10 μg / mL DAP. After 24 h of continued incubation, the culture medium was removed and the cells were collected for immunofluorescence staining. The immunofluorescence staining steps were as follows: 1) The supernatant was removed and the cells were washed 3 times with 2 mL of PBS (pH = 7.4), and 4% paraformaldehyde solution was added to each well for fixation for 10 min; 2) After fixation, the fixing solution was removed and the cells were washed 3 times with PBS, and 1 mL of cell permeation solution was added to each well for permeation at room temperature for 10 min, after which the cells were washed 3 times with PBS; 3) 1% BSA was prepared, the PBS in the wells was removed, and 1% BSA was added to each well for blocking at room temperature for 30 min, after which the wells did not need to be washed, 1% BSA-diluted p62 primary antibody (SOB0586, rabbit, 1:500, Sartorius, China) was added to each well, and the wells were placed at 4°C overnight; 4) The next day, the wells were removed and washed 3 times with PBS, and diluted rabbit secondary antibody (SA00003-2, goat, 1:300 dilution, Proteintech, USA) was added for incubation (1 h) in the dark, the secondary antibody was removed, and the cells were washed 3 times with PBS, and DAPI staining solution was added to each well for nuclear staining for 10 min. After the staining solution was removed, the cells were washed 3 times with PBS, and a certain amount of PBS was added to each well to maintain the sample state, and the samples were photographed under laser confocal microscopy in the dark. The results are shown in Figure 2 Figure 2 ​In the middle, A is the immunofluorescence image of the effect of DAP of different concentrations on the observation of mitochondrial autophagy in nucleus pulposus cells; B is the quantitative statistical analysis diagram of the immunofluorescence image results of A. It can be seen that P62 and cell mitochondrial autophagy are negatively correlated. It can be seen that the Rapa group (positive control) significantly induces autophagy, and the DAP group can significantly inhibit the mitochondrial autophagy of nucleus pulposus cells. The Rapa+DAP group shows the same trend, but due to the induction effect of Rapa, the autophagy content may be higher than that of the DAP group, but its inhibitory effect is basically the same as that of DAP. This experiment uses 10 μg / mL of DAP, which preliminarily shows the inhibitory effect on the mitochondrial autophagy of nucleus pulposus cells, but in the treatment of intervertebral disc degeneration, the mitochondrial autophagy homeostasis needs to be dynamically regulated according to the pathological stage. Early inhibition of autophagy can avoid cell apoptosis storm and be more beneficial, and later induction of autophagy to remove damaged / abnormal proteins is more beneficial. Therefore, the concentration-dependent effect of DAP on mitochondrial autophagy homeostasis regulation may exist. Combined with the slow-release effect of the drug delivery system designed in this experiment, the initial (low concentration) can inhibit the effect, and the later accumulation of drug release concentration may change to induction, so as to achieve the balanced regulation of the steady state, and achieve the apoptosis inhibition effect on nucleus pulposus cells, reduce the inflammatory microenvironment, and efficiently treat IDD.

[0052] Example 3

[0053] Effect of RosA on macrophage phenotype polarization at different concentrations

[0054] The RosA used in Example 1 was prepared under sterile conditions to 10 μg / mL RosA, stored at 4°C, and used as needed. Prepare a 12-well culture plate and place it in a biological safety cabinet for ultraviolet sterilization for 1 h. Prepare a cell suspension of RAW264.7 cells and inoculate it into a sterile 6-well cell culture plate with a seeding density of 5×10 4Cells / cone, add 1 mL complete medium (90% DMEM high glucose + 10% FBS + 1% PS); transfer the cell culture plate to a 37°C, 5% CO2 incubator and incubate for 4 h until the cells are fully adherent; divide the cells into 3 groups, each with 3 parallel samples, 3 are: Control (blank control), 1 μg / mL LPS (negative group), 1 μg / LPS + 10 μg / mL RosA. Continue to incubate for 24 h, remove the culture medium, and collect the cells for flow cytometry detection. The specific steps of flow cytometry detection are as follows: 1) wash twice with 1 mL PBS (pH = 7.4), centrifuge at 500 g for 5 min, and discard the supernatant; 2) resuspend in 200 μL PBS, add 0.5 μL CD80 (B306552, PE anti-mouse CD80, BioLegend) and 1 μL CD206 (B318302, APC anti-mouse CD206, BioLegend) respectively, and incubate on ice in the dark for 30 min; 3) after incubation, centrifuge at 500 g, discard the supernatant, wash twice with 1 mL PBS, centrifuge, and resuspend in 300 μL PBS; 4) use flow cytometry for data acquisition and analysis. The flow cytometry analysis results are shown in Figure 3 Figure 3 , where a is the FACS detection chart of the effect of different concentrations of rosmarinic acid (RosA) co-cultured with macrophages on phenotype polarization; and b is the FACS detection result quantitative statistical analysis chart of a. It can be seen that the content of M2 type macrophages (CD206 marker) in the blank control group is about 6.44%, the content in the LPS group (negative control group) is about 5.82%, and the content in the LPS + RosA group (treatment group) is about 46.21%, indicating that RosA can significantly regulate the polarization of macrophages to M2 type.

[0055] Example 4

[0056] The significant effect of different mass ratio daphnetin and rosmarinic acid (DR) administration group on the apoptosis of nucleus pulposus cells The DAP used in Example 1 was prepared under sterile conditions to a concentration of 10 μg / mL; RosA was prepared under sterile conditions to a concentration of 5, 10 μg / mL, and stored at 4°C until use. Prepare 2 12-well culture plates and place them in a biological safety cabinet for ultraviolet sterilization for 1 h, prepare rat nucleus pulposus cells (NPC) into a cell suspension, inoculate into sterilized 6-well cell culture plates, and the inoculation density is 5 × 10 4 ​Cells / cone, add 1 mL complete medium (90% DMEM / F12 + 10% FBS + 1% PS); transfer the cell culture plate to a 37°C, 5% CO2 incubator and incubate for 4 h until the cells are fully adherent; divide the cells into 6 groups, each with 3 parallel samples, 3 respectively: Control (blank control), 10 ng / mL IL-1β (negative group), 10 ng / mL IL-1β + 10 μg / mL DAP, 10 ng / mL IL-1β + 10 μg / mL RosA, 10 ng / mL IL-1β + 10 μg / mL DAP + 10 μg / mL RosA, 10 ng / mL IL-1β + 10 μg / mL DAP + 5 μg / mL RosA. Continue to incubate for 24 h, then remove the culture medium and collect the cells for flow cytometry detection. The specific steps of flow cytometry detection are as follows: 1) wash twice with 1 mL PBS (pH = 7.4) and centrifuge at 500 g for 5 min, then discard the supernatant; 2) add 300 uL of Binding buffer and gently blow it to a single cell suspension, then add 3 uL of Annexin V-FITC (KGA1102-100, Kaygen Biotech, China) and mix well in the dark, vortex, then add 3 uL of PI and mix well, vortex; incubate on ice for 30 min; 3) after incubation, use flow cytometry for data acquisition and analysis. The flow cytometry analysis results are shown in Figure 4 Figure 4 In the above table, A is the FACS detection chart of the effect of different mass ratio of daphnetin and rosmarinic acid (DR) administration group on the apoptosis of nucleus pulposus cells; B is the FACS detection result quantification statistical analysis chart of A. The single administration group and the different ratio of collaborative administration group can significantly inhibit the apoptosis of nucleus pulposus cells, and the DR (1:1) collaborative administration group has the best effect on inhibiting the apoptosis of nucleus pulposus cells, which further proves the significant effect of the two drugs on inhibiting the apoptosis of nucleus pulposus cells.

[0057] Example 5

[0058] Effect of E7 homing peptide on migration of bone marrow mesenchymal stem cells (BMSCs)

[0059] The E7 homing peptide used in Example 1 was prepared under sterile conditions to a concentration of 1, 5, and 10 μg / ml and stored at 4°C for later use. Two 6-well culture plates were placed in a biological safety cabinet and ultraviolet sterilized for 1 h. Rat bone marrow mesenchymal stem cells (BMSCs) were prepared into a cell suspension and inoculated into the sterilized culture plates at a seeding density of 5 x 10 5 ​Cells / well, 2 mL of complete medium (90% F12 + 10% FBS + 1% PS) was added. Subsequently, the cell culture plate was transferred to a 37°C, 5% CO2 incubator for 4 h, and the cells were allowed to adhere completely. The cells were then divided into 4 groups, each with 3 parallel samples, and the four groups were: 0 μg / ml E7 (blank control group), 1 μg / ml E7, 5 μg / ml E7 and 10 μg / ml E7. The four groups were incubated until the cell confluence reached 90-100%, at which time the cells were starved with serum-free medium for 6-12 h to reduce the influence of cell proliferation on migration. Subsequently, a straight line scratch was made in the middle of the well plate using a sterile 200 μl gun tip, and the cells were washed with PBS 3 times to remove the detached cell fragments. At this time, the medium was changed, the blank group did not contain E7, and the experimental groups were changed to three E7 concentrations. At 0, 12, 24 h, the scratch area was imaged using an inverted microscope. The results of the scratch test are shown in Figure 5 Figure 5 Figure 6 shows the results of the scratch test in which A is the effect of co-culture of E7 homing peptide and bone marrow mesenchymal stem cells (BMSCs) on cell migration observed in a scratch test, and B is a quantitative statistical analysis of the results of the scratch test in A. It can be seen that at the same time, the higher the concentration of E7 homing peptide, the higher the migration rate; at the same concentration, the longer the time, the higher the migration rate; when the migration area reaches a certain saturation, the migration rate tends to be stable. The results show that E7 homing peptide exhibits the ability to induce migration and homing of endogenous mesenchymal stem cells, and further plays an anti-inflammatory role. Figure 5

[0060] Example 6

[0061] The preparation of the dual-drug one-peptide drug delivery system is as follows:

[0062] (1) Preparation of Ce@MPBNPs drug carrier: 1 mg of CeO2 was added to 65 mL of sodium hydroxide solution (concentration: 0.5 mol / L), 200 μL of octadecyltrimethylammonium chloride cationic surfactant was added dropwise, and micelles were formed by stirring. Then PVP (2 g) and K3[Fe(CN)6] (100 mg) were added to the micelle solution, and the mixture was stirred vigorously and heated to 75°C for 22 h. Further, 10 mL of HCl (0.1 M) was added, and the mixture was stirred at room temperature for 3 h. Then the mixture was placed in a stainless steel autoclave at 0.1 MPa and heated to 145°C for 3 h. After cooling to room temperature, the mixture was centrifuged at 12000 rpm for 20 min, and the sample was collected. The sample was further washed with 75 wt% ethanol for 3 times to obtain the Ce@MPBNPs drug carrier.

[0063] ​​(2) Preparation of MM / DR-Ce@MPBNPs dual drug delivery unit: 7 mg of Ce@MPBNPs obtained in (1) was added to 17.5 mL of methanol, followed by further adding 2 mg of DAP and 5 mg of RosA, stirring at room temperature for 30 min, and then centrifuging at a speed of 12000 rpm for 20 min. The precipitate was collected and transferred to a 10 mL deionized water solution, 0.2 mg of extracted M1 macrophage membrane was added, and the mixture was stirred at room temperature for 15 min to obtain the MM / DR-Ce@MPBNPs dual drug delivery unit;

[0064] (3) Preparation of E7-MM / DR-Ce@MPBNPs dual drug and peptide delivery system: 0.2 mg of E7 homing peptide was dissolved in 2 mL of water, and then added to the MM / DR-Ce@MPBNPs solution (8 mL) prepared in step (2) and stirred at room temperature for 120 min to obtain the E7-MM / DR-Ce@MPBNPs dual drug and peptide delivery system;

[0065] (4) Preparation of E7-MM / DR-Ce@MPBNPs-DMNs dual drug and peptide drug delivery system: 5 mg of E7-MM / DR-Ce@MPBNPs powder prepared in (3) was dissolved in 7.5 mL of 30 wt% hyaluronic acid solution, and then transferred to a PDMS mold. The mold was centrifuged at 15000 rpm and 4°C for 30 min, and then taken out and transferred to a 37°C (0.01 MPa) oven for drying for 3 h. After drying, the microneedle patch DMNs was taken out with tweezers, which was the E7-MM / DR-Ce@MPBNPs-DMNs dual drug and peptide drug delivery system.

[0066] Example 7

[0067] The preparation of the dual drug and peptide drug delivery system is as follows:

[0068] (1) Preparation of Ce@MPBNPs drug carrier: 3 mg of CeO2 was added to 80 mL of sodium hydroxide solution (concentration: 0.5 mol / L), and 60 μL of dodecyl dimethyl benzyl ammonium chloride cationic surfactant was added dropwise to form micelles. Then, PVP (1 g) and K3[Fe(CN)6] (250 mg) were added to the micelle solution, and the mixture was stirred and heated to 85°C for 18 h. Further, 2 mL of HCl (1 M) was added, and the mixture was stirred at room temperature for 5 h. Then, the mixture was placed in a stainless steel autoclave at 3 MPa and heated to 135°C for 2 h. After cooling to room temperature, the mixture was centrifuged at 12000 rpm for 20 min, and the sample was collected and washed with 75 wt% ethanol for 3 times to obtain the Ce@MPBNPs drug carrier;

[0069] (2) Preparation of MM / DR-Ce@MPBNPs dual drug delivery unit: 10 mg of Ce@MPBNPs obtained in (1) was added to 15 mL of ethanol, followed by further addition of 5 mg of DAP and 2 mg of RosA. After stirring at room temperature for 30 min, centrifugation was performed at 12000 rpm for 20 min, and the precipitate was collected. The collected precipitate was transferred to 10 mL of deionized water solution, and 1 mg of extracted M1 macrophage membrane was added. After slight mixing and stirring at room temperature for 15 min, MM / DR-Ce@MPBNPs dual drug delivery unit was obtained.

[0070] (3) Preparation of E7-MM / DR-Ce@MPBNPs dual drug and peptide delivery system: 0.2 mg of E7 homing peptide was dissolved in 2 mL of isopropyl alcohol solution (50 wt%), which was then added to the MM / DR-Ce@MPBNPs solution (12 mL) prepared in step (2). After stirring at room temperature for 120 min, E7-MM / DR-Ce@MPBNPs dual drug and peptide delivery system was obtained.

[0071] (4) Preparation of E7-MM / DR-Ce@MPBNPs-DMNs dual drug and peptide drug delivery system: 5 mg of E7-MM / DR-Ce@MPBNPs powder prepared in (3) was dissolved in 12.5 mL of 10 wt% hyaluronic acid solution, which was then transferred to a PDMS mold. After centrifugation at 15000 rpm and 4°C for 30 min, the mold was removed and transferred to a 37°C (0.01 MPa) oven for drying for 3 h. After drying, the microneedle patch DMNs was removed with tweezers, and E7-MM / DR-Ce@MPBNPs-DMNs dual drug and peptide drug delivery system was obtained.

[0072] Comparative Example 1

[0073] The same as Example 1, except that in step (2), no MM was added, and the product of the previous step was changed accordingly in steps (3) and (4). In the comparative example step (2), 5 mg of Ce@MPBNPs obtained in (1) was added to 10 mL of methanol, followed by further addition of 1 mg of DAP and 1 mg of RosA. After stirring at room temperature for 30 min, centrifugation was performed at 12000 rpm for 20 min, and the precipitate was collected. The collected precipitate was transferred to 10 mL of deionized water solution, and slight mixing and stirring were performed at room temperature for 15 min. DR-Ce@MPBNPs dual drug delivery unit was obtained.

[0074] Example 8

[0075] Detection of E7-MM / DR-Ce@MPBNPs on Mitochondrial Autophagy of Nucleus Pulposus Cells

[0076] Prepare 2 12-well culture plates in a biosafety cabinet and ultraviolet sterilize for 1 h. Prepare a cell suspension of rat nucleus pulposus cells (NPCs) and inoculate into sterilized 6-well cell culture plates at a seeding density of 1 x 10 5 Cells / well, add 2 mL of complete culture medium (90% DMEM / F12 + 10% FBS + 1% PS); transfer the cell culture plate to a 37°C, 5% CO2 incubator and incubate for 4 h until the cells are completely adherent; divide the cells into 4 groups, each with 3 parallel samples, and add 4, respectively: Control (blank control), 90 ng / mL Rapa, 7.5 μg / mL DR-Ce@MPBNPs (prepared in Example 1, step (2)) + 90 ng / mL Rapa, 12.5 μg / mL E7-MM / DR-Ce@MPBNPs (prepared in Example 1, step (3)) + 90 ng / mL Rapa; continue to incubate for 24 h, then remove the culture medium and collect the cells for immunofluorescence staining. The steps for immunofluorescence staining are as follows: 1) remove the supernatant and wash 3 times with 2 mL of PBS (pH = 7.4); add 4% paraformaldehyde solution to each well to fix for 10 min; 2) after fixation, discard the fixing solution and wash 3 times with PBS; add 1 mL of cell permeation solution to each well and permeate at room temperature for 10 min, then wash 3 times with PBS; 3) prepare 1% BSA, remove the PBS in the well plate, add 1% BSA to each well for blocking at room temperature for 30 min, then do not wash the well plate; add 1% BSA-diluted p62 primary antibody (SOB0586, rabbit origin, 1:500, Sartorius, China) to each well, and place the well plate in a 4°C refrigerator overnight; 4) the next day, remove the well plate and wash 3 times with PBS; add diluted rabbit secondary antibody (SA00003-2, goat origin, 1:300 dilution, Proteintech, USA) and incubate in the dark (1 h); discard the secondary antibody and wash 3 times with PBS; add DAPI staining solution to each well to stain the nucleus for 10 min; discard the staining solution, wash 3 times with PBS, and then add a certain amount of PBS to each well to maintain the sample state and take a laser confocal microscope image in the dark.

[0077] Figure 6 In the figure, A is an immunofluorescence image of the effect of co-culture of different drug groups with nucleus pulposus cells on mitochondrial autophagy, and B is a quantitative statistical analysis image. Since the intensity of p62 immunofluorescence staining is negatively correlated with mitochondrial autophagy of the cells, as shown in the figure, the Rapa (rapamycin) group (positive control) significantly induced autophagy, the DR-Ce@MPBNPs group could significantly inhibit mitochondrial autophagy of the nucleus pulposus cells, the E7-MM / DR-Ce@MPBNPs group showed the same trend and was more significant, indicating that the E7-MM / DR-Ce@MPBNPs showed a significant inhibitory effect on mitochondrial autophagy of the nucleus pulposus cells. Figure 6 As can be seen, the Rapa (rapamycin) group (positive control) significantly induced autophagy, the DR-Ce@MPBNPs group could significantly inhibit mitochondrial autophagy of the nucleus pulposus cells, the E7-MM / DR-Ce@MPBNPs group showed the same trend and was more significant, indicating that the E7-MM / DR-Ce@MPBNPs showed a significant inhibitory effect on mitochondrial autophagy of the nucleus pulposus cells.

[0078] Example 9

[0079] Detection of the regulatory effect of E7-MM / DR-Ce@MPBNPs on the polarization of macrophages to M2:

[0080] Prepare a 12-well culture plate and place it in a biological safety cabinet for ultraviolet sterilization for 1 h. Prepare a cell suspension of RAW264.7 cells and inoculate it into a sterilized 6-well cell culture plate at a seeding density of 5 x 10 4 Cells / well, add 1 mL of complete culture medium (90% DMEM high glucose + 10% FBS + 1% PS), and transfer the cell culture plate to a 37°C, 5% CO2 incubator for incubation for 4 h until the cells are completely adherent. Divide the cells into 3 groups, each with 3 parallel samples, and 4 groups: Control (blank control), 1 μg / mL LPS (negative control), 7.5 μg / mL DR-Ce@MPBNPs (prepared in Example 1, step (2)) + 1 μg / mL LPS, and 12.5 μg / mL E7-MM / DR-Ce@MPBNPs (prepared in Example 1, step (3)) + 1 μg / mL LPS. Continue to incubate for 24 h, remove the culture medium, and collect the cells for flow cytometry detection. The specific steps for flow cytometry detection are as follows: 1) wash twice with 1 mL of PBS (pH = 7.4), centrifuge at 500 g for 5 min, and discard the supernatant; 2) resuspend in 200 μL of PBS, add 0.5 μL of CD86 (200307, BioLegend, USA) and 1 μL of CD206 (141708, BioLegend, USA), respectively, and incubate on ice in the dark for 30 min; 3) after incubation, centrifuge at 500 g, discard the supernatant, wash twice with 1 mL of PBS, centrifuge, and resuspend in 300 μL of PBS; 4) use a flow cytometer to collect and analyze the data.

[0081] Figure 7 In the present application, A is the FACS graph of the effect of co-culture of different drug groups with macrophages on phenotype polarization, and B is the M2 phenotype quantification analysis graph. The statistical results are shown in Figure 7 The content of M2 macrophages (CD206 marker) in the blank control group was about 18.66%, the content in the model group (negative control group) was about 13.78%, the content in the DR-Ce@MPBNPs group was about 70.4%, and the content in the E7-MM / DR-Ce@MPBNPs (treatment group) reached 86.5%, indicating that E7-MM / DR-Ce@MPBNPs can significantly regulate the polarization of macrophages to M2.

[0082] Example 10

[0083] Detection of the effect of E7-MM / DR-Ce@MPBNPs on the apoptosis of nucleus pulposus cells:

[0084] Prepare two 12-well culture plates in a biosafety cabinet and sterilize them under ultraviolet light for 1 h. Prepare a cell suspension of rat nucleus pulposus cells (NPCs) and inoculate them into the sterilized 6-well cell culture plates at a density of 5 x 10 4 Cells / well, add 1 mL of complete culture medium (90% DMEM / F12 + 10% FBS + 1% PS), and transfer the cell culture plates to a 37°C, 5% CO2 incubator for incubation for 4 h until the cells are completely adherent. Divide the cells into 6 groups, each with 3 parallel samples, and add 4, respectively: Control (blank control), 10 ng / mL IL-1β (negative group), 7.5 μg / mL DR-Ce@MPBNPs (prepared in Step (2) of Example 1) + 10 ng / mL IL-1β, 12.5 μg / mL E7-MM / DR-Ce@MPBNPs (prepared in Step (3) of Example 1) + 10 ng / mL IL-1β. Continue to incubate for 24 h, remove the culture medium, and collect the cells for flow cytometry detection. The specific steps for flow cytometry detection are as follows: 1) wash twice with 1 mL of PBS (pH = 7.4) and centrifuge at 500 g for 5 min, and discard the supernatant; 2) add 300 uL of Binding·buffer, gently blow to form a single cell suspension, and in a dark environment, add 3 uL of AnnexinV-FITC, mix, vortex, add 3 uL of PI (KGA1102-100, Annexin V-FITC / PI double staining cell apoptosis detection kit, Kaikai Biology), mix, and vortex; incubate on ice for 30 min; 3) after incubation, use a flow cytometer to collect data and analyze.

[0085] Figure 8 In the present application, A is the FACS detection chart of the effect of different drug groups on the apoptosis of nucleus pulposus cells, and B is the quantitative statistical analysis chart. The research results are shown in Figure 8 As shown in the results, both the DR-Ce@MPBNPs group and the E7-MM / DR-Ce@MPBNPs group can significantly inhibit the apoptosis of nucleus pulposus cells, and the E7-MM / DR-Ce@MPBNPs group has a better effect on inhibiting the apoptosis of nucleus pulposus cells.

[0086] The E7-MM / DR-Ce@MPBNPs prepared in Examples 6-7 have performance comparable to that of Example 1, preliminarily show an inhibitory effect on the mitochondrial autophagy of nucleus pulposus cells, can regulate the M2 polarization of macrophages, and have a significant effect on the apoptosis of nucleus pulposus cells.

[0087] The above merely describes preferred specific embodiments of the present application, and the protection scope of the present application is not limited thereto, and any person skilled in the art, according to the technical scheme and inventive concept of the present application, makes equivalent replacement or change within the technical range disclosed by the present application, should be covered within the protection scope of the present application.

Claims

1. A dual-drug and one-peptide drug delivery system, characterized in that: It includes mesoporous Prussian blue nanoparticles Ce@MPBMPs with built-in ceria, daphnetin DAP, rosmarinic acid RosA, M1 macrophage membrane MM and E7 homing peptide.

2. The dual-drug and one-peptide drug delivery system according to claim 1, characterized in that: The Ce@MPBMPs include cerium dioxide and mesoporous Prussian blue nanoparticles; the mass ratio of the Ce@MPBMPs, DAP, RosA, MM and E7 homing peptide is (5-10):(1-5):(1-5):(0.2-1):(0.1-0.5); the built-in cerium dioxide is a nanoparticle structure with a particle size of 2-10 nm, and the particle size of the mesoporous Prussian blue nanoparticles is 70-120 nm; the MM is taken from RAW264.7 cells and obtained by ultrasonic fragmentation.

3. A method for preparing a dual-drug and one-peptide drug delivery system according to claim 1 or 2, characterized in that: The following steps are included: S1. Preparation of Ce@MPBMPs drug carrier: CeO2 and cationic surfactant are reacted in an alkaline solvent to form micelles, K3[Fe(CN)6] and polyvinyl pyrrolidone are added, heated and stirred, hydrochloric acid is added to the resulting mixture, stirring is continued, and then heated again, cooled, centrifuged, the precipitate is collected, and washed to obtain the Ce@MPBMPs drug carrier; S2. Preparation of MM / DR-Ce@MPBNPs dual drug loading unit: DAP, RosA and Ce@MPBNPs were added to a benign solvent, stirred, centrifuged, and the precipitate was collected and dissolved in water. MM was then added, stirred, and mixed to obtain the MM / DR-Ce@MPBNPs dual drug loading unit solution. S3. Preparation of E7-MM / DR-Ce@MPBNPs dual-drug and one-peptide drug delivery system: dissolve the E7 homing peptide in a solvent, then add the MM / DR-Ce@MPBNPs dual-drug delivery unit solution obtained in step S2, and stir to obtain the E7-MM / DR-Ce@MPBNPs dual-drug and one-peptide drug delivery system.

4. The preparation method according to claim 3, characterized in that In S1, the amount ratio of CeO2, alkaline solvent, K3[Fe(CN)6], polyvinyl pyrrolidone and hydrochloric acid is (1-5) mg: (50-80) mL: (100-250) mg: (1-3) g: (2-10) mL; the cationic surfactant includes one or more of hexadecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, dodecyldimethylbenzylammonium chloride and dodecyldimethylamine oxide; the alkaline solvent includes sodium hydroxide or potassium hydroxide solution with a concentration of 0.5 mol / L; the hydrochloric acid concentration is 0.1-1 M, and the mixture is heated and stirred at 80-140° C. for 5-20 h; cooled to 20-30 ℃; centrifugation is performed at a speed of 10000-12000 rpm for 15-30 min; washing is performed 3-6 times with ethanol, and the ethanol concentration is 50-100 wt%; in S2, the benign solvent is 20-50 mL ethanol or methanol, and the dosage ratio of the benign solvent to Ce@MPBNPs is (1.5-2.5) mL:1 mg; the mass ratio of DAP, RosA, Ce@MPBNPs and MM is (1-5):(1-5):(5-10):(0.2-1); stirring is performed at 20-25 ℃ for 30-60 min; centrifugation is performed at a speed of 10000-12000 rpm for 15-30 min.

5. The preparation method according to claim 3, characterized in that In S3, the solvent includes water, DMSO or isopropanol, the concentration of DMSO is 50-70wt%, and the concentration of isopropanol is 30-70wt%; the stirring is at 20-25°C for 60-300min; the dosage ratio of E7 homing peptide, solvent and MM / DR-Ce@MPBNPs dual drug loading unit solution is 1mg:10mL:(40-60)mL.

6. Use of the dual-drug and one-peptide drug delivery system according to claim 1 or 2 in the preparation of anti-inflammatory drugs and drugs for treating intervertebral disc degeneration.

7. A "dual-drug, one-peptide" drug delivery system, characterized in that: It comprises the dual-drug and one-peptide drug delivery system according to claim 1 or 2 and soluble microneedle DMNs.

8. A method for preparing the "dual-drug, one-peptide" drug delivery system according to claim 7, characterized in that: The following steps are involved: The E7-MM / DR-Ce@MPBNPs dual-drug and one-peptide drug delivery system was dissolved in the microneedle matrix, placed on the PDMS template, centrifuged, and dried to obtain the E7-MM / DR-Ce@MPBNPs-DMNs "dual-drug and one-peptide" drug delivery system.

9. The preparation method according to claim 8, characterized in that The microneedle matrix includes one or more of hyaluronic acid, polyvinyl alcohol and polyvinyl pyrrolidone with a concentration of 10 to 30 wt%; the dosage ratio of the E7-MM / DR-Ce@MPBNPs dual-drug and one-peptide drug delivery system to the microneedle matrix is ​​1 mg: (1.5 to 2.5) mL; centrifugation is performed at 4°C and a speed of 10,000 to 12,000 rpm for 15 to 30 minutes; and drying is performed in a vacuum drying oven at 37 to 50°C and 0.01 to 0.05 MPa for 2 to 5 hours.

10. Use of the "dual-drug, one-peptide" drug delivery system according to claim 7 in the preparation of anti-inflammatory drugs and drugs for treating intervertebral disc degeneration.

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