A dual-drug one-peptide drug-loading system, a preparation method and application thereof

Abstract

The application discloses a double-drug one-peptide drug delivery system, a preparation method and application thereof, and belongs to the technical field of biological medicines. The drug delivery system comprises Ce@MPBMPs, DAP, RosA, MM and E7 homing peptides. The drug delivery system can be in situ attached to the corresponding intervertebral disc segment of waist pain, and oxygen vortex generated by CeO2 is triggered in an inflammatory acidic environment by means of microneedle transdermal drug delivery technology, so as to realize the response release of double drugs and the synergistic inhibition of pathological microenvironment deterioration. Meanwhile, the homing peptide E7 modified on the outer layer can specifically recruit endogenous mesenchymal stem cells, and activate the anti-inflammatory outer vesicle secreted by the mesenchymal stem cells in a time and space specific manner, so as to play an anti-inflammatory role. Therefore, the drug delivery system can effectively inhibit the deterioration of the inflammatory microenvironment, a key pathological factor of intervertebral disc degeneration, and realize the delay treatment of intervertebral disc degeneration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a dual-drug peptide delivery system, its preparation method, and its application. Background Technology

[0002] Intervertebral disc degeneration (IDD) causes approximately 40% of low back pain worldwide, severely impacting patients' physical and mental health. A pathological inflammatory microenvironment is likely a key factor influencing IDD, characterized by the production of inflammatory mediators, gradual loss of extracellular matrix, increased cellular senescence and apoptosis, and phenotypic changes in nucleus pulposus cells. Studies have shown that a prolonged inflammatory microenvironment recruits more inflammatory cells, further exacerbating the condition. Therefore, inhibiting the deterioration of the pathological microenvironment caused by IDD is one of the main strategies for treating and alleviating IDD. Nucleus pulposus cell apoptosis is a crucial indicator of the worsening inflammatory microenvironment, making the delay of nucleus pulposus cell apoptosis significant for the prevention and treatment of IDD. Mitochondrial autophagy homeostasis imbalance and M1 macrophage recruitment are two key factors leading to nucleus pulposus cell apoptosis. Targeted design of a dual-pathway drug regulation strategy holds promise for improving the therapeutic efficiency of IDD. Furthermore, research has shown that bone marrow mesenchymal stem cells (BMSCs) are deeply involved in the repair process of intervertebral disc degeneration (IDD). By secreting various factors, including growth factors, cytokines, and chemokines, they interact with the surrounding environment, regulate the high expression of type II collagen, and slow down apoptosis of nucleus pulposus cells within the intervertebral disc. Simultaneously, BMSCs play an immunomodulatory role in the inflammatory microenvironment of IDD. While BMSCs possess the potential for induced differentiation, they are distributed in small quantities and are difficult to autonomously aggregate. Therefore, how to construct strategies that can induce endogenous BMSCs to aggregate in the inflammatory region of degenerated intervertebral discs, thereby secreting beneficial cytokines to regulate the microenvironment, counteract inflammatory responses, and synergistically treat or delay IDD, is a pressing technical problem that needs to be solved in this field. Summary of the Invention

[0003] To address the aforementioned problems in the prior art, this invention provides a dual-drug, single-peptide delivery system, its preparation method, and its applications. The drug delivery system provided by this invention employs a target-site-on-demand drug release strategy based on a transdermal drug delivery route. This delivers the drug combination to the lesion site in a convenient and efficient manner, and synergistically regulates the endogenous stem cell microenvironment (a "triple synergistic" strategy of dual-drug, single-peptide delivery) to inhibit the inflammatory microenvironment and delay the occurrence and development of IDD.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] One of the technical solutions of the present invention is a dual-drug, single-peptide delivery system comprising mesoporous Prussian blue nanoparticles Ce@MPBMPs containing cerium dioxide, daphne DAP, rosmarinic acid RosA, M1 macrophage membrane MM, and E7 homing peptide.

[0006] As a preferred embodiment of the present invention, the Ce@MPBMPs comprises cerium dioxide and mesoporous Prussian blue nanoparticles; the mass ratio of Ce@MPBMPs, DAP, RosA, MM and E7 homing peptide is (5-10):(1-5):(1-5):(0.2-1):(0.1-0.5); the embedded cerium dioxide has a nanoparticle structure with a particle size of 2-10 nm; the mesoporous Prussian blue nanoparticles have a particle size of 70-120 nm; the MM is obtained from RAW264.7 cells by ultrasonic disruption.

[0007] E7 homing peptide has a high affinity for bone marrow mesenchymal stem cells (BMSCs) and can effectively induce BMSCs to hom.

[0008] The second technical solution of the present invention: a method for preparing a dual-drug peptide delivery system according to the above description, comprising the following steps;

[0009] S1. Preparation of Ce@MPBMPs drug carrier: CeO2 and cationic surfactant are mixed in an alkaline solvent to form micelles. K3[Fe(CN)6] and polyvinylpyrrolidone (PVP) are added and heated to 75-85°C and stirred for 18-22 h. After cooling to room temperature, hydrochloric acid is added to the resulting mixture and stirring is continued for 3-5 h. Then, the mixture is placed in a stainless steel autoclave and heated to 135-145°C and 0.1-3 MPa for 2-3 h. After cooling, the mixture is centrifuged, the precipitate is collected, and washed to obtain the Ce@MPBMPs drug carrier.

[0010] S2. Preparation of MM / DR-Ce@MPBNPs dual drug-loaded units: In a benign solvent, add DAP, RosA and Ce@MPBNPs, stir, centrifuge, collect the precipitate, dissolve it in water, then add MM, stir and mix to obtain the MM / DR-Ce@MPBNPs dual drug-loaded unit solution.

[0011] S3. Preparation of E7-MM / DR-Ce@MPBNPs dual-drug-one-peptide 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, stir, and the E7-MM / DR-Ce@MPBNPs dual-drug-one-peptide delivery system can be obtained.

[0012] In a preferred embodiment of the present invention, in S1, the ratio of CeO2, 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 ratio of CeO2 to cationic surfactant is 1 mg: (20-200) μL, wherein the cationic surfactant includes hexadecyltrimethylammonium chloride and octadecyltrimethylammonium chloride. One or more of dodecyl dimethyl benzyl ammonium chloride and dodecyl dimethyl amine oxide; the alkaline solvent includes a sodium hydroxide or potassium hydroxide solution with a concentration of 0.5 mol / L; the hydrochloric acid concentration is 0.1-1 M; the heating and stirring are carried out at 80-140°C for 5-20 h; the mixture is cooled to 20-30°C; the centrifugation is carried out at 10000-12000 rpm for 15-30 min; the washing is carried out with ethanol 3-6 times, with an ethanol concentration of 50-100 wt%.

[0013] Hydrochloric acid functions to adjust pH, catalyze reactions, and promote precipitation.

[0014] After heating and stirring, the mixture should be placed in a sealed container in an autoclave and further heated to 140°C for 2.5-10 hours; after heating, it should be cooled to room temperature. This invention utilizes an improved hydrothermal synthesis method to prepare mesoporous Prussian blue nanoparticles with embedded cerium dioxide. By controlling the raw material ratio, reagent type, and heat treatment time, Prussian blue nanoparticles with good monodispersity, uniform particle size, and mesoporous structure with embedded cerium dioxide are synthesized. Subsequently, after centrifugation, precipitation, and washing, Ce@MPBNPs with uniform structure and stable performance are obtained and used as drug carriers for thymol and rosmarinic acid.

[0015] As a preferred embodiment of the present invention, in S2, the benign solvent is 20-50 mL of ethanol or methanol, and the ratio of 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 carried out at 20-25°C for 30-60 min; centrifugation is carried out at 10000-12000 rpm for 15-30 min.

[0016] This invention first loads drugs onto drug carriers Ce@MPBNPs, DAP, and RosA via a self-assembly method, and then encapsulates them onto the M1 macrophage membrane, which can increase the drug loading rate and the stability of the membrane structure.

[0017] As a preferred embodiment of the present invention, in S3, the solvent includes water, DMSO or isopropanol, wherein the concentration of DMSO is 50-70 wt% and the concentration of isopropanol is 30-70 wt%; stirring is carried out at 20-25°C for 60-300 min; and the ratio of E7 homing peptide, solvent and MM / DR-Ce@MPBNPs dual drug-carrying unit solution is 1 mg: 10 mL: (40-60) mL.

[0018] E7 homing peptide is grafted onto the surface of MM via non-covalent bonds; the non-covalent bonds are one or more of hydrogen bonds, ionic bonds, and intermolecular forces.

[0019] The third technical solution of the present invention: the application of the dual-drug peptide delivery system described above in the preparation of anti-inflammatory drugs and drugs for treating intervertebral disc degeneration.

[0020] The fourth technical solution of the present invention: a "dual-drug-peptide" drug delivery system, comprising the above-mentioned dual-drug-peptide drug delivery system and soluble microneedles DMNs.

[0021] The fifth technical solution of the present invention: A method for preparing the "dual-drug-one-peptide" drug delivery system according to the above description, comprising the following steps: dissolving the E7-MM / DR-Ce@MPBNPs dual-drug-one-peptide drug delivery system in a microneedle matrix, placing it in a PDMS template, centrifuging, and drying to obtain the E7-MM / DR-Ce@MPBNPs-DMNs "dual-drug-one-peptide" drug delivery system.

[0022] As a preferred embodiment of the present invention, the microneedle matrix comprises one or more of hyaluronic acid, polyvinyl alcohol, and polyvinylpyrrolidone at a concentration of 10-30 wt%; the ratio of the E7-MM / DR-Ce@MPBNPs dual-drug peptide loading system to the microneedle matrix is ​​1 mg:(1.5-2.5) mL; centrifugation is performed at 4°C and 10000-12000 rpm for 15-30 min; drying is performed in a vacuum drying oven at 37-50°C and 0.01-0.05 MPa for 2-5 h.

[0023] The sixth technical solution of the present invention: the application of the "dual-drug, single-peptide" drug delivery system described above in the preparation of anti-inflammatory drugs and drugs for treating intervertebral disc degeneration.

[0024] This invention utilizes micro-molding to prepare DMNs, and selects the optimal DMNs microneedle matrix by controlling conditions such as hardness, morphology, drug loading, and dissolution rate.

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

[0026] The drug delivery system provided by this invention can be applied in situ to the intervertebral disc segment corresponding to low back pain, and by using microneedle percutaneous drug delivery technology, it triggers the generation of oxygen vortex in CeO2 in an inflammatory acidic environment, thereby achieving the responsive release of dual-loaded drugs. These two drugs synergistically inhibit the deterioration of the pathological microenvironment by regulating the mitochondrial autophagy homeostasis of nucleus pulposus cells and inducing M2 polarization of macrophages, respectively. At the same time, the outer layer modified homing peptide E7 can specifically recruit endogenous mesenchymal stem cells, and spatiotemporally activate them to secrete anti-inflammatory extracellular vesicles carrying regulatory factors, thereby achieving gradient reconstruction of the inflammatory microenvironment and exerting an anti-inflammatory effect. Therefore, by grafting homing peptide E7 onto drugs, drugs are released into the inflammatory area of ​​the lumbar intervertebral disc. Because E7 has a high affinity for BMSCs, it can induce BMSCs to aggregate and secrete more beneficial cytokines through proliferation and differentiation to participate in regulation. This drug delivery system can conveniently and efficiently deliver drug combinations to the lesion site and synergistically recruit endogenous stem cells to hom, forming a "two-drug-one-peptide" treatment strategy. This inhibits the inflammatory microenvironment and effectively suppresses the deterioration of the inflammatory microenvironment, a key pathological factor in intervertebral disc degeneration, thereby achieving a delayed therapeutic effect on intervertebral disc degeneration. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0029] Figure 2 In Figure A, the immunofluorescence graph shows the effect of co-culturing daphne (DAP) with nucleus pulposus cells at different concentrations on mitophagy in Example 2; Figure B is a quantitative statistical analysis of the immunofluorescence results in Figure A.

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

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

[0032] Figure 5 In the diagram, A is a scratch assay diagram showing the effect of co-culturing E7 homing peptide with bone marrow mesenchymal stem cells (BMSCs) on cell migration in Example 5; B is a quantitative statistical analysis diagram of the scratch assay results in A.

[0033] Figure 6 In Figure A, the immunofluorescence graph shows the effect of co-culturing different drug groups with nucleus pulposus cells on mitophagy in Example 8; Figure B is a quantitative statistical analysis of the immunofluorescence results in Figure A.

[0034] Figure 7 In the figure, A is the FACS diagram of the effect of co-culturing different drug groups with macrophages on phenotypic polarization in Example 9, and B is the FACS detection result of A, M2 phenotypic quantitative statistical analysis diagram.

[0035] Figure 8 In the figure, A is the FACS detection graph of the effect of co-culturing different drug groups with nucleus pulposus cells on cell apoptosis in Example 10, and B is the quantitative statistical analysis graph of the FACS detection results of A. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0037] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] The raw materials used in the following examples are all commercially available and conventional, and are not particularly limited; the room temperature is 20-25℃. The preparation method of M1 macrophage membranes is as follows: First, RAW264.7 cells are cultured and collected, and resuspended in hypotonic buffer. Protease inhibitors and phosphatase inhibitors are added to prevent membrane protein degradation. Next, the cell suspension is placed in an ultrasonic disruptor. When using a probe-type disruptor, it is recommended to use a 3mm (1 / 8 inch) probe to process 1-5 mL of sample, setting the power to 35%-40%, and performing ultrasonic disruption with 10-second pulses and 10-second intervals. Alternatively, parameters can be set to no more than 5 seconds per pulse and an interval no less than the ultrasonic time. During disruption, the sample should be kept on ice to prevent overheating. After disruption, the cell suspension is transferred to centrifuge tubes and centrifuged at 4℃ and 12000 rpm for 10 min. The supernatant contains cell membrane fragments, and the precipitate consists of undisrupted cells and contents. If membrane proteins are required, centrifuge the supernatant at 4°C for 1 hour, discard the precipitate, wash once with PBS, resuspend, determine the protein concentration using a kit, and store at -80°C for later use. This will not be repeated below.

[0042] Example 1

[0043] The preparation steps of the dual-drug single-peptide drug delivery system are as follows:

[0044] (1) Preparation of Ce@MPBNPs drug carrier: Weigh 5 mg of CeO2 and add it to 50 mL of sodium hydroxide solution (concentration: 0.5 mol / L). Add 500 μL of hexadecyltrimethylammonium chloride cationic surfactant and stir to form micelles. Then add PVP (3 g) and K3[Fe(CN)6] (150 mg) to the micelle solution. Stir vigorously and heat to 80 °C for 20 h. Add 10 mL of HCl (0.2 M) and stir at room temperature for 4 h. Then put it into a stainless steel autoclave and heat to 140 °C at 2 MPa for 2.5 h. Cool to room temperature and then centrifuge at 12000 rpm for 20 min. Collect the sample and wash it three times with 75 wt% ethanol to obtain Ce@MPBNPs drug carrier.

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

[0046] (3) E7-MM / DR-Ce@MPBNPs dual-drug single-peptide loading unit: Weigh 0.2 mg of E7 homing peptide and dissolve it in 2 mL of DMSO solution (50 wt%), then add it to the MM / DR-Ce@MPBNPs solution (10 mL) in step (2), stir at room temperature for 120 min, and the E7-MM / DR-Ce@MPBNPs dual-drug single-peptide loading system can be obtained;

[0047] (4) Preparation of E7-MM / DR-Ce@MPBNPs-DMNs dual-drug single-peptide drug delivery system: Weigh 5mg of E7-MM / DR-Ce@MPBNPs powder prepared in (3), then dissolve it in 10mL of 15wt% hyaluronic acid solution, then transfer it to a PDMS mold, centrifuge at 15000rpm at 4℃ for 30min, then remove the mold and transfer it to an oven at 37℃ (0.01MPa) to dry for 3h. After drying, remove the microneedle patch DMNs with tweezers, which is the E7-MM / DR-Ce@MPBNPs-DMNs dual-drug single-peptide drug delivery system.

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

[0049] Example 2

[0050] Effects of DAP at set concentrations on mitochondrial autophagy in nucleus pulposus cells

[0051] The DAP used in Example 1 was prepared under aseptic conditions to a concentration of 10 μg / mL and stored at 4°C for later use. Two 12-well culture plates were prepared and sterilized under UV light in a biosafety cabinet for 1 hour. Rat nucleus pulposus (NPC) cells were prepared into a cell suspension and seeded into sterilized 6-well cell culture plates at a seeding density of 1 × 10⁻⁶. 5 Add 2 mL of complete culture medium (90% DMEM / F12 + 10% FBS + 1% PS) to each cell / well. Transfer the cell culture plate to a 37°C, 5% CO2 incubator and incubate for 4 h until the cells are fully adhered. Divide the cells into 4 groups, with 3 replicates per group. The 4 groups are: Control (blank control), 90 ng / mL rapamycin (Rapa), 10 μg / mL DAP, and 90 ng / mL Rapa + 10 μg / mL DAP. After incubation for another 24 h, remove the culture medium and collect the cells for immunofluorescence staining. Immunofluorescence staining procedure: 1) Aspirate the supernatant, add 2 mL of PBS (pH=7.4), wash 3 times, and add 4% paraformaldehyde solution to each well for fixation for 10 min; 2) After fixation, discard the fixative, wash 3 times with PBS, add 1 mL of cell permeation buffer to each well, and permeate at room temperature for 10 min. After fixation, wash 3 times with PBS; 3) Prepare 1% BSA, aspirate the PBS from the well plate, add 1% BSA to each well for blocking, and block at room temperature for 30 min. After blocking, the well plate does not need to be washed. Add p62 primary antibody (SOB0586, rabbit source, 1:500, Startec, China) diluted with 1% BSA to each well, and incubate the well plate at 4°C overnight. 4) On the second day, remove the plate, wash three times with PBS, add diluted rabbit secondary antibody (SA00003-2, sheep-derived, 1:300 dilution, Proteintech, USA), incubate in the dark (1 h), discard the secondary antibody, wash three times with PBS, add DAPI staining solution to each well for nuclear staining for 10 min. After the staining, discard the staining solution, wash three times with PBS, and after washing, add a certain amount of PBS to each well to maintain the sample condition. Perform laser confocal microscopy imaging in the dark. The results are as follows: Figure 2 As shown, Figure 2Figure A shows the immunofluorescence images of the effects of co-culturing DAP with nucleus pulposus cells at different concentrations on mitophagy; Figure B shows the quantitative statistical analysis of the immunofluorescence results in Figure A. It can be seen that since P62 and mitophagy are negatively correlated, the Rapa group (positive control) significantly induced autophagy, while the DAP group significantly inhibited mitophagy in nucleus pulposus cells. The Rapa+DAP group showed the same trend, but due to the induced effect of Rapa, the autophagy content might be higher than that in the DAP group, although its inhibitory effect was basically the same as that of DAP. This experiment used 10 μg / mL DAP, which initially showed an inhibitory effect on mitophagy in nucleus pulposus cells. However, in the treatment of intervertebral disc degeneration, it is necessary to dynamically regulate mitophagy homeostasis according to the pathological stage. Early inhibition of autophagy is more beneficial in avoiding apoptotic storms, while late-stage induction of autophagy to clear damaged / abnormal proteins is more beneficial. Therefore, the regulatory effect of DAP on mitophagy homeostasis may be concentration-dependent. The sustained-release effect designed in this drug delivery system allows for initial (low concentration) inhibition, while later, as the drug release accumulates and the concentration increases, it may transform into an inducing effect, thereby achieving homeostatic balance regulation, inhibiting apoptosis of nucleus pulposus cells, reducing the inflammatory microenvironment, and effectively treating IDD.

[0052] Example 3

[0053] Effect of RosA at set concentrations on macrophage phenotypic polarization

[0054] The RosA used in Example 1 was aseptically prepared to 10 μg / mL and stored at 4°C for later use. A 12-well culture plate was prepared and sterilized under UV light in a biosafety cabinet for 1 hour. RAW264.7 cells were prepared into a cell suspension and seeded into a sterilized 6-well cell culture plate at a seeding density of 5 × 10⁻⁶ cells / well. 4Cells / well were added to 1 mL of complete culture medium (90% DMEM high glucose + 10% FBS + 1% PS); the cell culture plate was transferred to a 37°C, 5% CO2 incubator and incubated for 4 h until the cells were fully adhered; the cells were divided into 3 groups, with 3 replicates per group: Control (blank control), 1 μg / mL LPS (negative group), and 1 μg / LPS + 10 μg / mL RosA. After incubation for another 24 h, the culture medium was removed, and the cells were collected for flow cytometry analysis. The specific steps for 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), 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 at 500 g, and resuspend in 300 μL PBS; 4) Collect and analyze data using a flow cytometer. The flow cytometry statistical analysis results are as follows: Figure 3 As shown, Figure 3 In the figure, a is the FACS graph showing the effect of co-culturing macrophages with different concentrations of rosmarinic acid (RosA) on phenotypic polarization; b is the quantitative statistical analysis graph of the FACS results of A. It can be seen that the content of M2 macrophages (CD206 labeled) 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 macrophage polarization towards M2 type.

[0055] Example 4

[0056] The significant effects of different mass ratios of daphne and rosmarinic acid (DR) on nucleus pulposus cell apoptosis were investigated. DAP used in Example 1 was aseptically prepared at a concentration of 10 μg / mL; RosA was aseptically prepared at concentrations of 5 and 10 μg / mL and stored at 4°C for later use. Two 12-well culture plates were prepared and sterilized under UV light in a biosafety cabinet for 1 hour. Rat nucleus pulposus (NPC) cells were prepared into cell suspensions and seeded into sterilized 6-well cell culture plates at a seeding density of 5 × 10⁶ cells / well. 4Cells / well were added to 1 mL of complete culture medium (90% DMEM / F12 + 10% FBS + 1% PS); the cell culture plate was transferred to a 37°C, 5% CO2 incubator and incubated for 4 h until the cells were fully adhered; the cells were divided into 6 groups, with 3 replicates per group, namely: 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, and 10 ng / mL IL-1β + 10 μg / mL DAP + 5 μg / mL RosA. After incubation for another 24 h, the culture medium was removed, and the cells were collected for flow cytometry analysis. The specific steps for 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) Add 300 μL of Binding buffer and gently mix to form a single-cell suspension. Under light-protected conditions, add 3 μL of Annexin V-FITC (KGA1102-100, Kaiji Biotechnology, China), mix well, vortex, then add 3 μL of PI, mix well, and vortex; incubate on ice for 30 min; 3) After incubation, collect and analyze data using flow cytometry. The flow cytometry statistical analysis results are as follows: Figure 4 As shown, Figure 4 In the figure, A shows the FACS (Fluorescence Archiving and Co-culture) results of nucleus pulposus cells co-cultured with different mass ratios of daphne and rosmarinic acid (DR) to observe the effect on cell apoptosis; B shows the quantitative statistical analysis of the FACS results in A. Both the single-drug group and the synergistic drug group at different ratios significantly inhibited nucleus pulposus cell apoptosis, while the DR (1:1) synergistic drug group showed the best inhibitory effect on nucleus pulposus cell apoptosis, further demonstrating the significant effect of the two drugs in synergistically inhibiting nucleus pulposus cell apoptosis.

[0057] Example 5

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

[0059] The E7 homing peptide used in Example 1 was aseptically prepared to concentrations of 1, 5, and 10 μg / ml and stored at 4°C for later use. Two 6-well culture plates were sterilized under UV light in a biosafety cabinet for 1 hour. Rat bone marrow mesenchymal stem cells (BMSCs) were prepared into a cell suspension and seeded into the sterilized culture plates at a seeding density of 5 × 10⁶ cells / well. 5Cells / well were added to 2 mL of complete culture medium (90% F12 + 10% FBS + 1% PS). The cell culture plates were then transferred to a 37°C, 5% CO2 incubator and incubated for 4 hours until complete cell adhesion. Four groups were then incubated, with three replicates per group: 0 μg / ml E7 (blank control), 1 μg / ml E7, 5 μg / ml E7, and 10 μg / ml E7. Incubation continued until cell confluence reached 90-100%. Simultaneously, the cells were starved with serum-free medium for 6-12 hours to reduce the impact of cell proliferation on migration. Then, a straight scratch was made at a uniform speed in the center of the well using a sterile 200 μl pipette tip, and the cells were washed three times with PBS to remove detached cell debris. The culture medium was then changed; the blank control group contained no E7, while the experimental groups used three different E7 concentrations. Images of the scratched areas were acquired using an inverted microscope at 0, 12, and 24 hours. The scratch experiment results are shown below. Figure 5 As shown, Figure 5 In the diagram, A represents a scratch assay showing the effect of co-culturing E7 homing peptide with bone marrow mesenchymal stem cells (BMSCs) on cell migration; B represents a quantitative statistical analysis of the scratch assay results in A. Figure 5 It can be seen that, within the same time frame, 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 region reaches a certain saturation, the migration rate tends to stabilize. The overall results indicate that E7 homing peptide exhibits the ability to induce migration and homing of endogenous mesenchymal stem cells, further exerting its anti-inflammatory effect.

[0060] Example 6

[0061] The preparation steps of the dual-drug single-peptide drug delivery system are as follows:

[0062] (1) Preparation of Ce@MPBNPs drug carrier: Weigh 1 mg of CeO2 and add it to 65 mL of sodium hydroxide solution (concentration: 0.5 mol / L). Add 200 μL of octadecyltrimethylammonium chloride cationic surfactant and stir to form micelles. Then add PVP (2 g) and K3[Fe(CN)6] (100 mg) to the micelle solution. Stir vigorously and heat to 75 °C for 22 h. Add 10 mL of HCl (0.1 M) and stir at room temperature for 3 h. Then put it into a stainless steel autoclave and heat to 145 °C at 0.1 MPa for 3 h. Cool to room temperature and then centrifuge at 12000 rpm for 20 min. Collect the sample and wash it three times with 75 wt% ethanol to obtain Ce@MPBNPs drug carrier.

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

[0064] (3) E7-MM / DR-Ce@MPBNPs dual-drug single-peptide delivery system: Weigh 0.2 mg of E7 homing peptide and dissolve it in 2 mL of water, then add it to the MM / DR-Ce@MPBNPs solution (8 mL) in step (2), stir at room temperature for 120 min, and the E7-MM / DR-Ce@MPBNPs dual-drug single-peptide delivery system can be obtained.

[0065] (4) Preparation of E7-MM / DR-Ce@MPBNPs-DMNs dual-drug single-peptide drug delivery system: Weigh 5mg of E7-MM / DR-Ce@MPBNPs powder prepared in (3), then dissolve it in a preferred 7.5mL 30wt% hyaluronic acid solution, then transfer it to a PDMS mold, centrifuge at 15000rpm at 4℃ for 30min, then remove the mold and transfer it to a 37℃ (0.01MPa) oven to dry for 3h. After drying, use tweezers to remove the microneedle patch DMNs, which is the E7-MM / DR-Ce@MPBNPs-DMNs dual-drug single-peptide drug delivery system.

[0066] Example 7

[0067] The preparation steps of the dual-drug single-peptide drug delivery system are as follows:

[0068] (1) Preparation of Ce@MPBNPs drug carrier: Weigh 3 mg of CeO2 and add it to 80 mL of sodium hydroxide solution (concentration: 0.5 mol / L). Add 60 μL of dodecyl dimethyl benzyl ammonium chloride cationic surfactant and stir to form micelles. Then add PVP (1 g) and K3[Fe(CN)6] (250 mg) to the micelle solution, stir vigorously and heat to 85 °C for 18 h. Add 2 mL of HCl (1 M) and stir at room temperature for 5 h. Then put it into a stainless steel autoclave, heat to 135 °C at 3 MPa for 2 h, cool to room temperature, and then centrifuge at 12000 rpm for 20 min. Collect the sample and wash it three times with 75 wt% ethanol to obtain Ce@MPBNPs drug carrier.

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

[0070] (3) E7-MM / DR-Ce@MPBNPs dual-drug single-peptide delivery system: Weigh 0.2 mg of E7 homing peptide and dissolve it in 2 mL of isopropanol solution (50 wt%), then add it to the MM / DR-Ce@MPBNPs solution (12 mL) in step (2), stir at room temperature for 120 min to obtain the E7-MM / DR-Ce@MPBNPs dual-drug single-peptide delivery system;

[0071] (4) Preparation of E7-MM / DR-Ce@MPBNPs-DMNs dual-drug single-peptide drug delivery system: Weigh 5mg of E7-MM / DR-Ce@MPBNPs powder prepared in (3), then dissolve it in a preferred 12.5mL 10wt% hyaluronic acid solution, then transfer it to a PDMS mold, centrifuge at 15000rpm at 4℃ for 30min, then remove the mold and transfer it to a 37℃ (0.01MPa) oven to dry for 3h. After drying, remove the microneedle patch DMNs with tweezers, which is the E7-MM / DR-Ce@MPBNPs-DMNs dual-drug single-peptide drug delivery system.

[0072] Comparative Example 1

[0073] Same as Example 1, except that MM is not added in step (2), and the product used in steps (3) and (4) is changed accordingly. Step (2) of this comparative example is as follows: 5 mg of Ce@MPBNPs obtained in (1) is added to 10 mL of methanol, followed by 1 mg of DAP and 1 mg of RosA. After stirring at room temperature for 30 min, the mixture is centrifuged at 12000 rpm for 20 min, the precipitate is collected, and the collected precipitate is transferred to 10 mL of deionized water solution. The mixture is then gently stirred at room temperature for 15 min to obtain the DR-Ce@MPBNPs dual drug-loaded unit.

[0074] Example 8

[0075] E7-MM / DR-Ce@MPBNPs for detecting mitochondrial autophagy in nucleus pulposus cells:

[0076] Prepare two 12-well culture plates and sterilize them under UV light in a biosafety cabinet for 1 hour. Prepare rat nucleus pulposus (NPC) cells into a cell suspension and inoculate them into sterilized 6-well cell culture plates at a density of 1 × 10⁻⁶ cells / well. 5 Add 2 mL of complete culture medium (90% DMEM / F12 + 10% FBS + 1% PS) to each cell / well. Transfer the cell culture plate to a 37°C, 5% CO2 incubator and incubate for 4 h until the cells are fully attached. Divide the cells into 4 groups, with 3 replicates per group. The 4 groups are: Control (blank control), 90 ng / mL Rapa, 7.5 μg / mL DR-Ce@MPBNPs (prepared in step (2) of Comparative Example 1) + 90 ng / mL Rapa, and 12.5 μg / mL E7-MM / DR-Ce@MPBNPs (prepared in step (3) of Example 1) + 90 ng / mL Rapa. After incubation for 24 h, remove the culture medium and collect the cells for immunofluorescence staining. Immunofluorescence staining procedure: 1) Aspirate the supernatant, add 2 mL of PBS (pH=7.4), wash 3 times, and add 4% paraformaldehyde solution to each well for fixation for 10 min; 2) After fixation, discard the fixative, wash 3 times with PBS, add 1 mL of cell permeation buffer to each well, and permeate at room temperature for 10 min. After fixation, wash 3 times with PBS; 3) Prepare 1% BSA, aspirate the PBS from the well plate, add 1% BSA to each well for blocking, and block at room temperature for 30 min. After blocking, the well plate does not need to be washed. Add p62 primary antibody (SOB0586, rabbit, 1:500, Startech, China) diluted with 1% BSA to each well, and incubate the well plate at 4°C overnight. 4) On the second day, remove the plate, wash three times with PBS, add diluted rabbit secondary antibody (SA00003-2, sheep-derived, 1:300 dilution, Proteintech, USA), incubate in the dark (1 h), discard the secondary antibody, wash three times with PBS, add DAPI staining solution to each well for nuclear staining for 10 min. After the staining is complete, discard the staining solution, wash three times with PBS, and after washing, add a certain amount of PBS to each well to maintain the sample condition. Perform laser confocal microscopy imaging in the dark.

[0077] Figure 6 In the diagram, A shows the immunofluorescence images of the effects of co-culturing different drug groups with nucleus pulposus cells on mitophagy, and B is the quantitative statistical analysis graph. Because the intensity of P62 immunofluorescence staining is negatively correlated with cellular mitophagy, as... Figure 6 It can be seen that the Rapa (rapamycin) group (positive control) significantly induced autophagy, the DR-Ce@MPBNPs group could significantly inhibit mitochondrial autophagy in nucleus pulposus cells, and the E7-MM / DR-Ce@MPBNPs group showed the same trend, and was even more significant, indicating that E7-MM / DR-Ce@MPBNPs showed a significant inhibitory effect on mitochondrial autophagy in nucleus pulposus cells.

[0078] Example 9

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

[0080] Prepare a 12-well culture plate and sterilize it under UV light in a biosafety cabinet for 1 hour. Prepare a cell suspension of RAW264.7 cells and seed them into a sterilized 6-well cell culture plate at a seeding density of 5 × 10⁻⁶ cells / well. 4 Cells / well were added to 1 mL of complete culture medium (90% DMEM high glucose + 10% FBS + 1% PS); the cell culture plate was transferred to a 37°C, 5% CO2 incubator and incubated for 4 h until the cells were fully adhered; the cells were divided into 3 groups, with 3 replicates in each group, and the 4 groups were: Control (blank control), 1 μg / mL LPS (negative group), 7.5 μg / mL DR-Ce@MPBNPs (prepared in step (2) of Comparative Example 1) + 1 μg / mL LPS, and 12.5 μg / mL LE7-MM / DR-Ce@MPBNPs (prepared in step (3) of Example 1) + 1 μg / mL LPS. After incubation for another 24 h, the culture medium was removed and the cells were collected for flow cytometry analysis. The specific steps for flow cytometry detection are as follows: 1) Wash twice with 1 mL PBS (pH=7.4), centrifuge at 500g for 5 min, and discard the supernatant; 2) Resuspend in 200 μL PBS, add 0.5 μL CD86 (200307, ​​BioLegend, USA) and 1 μL CD206 (141708, BioLegend, USA) respectively, and incubate on ice in the dark for 30 min; 3) After incubation, centrifuge at 500g, discard the supernatant, wash twice with 1 mL PBS, centrifuge at 500g, and resuspend in 300 μL PBS; 4) Use a flow cytometer for data acquisition and analysis.

[0081] Figure 7 In the diagram, A shows the FACS graph of the effect of co-culturing different drug groups with macrophages on phenotypic polarization, and B is the quantitative analysis graph of the M2 phenotype. Statistical results are as follows: Figure 7 As shown, the content of M2 macrophages (CD206 labeled) in the blank control group was approximately 18.66%, the content in the model group (negative control group) was approximately 13.78%, the content in the DR-Ce@MPBNPs group was approximately 70.4%, while the content in the E7-MM / DR-Ce@MPBNPs (treatment group) reached 86.5%, indicating that E7-MM / DR-Ce@MPBNPs can significantly regulate macrophage polarization towards M2.

[0082] Example 10

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

[0084] Prepare two 12-well culture plates and sterilize them under UV light in a biosafety cabinet for 1 hour. Prepare rat nucleus pulposus (NPC) cells into a cell suspension and inoculate them into sterilized 6-well cell culture plates at a density of 5 × 10⁶ cells / well. 4 Cells / well were added to 1 mL of complete culture medium (90% DMEM / F12 + 10% FBS + 1% PS); the cell culture plate was transferred to a 37°C, 5% CO2 incubator and incubated for 4 h until the cells were fully adhered; the cells were divided into 6 groups, with 3 replicates per group and 4 replicates per group: Control (blank control), 10 ng / mL IL-1β (negative group), 7.5 μg / mL DR-Ce@MPBNPs (prepared in step (2) of Comparative Example 1) + 10 ng / mL IL-1β, and 12.5 μg / mL E7-MM / DR-Ce@MPBNPs (prepared in step (3) of Example 1) + 10 ng / mL IL-1β. After incubation for another 24 h, the culture medium was removed and the cells were collected for flow cytometry analysis. The specific steps for 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) Add 300 μL of Binding buffer and gently mix to form a single-cell suspension. Under light-protected conditions, add 3 μL of Annexin V-FITC, mix, vortex, then add 3 μL of PI (KGA1102-100, Annexin V-FITC / PI double staining apoptosis detection kit, Kaiji Biotechnology), mix, and vortex; incubate on ice for 30 min; 3) After incubation, use flow cytometry to collect and analyze data.

[0085] Figure 8 In the diagram, A shows the FACS (Fluorescence Archiving and Co-culture of Different Drug Groups with Nucleus Pulp Cells) on the effect on apoptosis, and B shows the quantitative statistical analysis. The research results are as follows: Figure 8 As shown, both the DR-Ce@MPBNPs group and the E7-MM / DR-Ce@MPBNPs group significantly inhibited nucleus pulposus cell apoptosis, while the E7-MM / DR-Ce@MPBNPs group showed better inhibition of nucleus pulposus cell apoptosis.

[0086] The E7-MM / DR-Ce@MPBNPs prepared in Examples 6 and 7 have comparable performance to those in Example 1. They show preliminary inhibitory effects on mitochondrial autophagy in nucleus pulposus cells, can regulate macrophage polarization toward M2, and have a significant impact on nucleus pulposus cell apoptosis.

[0087] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A dual-drug peptide delivery system, characterized in that, Includes mesoporous Prussian blue nanoparticles Ce@MPBMPs with built-in cerium dioxide, daphne DAP, rosmarinic acid RosA, M1 macrophage membrane MM, and E7 homing peptide; The mass ratio of Ce@MPBMPs, DAP, RosA, MM and E7 homing peptide is (5-10): (1-5): (1-5): (0.2-1): (0.1-0.5); The drug carriers Ce@MPBNPs, DAP, and RosA were first loaded with drugs via self-assembly, and then encapsulated on the M1 macrophage membrane; the E7 homing peptide was non-covalently grafted onto the surface of MM.

2. The dual-drug peptide delivery system according to claim 1, characterized in that, The Ce@MPBMPs comprise cerium dioxide and mesoporous Prussian blue nanoparticles; the embedded cerium dioxide is a nanoparticle structure with a particle size of 2–10 nm, and the mesoporous Prussian blue nanoparticles have a particle size of 70–120 nm; the MM is obtained from RAW264.7 cells by ultrasonic disruption.

3. A method for preparing a dual-drug peptide delivery system according to claim 1 or 2, characterized in that, Includes the following steps; S1. Preparation of Ce@MPBMPs drug carrier: CeO2 and cationic surfactant are mixed in an alkaline solvent to form micelles. K3[Fe(CN)6] and polyvinylpyrrolidone are added, and the mixture is heated and stirred. Hydrochloric acid is added to the resulting mixture, and the mixture is stirred again. Then the mixture is heated, cooled, centrifuged, the precipitate is collected, and washed to obtain the Ce@MPBMPs drug carrier. S2. Preparation of MM / DR-Ce@MPBNPs dual drug-loaded units: In a benign solvent, add DAP, RosA and Ce@MPBNPs, stir, centrifuge, collect the precipitate, dissolve it in water, then add MM, stir and mix to obtain the MM / DR-Ce@MPBNPs dual drug-loaded unit solution. S3. Preparation of E7-MM / DR-Ce@MPBNPs dual-drug-one-peptide 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, stir, and the E7-MM / DR-Ce@MPBNPs dual-drug-one-peptide delivery system can be obtained.

4. The preparation method according to claim 3, characterized in that, In S1, the ratio of CeO2, 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 cationic surfactant includes one or more of hexadecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, dodecyldimethylbenzylammonium chloride, and dodecyldimethylamine oxide; the alkaline solvent includes a sodium hydroxide or potassium hydroxide solution with a concentration of 0.5 mol / L; the hydrochloric acid concentration is 0.1-1 M; the heating and stirring are carried out at 80-140 °C for 5-20 h; the temperature is cooled to 20-30 °C; and the centrifugation is carried out at 10000-12000 rpm. Centrifuge at rpm for 15–30 min; wash 3–6 times with ethanol at a concentration of 50–100 wt%; in S2, the benign solvent is 20–50 mL of ethanol or methanol, and the ratio of 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); stir at 20–25 °C for 30–60 min; centrifuge at 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, wherein the concentration of DMSO is 50-70 wt% and the concentration of isopropanol is 30-70 wt%; stirring is carried out at 20-25°C for 60-300 min; the ratio of E7 homing peptide, solvent and MM / DR-Ce@MPBNPs dual drug-carrying unit solution is 1 mg: 10 mL: (40-60) mL.

6. The use of a dual-drug peptide delivery system according to claim 1 or 2 in the preparation of a drug for treating intervertebral disc degeneration.

7. A dual-drug, single-peptide drug delivery system, characterized in that, Includes the dual-drug peptide delivery system and soluble microneedles DMNs as described in claim 1 or 2.

8. A method for preparing a dual-drug peptide drug delivery system according to claim 7, characterized in that, The process includes the following steps: dissolving the dual-drug peptide delivery system in a microneedle matrix, placing it in a PDMS template, centrifuging, and drying to obtain the dual-drug peptide drug delivery system.

9. The preparation method according to claim 8, characterized in that, The microneedle matrix comprises one or more of hyaluronic acid, polyvinyl alcohol, and polyvinylpyrrolidone at a concentration of 10–30 wt%; the ratio of the dual-drug peptide loading system to the microneedle matrix is ​​1 mg: (1.5–2.5) mL; centrifugation is performed at 4°C and 10,000–12,000 rpm for 15–30 min; drying is performed in a vacuum drying oven at 37–50°C and 0.01–0.05 MPa for 2–5 h.

10. The use of the dual-drug peptide drug delivery system according to claim 7 in the preparation of drugs for treating intervertebral disc degeneration.

Images