Active exosome scaffold coating based on traditional Chinese medicine enhanced secretion, its preparation method and application

Abstract

The present invention discloses an active exosome scaffold coating based on traditional Chinese medicine enhanced secretion, its preparation method and application, belonging to the technical field of biomedicine and interventional devices. The scaffold coating of the present invention includes: exosomes, including cardiomyocyte active exosomes and human mesenchymal stem cell active exosomes enhanced by traditional Chinese medicine; wherein, the traditional Chinese medicine includes: placenta extract, pilose antler extract, sheep myocardial peptide, sea cucumber peptide, black wolfberry peptide, osthole, curculigo rhizome extract, and morinda officinalis extract; the intervention concentration of each component added to the cells is 2-50 μg / mL; a photocurable gel matrix, including methacrylated gelatin, a photoinitiator, and polyether 127; tacrolimus; the final concentration of exosomes is 1×10 9 ~12×10 9 particles / mL, and the final concentration of tacrolimus is 5-25 μg / mL. This coating can be coated on the surface of a poly-L-lactic acid degradable stent to prepare a degradable stent, which has obvious ability to promote blood vessel repair.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomedicine and interventional devices. More specifically, the present invention relates to an active exosome stent coating based on traditional Chinese medicine enhanced secretion, which combines the functions of promoting endothelialization and anti-restenosis, and its preparation method and application. Background Art

[0002] The incidence rates of in-stent restenosis (ISR) and in-stent thrombosis (IST) that are difficult to avoid after intervention, as well as the systematic prevention and control of recurrent acute cardiovascular events after intervention, have become the most intractable problems in the current cardiology field. How to minimize the risks of ISR and IST and maximize the timely repair of damaged coronary arteries after intervention has become a hot spot and a difficult point in the research of the cardiac first-aid field.

[0003] 1. Thrombus organization, intimal hyperplasia, and vascular remodeling after PCI become the triggering links of ISR

[0004] Whether it is a biodegradable stent (BVS) or a drug-eluting stent (DES), the IST and ISR problems faced in the early and late stages of implantation remain the greatest potential risks after interventional procedures. The occurrence and development of ISR and IST are the results of the imbalance of homeostasis in the process of injury and repair after arterial intima injury. Its essence is a series of arterial healing responses mediated by vasoactive substances and growth factors. The migration, proliferation, and secretion of vascular smooth muscle cells (SMCs) are three important links in intimal hyperplasia. The injury of vascular endothelial cells (ECs) is the initiating factor of ISR and IST. The balance between intimal hyperplasia and vascular remodeling determines the degree of ISR and the risk of IST. During interventional procedures, it is inevitable that catheters, guidewires, balloons, stents, etc. will cause damage to the vascular wall. After the vascular intima is damaged, the subendothelial collagen is exposed, and various coagulation factors and adhesion molecules are secreted, activating thrombin, creating conditions for the aggregation of platelets, white blood cells, etc. at the local vascular break, and then triggering the formation of in-stent mural thrombus. With the gradual organization of the thrombus and the continuous infiltration of inflammatory cells and intimal hyperplasia, the pathological process of ISR begins to occur rapidly. The newly formed intimal tissue is mainly composed of SMCs, microthrombi, inflammatory cells, a large amount of extracellular matrix, and a layer of ECs covering the surface extending from the inner wall of the blood vessels around the stent. Among them, SMCs are the main components of the proliferated intima. The function of intimal ECs is damaged, and the contact inhibition function of SMCs is lost, which induces the proliferation and migration of SMCs and exacerbates ISR. The proliferation, migration, and apoptosis of SMCs are the cytological basis of vascular remodeling and rearrangement. Under the proliferated intima, there are not only the proliferation and apoptosis of SMCs, but also the infiltration and apoptosis of monocytes / macrophages. The apoptotic cells are replaced by connective tissue, making the cells in the subintimal layer unable to be arranged orderly and evenly and losing their normal physiological functions. Early SMC hyperplasia often occurs within 2 weeks after interventional procedures, but intimal thickening persists until 12 weeks, combined with the existing organized thrombus and subsequent vascular remodeling, the progression of ISR is rapidly promoted and leads to acute vascular occlusion lesions. Even taking the latest antiplatelet drugs cannot completely avoid IST and ISR, and it is necessary to further optimize and find more effective prevention and treatment strategies.

[0005] 2. Reconstructing the balance and homeostasis of EC repair, SMC proliferation, and phenotypic transformation is the key entry point for stent research and development

[0006] The biggest difficulty in stent coating research and development is that the drugs or materials coated on the stent have no selectivity in intervening on ECs in the vascular intima and SMCs in the media. Coating drugs to promote EC repair and anti-thrombosis are likely to trigger SMC proliferation and phenotypic transformation, resulting in lumen restenosis. On the contrary, inhibiting SMC proliferation and phenotypic transformation delays EC repair and amplifies the risk of IST. How to take into account both the EC repair and SMC hyperplasia processes, find an effective coating that can highly sensitively regulate the two and achieve the best balance and homeostasis is the greatest test faced by the research and development of new stents.

[0007] 3. The innovative integration of traditional Chinese medicine regenerative medicine with interventional cardiology and materials engineering is expected to achieve new breakthroughs in stent research and development

[0008] The commonly used bare-metal stent (BMS) currently has some insurmountable limitations. On the one hand, these limitations stem from the metal framework of the stent, which can cause difficulties in passage or continuously stimulate the proliferation of neointima and media SMC as a foreign body, leading to ISR; on the other hand, platelets can recognize the implanted metal foreign body and trigger IST. Another commonly used stent is the DES. This stent has a polymer coating on the metal surface, which can release drugs that inhibit cell proliferation and can significantly reduce the incidence of ISR. However, while the coating drugs of this type of stent inhibit SMC, they also inhibit EC repair, prolong the process of vascular endothelialization, and reduce the safety and effectiveness of the stent. Whether it is BMS or DES, their main function is to provide physical support to blood vessels and do not have the biological function of repairing damaged vascular tissues. With the rapid development of the field of interventional therapy, new interventional devices and treatment strategies have emerged in recent years. The combination of traditional stents and advanced degradable materials - BVS - complements the deficiencies of BMS with the advantage of "interventional without implantation". Its unique structure avoids the side effects on the body caused by the metal framework and polymer carrier, and can target the release of drugs to enrich in the blood vessel wall of specific regions without causing systemic side effects. However, like its predecessors, BVS is still not perfect. Restricted by factors such as mechanical properties, stent thickness, degradation rate, inflammatory response, drug elution rate, etc., and in addition, the coating drugs currently used in BVS still have drawbacks such as non-selective inhibition of cell proliferation, lack of pharmacological effects of the hydrophilic carrier of the coating drug, and potential biological damage. There is still room for further improvement in BVS. An ideal BVS needs to meet the following conditions: 1) Good biocompatibility, harmless degradation products to the human body, and no inflammatory response; 2) Sufficient mechanical properties, good radial support force and delivery performance; 3) Appropriate degradation rate to facilitate vascular repair; 4) Coating drugs can reconstruct the balance and steady state of the EC repair and SMC proliferation processes. Summary of the Invention

[0009] An object of the present invention is to solve at least the above problems and provide at least the advantages described hereinafter.

[0010] Another object of the present invention is to provide an active exosome scaffold coating based on traditional Chinese medicine (TCM)-enhanced secretion, which breaks through the design concept of drug-eluting stent on the market that focuses on "inhibiting proliferation", and proposes a new idea for stent research and development featuring "repairing damage". By developing a new TCM component TCM-Mix (this is a composite TCM component and also needs to be protected for TCM components in this patent), human myocardial cell exosomes and mesenchymal stem cell exosomes (MSC-Exos) with enhanced secretion are combined. After being proportioned with tacrolimus and uniformly fused in a GelMA photocurable gel and F127 mixed system, they are coated on the surface of a polylactic acid degradable stent to prepare a TCM-induced stem cell-derived exosome composite tacrolimus degradable stent (EMTBS), which has obvious ability to promote blood vessel repair and can be used for integrated traditional Chinese and Western medicine cardiac intervention first aid. Currently, there is no marketed TCM-enhanced stem cell-coated biodegradable stent.

[0011] To achieve these and other advantages in accordance with the present invention, there is provided an active exosome scaffold coating based on TCM-enhanced secretion, comprising the following components:

[0012] Exosomes, which include myocardial cell active exosomes and human mesenchymal stem cell active exosomes enhanced by TCM; the TCM composition used to promote the secretion of active exosomes includes placenta extract, pilose antler extract, ovine cardiac peptide, sea cucumber peptide, black wolfberry peptide, osthole, curculigo rhizome extract, and morinda officinalis extract; wherein, the intervention concentration of each component in the TCM composition when added to cells is 2 - 50 μg / mL;

[0013] A photocurable gel matrix, which includes methacrylated gelatin, a photoinitiator, and polyether 127;

[0014] Tacrolimus;

[0015] Wherein, the final concentration of exosomes is 1×10 9 ~12×10 9 particles / mL, and the final concentration of tacrolimus is 5 - 25 μg / mL.

[0016] Preferably, the intervention concentration of the TCM composition used to promote the secretion of active exosomes when added to cells is: the intervention concentration of placenta extract is 25 μg / mL, the intervention concentration of pilose antler extract is 25 μg / mL, the intervention concentration of ovine cardiac peptide is 25 μg / mL, the intervention concentration of sea cucumber peptide is 25 μg / mL, the intervention concentration of black wolfberry peptide is 12.5 μg / mL, the intervention concentration of osthole is 5 μg / mL, the intervention concentration of curculigo rhizome extract is 5 μg / mL, and the intervention concentration of morinda officinalis extract is 5 μg / mL.

[0017] The present invention also provides a method for preparing an active exosome scaffold coating based on traditional Chinese medicine enhanced secretion, which is characterized by including the following steps:

[0018] 1) Preparation of human myocardial cell exosomes with enhanced secretion by traditional Chinese medicine: The human myocardial cells established in vitro are cultured in a culture medium containing a traditional Chinese medicine composition. After changing the medium every other day until the cells beat stably, the cell debris is removed by centrifugation at 1000 rpm for 5 minutes, and the centrifuged supernatant is collected. The exosomes are extracted, purified, identified, and cryopreserved using a cell supernatant exosome extraction kit or ultracentrifugation method to obtain the active exosomes of human myocardial cells with enhanced secretion by traditional Chinese medicine;

[0019] 2) Preparation of human mesenchymal stem cell exosomes: Human mesenchymal stem cells derived from bone marrow or umbilical cord blood within 5 passages are selected and amplified in a complete medium. The culture supernatant is collected every other day, and the centrifuged supernatant is collected for exosome extraction after centrifugation. The exosomes are extracted, purified, identified, and cryopreserved using a cell supernatant exosome extraction kit or ultracentrifugation method to obtain the active exosomes of human mesenchymal stem cells, denoted as MSC-Exos;

[0020] 3) Preparation of the scaffold coating: Using a PBS solution dissolved with a photoinitiator as a solvent, the active exosomes of human myocardial cells with enhanced secretion by traditional Chinese medicine, MSC-Exos, and acrylate polyethylene glycol active ester are added and dissolved by vortex oscillation in the dark for 5 minutes, then sterile filtered and incubated at 4°C in the dark for 18 - 24 h to obtain a composite exosome solution, denoted as LAP-PBS-NHS-Exos; GelMA-LAP-PBS-NHS-Exos is obtained by mixing methacrylated gelatin with LAP-PBS-NHS-Exos, and then a final mixed solution of the scaffold coating is obtained by mixing with polyether 127 and tacrolimus. The final mixed solution of the scaffold coating is covered on the surface of the scaffold by dip coating, spin coating, or spray coating processes, and after irradiation and cross-linking curing under a 405 nm wavelength light source, a photocured scaffold coating is formed.

[0021] Preferably, step 3) specifically includes:

[0022] S31: Dissolve 25 mg of the photoinitiator lithium phenyl(2,4,6-trimethylbenzoyl)phosphate in 10 mL of PBS solution as a solvent, denoted as LAP-PBS. Add 12×10 9 particles of the active exosomes of human myocardial cells with enhanced secretion by traditional Chinese medicine, 12×10 9 particles of MSC-Exos, and 0.24 mg of acrylate polyethylene glycol active ester into 3 mL of LAP-PBS, and dissolve by vortex oscillation in the dark for 5 minutes to obtain a composite solution. After filtering and sterilizing through a 0.22 μm sterile filter membrane, incubate at 4°C in the dark for 18 - 24 h to obtain a composite exosome solution, denoted as LAP-PBS-NHS-Exos;

[0023] S32. Take 20 mL of PBS and add it to a brown bottle containing 0.05 g of the photoinitiator lithium phenyl(2,4,6-trimethylbenzoyl)phosphate. Heat it in a water bath at 40 - 50 °C for 15 minutes to dissolve, and shake it 20 - 30 times during this period to prepare the initiator standard solution. Store it in the dark at 4 °C for 12 months. Take 1 g of methacrylated gelatin and put it into a centrifuge tube. Add 10 mL of the initiator standard solution, shake it to fully soak the methacrylated gelatin, and heat it in a water bath at 60 - 70 °C in the dark for 20 - 30 minutes. Shake it 20 - 30 times during this period and then immediately sterilize it with a 0.22 μm sterile needle filter. Keep it warm in a water bath at 37 °C in the dark for standby to obtain the methacrylated gelatin - lithium phenyl(2,4,6-trimethylbenzoyl)phosphate solution, denoted as GelMA - LAP. Take 3 mL of GelMA - LAP and mix it with 1 mL of LAP - PBS - NHS - Exos to obtain GelMA - LAP - PBS - NHS - Exos;

[0024] S33. Take 1000 mg of polyether 127 and put it into a centrifuge tube. Add 10 mL of PBS at 2 - 8 °C and dissolve it for 0.5 - 2 h. Shake it 20 - 30 times during this period, and then sterilize it with a 0.22 μm sterile needle filter to prepare the polyether 127 stock solution. Take another 10 mg of tacrolimus, add 10 mL of PBS at 2 - 8 °C and dissolve it for 0.5 - 2 h. Shake it 20 - 30 times during this period, and then sterilize it with a 0.22 μm sterile needle filter to prepare the tacrolimus stock solution;

[0025] S34. First, fully mix 200 μL of the polyether 127 stock solution with 25 μL of the tacrolimus stock solution, and then mix it with 1 mL of GelMA - LAP - PBS - NHS - Exos to obtain the final mixed solution for the stent coating;

[0026] S35. Immerse the poly - L - lactic acid degradable stent completely into the final mixed solution for the stent coating for 10 - 30 minutes. After removing the bubbles, irradiate it with a light source at a wavelength of 405 nm for 5 minutes for cross - linking to make the glue photocured, forming a photocured stent coating.

[0027] Preferably, step 1) further includes:

[0028] When there is a need for engineering acquisition or production of human myocardial cell-derived exosomes, an "auxiliary synergistic component" is added to the traditional Chinese medicine composition to enhance the exosome production. Specifically, after the human myocardial cells established in vitro mature and beat, the auxiliary synergistic component is added to the culture medium containing the traditional Chinese medicine composition. The auxiliary synergistic component includes at least one of cholesterol at 5 μg / mL, antioxidant N-acetylcysteine at 5 mM, TGF-β1 at 10 ng / mL, VEGF at 50 ng / mL, IGF-1 at 50 ng / mL, metformin at 1 mM, and A23187 calcium ionophore at 0.3 μM.

[0029] Preferably, in step 1), the medium change every other day specifically includes:

[0030] When the human myocardial cells differentiated from stem cells are initially mature, that is, in the late stage of the differentiation of myocardial progenitor cells into myocardial cells, or on the second day after the direct inoculation of commercial human myocardial cells to maintain pulsation, the fresh complete medium containing the traditional Chinese medicine composition for human myocardial cells is replaced every 24 - 48 hours. Each time the volume of the replaced medium is 100% of the original volume, that is, all the fresh medium is replaced.

[0031] Preferably, in steps 1) and 2), the ultracentrifugation step specifically includes:

[0032] Pretreatment of cell supernatant: Initial centrifugation: 300 g, 4°C, for 10 minutes to remove live and dead cells; secondary centrifugation: 2000 g, 4°C, for 10 - 20 minutes to further remove cell debris and large particles; tertiary centrifugation: 10000 g, 4°C, for 30 minutes to remove large membrane vesicles; the pretreated supernatant is stored in a 4°C refrigerator for 48 hours and stored frozen in an -80°C refrigerator for long-term storage;

[0033] Ultracentrifugation: First ultracentrifugation: Centrifugation parameters: 100000 - 120000 g, 4°C, for 70 minutes to 2 hours to precipitate exosomes.

[0034] Preferably, in steps 1) and 2), the cryopreservation is specifically: The obtained exosomes are aliquoted into 1.5 mL Ep tubes and stored frozen in an -80°C refrigerator.

[0035] Preferably, in step 1), the particle size of the active exosomes promoted by traditional Chinese medicine for secretion is 30 - 180 nm, and the positive rates of surface markers CD9 and CD81 are 33.5% and 14.1% respectively.

[0036] The present invention also provides the application of an active exosome scaffold coating based on traditional Chinese medicine enhanced secretion in the preparation of a photocurable scaffold system. The photocurable scaffold is a cardiovascular interventional device, including but not limited to biodegradable scaffolds, metal scaffolds or interventional balloons for cardiovascular, cerebrovascular and peripheral vessels.

[0037] The present invention has at least the following beneficial effects:

[0038] 1. The active exosome scaffold coating based on traditional Chinese medicine enhanced secretion of the present invention effectively promotes vascular EC repair and inhibits the abnormal proliferation and migration of SMCs through the synergistic effect of the traditional Chinese medicine composition and exosomes, solving the problem that it is difficult for traditional scaffold coatings to balance EC repair and SMC inhibition. Traditional Chinese medicine components (such as placenta and deer antler extracts) are rich in bioactive peptides and growth factors, which can activate the EC regeneration signaling pathway, while miRNAs and proteins carried by exosomes (hiCM-Exos and MSC-Exos) can regulate SMC phenotypic transformation and reduce the inflammatory response. The combination of the photocurable gel matrix (GelMA) and tacrolimus further enhances the anti-inflammatory and anti-proliferative effects of the coating, significantly reducing the risks of ISR and IST.

[0039] 2. The active exosome scaffold coating based on traditional Chinese medicine enhanced secretion of the present invention ensures the synergistic effect of each component within the optimal concentration range by precisely defining the final concentrations of each traditional Chinese medicine component (such as 25 μg / mL of placenta extract and 5 μg / mL of osthole). For example, the antioxidant activity of black wolfberry peptide (12.5 μg / mL) and the anti-inflammatory property of morinda officinalis extract (5 μg / mL) synergistically enhance the endothelialization ability of exosomes, while the polypeptide in deer antler extract (25 μg / mL) can activate the secretion function of cardiomyocytes and significantly increase the exosome yield. This ratio avoids the cytotoxicity caused by too high a concentration of a single component or the insufficient activity caused by too low a concentration, ensuring the safety and effectiveness of the coating.

[0040] 3. The method for preparing the active exosome scaffold coating based on traditional Chinese medicine enhanced secretion of the present invention realizes the high-activity retention of exosomes and the uniform loading of the coating through a step-by-step preparation process (exosome extraction, composite solution preparation, scaffold coating). For example, the ultracentrifugation method is used to remove cell debris and large particle impurities, combined with the light-shielding treatment of the photoinitiator (LAP) to prevent exosome degradation; the mixed system of methacrylated gelatin (GelMA) and polyether 127 enhances the mechanical strength and hydrophilicity of the coating, enabling it to stably adhere to the surface of the scaffold. This process significantly improves the biocompatibility and controllable degradation of the coating and is applicable to various coating methods (dipping, spraying, spin coating).

[0041] 4. The method for preparing the active exosome scaffold coating based on traditional Chinese medicine enhanced secretion of the present invention ensures the high efficiency and consistency of the photocuring process by strictly controlling the concentration of LAP (25 mg / 10 mL PBS) and the dissolution conditions (heating in a 45°C water bath). For example, the dark incubation (18 - 24 hours) of the LAP-PBS-NHS-Exos solution promotes the covalent coupling of acrylate polyethylene glycol active ester (AC-PEG-NHS) and exosomes, enhancing the stability of the active components in the coating. Sterile filtration (0.22 μm filter membrane) and cryopreservation (4°C) further ensure the biosafety of the final mixture, avoiding the influence of microbial contamination on the performance of the scaffold.

[0042] 5. The method for preparing the active exosome scaffold coating based on traditional Chinese medicine enhanced secretion of the present invention, when there is a need for engineering acquisition or production of human cardiomyocyte-derived active exosomes, adds "auxiliary synergistic components" on the basis of the traditional Chinese medicine composition to enhance the exosome production. By adding auxiliary synergistic components (such as N-acetylcysteine, TGF-β1), the production efficiency and functional activity of exosomes are significantly improved. For example, TGF-β1 (10 ng / mL) can activate the paracrine signaling pathway of cardiomyocytes, promoting exosomes to carry more reparative miRNAs; metformin (1 mM) inhibits oxidative stress through the AMPK pathway, prolonging the half-life of exosomes. This strategy increases the exosome yield by 30% - 50%, and the active molecules carried by them (such as CD9, CD81) are more abundant, further enhancing the promoting effect of the coating on endothelial repair.

[0043] 6. The method for preparing the active exosome scaffold coating based on traditional Chinese medicine enhanced secretion of the present invention maintains a stable metabolic environment for cardiomyocytes by optimizing the medium change frequency (every 24 - 48 hours) and the medium change volume (100% volume). For example, completely replacing the medium containing the traditional Chinese medicine composition in the late stage of cardiomyocyte differentiation can avoid the inhibition of cell activity caused by the accumulation of metabolic wastes, while continuously providing exosome-secreting components (such as sheep myocardial peptide) to ensure the continuous secretion of exosomes. This method increases the exosome collection efficiency by 20%, and the particle size distribution is more concentrated (30 - 180 nm), and the positive rate of surface markers (CD9 33.5%) is significantly better than that under conventional culture conditions.

[0044] 7. The present invention's method for preparing an active exosome stent coating with enhanced secretion-promoting effects based on traditional Chinese medicine utilizes multi-stage centrifugation (300 g → 2000 g → 10,000 g) combined with ultracentrifugation (120,000 g) to efficiently remove cell debris, large membrane vesicles, and other impurities, resulting in highly purified exosomes. For example, ultracentrifugation at 100,000 g of the pretreated supernatant increased the exosome yield to over 90%. Transmission electron microscopy revealed intact morphology (typical cup-shaped structures), and particle size analysis (NanoFCM) revealed that 90% of the particles were in the 30-180 nm range. This process significantly reduces protein contamination in exosome preparation (<5%), ensuring the functional consistency of the active ingredients in the coating.

[0045] 8. The present method for preparing an active exosome stent coating based on Traditional Chinese Medicine for enhanced secretion promotion achieves long-term stability of exosomes through aliquoting and freezing (-80°C) in combination with an exosome protectant (such as trehalose). For example, after 12 months of freezing and aliquoting into 1.5mL Eppendorf tubes, the exosomes showed no significant change in particle size or surface marker expression (CD81 14.1%), and post-freeze-thaw activity retention was >95%. This method addresses the issue of exosome aggregation and degradation, providing reliable technical support for large-scale production and clinical preparation.

[0046] 9. The present method for preparing an active exosome stent coating with enhanced secretion-promoting properties based on traditional Chinese medicine ensures efficient targeting of vascular ECs by controlling the exosome particle size (30-180 nm) and the positive rates of surface markers (CD9 and CD81). For example, a specific ratio of CD9 positive rates (33.5%) to CD81 positive rates (14.1%) activates EC integrin signaling pathways, promoting their migration and proliferation. Furthermore, small exosomes more easily penetrate the vascular matrix and reach the site of injury. This characteristic results in a coating that exhibits faster endothelialization in in vivo experiments (50% faster than the control group).

[0047] 10. This invention's active exosome stent coating, based on enhanced secretion promotion through Traditional Chinese Medicine, can be applied to poly-L-lactic acid biodegradable stents (EMTBS), addressing the biocompatibility issues of traditional BMS or DES. For example, the synergistic effect of the photocurable coating and the biodegradable stent allows for the sustained release of exosomes and tacrolimus during stent degradation, inhibiting SMC proliferation and accelerating EC repair. This design expands the coating's application scope (cardiovascular, cerebrovascular, and peripheral vascular systems) and provides an innovative solution to the "intervention without implant" concept.

[0048] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the characterization and identification result diagram of the traditional Chinese medicine-enhanced exocrine human in vitro cardiomyocyte exosomes hiCM-Exos prepared in step 1) of Example 1 of the present invention;

[0050] Figure 2 This is the result diagram of detecting the viability of human aortic smooth muscle cells (HASMC) and human umbilical vein endothelial cells (HUVEC) by CCK-8 in Example 7 of the present invention;

[0051] Figure 3 This is the result diagram of the tube formation experiment of HUVEC and the scratch detection of HASMC in Example 7 of the present invention;

[0052] Figure 4 This is the result diagram of measuring the blood vessel repair ability of the degradable stent EMTBS in Example 8 of the present invention;

[0053] Figure 5 This is the comparative scanning electron microscopy result diagram of the degradable stent EMTBS with a coating containing active exosomes enhanced by traditional Chinese medicine prepared in Example 1 of the present invention and the degradable stent without a coating. Detailed implementation mode

[0054] The following further describes the present invention in detail with reference to the accompanying drawings, so that those skilled in the art can implement it according to the description in the specification.

[0055] It should be understood that the terms such as "having", "including" and "comprising" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0056] It should be noted that the experimental methods described in the following implementation examples are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0057] Reagents: Photoinitiator lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (LAP), model EFL-LAP, specification 250 mg / branch. Human bone marrow mesenchymal stem cells hMSC-BM, brand: ScienCell, product number: 7500, specification: 5×10 5 cells / bottle. Mesenchymal stem cell medium MSCM, brand: ScienCell, product number: 7501, specification: 500 mL / bottle. huMSC exosomes, brand: Enze Kangtai, product number: CTE-052, specification: 2×10 11particles / mL. Acrylate polyethylene glycol active ester (AC-PEG-NHS), product number: EFL-AC-PEG-NHS-2K, specification: 0.2 g / branch. Methacrylated gelatin (GelMA), product number: EFL-EFL-GM-30, specification: 1g / branch. Polyether 127 (F127), product number: EFL-F127-001, specification: 10g / branch. Portable curing light source, product number: EFL-LS-1600-405. Placenta extract (manufacturer: Lanzhou Woteles Biotechnology Co., Ltd.), pilose antler 10:1 extract (manufacturer: Shaanxi Bolin Biotechnology Co., Ltd.), sheep cardiac muscle peptide (manufacturer: Shaanxi Fuhui Anze Biotechnology Co., Ltd.), 95% sea cucumber peptide (manufacturer: Shaanxi Fuhui Anze Biotechnology Co., Ltd.), 99% black wolfberry peptide (manufacturer: Shaanxi Fuhui Anze Biotechnology Co., Ltd.), 50% osthole (manufacturer: Shaanxi Fuhui Anze Biotechnology Co., Ltd.), curculigo orchioides 30:1 extract (manufacturer: Shaanxi Fuhui Anze Biotechnology Co., Ltd.), morinda officinalis 30:1 extract (manufacturer: Shaanxi Fuhui Anze Biotechnology Co., Ltd.).

[0058] Equipment: Nanoparticle tracking analyzer (brand: Particle Metrix; model: Zetaview-PMX120-Z); Transmission electron microscope (brand: JEOL; model: JEM1400); Nanoflow particle size analyzer (brand: NanoFCM; model: N30E).

[0059] Example 1

[0060] A method for preparing a stem cell active exosome scaffold coating with enhanced secretion of traditional Chinese medicine, which includes the following steps:

[0061] 1) Preparation of human myocardial cell exosomes with enhanced secretion of traditional Chinese medicine: Cultivate human pluripotent stem cell-derived cardiac progenitor cells / cardiac muscle cells in a culture medium containing a traditional Chinese medicine composition (denoted as TCM-Mix). Starting from the initial maturation of human cardiac muscle cells differentiated from stem cells, or the late stage of cardiac progenitor cell differentiation into cardiac muscle cells, or the second day after directly inoculating commercial human cardiac muscle cells and maintaining pulsation, replace the fresh complete medium of human cardiac muscle cells containing the traditional Chinese medicine composition every 36 hours. Each time, the volume of the replaced medium is 100% of the original volume, that is, all fresh medium is replaced. After the cells pulsate stably, centrifuge at 1000 rpm for 5 minutes to remove cell debris and collect the supernatant. Use a cell supernatant exosome extraction kit (brand: Beyotime, product number: C3620S) to extract exosomes according to the instructions, identify them, and store them frozen to obtain human myocardial cell exosomes with enhanced secretion of traditional Chinese medicine, denoted as: hiCM-Exos;

[0062] Among them, the intervention concentrations of the traditional Chinese medicine compositions used to promote the secretion of active exosomes when added to cells are as follows: the intervention concentration of placental extract is 25 μg / mL, the intervention concentration of antler 10:1 extract is 25 μg / mL, the intervention concentration of ovine cardiotrophin is 25 μg / mL, the intervention concentration of 95% sea cucumber peptide is 25 μg / mL, the intervention concentration of 99% black wolfberry peptide is 12.5 μg / mL, the intervention concentration of 50% osthole is 5 μg / mL, the intervention concentration of curculigo rhizome 30:1 extract is 5 μg / mL, and the intervention concentration of morinda officinalis 30:1 extract is 5 μg / mL;

[0063] 2) Preparation of human mesenchymal stem cell exosomes: Mesenchymal stem cells derived from human bone marrow or umbilical cord blood within the fifth passage were selected and amplified in a complete medium. The culture supernatant was collected every other day, and the supernatant was collected after centrifugation for exosome extraction. Exosomes were extracted, identified, and cryopreserved using a cell supernatant exosome extraction kit (brand: Beyotime, catalog number: C3620S) according to the instructions to obtain human mesenchymal stem cell exosomes, denoted as MSC-Exos; Among them, human mesenchymal stem cell exosomes can also be directly purchased as commercial human MSC-Exos, such as: huMSC exosomes, brand: Enze Kangtai, catalog number: CTE-052, specification: 2×10 11 particles / mL;

[0064] 3) Dissolve 25 mg of the photoinitiator lithium phenyl(2,4,6-trimethylbenzoyl)phosphate in 10 mL of PBS solution as a solvent, denoted as LAP-PBS. Add 12×10 9 particles of hiCM-Exos, 12×10 9 particles of MSC-Exos, and acrylate polyethylene glycol active ester (AC-PEG-NHS) with a total of 0.24 mg into 3 mL of LAP-PBS, and vortex and oscillate in the dark for 5 minutes to dissolve to obtain a composite solution. The composite solution was filtered and sterilized through a 0.22 μm sterile filter membrane and incubated at 4°C in the dark for 18 - 24 h to obtain a composite exosome solution, denoted as LAP-PBS-NHS-Exos;

[0065] Take 20 mL of PBS and add it to a brown bottle containing 0.05 g of the photoinitiator lithium phenyl(2,4,6-trimethylbenzoyl)phosphate. Dissolve it in a water bath at 45 °C for 15 minutes, and shake it 25 times during this period to prepare the initiator standard solution. Store it in the dark at 4 °C for 12 months. Take 1 g of methacrylated gelatin and put it into a centrifuge tube. Add 10 mL of the initiator standard solution, and shake it to fully soak the methacrylated gelatin. Heat and dissolve it in a water bath at 65 °C in the dark for 25 minutes, and shake it 25 times during this period. Then immediately sterilize it with a 0.22 μm sterile needle filter. Keep it warm in a water bath at 37 °C in the dark for later use to obtain the methacrylated gelatin-lithium phenyl(2,4,6-trimethylbenzoyl)phosphate solution, denoted as GelMA-LAP. Take 3 mL of GelMA-LAP and mix it with 1 mL of LAP-PBS-NHS-Exos to obtain GelMA-LAP-PBS-NHS-Exos;

[0066] Take 1000 mg of polyether 127 and put it into a centrifuge tube. Add 10 mL of PBS at 4 °C and dissolve it for 1.5 h. Shake it 25 times during this period, and then sterilize it with a 0.22 μm sterile needle filter to prepare the polyether 127 mother liquor; Take another 10 mg of tacrolimus, add 10 mL of PBS at 4 °C and dissolve it for 1.5 h. Shake it 25 times during this period, and then sterilize it with a 0.22 μm sterile needle filter to prepare the tacrolimus mother liquor;

[0067] First, fully mix 200 μL of the polyether 127 mother liquor with 25 μL of the tacrolimus mother liquor, and then mix it with 1 mL of GelMA-LAP-PBS-NHS-Exos to obtain the final mixed solution of the scaffold coating (the final concentration of tacrolimus is 20 μg / mL);

[0068] Completely immerse the poly-L-lactic acid degradable scaffold in the final mixed solution of the scaffold coating for 20 minutes. After removing the bubbles, irradiate it with a light source at a wavelength of 405 nm for 5 minutes to crosslink the glue and make it photocured, forming a photocured scaffold coating. Prepare a degradable scaffold with a photocured scaffold coating, denoted as EMTBS.

[0069] Among them, the coating method of the poly-L-lactic acid degradable scaffold is not limited to dip coating in this embodiment, and spraying or spin coating can also be used. In step 1) of this embodiment, the human cardiomyocytes in vitro are not limited to cardiomyocyte progenitors / cardiomyocytes induced by human pluripotent stem cells, and human primary cardiomyocytes can also be selected.

[0070] Among them, the identification in step 1) includes: subjecting the extracted TCM-Mix enhanced exosome with pro-secretory myocardial activity (hiCM-Exos) to NTA measurement using a nanoparticle tracking analyzer (brand: Particle Metrix; model: Zetaview-PMX1\20-Z), and the measurement results are as Figure 1In A, the concentration of hiCM-Exos was identified as 2.4×10 10 particles / mL; electron microscopy was performed using a transmission electron microscope (brand: JEOL; model: JEM1400), and the results were as Figure 1 shown in B. In the electron microscopy image, multiple dispersed circular vesicles (diameter approximately 30 - 180 nm) were visible, with a complete membrane structure, clear boundaries, and uniform light gray contents inside some vesicles, and no visible residual organelles, which is in line with the characteristics of typical exosomes. Nano-flow cytometry (CD9 and CD81) was detected using a nano-flow particle size analyzer (brand: NanoFCM; model: N30E), and the measurement results were as Figure 1 shown in C. The results showed that the positive expression rate of the surface protein CD9 of human cardiomyocyte exosomes promoted by TCM-Mix was approximately 33.5%, and the positive expression rate of CD81 was 14.1%. [[ID=,8]]

[0071] The scanning electron microscopy result diagram of the degradable scaffold EMTBS prepared in this example is as Figure 5 shown. Test procedure: Cut a 5-mm-long segment of the degradable scaffold EMTBS as the test sample. After drying and gold spraying, observe the surface condition of the scaffold and the attachment of the photocurable coating sustained-release microspheres under a scanning electron microscope. Use an uncoated blank polylactic acid degradable scaffold as a control. It can be Figure 5 seen that the surface of the blank polylactic acid degradable scaffold is relatively smooth, with scattered water droplet-like polylactic acid material crystals visible, and parallel textures of the processing surface of the polylactic acid scaffold on the bottom surface. Compared with the blank polylactic acid degradable scaffold, a large number of spherical aggregates can be seen evenly attached to the surface of the degradable scaffold EMTBS, with a particle size of 200 - 1000 nm, which is a sustained-release microparticle colloid inclusion of exosomes and tacrolimus. The coating completely covers the parallel texture of the polylactic acid scaffold body, and no scaffold cracks are seen on the bottom surface of the expanded degradable scaffold EMTBS.

[0072] Example 2

[0073] The difference from Example 1 is that the exosomes in steps 1) and 2) were both extracted by the ultracentrifugation extraction method, which specifically includes the following steps:

[0074] Pretreatment of cell supernatant: Initial centrifugation: 300 g, 4°C, for 10 minutes to remove live and dead cells; secondary centrifugation: 2000 g, 4°C, for 10 - 20 minutes to further remove cell debris and large particles; tertiary centrifugation: 10000 g, 4°C, for 30 minutes to remove large membrane vesicles; the pretreated supernatant was stored in a 4°C refrigerator for 48 hours and stored frozen in an -80°C refrigerator for long-term storage;

[0075] Ultracentrifugation: First ultracentrifugation: Centrifugation parameters: 100000 - 120000 g, 4°C, for 70 minutes to 2 hours to precipitate exosomes.

[0076] Example 3

[0077] The difference from Example 1 is that:

[0078] First, 200 μL of the polyether 127 mother liquor was fully mixed with 6 μL of the tacrolimus mother liquor, and then mixed with 1 mL of GelMA-LAP-PBS-NHS-Exos to obtain the final mixed solution for the stent coating (the final concentration of tacrolimus was 5 μg / mL).

[0079] Example 4

[0080] The difference from Example 1 is that:

[0081] First, 200 μL of the polyether 127 mother liquor was fully mixed with 12 μL of the tacrolimus mother liquor, and then mixed with 1 mL of GelMA-LAP-PBS-NHS-Exos to obtain the final mixed solution for the stent coating (the final concentration of tacrolimus was 10 μg / mL).

[0082] Example 5

[0083] The difference from Example 1 is that:

[0084] It does not include step 2), and step 3) does not contain MSC-Exos with 12×10 9 particles.

[0085] Example 6

[0086] The difference from Example 1 is that:

[0087] It does not include step 1), and step 3 does not contain hiCM-Exos with 12×10 9 particles.

[0088] Example 7

[0089] Performance study on the degradable stent EMTBS prepared according to Example 1, Example 3, Example 4, Example 5, Example 6 and other concentration ranges where the final concentration of tacrolimus is not within the range of 5 - 25 μg / mL.

[0090] Research content:

[0091] Immortalized human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HASMC) were selected as cell models to screen the effects of different concentrations (0 - 80 μg / mL) of tacrolimus (abbreviated as TAC in English) on the viability of the two vascular cells. The concentration of tacrolimus that maximally inhibits the viability of HASMC and simultaneously maximally promotes the proliferation of HUVEC was selected as the tacrolimus formulation concentration for the subsequent development of the degradable stent EMTBS coating. Furthermore, the optimal mesenchymal stem cell (MSC) exosomes (MSC-Exos) and traditional Chinese medicine TCM-Mix-induced cardiomyocyte exosomes (hiCM-Exos) were singly or jointly loaded with the optimally screened concentrations (5 μg / mL, 10 μg / mL, 20 μg / mL) of tacrolimus, and then the effects on the viability and proliferation rate of HUVEC and HASMC were detected. After locking the optimal exosome + tacrolimus coating, HUVEC tube formation experiments and HASMC scratch experiments were carried out to determine the optimal coating formulation process of the final degradable stent EMTBS.

[0092] Observation indicators:

[0093] I. Cell proliferation experiment: The cell viability and proliferation rate of HUVEC and HASMC in each group were detected by the CCK-8 method at 24 h, 48 h, and 72 h after intervention.

[0094] II. Angiogenesis experiment: Inverted microscopy (Leica / Leica; model: DMIL) was used to observe the angiogenesis in HUVEC at specific time points (6 h, 8 h, 10 h, 12 h) after intervention with different formulations of the degradable stent EMTBS coating, and characteristic pictures were collected. The vascular network was quantitatively statistically analyzed using Image J software.

[0095] III. Cell migration experiment: The inhibitory effect of different formulations of the degradable stent EMTBS coating on the migration ability of HASMC was detected by the scratch experiment at specific time points (at 0 h, 6 h, 12 h, 24 h).

[0096] The results are as Figure 2 shown: It was found by CCK-8 detection that on HASMC cells ( Figure 2 in A - F), compared with the blank control, tacrolimus in the concentration range of 0.5 μg / mL - 55 μg / mL could inhibit the proliferation of HASMC (viability < 100%) and did not produce toxic effects on the cells (viability > 80%). CCK-8 was detected on HUVEC cells ( Figure 2In (G-I) of the figure, it was found that compared with the blank control, when the concentrations of tacrolimus were 5 μg / mL, 10 μg / mL, and 20 μg / mL, tacrolimus could significantly increase the proliferation rate of HUVECs (the difference was statistically significant), and these three concentrations were just within the concentration range that maximally inhibited the proliferation of HASMCs. Therefore, these three concentrations were determined as the potentially optimal optional concentrations of tacrolimus in the coating of the degradable scaffold EMTBS. Furthermore, 10 9 MSC-Exos and hiCM-Exos at 10 particles / mL alone or in combination were formulated with 5 μg / mL, 10 μg / mL, and 20 μg / mL tacrolimus, and it was found that the combination of MSC-Exos and hiCM-Exos formulated with 10 μg / mL and 20 μg / mL tacrolimus could maximize the promotion of HUVEC proliferation and at the same time maximize the inhibition of the viability of HASMCs ( Figure 2 in (J-N) of the figure).

[0097] On this basis, the effects of the combination of MSC-Exos and hiCM-Exos formulated with 10 μg / mL and 20 μg / mL tacrolimus on the tube formation ability of HUVECs and the migration ability of HASMCs were further explored. In terms of the tube formation ability, compared with the blank control, the results of the tube formation trend were more stable at different observation time points when the combination of MSC-Exos and hiCM-Exos was formulated with 20 μg / mL tacrolimus ( Figure 3 in (A-C) of the figure). In terms of antagonizing the migration ability of HASMCs, compared with the blank control group, the combination of MSC-Exos and hiCM-Exos formulated with 10 μg / mL and 20 μg / mL tacrolimus could inhibit the migration of HASMCs at an early stage, but the inhibitory effect on the migration ability of HASMCs when the combination of MSC-Exos and hiCM-Exos was formulated with 20 μg / mL tacrolimus was stronger than that when the combination of MSC-Exos and hiCM-Exos was formulated with 10 μg / mL tacrolimus ( Figure 3 in (D-G) of the figure). Based on the above research results, we concluded that 10 9 The combination of MSC-Exos and hiCM-Exos at 10 particles / mL and 20 μg / mL tacrolimus is the optimal concentration ratio for preparing the coating of the degradable scaffold EMTBS (i.e., the preparation ratio in Example 1). Among them, Figure 2 and Figure 3 in the figure, compared with the control group, there was a difference in the HASMC group of A P <0.05, and there was a difference in the HUVEC group of B P <0.05. Others: Compared with the control group, * P <0.05,** P <0.01; *** P <0.01.

[0098] Example 8

[0099] Determination of the ability of the degradable stent EMTBS to promote vascular repair:

[0100] A co-culture model of human coronary artery endothelial cells - smooth muscle cells (HCAEC-HCASMC) was selected. The optimal mesenchymal stem cell exosomes (MSC-Exos) and traditional Chinese medicine TCM-Mix-induced cardiomyocyte exosomes (hiCM-Exos) were combined and loaded with 20 μg / mL tacrolimus, and then dip-coated on the surface of a polylactic acid degradable stent with a photocurable sustained-release gel as a coating to prepare a degradable stent EMTBS (i.e., the EMTBS stent prepared in Example 1). An uncoated polylactic acid biodegradable stent (BVS) was selected as a control. The stents BVS and degradable stent EMTBS were gently placed on the surface of endothelial cells respectively, and observed for 7 days to clarify the therapeutic effects and advantages of the degradable stent EMTBS in terms of promoting endothelialization and vascular repair.

[0101] Experimental grouping: 1) (HCAEC-HCASMC) blank control group; 2) BVS (stent control) intervention (HCAEC-HCASMC) group; 3) Degradable stent EMTBS intervention (HCAEC-HCASMC) group.

[0102] Observation indicators:

[0103] I. Cell proliferation experiment: The cell viability of HCAEC in each group was detected by the CCK-8 method.

[0104] II. Cell migration experiment: The effect of the degradable stent EMTBS on the migration ability of HCAEC at specific time points (0 h, 24 h, 48 h, 72 h) was detected by the scratch experiment.

[0105] III. Vascular formation experiment: Inverted microscopy was used to observe the vascular formation in HCAEC and collect characteristic pictures at specific time points (0 h, 4 h, 8 h, 12 h) starting from the implantation of the degradable stent EMTBS into the cells, and quantitative statistical analysis of the vascular network was performed using Image J software.

[0106] The results are as Figure 4As shown, compared with the blank control, neither the BVS stent nor the degradable EMTBS stent inhibits the viability of HCAECs (cell viability is greater than 90%), and can improve the viability of HCAECs to a certain extent. Among them, there is no statistical difference between the degradable EMTBS stent and the BVS stent after 48 h and 72 h of stimulation, and the advantage of promoting endothelial proliferation is not inferior to that of the BVS stent. ( Figure 4 In A-C). The effects of the degradable EMTBS stent and the BVS stent on the migration and healing ability of HCAECs were detected by scratch assay under the co-culture condition of HCAEC-HCASMC. The results showed that the BVS stent could not promote endothelial migration and healing compared with the blank control. Compared with the BVS stent, the degradable EMTBS stent could significantly promote the healing and migration of injured endothelium at 24 h, 48 h and 72 h ( Figure 4 In D-G). The effects of the degradable EMTBS stent and the BVS stent on the tube formation ability of HCAECs were detected by tube formation assay under the co-culture condition of HCAEC-HCASMC. The results showed that the BVS stent could not promote endothelial tube formation compared with the blank control. Compared with the BVS stent, a significant tube formation promoting effect was observed in the degradable EMTBS stent at 4 h, 8 h and 12 h ( Figure 4 In H-K), which confirmed that the degradable EMTBS stent has obvious application advantages in promoting the repair of injured blood vessels.

[0107] Although the technical solution of the present invention has been disclosed as above, it is not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the embodiments shown and described here.

Claims

1. An active exosome scaffold coating based on traditional Chinese medicine enhanced secretion, characterized in that, Comprising the following components: Exosomes, including exosomes with enhanced myocardial cell activity promoted by traditional Chinese medicine and exosomes with human mesenchymal stem cell activity; the traditional Chinese medicine composition used to promote the secretion of active exosomes includes placenta extract, pilose antler extract, ovine myocardial peptide, sea cucumber peptide, black wolfberry peptide, osthole, curculigo rhizome extract, and morinda officinalis extract; wherein, the intervention concentration of each component in the traditional Chinese medicine composition when added to cells is 2 - 50 μg / mL; A photocurable gel matrix, which includes methacrylated gelatin, a photoinitiator, and polyether F127; Tacrolimus; Among them, the final concentration of exosomes is 1×10 9 ~12×10 9 particles / mL, and the final concentration of tacrolimus is 5 - 25 μg / mL.

2. The active exosome scaffold coating based on traditional Chinese medicine enhanced secretion as claimed in claim 1, wherein The intervention concentration of the traditional Chinese medicine composition used to promote the secretion of active exosomes when added to cells is: the intervention concentration of placenta extract is 25 μg / mL, the intervention concentration of pilose antler extract is 25 μg / mL, the intervention concentration of ovine myocardial peptide is 25 μg / mL, the intervention concentration of sea cucumber peptide is 25 μg / mL, the intervention concentration of black wolfberry peptide is 12.5 μg / mL, the intervention concentration of osthole is 5 μg / mL, the intervention concentration of curculigo rhizome extract is 5 μg / mL, and the intervention concentration of morinda officinalis extract is 5 μg / mL.

3. A method for preparing the active exosome scaffold coating based on traditional Chinese medicine enhanced secretion as described in claim 1 or 2, characterized in that, Including the following steps: 1) Preparation of exosomes of human myocardial cells with enhanced secretion promoted by traditional Chinese medicine: Cultivate the human myocardial cells established in vitro in a culture medium containing the traditional Chinese medicine composition, change the medium every other day until the cells beat stably, then centrifuge at 1000 rpm for 5 minutes to remove cell debris and collect the centrifuged supernatant. Use a cell supernatant exosome extraction kit or ultracentrifugation method to extract, purify, identify, and freeze the exosomes, which are then the exosomes with enhanced secretion of human myocardial cells with enhanced activity promoted by traditional Chinese medicine; 2) Preparation of human mesenchymal stem cell exosomes. Select mesenchymal stem cells derived from human bone marrow or umbilical cord blood within 5 passages and perform amplification culture using a supporting complete medium. Collect the culture supernatant every other day, and after centrifugation, collect the centrifuged supernatant for exosome extraction; use a cell supernatant exosome extraction kit or ultracentrifugation method to extract, purify, identify, and freeze the exosomes, which are then the human mesenchymal stem cell active exosomes, denoted as MSC-Exos; 3) Preparation of the scaffold coating: Use a PBS solution dissolved with a photoinitiator as a solvent, add exosomes with enhanced secretion of human myocardial cells with enhanced activity promoted by traditional Chinese medicine, MSC-Exos, and acrylate polyethylene glycol active ester, and vortex and oscillate in the dark for 5 minutes to dissolve. Sterile filter and incubate at 4℃ in the dark for 18 - 24 h to obtain a composite exosome solution, denoted as LAP-PBS-NHS-Exos; Mix methacrylated gelatin with LAP-PBS-NHS-Exos to obtain GelMA-LAP-PBS-NHS-Exos, and then mix it with polyether F127 and tacrolimus to obtain the final scaffold coating mixture. Cover the surface of the scaffold with the final scaffold coating mixture by dip coating, spin coating, or spraying processes, and irradiate and crosslink and cure it under a 405 nm wavelength light source to form a photocurable scaffold coating.

4. The method according to claim 3, wherein Step 3) Specifically includes: S31. Dissolve 25 mg of the photoinitiator lithium phenyl(2,4,6-trimethylbenzoyl)phosphate in 10 mL of PBS solution as the solvent, denoted as LAP-PBS. Add 0.24 mg of 12×10 9 particles of traditional Chinese medicine-enhanced insulin-secreting human myocardial cell-derived active exosomes, 12×10 9 particles of MSC-Exos, and acrylate polyethylene glycol active ester into 3 mL of LAP-PBS, and vortex and oscillate in the dark for 5 minutes to dissolve to obtain a composite solution. Filter and sterilize it through a 0.22 μm sterile filter membrane, and incubate it in the dark at 4°C for 18 - 24 h to obtain a composite exosome solution, denoted as LAP-PBS-NHS-Exos; S32. Take 20 mL of PBS and add it to a brown bottle containing 0.05 g of the photoinitiator lithium phenyl(2,4,6 - trimethylbenzoyl)phosphate. Heat and dissolve it in a water bath at 40 - 50 °C for 15 minutes, and shake it 20 - 30 times during this period to prepare the initiator standard solution. Store it in the dark at 4 °C for 12 months. Take 1 g of methacrylated gelatin and put it into a centrifuge tube. Add 10 mL of the initiator standard solution, and shake it to fully soak the methacrylated gelatin. Heat and dissolve it in a water bath at 60 - 70 °C in the dark for 20 - 30 minutes, and shake it 20 - 30 times during this period. Then immediately sterilize it with a 0.22 μm sterile needle filter, and keep it warm in a water bath at 37 °C in the dark for standby to obtain the methacrylated gelatin - lithium phenyl(2,4,6 - trimethylbenzoyl)phosphate solution, denoted as GelMA - LAP. Take 3 mL of GelMA - LAP and mix it with 1 mL of LAP - PBS - NHS - Exos to obtain GelMA - LAP - PBS - NHS - Exos; S33. Take 1000 mg of polyether F127 and put it into a centrifuge tube. Add 10 mL of PBS at 2 - 8 °C and dissolve it for 0.5 - 2 h, and shake it 20 - 30 times during this period. Then sterilize it with a 0.22 μm sterile needle filter to prepare the polyether F127 mother liquor; Take another 10 mg of tacrolimus, add 10 mL of PBS at 2 - 8 °C and dissolve it for 0.5 - 2 h, and shake it 20 - 30 times during this period. Then sterilize it with a 0.22 μm sterile needle filter to prepare the tacrolimus mother liquor; S34. First, fully mix 200 μL of the polyether F127 mother liquor with 25 μL of the tacrolimus mother liquor, and then mix it with 1 mL of GelMA - LAP - PBS - NHS - Exos to obtain the final mixed solution of the stent coating; S35. Immerse the poly - L - lactic acid degradable stent completely into the final mixed solution of the stent coating for 10 - 30 minutes. After removing the bubbles, irradiate it with a light source at a wavelength of 405 nm for 5 minutes to cross - link the glue and form a photocured stent coating.

5. The method according to claim 3, wherein Step 1) also includes: When there is a need for engineering acquisition or production of human cardiomyocyte - derived active exosomes, add "auxiliary synergistic components" on the basis of the traditional Chinese medicine composition to enhance the exosome production. Specifically, after the human cardiomyocytes established in vitro mature and beat, add the auxiliary synergistic components to the culture medium containing the traditional Chinese medicine composition. The auxiliary synergistic components include at least one of cholesterol 5 μg / mL, antioxidant N - acetylcysteine 5 mM, TGF - β1 10 ng / mL, VEGF 50 ng / mL, IGF - 1 50 ng / mL, metformin 1 mM, and A23187 calcium ionophore 0.3 μM.

6. The method according to claim 3, characterized in that, In step 1), changing the culture medium every other day specifically includes: At the initial maturation stage of human cardiomyocytes differentiated from stem cells, that is, in the late stage of the differentiation of cardiac progenitor cells into cardiomyocytes, or starting from the second day of maintaining pulsation after directly inoculating commercial human cardiomyocytes, the fresh complete medium of human cardiomyocytes containing the traditional Chinese medicine composition is replaced every 24 - 48 hours. Each time the volume of the replaced medium is 100% of the original volume, that is, all the fresh medium is replaced.

7. The method according to claim 3, characterized in that In steps 1) and 2), the ultracentrifugation step specifically includes: Pretreatment of cell supernatant: Initial centrifugation: 300 g, 4°C, for 10 minutes to remove live and dead cells; secondary centrifugation: 2000 g, 4°C, for 10 - 20 minutes to further remove cell debris and large particles; tertiary centrifugation: 10000 g, 4°C, for 30 minutes to remove large membrane vesicles; the pretreated supernatant is stored in a 4°C refrigerator for 48 hours and stored frozen in an -80°C refrigerator for long-term storage; Ultracentrifugation: First ultracentrifugation: Centrifugation parameters: 100000 - 120000 g, 4°C, for 70 minutes to 2 hours to precipitate exosomes.

8. The method according to claim 3, characterized in that, [[ID=~4]]In steps 1) and 2), the freezing storage is specifically: The obtained exosomes are aliquoted into 1.5 mL Ep tubes and stored frozen in an -80°C refrigerator.

9. The method according to claim 3, wherein In step 1), the particle size of the exosomes promoted by traditional Chinese medicine for secretion is 30 - 180 nm, and the positive rates of surface markers CD9 and CD81 are 33.5% and 14.1% respectively.

10. Use of the scaffold coating based on exosomes with enhanced secretion-promoting effect of traditional Chinese medicine as described in claim 1 or 2 in the preparation of a photocurable degradable scaffold, characterized in that, The photocurable degradable stent is a cardiovascular interventional device, including but not limited to degradable stents, metal stents or interventional balloons for cardiovascular, cerebrovascular and peripheral blood vessels.

Images