Search

CN-121971711-A - Active exosome stent coating, vascular stent, preparation method and application

CN121971711ACN 121971711 ACN121971711 ACN 121971711ACN-121971711-A

Abstract

The invention provides an active exosome stent coating, a vascular stent, a preparation method and application thereof, and belongs to the technical field of biological medicines and interventional instruments. The active exosome stent coating adopts stem cell source myocardial cell active exosome which is enhanced and promoted by traditional Chinese medicine active components. Compared with commercial myocardial cells, the stem cell-derived myocardial cells have more remarkable vascular and tissue repair regeneration promotion effects and stronger cytokine secretion capacity. The preparation method of the active exosome stent coating adjusts the introduction sequence and premixing mode of active ingredients such as exosome, medicine and the like, and enhances the homogeneity and stability. The vascular stent prepared by the active exosome stent coating has reasonable sequential release dynamics design, can realize the accurate targeted regulation and control of human coronary smooth muscle cells (HCASMC) in vitro, does not influence the restoration potential of endothelial cells, and solves the contradiction between 'antiproliferation and healing promotion' of the traditional stent.

Inventors

  • LI HONGMEI

Assignees

  • 北京中医药大学

Dates

Publication Date
20260505
Application Date
20260409

Claims (10)

  1. 1. An active exosome stent coating comprises exosome, a photoinitiator LAP, methacryloylated gelatin, polyether F127 and tacrolimus, wherein the exosome comprises a cardiomyocyte active exosome and a human mesenchymal stem cell active exosome which are enhanced and promoted by traditional Chinese medicine active components; The traditional Chinese medicine active components comprise placenta extract, hairy antler extract, sheep myocardial peptide, sea cucumber peptide, lycium ruthenicum murr peptide, osthole, curculigo rhizome extract and morinda officinalis extract, wherein the intervention concentration of each component added into myocardial cells is 2-50 mug/mL, the final concentration of exosomes in the active exosome stent coating is 1X 10 9 ~ 12×10 9 particles/mL, and the final concentration of tacrolimus in the active exosome stent coating is 5-25 mug/mL.
  2. 2. The active exosome stent coating according to claim 1, wherein the method for preparing the cardiomyocyte active exosome for enhanced secretion by using the active ingredients of the traditional Chinese medicine comprises the following steps: 1) Inoculating the pluripotent stem cells into a six-hole plate pre-paved with Matrigel matrix, adding mTESR stem cell culture medium containing Y27632 factors into each hole, performing first culture, replacing mTESR stem cell culture medium without Y27632 factors after the first culture, and performing second culture until the cell density in the six-hole plate reaches 100%; 2) After the cell density in the six-hole plate reaches 100%, carrying out directional differentiation on myocardial cells; The directional differentiation of the myocardial cells comprises the steps of sequentially adopting a culture medium containing chemical small molecules and a culture medium containing traditional Chinese medicine active components for differentiation culture; 3) Collecting supernatant of the myocardial cells subjected to directional differentiation after the myocardial cells are subjected to directional differentiation, centrifuging, and collecting supernatant after centrifuging for filtering and sterilizing to obtain stem cell-derived myocardial cell extract; 4) Extracting exosomes from the stem cell-derived cardiomyocyte extract to obtain the cardiomyocyte active exosomes with the Chinese medicinal active ingredient for strengthening and promoting the secretion.
  3. 3. The active exosome stent coating according to claim 2, wherein the first culturing time is 10-12 hours, the medium is replaced every 24 h times during the second culturing, the time for directional differentiation of cardiomyocytes is 15 days, and the procedure for directional differentiation of cardiomyocytes comprises: On day 1, the culture was performed by replacing CDM medium containing 10. Mu.M small molecule CHIR 99021; on day 2, the CDM medium containing 5 ng/ml small molecule bFGF is replaced for culture; On day 3, the CDM medium is replaced for culture; On day 4, half of CDM medium was aspirated and half of CDM medium containing 10. Mu.M small molecule IWP2 was added for cultivation; Culturing for 1 day without changing liquid; On day 6, replacing the RPMI-1640 culture medium containing 3% serum substitute and 0.2% Chinese medicinal active component solution, and culturing; On day 7, replacing RPMI-1640 culture medium containing 0.2% of the Chinese medicinal active component solution, and culturing; changing an RPMI-1640 culture medium containing a Chinese medicinal active component solution with the volume concentration of 0.2% every 3 days for culturing; The concentration of placenta extract, cornu Cervi Pantotrichum extract, sheep myocardial peptide and sea cucumber peptide in the active component solution of the traditional Chinese medicine is 100 mg/mL, the concentration of black matrimony vine peptide in the active component solution of the traditional Chinese medicine is 50 mug/mL, and the concentration of osthole, rhizoma Curculiginis extract and radix Morindae officinalis extract in the active component solution of the traditional Chinese medicine is 20 mug/mL.
  4. 4. A method for preparing an active exosome stent coating according to any one of claims 1 to 3, comprising the steps of: mixing the exosomes with a solution of part of the photoinitiator LAP to obtain a first solution; Mixing the methacryloylated gelatin with the solution of the remaining photoinitiator LAP to obtain a second solution; mixing the first solution and the second solution to obtain a third solution; mixing a polyether F127 solution and a tacrolimus solution to obtain a fourth solution; And mixing the third solution and the fourth solution to obtain the active exosome stent coating.
  5. 5. The method according to claim 4, wherein the total number of the myocardial cell active exosomes and the human mesenchymal stem cell active exosomes intensified by the active ingredients of the traditional Chinese medicine is 2.4x10 10 particles, the volume of the solution of the partial photoinitiator LAP is 3 mL, and the concentration of LAP in the solution of the partial photoinitiator LAP is 2.5 mg/mL.
  6. 6. The method according to claim 4, wherein the concentration of LAP in the solution of the remaining photoinitiator LAP is 2.5 mg/mL and the concentration of the methacryloylated gelatin in the second solution is 100 mg/mL.
  7. 7. The method of claim 4, wherein the first solution and the second solution are present in a volume ratio of 1:3.
  8. 8. The use of an active exosome stent coating according to any one of claims 1 to 3 or an active exosome stent coating prepared by the preparation method according to any one of claims 4 to 7 in the preparation of vascular stents.
  9. 9. A vascular stent comprising a stent, characterized in that the outer surface of the stent is coated with the active exosome stent coating according to any one of claims 1 to 3 or the active exosome stent coating prepared by the preparation method according to any one of claims 4 to 7.
  10. 10. A method of preparing a vascular stent as defined in claim 9, comprising the steps of: Coating the active exosome stent coating according to any one of claims 1-3 or the active exosome stent coating prepared by the preparation method according to any one of claims 4-7 on the outer surface of the stent, and irradiating and crosslinking for 5-10 min under a 405 nm-wavelength light source to form a stable bioactive hydrogel coating, thereby obtaining the vascular stent.

Description

Active exosome stent coating, vascular stent, preparation method and application Technical Field The invention belongs to the technical field of biological medicine and interventional instruments, and particularly relates to an active exosome stent coating, a vascular stent, a preparation method and application. Background The vascular stent is a stent which is placed into an inner stent on the basis of the expansion and the formation of a lumen balloon so as to achieve the purposes of supporting a narrow occlusion section blood vessel, reducing the elastic retraction and the reshaping of the blood vessel and keeping the lumen blood flow smooth. Common vascular stents are metallic stents (BMS) and Drug Eluting Stents (DES). However, platelets can recognize implanted metallic foreign objects and trigger thrombus formation within the stent. In addition, vascular smooth muscle cells also proliferate excessively upon stimulation by the metallic stent. These factors lead to a higher risk of restenosis in BMS. The DES is formed by adding a layer of polymer coating on the surface of metal, and can release drugs for inhibiting cell proliferation. The DES reduces the occurrence probability of stent restenosis, but the medicines for inhibiting cell proliferation not only inhibit smooth muscle cells, but also inhibit vascular endothelial cells, prolong the covering process of the medicines on the stent, and reduce the safety and effectiveness of the stent. The main function of the BMS or the DES is to provide physical support for blood vessels, and the BMS or the DES has no biological function of repairing ischemia damaged tissues. To address the limitations of current BMS and DES, researchers have developed Exosome Eluting Stents (EES). The EES outer surface is coated with an exosome coating, which can improve the biocompatibility of the metal stent, promote vascular endothelialization, inhibit migration of vascular smooth muscle cells and reduce inflammatory reaction, thereby reducing the occurrence probability of restenosis. The biological activity of the exosome coating directly affects the function of the EES, and thus, increasing the biological activity of the exosome coating is an important point in EES development. Disclosure of Invention The invention aims to provide an active exosome stent coating, a vascular stent, a preparation method and application thereof, and has better vascular and tissue repair promoting and regenerating effects. The invention provides an active exosome stent coating, which comprises exosome, a photoinitiator LAP, methacryloylated gelatin, polyether F127 and tacrolimus, wherein the exosome comprises a traditional Chinese medicine active component for strengthening and promoting myocardial cell active exosome and human mesenchymal stem cell active exosome, the traditional Chinese medicine active component comprises placenta extract, pilose antler extract, sheep myocardial peptide, sea cucumber peptide, lycine, osthole, curculine extract and morinda officinalis extract, the intervention concentration of each component added into myocardial cells is 2-50 mug/mL, the final concentration of the exosome in the active exosome stent coating is 1X 10 9~ 12×109 -25 mug/mL, and the final concentration of tacrolimus in the active exosome stent coating is 5-25 mug/mL. Preferably, the preparation method of the myocardial cell active exosome for strengthening and promoting the secretion by the active components of the traditional Chinese medicine comprises the following steps: 1) Inoculating the pluripotent stem cells into a six-hole plate pre-paved with Matrigel matrix, adding mTESR stem cell culture medium containing Y27632 factors into each hole, performing first culture, replacing mTESR stem cell culture medium without Y27632 factors after the first culture, and performing second culture until the cell density in the six-hole plate reaches 100%; 2) After the cell density in the six-hole plate reaches 100%, carrying out directional differentiation on myocardial cells; The directional differentiation of the myocardial cells comprises the steps of sequentially adopting a culture medium containing chemical small molecules and a culture medium containing traditional Chinese medicine active components for differentiation culture; 3) Collecting supernatant of the myocardial cells subjected to directional differentiation after the myocardial cells are subjected to directional differentiation, centrifuging, and collecting supernatant after centrifuging for filtering and sterilizing to obtain stem cell-derived myocardial cell extract; 4) Extracting exosomes from the stem cell-derived cardiomyocyte extract to obtain the cardiomyocyte active exosomes with the Chinese medicinal active ingredient for strengthening and promoting the secretion. Preferably, the first culture time is 10-12 h, the medium is replaced every 24 h times in the second culture process, the directional differentiation time of the myocar