A composition for coating a surface of a stent and a stent comprising the same

Abstract

The present invention relates to a composition for coating a stent and a stent comprising the same. Specifically, the invention relates to a composition for coating the surface of a stent that includes nanovesicles utilizing mesenchymal stem cells, thereby increasing the absorption rate by cells and simultaneously alleviating inflammation at the site where the stent is implanted, and a stent comprising the same.

Description

Technology Field

[0001] The present invention relates to a composition for coating a stent and a stent comprising the same. Specifically, by including nanovesicles utilizing mesenchymal stem cells in a composition for coating the surface of a stent, the invention relates to a composition for coating a stent and a stent comprising the same that can increase the local delivery rate of a drug (NV) while simultaneously alleviating inflammation at the site where the stent is implanted. Background Technology

[0002] In the treatment of cardiovascular diseases and other vascular stenosis, stent implantation has established itself as an essential method for expanding blocked blood vessels and mechanically supporting them to restore blood flow. Initially, bare metal stents (BMS) made of materials such as stainless steel were primarily used; however, the damage to the blood vessel walls and inflammatory responses inevitably caused by stent insertion ultimately led to excessive proliferation and migration of smooth muscle cells, resulting in a serious side effect known as in-stent restenosis, where the inside of the stent narrows again.

[0003] To address this restenosis problem, drug-eluting stents (DES) were developed by coating the surface of a stent with an antiproliferative drug that inhibits the proliferation of vascular smooth muscle cells and releasing it locally. DES have brought about a revolutionary advancement in the field of cardiovascular intervention by dramatically reducing the restenosis rate.

[0004] However, the permanent polymer coating layer primarily used in first-generation DES remains in the body even after drug release is complete, and this has been identified as a cause of persistent inflammatory responses and blood clot formation, revealing limitations that increase the risk of fatal complications such as Very Late Stent Thrombosis (VLST). In addition, due to technical issues in the coating process, non-uniform coating thickness or delamination from the stent struts can hinder the stable release of the drug and pose a potential risk of post-procedure problems.

[0005] Ultimately, while current stent technology has largely achieved the goal of preventing restenosis, it faces long-term challenges such as the risk of stent thrombosis and incomplete vascular healing. Therefore, the present invention aims to overcome the limitations of existing stent coating technologies and contribute to improving the long-term clinical prognosis of patients by providing an innovative stent surface coating technology that maintains stable drug release behavior in vivo while possessing excellent biocompatibility and durability. The problem to be solved

[0006] The technical problem to be solved by the present invention is to provide a stent coating composition and a stent comprising the same, wherein a composition containing nanovesicles utilizing mesenchymal stem cells is coated on the surface of a stent to increase the efficiency of absorption of substances generated from mesenchymal stem cells by the cells and to alleviate inflammation at the site where the stent is implanted.

[0007] However, the problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0008] One embodiment of the present invention provides a composition for stent coating comprising mesenchymal stem cell-derived nanovesicles.

[0009] According to one embodiment of the present invention, the mesenchymal stem cell-derived nanovesicles may be formed from an extract obtained by applying pressure to mesenchymal stem cells.

[0010] According to one embodiment of the present invention, the extract may be passed through a filter having an average pore size of 0.1 μm or more and 20 μm or less.

[0011] According to one embodiment of the present invention, the extract may be passed sequentially through a first filter having an average pore size of 9 μm or more and 11 μm or less, a second filter having an average pore size of 4 μm or more and 6 μm or less, and a third filter having an average pore size of 0.1 μm or more and 1.0 μm or less.

[0012] According to one embodiment of the present invention, the concentration of the protein derived from the mesenchymal stem cell may be 0.5 mg / ml or more and 2.0 mg / ml or less.

[0013] According to one embodiment of the present invention, the number of particles of the mesenchymal stem cell-derived nanovesicles is 1 x 10 10 Pieces / ml or more 1 X 10 11 It may be less than or equal to one / ml.

[0014] According to one embodiment of the present invention, it may further include trehalose.

[0015] According to one embodiment of the present invention, the concentration of the trehalose may be 3 (w / v)% or more and 5 (w / v)% or less.

[0016] According to one embodiment of the present invention, it may further include phosphate-buffered physiological saline.

[0017] One embodiment of the present invention provides a stent comprising: a substrate comprising a biodegradable polymer material; and a mesenchymal stem cell-derived nanovesicle coating layer provided on the substrate and comprising the composition for stent coating.

[0018] According to one embodiment of the present invention, a hydrophilic coating layer containing a hydrophilic polymer may be provided between the substrate and the mesenchymal stem cell-derived nanovesicle coating layer.

[0019] According to one embodiment of the present invention, the hydrophilic polymer may be a polymer comprising phosphocholine groups.

[0020] According to one embodiment of the present invention, the biodegradable polymer material may comprise one or more selected from the group consisting of poly(L-lactic acid, PLLA), polyglycolide, poly p-dioxanone, polycaprolactone, trimethylene carbonate, polyhydroxyalkanoates, polypropylene fumarate, polyortho esters, other polyesters, polyanhydride, polyphosphazenes, polyalkyl cyanoacrylates, poloxamers, polyamino L-tyrosine, modified polysaccharides, oxidized cellulose, gelatin, and collagen. Effects of the invention

[0021] A stent coating composition according to one embodiment of the present invention can increase the efficiency of absorption of a substance derived from mesenchymal stem cells into cells, while simultaneously alleviating inflammation at the site where the stent is implanted.

[0022] A stent according to one embodiment of the present invention can promote the re-endothelialization process after the procedure, has an endothelial cell protective effect, and can improve safety regarding heat treatment applied during the crimping process. Brief explanation of the drawing

[0023] FIG. 1 is a flowchart of a method for manufacturing mesenchymal stem cell-derived nanovesicles in one embodiment of the present invention. FIG. 2(a) is a TEM image of a mesenchymal stem cell-derived nanovesicle according to one embodiment of the present invention. FIG. 2(b) is a tetraspanin marker analysis image of a mesenchymal stem cell-derived nanovesicle according to one embodiment of the present invention. FIG. 3(a) is a graph showing the protein content according to the additives included in a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention and whether freeze-drying is performed. FIG. 3(b) is a graph showing the particle size distribution of mesenchymal stem cell-derived nanovesicles according to the additives included in a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention and whether freeze-drying is performed. FIG. 3(c) is a graph showing the number of mesenchymal stem cell-derived nanovesicles according to the additives included in a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention and whether freeze-drying is performed. Figure 4 is a graph analyzing the internal genomic expression of mesenchymal stem cell-derived nanovesicles according to an additive included in a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 5(a) is a graph showing protein concentration after heat treatment of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 5(b) is a graph showing particle number distribution after heat treatment of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 5(c) is a graph showing RNA quantification inside mesenchymal stem cell-derived nanovesicles after heat treatment of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. Figure 6(a) is a photograph showing the shape of a water droplet on a surface according to the material of the substrate and the composition of the hydrophilic coating layer. Figure 6(b) is a graph showing the contact angle according to the material of the substrate and the composition of the hydrophilic coating layer. FIG. 7(a) is an image obtained by fluorescence staining and confocal microscopy analysis to confirm cell uptake in a human monocyte cell line of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 7(b) is a graph showing the expression level of TNF-α in a human monocyte cell line of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 7(c) is a graph showing the expression level of IL-1β in a human monocyte cell line of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 7(d) is a graph showing the expression level of IL-6 in a human monocyte cell line of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. Figure 7(e) is a graph showing the expression level of IL-13 in a human monocyte cell line of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 8(a) is an image obtained by fluorescent staining and confocal microscopy analysis to confirm oxidized LDL (low-density lipoprotein cholesterol) uptake in THP-1 macrophages of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 8(b) is a graph showing the amount of oxidized LDL uptake in THP-1 macrophages of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 9(a) is an image obtained by fluorescence staining and confocal microscopy analysis to confirm uptake in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 9(b) is a graph showing the expression level of TNF-α in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 9(c) is a graph showing the expression level of IL-1β in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 9(d) is a graph showing the expression level of IL-6 in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 9(e) is a graph showing the expression level of ICAM-1 in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 9(f) is a graph showing the expression level of VCAM-1 in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 9(g) is a graph showing the expression level of E-selectin in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 10(a) is an image showing the expression of VCAM-1 in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention, obtained by fluorescent staining and confocal microscopy analysis. FIG. 10(b) is a graph showing the expression amount of VCAM-1 in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. Figure 11(a) is a photograph showing migration over time when a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention is stimulated with LPS in human coronary artery endothelial cells (hCAECs). Figure 11(b) is a graph showing the relative amount of migration when a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention is stimulated with LPS in human coronary artery endothelial cells (hCAECs). FIG. 12(a) is a photograph showing the formation of blood vessels in human umbilical cord blood-derived endothelial cells (HUVECs) using a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 12(b) is a graph showing the number of junctions of blood vessels formed in human umbilical cord blood-derived endothelial cells (HUVECs) using a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 12(c) is a graph showing the number of master junctions of blood vessels formed in human umbilical cord blood-derived endothelial cells (HUVECs) using a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 12(d) is a graph showing the number of master segments of blood vessels formed in human umbilical cord blood-derived endothelial cells (HUVECs) using a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. Figure 12 (e) is a graph showing the total length of a blood vessel formed in human umbilical cord blood-derived vascular endothelial cells (HUVEC) using a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 13(a) is an image obtained by fluorescence staining and confocal microscopy analysis to confirm uptake in human aortic smooth muscle cells (hAoSMC) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 13(b) is a graph showing the expression level of TNF-α in human aortic smooth muscle cells (hAoSMC) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 13(c) is a graph showing the expression level of IL-1β in human aortic smooth muscle cells (hAoSMC) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 13(d) is a graph showing the expression level of IL-6 in human aortic smooth muscle cells (hAoSMC) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 13(e) is an image confirming that nanovesicles are absorbed into the intima (vascular endothelial cells) and delivered to smooth muscle cells in the media through an organ-on-a-chip of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 13(f) is a schematic diagram of an organ-on-a-chip. FIG. 14(a) is a photograph showing migration over time when a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention is stimulated with PDGF-BB, TNF-α, and INF-γ in human aortic smooth muscle cells (hAoSMC). FIG. 14(b) is a graph showing the relative amount of migration when a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention is stimulated with PDGF-BB, TNF-α, and INF-γ in human aortic smooth muscle cells (hAoSMC). FIG. 15(a) is an image obtained by fluorescence staining and confocal microscopy analysis to confirm α-smooth muscle actin expression in human aortic smooth muscle cells (hAoSMC) after stimulating them with PDGF-BB, TNF-α, and INF-γ using a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 15(b) is a graph showing the amount of α-smooth muscle actin expression in human aortic smooth muscle cells (hAoSMC) after stimulating them with PDGF-BB, TNF-α, and INF-γ using a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 16 (a) is a photograph showing the release of mesenchymal stem cell-derived nanovesicles over time from a stent according to one embodiment of the present invention. FIG. 16 (b) is a graph showing the release amount of mesenchymal stem cell-derived nanovesicles over time from a stent according to one embodiment of the present invention. Specific details for implementing the invention

[0024] Hereinafter, various embodiments of the present invention are described in detail to enable those skilled in the art to easily practice the present invention. However, these are merely examples provided for illustrative purposes, and the scope of the present invention is not limited by the following.

[0025] Unless otherwise limited, the detailed description defining or specifying embodiments may apply to all inventions and is not limited to the description of specific inventions. That is, the present disclosure also refers to combinations of embodiments disclosed separately. Additionally, unless otherwise specified, the singular form includes the plural form throughout the detailed description and appended claims.

[0026] In this specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components. However, unless explicitly stated otherwise, terms such as "composition" or "comprising" may include "essentially composition" and "composition."

[0027] In this specification, "A and / or B" means "A and B, or A or B".

[0028] In this specification, when a component is described as being "on" one component, it means that other components may be placed therein, rather than excluding the placement of other components between them, unless specifically stated otherwise. However, unless explicitly stated otherwise, "on" means "directly on," that is, encompassing a situation where no other components can be placed between them.

[0029] In this specification, 'nanovesicle' may mean a material in the form of a nano-sized vesicle extruded from a cell, including exosomes.

[0030] The drawings attached to this specification illustrate preferred embodiments of the invention and explain the principles of the invention together with the description of the invention, but the scope of the invention is not limited thereto. Meanwhile, the shapes, sizes, scales, or proportions of elements in the drawings included in this specification may be exaggerated to emphasize clearer explanations.

[0031] The present invention will be described in more detail below.

[0032] One embodiment of the present invention provides a composition for stent coating comprising mesenchymal stem cell-derived nanovesicles.

[0033] A stent coating composition according to one embodiment of the present invention can increase the absorption rate by cells while simultaneously alleviating inflammation at the site where the stent is implanted.

[0034] According to one embodiment of the present invention, the mesenchymal stem cell-derived nanovesicles may be formed from an extract obtained by applying pressure to mesenchymal stem cells. As described above, by forming the mesenchymal stem cell-derived nanovesicles from an extract obtained by applying pressure to mesenchymal stem cells, the active ingredients contained within the mesenchymal stem cells can be effectively delivered while the stent is inserted.

[0035] According to one embodiment of the present invention, the nanovesicle may be an exosome. As described above, by selecting the nanovesicle as an exosome, the absorption efficiency of the active ingredients included in the stent coating composition to the cells surrounding the stent can be improved.

[0036] According to one embodiment of the present invention, the extract may be passed through a filter having an average pore size of 0.1 μm or more and 20 μm or less. Specifically, the extract may be passed through a filter having an average pore size of 0.4 μm or more and 10 μm or less. As described above, by passing the extract through a filter having an average pore size within the above range, the formation of nanovesicles can be facilitated, and by forming nanovesicles of uniform size, the absorption efficiency for surrounding cells where the stent is inserted can be improved.

[0037] According to one embodiment of the present invention, the extract may be passed sequentially through a first filter having an average pore size of 9 μm or more and 11 μm or less, a second filter having an average pore size of 4 μm or more and 6 μm or less, and a third filter having an average pore size of 0.1 μm or more and 1.0 μm or less. Specifically, the first filter may have an average pore size of 9 μm or more and 11 μm or less. More specifically, the first filter may have an average pore size of 9.5 μm or more and 10.5 μm or less. Preferably, the first filter may have an average pore size of 10 μm. Furthermore, the second filter may have an average pore size of 4 μm or more and 6 μm or less. Specifically, the second filter may have an average pore size of 4.5 μm or more and 5.5 μm or less. Preferably, the second filter may have an average pore size of 5 μm. In addition, the third filter may have an average pore size of 0.1 μm or more and 1.0 μm or less. Specifically, the third filter may have an average pore size of 0.3 μm or more and 0.6 μm or less. Preferably, the third filter may have an average pore size of 0.4 μm. As described above, by controlling the average pore sizes of the first to third filters and passing through the first filter, the second filter, and the third filter in that order, the formation of nanovesicles can be facilitated, and nanovesicles of uniform size can be formed to improve the absorption efficiency of surrounding cells into which the stent is inserted.

[0038] According to one embodiment of the present invention, the concentration of the protein derived from the mesenchymal stem cells may be 0.5 mg / ml or more and 2.0 mg / ml or less. Specifically, the concentration of the protein derived from the mesenchymal stem cells may be 0.6 mg / ml or more and 1.9 mg / ml or less, 0.7 mg / ml or more and 1.8 mg / ml or less, 0.8 mg / ml or more and 1.7 mg / ml or less, 0.9 mg / ml or more and 1.6 mg / ml or less, 0.9 mg / ml or more and 1.5 mg / ml or less, 0.9 mg / ml or more and 1.4 mg / ml or less, 0.9 mg / ml or more and 1.3 mg / ml or less, 0.9 mg / ml or more and 1.2 mg / ml or less, or 0.9 mg / ml or more and 1.1 mg / ml or less. By controlling the concentration of the protein derived from the mesenchymal stem cells within the above-described range, inflammation at the site where the stent is implanted can be alleviated.

[0039] According to one embodiment of the present invention, the number of particles of the mesenchymal stem cell-derived nanovesicles is 1 x 10 10 Pieces / ml or more 1 X 10 11 It may be less than or equal to a number / ml. By controlling the number of nanovesicles derived from the mesenchymal stem cells within the above-mentioned range, inflammation at the site where the stent is implanted can be alleviated.

[0040] According to one embodiment of the present invention, the stent coating composition may further comprise trehalose. As described above, by further comprising trehalose, the stent coating composition can minimize the reduction of nanovesicle and protein concentrations upon freeze-drying. Furthermore, the reduction of miRNA upon freeze-drying can be minimized.

[0041] According to one embodiment of the present invention, the concentration of trehalose may be 3 (w / v)% or more and 5 (w / v)% or less. Specifically, the concentration of trehalose may be 3.5 (w / v)% or more and 4.5 (w / v)% or less. Preferably, the concentration of trehalose may be 4 (w / v)%. By controlling the concentration of trehalose as described above, the reduction in nanovesicle and protein concentrations due to freeze-drying can be minimized. Furthermore, the reduction in miRNA due to freeze-drying can be minimized.

[0042] According to one embodiment of the present invention, the stent coating composition may further comprise phosphate-buffered physiological saline. As described above, by further comprising phosphate-buffered physiological saline in the stent coating composition, the reduction in nanovesicle and protein concentrations due to freeze-drying can be minimized. Furthermore, the reduction in miRNA due to freeze-drying can be minimized.

[0043] According to one embodiment of the present invention, the stent coating composition may comprise the mesenchymal stem cell-derived nanovesicles, trehalose, and phosphate-buffered physiological saline. More specifically, the stent coating composition may comprise only the mesenchymal stem cell-derived nanovesicles, trehalose, and phosphate-buffered physiological saline. As described above, by including the mesenchymal stem cell-derived nanovesicles, trehalose, and phosphate-buffered physiological saline, the reduction in nanovesicle and protein concentrations due to freeze-drying can be minimized. Furthermore, the reduction in miRNA due to freeze-drying can be minimized.

[0045] One embodiment of the present invention provides a stent comprising: a substrate comprising a biodegradable polymer material; and a mesenchymal stem cell-derived nanovesicle coating layer provided on the substrate and comprising the composition for stent coating.

[0046] A stent according to one embodiment of the present invention can promote the re-endothelialization process after the procedure, has an endothelial cell protective effect, and can improve safety regarding heat treatment applied during the crimping process.

[0047] According to one embodiment of the present invention, the stent comprises a substrate containing a biodegradable polymer material. As described above, by including a substrate containing a biodegradable polymer material, the stent is maintained for the time required for blood vessel regeneration and then degrades, and has high crystallinity and excellent strength, thereby improving blood vessel support function.

[0048] According to one embodiment of the present invention, the nanovesicle coating layer is provided on the substrate. Specifically, the nanovesicle coating layer may be located at the outermost side of the stent, which is exposed to the outside. Furthermore, a separate layer may be provided between the substrate and the nanovesicle coating layer. As described above, by providing the nanovesicle coating layer on the substrate, the re-endothelialization process after the procedure can be promoted, and there is an endothelial cell protective effect.

[0049] According to one embodiment of the present invention, the nanovesicle coating layer comprises the stent coating composition. As described above, by the nanovesicle coating layer comprising the stent coating composition, the absorption rate by cells can be increased, and at the same time, inflammation at the site where the stent is implanted can be alleviated.

[0050] According to one embodiment of the present invention, a hydrophilic coating layer containing a hydrophilic polymer may be provided between the substrate and the mesenchymal stem cell-derived nanovesicle coating layer. As described above, by further including a hydrophilic coating layer containing a hydrophilic polymer provided between the substrate and the mesenchymal stem cell-derived nanovesicle coating layer, it is possible to prevent the substrate and the nanovesicle coating layer from peeling off.

[0051] According to one embodiment of the present invention, the hydrophilic polymer may be a polymer containing phosphocholine groups. By including the hydrophilic polymer as a polymer containing phosphocholine groups as described above, the adhesion between the non-hydrophilic layer and the hydrophilic layer is improved by the amphoteric phosphocholine groups, and the nanovesicle coating layer can be prevented from peeling off.

[0052] According to one embodiment of the present invention, the polymer comprising the phosphocholine group comprises methyl methacrylate (MMA), ethyl methacrylate (EMA), butyl methacrylate (BMA), 2-ethyl-hexyl methacrylate, lauryl methacrylate (LMA), hydroxyl-ethyl methacrylate (HEMA), choline methacrylate (CMA), PEG acrylate (PEGA), PEG methacrylate, 2-methacryloxyethylphosphorylcholine (MFC), n-vinylpyrrolidone (VP), methacrylic acid (MA), acrylic acid (AA), hydroxypropyl methacrylate (HPMA), hydroxypropyl methacrylamide, 3-trimethylsilyl-propyl methacrylate (TMSPMA), p-nitrophenioxycarbonyl (polyoxyethylene) methacrylate (MEONP), N-isopropylacrylamide (NIPAM), and n-octylacrylamide. Monomers selected from the group and MPC units may be polymerized. By selecting a polymer containing the phosphocholine group from the above, the adhesion between the substrate and the nanovesicle coating layer can be improved.

[0053] According to one embodiment of the present invention, the polymer containing the phosphocholine group may be poly(2-methacryloyloxyethyl phosphorylcholine co-n-butyl methacrylate, poly(MPC-co-BMA-co-methacrylic acid (MA); 40, 40, 20), poly(MPC-coBMA; 80, 20), poly(MPC-co-BMA-co-p-nitrophenioxycarbonyl (polyoxyethylene) methacrylate (MEONP); 30, 60, 10), or poly(MPC-co-n-octylacrylamide-co-NIPAM; 40, 30, 30). Preferably, the polymer containing the phosphocholine group may be poly(2-methacryloyloxyethyl phosphorylcholine co-n-butyl methacrylate). By selecting the polymer containing the phosphocholine group from the above, the substrate and the nanovesicle coating layer It can improve adhesion.

[0054] According to one embodiment of the present invention, the biodegradable polymer material may comprise one or more selected from the group consisting of poly(L-lactic acid, PLLA), polyglycolide, poly p-dioxanone, polycaprolactone, trimethylene carbonate, polyhydroxyalkanoates, polypropylene fumarate, polyortho esters, other polyesters, polyanhydride, polyphosphazenes, polyalkylcyanoacrylates, poloxamers, polyamino L-tyrosine, modified polysaccharides, oxidized cellulose, gelatin, and collagen. Specifically, the The biodegradable polymer material may be poly(L-lactic acid, PLLA). In particular, since the PLLA degrades slowly at a rate of about 2 to 5 years, it is maintained for the time required for blood vessel regeneration before degrading. Compared to other biodegradable polymer materials (e.g., poly(glycolide), poly(lactide-co-glycolide, polycaprolactone, etc.), it has high crystallinity and excellent strength, resulting in excellent blood vessel support function. In addition, the PLLA is L-type lactic acid present in the body, so it is highly biocompatible and safe, and has the advantage of being able to achieve desired physical properties by controlling amorphousness and crystallinity.

[0055] According to one embodiment of the present invention, the weight-average molecular weight of the biodegradable polymer material may be 250,000 or more and 530,000 g / mol or less. When the weight-average molecular weight of the biodegradable polymer material satisfies the above range, the required radial force strength and biodegradation period can be achieved when the stent processing process, including laser cutting, is performed and post-sterilization treatment is carried out.

[0056] One embodiment of the present invention provides a method for preparing a stent coating composition comprising the steps of: pressurizing mesenchymal stem cells and passing them through a filter (S10); and forming nanovesicles from the mesenchymal stem cell extract that has passed through (S30).

[0057] A method for preparing a stent coating composition according to one embodiment of the present invention can easily form nanovesicles from mesenchymal stem cell extracts.

[0058] According to one embodiment of the present invention, the filter may include pores of 0.1 μm or more and 20 μm or less. Specifically, the extract may be obtained by passing the mesenchymal stem cell extract through a filter having an average pore size of 0.4 μm or more and 10 μm or less. As described above, by passing the extract through a filter having an average pore size within the above range, the formation of nanovesicles can be facilitated, and by forming nanovesicles of uniform size, the absorption efficiency for surrounding cells where the stent is inserted can be improved.

[0059] According to one embodiment of the present invention, the step (S30) of passing through the filter may sequentially pass through a first filter having pores of 9 μm or more and 11 μm or less, a second filter having pores of 4 μm or more and 6 μm or less, and a third filter having pores of 0.1 μm or more and 1.0 μm or less. Specifically, the first filter may have an average pore size of 9 μm or more and 11 μm or less. More specifically, the first filter may have an average pore size of 9.5 μm or more and 10.5 μm or less. Preferably, the first filter may have an average pore size of 10 μm. Furthermore, the second filter may have an average pore size of 4 μm or more and 6 μm or less. Specifically, the second filter may have an average pore size of 4.5 μm or more and 5.5 μm or less. Preferably, the second filter may have an average pore size of 5 μm. Additionally, the third filter may have an average pore size of 0.1 μm or more and 1.0 μm or less. Specifically, the third filter may have an average pore size of 0.3 μm or more and 0.6 μm or less. Preferably, the third filter may have an average pore size of 0.4 μm. As described above, by controlling the average pore sizes of the first to third filters and passing through the first filter, the second filter, and the third filter in that order, the formation of nanovesicles can be facilitated, and nanovesicles of uniform size can be formed to improve the absorption efficiency of surrounding cells into which the stent is inserted.

[0060] According to one embodiment of the present invention, the method may further include a step (S20) of mixing trehalose, phosphate-buffered physiological saline, and a mixture thereof after the step (S10) of passing through the filter and before the step (S30) of forming the nanovesicles. More specifically, the stent coating composition may comprise only the mesenchymal stem cell-derived nanovesicles, trehalose, and phosphate-buffered physiological saline. As described above, by including the mesenchymal stem cell-derived nanovesicles, trehalose, and phosphate-buffered physiological saline, the reduction in nanovesicle and protein concentrations due to freeze-drying can be minimized. Furthermore, the reduction in miRNA due to freeze-drying can be minimized.

[0061] According to one embodiment of the present invention, the concentration of the protein derived from the mesenchymal stem cells may be 0.5 mg / ml or more and 2.0 mg / ml or less. Specifically, the concentration of the protein derived from the mesenchymal stem cells may be 0.6 mg / ml or more and 1.9 mg / ml or less, 0.7 mg / ml or more and 1.8 mg / ml or less, 0.8 mg / ml or more and 1.7 mg / ml or less, 0.9 mg / ml or more and 1.6 mg / ml or less, 0.9 mg / ml or more and 1.5 mg / ml or less, 0.9 mg / ml or more and 1.4 mg / ml or less, 0.9 mg / ml or more and 1.3 mg / ml or less, 0.9 mg / ml or more and 1.2 mg / ml or less, or 0.9 mg / ml or more and 1.1 mg / ml or less. By controlling the concentration of the protein derived from the mesenchymal stem cells within the above-described range, inflammation at the site where the stent is implanted can be alleviated.

[0062] According to one embodiment of the present invention, the concentration of trehalose may be 3 (w / v)% or more and 5 (w / v)% or less. Specifically, the concentration of trehalose may be 3.5 (w / v)% or more and 4.5 (w / v)% or less. Preferably, the concentration of trehalose may be 4 (w / v)%. By controlling the concentration of trehalose as described above, the reduction in nanovesicle and protein concentrations due to freeze-drying can be minimized. Furthermore, the reduction in miRNA due to freeze-drying can be minimized.

[0063] According to one embodiment of the present invention, a freeze-drying step (S50) may be further included after the step of forming nanovesicles (S30). By further including the freeze-drying step as described above, workability for coating on a stent can be improved.

[0064] According to one embodiment of the present invention, the freeze-drying step may be performed by freezing at -70 to -90 ℃ and then freeze-drying for 1 hour to 100 hours. Specifically, the freeze-drying may be performed for 12 hours to 36 hours. More specifically, the freezing may be performed for 24 hours, the freeze-drying step may be performed under a pressure of 1 to 7 mTorr, and the first, second, or third freeze-drying process may be performed.

[0065] Hereinafter, the present invention will be described in detail with reference to examples to specifically explain the invention. However, the embodiments according to the present invention may be modified in various different forms, and the scope of the present invention is not to be interpreted as being limited to the embodiments described below. The embodiments of this specification are provided to more completely explain the present invention to those with average knowledge in the art.

[0067] <실시예>

[0068] Mesenchymal stem cells were cultured. Specifically, human adipose-derived mesenchymal stem cells (hASC) (Lonza, PT-5006) were used and cultured in 10% FBS (Gibco, 16000-044), 1% P / S (Cytiva, SV30010), and DMEM-low (Gibco, 11885-084). The hASCs were cultured in a humidified 5% CO2 incubator at 37°C, and subculture was performed when the cells reached 80-90% confluency.

[0069] The hASCs (150 pi dish) that had reached 80-90% confluency were washed once with DPBS, and the cells were detached by treating with 0.25% trypsin for 3 minutes. Subsequently, the trypsin was neutralized using DMEM-low media (containing FBS), and a cell pellet was obtained by centrifuging at 1200 rpm for 3 minutes. The cell pellet was resuspended in DPBS, and after cell counting, the cells were extruded by sequentially passing through a first filter of 10 μm, a second filter of 5 μm, and a third filter of 0.4 μm to prepare an extract.

[0070] For washing the above extract, centrifugation was performed three times at 21,400 g for 30 minutes, and after removing the supernatant during the third washing, the pellet of the extract was resuspended in a 4(w / v)% trehalose solution to prepare a stent coating composition containing mesenchymal stem cell-derived nanovesicles.

[0071] Subsequently, the above stent coating composition was freeze-dried at -70 to -90 ℃ for 24 hours.

[0072] A stent-shaped substrate made of the biodegradable polymer poly(L-lactic acid, PLLA, molecular weight 400,000 g / mol) was coated with a composition containing poly(2-methacryloyloxyethyl phosphorylcholine co-n-butyl methacrylate), a polymer containing phosphocholine groups, and dried to form a hydrophilic coating layer.

[0073] Subsequently, the freeze-dried stent coating composition, which was resuspended in DPBS on the hydrophilic coating layer, was applied and dried to form a stent.

[0075] <실험예 1: 중간엽 줄기세포 유래 나노베지클을 포함하는 스텐트 코팅용 조성물의 특성 분석>

[0076] The characteristics of a stent coating composition containing mesenchymal stem cell-derived nanovesicles prepared through extruding were analyzed. The morphology and size of the stent coating composition containing the mesenchymal stem cell-derived nanovesicles were analyzed using transmission electron microscopy (TEM).

[0077] In addition, exosome-specific markers (CD81, CD63, CD9) were analyzed using a super-resolution microscope (dSTORM). Specifically, it was confirmed whether a stent coating composition containing extracted mesenchymal stem cell-derived nanovesicles possesses exosome characteristics.

[0078] FIG. 2(a) is a TEM image of a mesenchymal stem cell-derived nanovesicle according to one embodiment of the present invention. FIG. 2(b) is a tetraspanin marker analysis image of a mesenchymal stem cell-derived nanovesicle according to one embodiment of the present invention.

[0079] Referring to Figure 2 above, it was confirmed that mesenchymal stem cell-derived nanovesicles were formed according to an embodiment of the present invention, and it was confirmed that they have characteristics similar to exosomes from the expression of exosome-specific markers (CD81, CD63, CD9) from the mesenchymal stem cell-derived nanovesicles.

[0081] <실험예 2: 동결 건조 안전성 평가-입자 크기 및 수와 단백질 농도>

[0082] We analyzed whether there were any changes in protein concentration and particle size of the stent coating composition containing the mesenchymal stem cell-derived nanovesicles even after freeze-drying. The experiment was conducted by dividing the samples into a Saline group (control), a Saline (w / o. trehalose) group, and a 4% trehalose group. After freeze-drying, rehydration was performed, and 5 μL of each sample was recovered, mixed with a BCA solution, and reacted at 37°C for 30 minutes. After the reaction, the protein concentration was determined by measuring the absorbance at 562 nm. For nanoparticle tracking analysis (NTA), the corresponding samples were diluted 50–100-fold in DPBS and analyzed at a flow rate of 1–2 μL / min.

[0083] FIG. 3(a) is a graph showing the protein content according to additives and freeze-drying in a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 3(b) is a graph showing the particle size distribution of mesenchymal stem cell-derived nanovesicles according to additives and freeze-drying in a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 3(c) is a graph showing the number of mesenchymal stem cell-derived nanovesicles according to additives and freeze-drying in a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. In FIG. 3, "Saline" means not containing trehalose, and "Lyophilized" means that freeze-drying has been performed.

[0084] Referring to Figure 3 above, the stent coating composition (4% trehalose, Lyophilized) according to one embodiment of the present invention, with 4 (w / v)% trehalose added, showed particle size and protein concentration at the same level as the composition without freeze-drying (Saline). In contrast, it was confirmed that the particle size and protein concentration decreased when freeze-drying was performed without trehalose (Saline, Lyophilized). Ultimately, it was confirmed that the freeze-drying safety of the stent coating composition according to one embodiment of the present invention, with 4 (w / v)% trehalose added, was improved.

[0086] <실험예 3: 동결 건조 안전성 평가-miRNA 유전체 발현 분석>

[0087] 200 μL aliquots of a stent coating composition containing PBS without trehalose (Saline: control) and a stent coating composition containing 4 (w / v)% trehalose (4% trehalose: control) were each stored at -80 ℃ until immediately before analysis. Subsequently, miRNA prep was performed, and miRNA array analysis was conducted using an Affymetrix chip. Specific values ​​for sample volume and concentration were not set, and analysis was performed after confirming the miRNA prep QC results.

[0088] Figure 4 is a graph analyzing the internal genomic expression of mesenchymal stem cell-derived nanovesicles according to an additive included in a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention.

[0089] Referring to Figure 4 above, a stent coating composition containing PBS other than trehalose (Saline: control) shows a change in miRNA composition of about 80% after freeze-drying (a ratio including miRNA that increased / decreased by more than 1.5 fold and miRNA that was lost and not detected), while a stent coating composition containing 4 (w / v)% trehalose (4% trehalose: control) shows a loss of miRNA of about 25% after freeze-drying, thereby confirming that the stent coating composition according to one embodiment of the present invention can reduce miRNA loss.

[0091] <실험예 4: 열처리 안전성 분석- 입자 크기 및 수와 단백질 농도>

[0092] A stent coating composition containing mesenchymal stem cell-derived nanovesicles that were not freeze-dried (Fresh-NV, control group) was heat-treated in solution form (heated at 40°C for 300 seconds), and a stent coating composition containing mesenchymal stem cell-derived nanovesicles that were freeze-dried (Lyo-NV) was heat-treated in powder form (heated at 40°C for 300 seconds).

[0093] BCA and NTA analyses were performed in the same manner as in Experimental Example 2 above. After heat treatment, total RNA of the stent coating composition containing the mesenchymal stem cell-derived nanovesicles was extracted using the RNeasy mini kit (QIAGEN). Total RNA was quantified using RNA quantifour (Promega). Fluorescence intensity was measured using a microplate reader at excitation: 492 nm and emission: 540 nm according to the provided protocol.

[0094] FIG. 5(a) is a graph showing protein concentration after heat treatment of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 5(b) is a graph showing particle number distribution after heat treatment of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 5(c) is a graph showing RNA quantification inside mesenchymal stem cell-derived nanovesicles after heat treatment of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. In FIG. 5, Fresh-NV means that freeze-drying was not performed, and Lyo-NV means that freeze-drying was performed.

[0095] Referring to Figure 5 above, the degradation of the nanovesicle coating layer that may occur under the heat treatment conditions of the stent clamping process was confirmed, and no significant difference was observed in the experimental group heated at 40°C for 300 seconds under the heat treatment conditions.

[0097] <실험예 5: 친수성 코팅층의 친수성 분석-접촉각>

[0098] Distilled water was filled into a syringe while maintaining 25 ℃ and a relative humidity (RH) of 40%, and a water drop was dropped onto one surface of a hydrophilic coating layer. After waiting for 5 minutes for the water drop to spread, the contact angle formed between one surface of the hydrophilic coating layer and the water drop was measured after 5 minutes.

[0099] Figure 6(a) is a photograph showing the shape of a water droplet on a surface according to the material of the substrate and the composition of the hydrophilic coating layer. Figure 6(b) is a graph showing the contact angle according to the material of the substrate and the composition of the hydrophilic coating layer. In Figure 6, PLLA is poly(L-lactic acid), and PMB30W is poly(2-methacryloyloxyethyl phosphorylcholine co-n-butyl methacrylate).

[0100] Referring to Figure 6 above, it was confirmed that the contact angle of the PLLA surface is larger than the contact angle of the poly(2-methacryloyloxyethyl phosphorylcholine co-n-butyl methacrylate surface, and through this, it was confirmed that the poly(2-methacryloyloxyethyl phosphorylcholine co-n-butyl methacrylate surface is hydrophilic.

[0102] <실험예 6: 인간 단핵구 세포주 THP-1에서의 uptake 및 항염증 효과 확인>

[0103] THP-1 is a monocyte-derived suspension cell, and differentiation into macrophage-like cells (adherent cells) can be induced through treatment with phorbol 12-myristate 13-acetate (PMA). Using THP-1 in vitro All experiments were conducted by differentiating into adherent cells through PMA treatment.

[0104] To confirm uptake in the human monocyte cell line THP-1, experiments were performed using the following procedure.

[0105] Prepare media containing 100 nM PMA, and 2.4 X 10 6 Mix cells with media supplemented with PMA and seed into 24 wells (1 x 10⁶ 5 Differentiation was carried out for 24 hours after cell / well. Afterward, a resting period of 24 hours was conducted in media not containing PMA.

[0106] After the above resting period, DiO-labeled mesenchymal stem cell-derived nanovesicles (DiO-labeled NV) were mixed into media and treated with cells for 4 hours.

[0107] Afterward, the cells were washed with DPBS, fixed with 4% paraformaldehyde for 5 minutes, and then washed again with DPBS.

[0108] Didium-Did solution was diluted in media and cells were labeled for 30 minutes. After staining the nuclei with DAPI solution, the cells were analyzed using a confocal microscope.

[0109] In addition, qPCR to confirm mRNA expressed in the human monocyte cell line THP-1 was performed using the following procedure.

[0110] Prepare media containing 100 nM PMA, and 2.4 X 10 6 Mix cells with media supplemented with PMA and seed into 24 wells (1 x 10⁶ 5 Differentiation was carried out for 24 hours after cell / well. Afterward, it was carried out for a 24-hour resting period in media not containing PMA.

[0111] Subsequently, inflammation was induced by treatment with LPS (1 μg / mL) and interferon-γ (20 ng / mL), and a stent coating composition containing mesenchymal stem cell-derived nanovesicles was simultaneously treated with LPS and interferon-γ. After 24 hours, RNA prep was performed using the RNeasy mini kit, and cDNA was synthesized to proceed with qPCR.

[0112] FIG. 7(a) is an image obtained by fluorescence staining and confocal microscopy analysis to confirm cell uptake in a human monocyte cell line of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 7(b) is a graph showing the expression level of TNF-α in a human monocyte cell line of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 7(c) is a graph showing the expression level of IL-1β in a human monocyte cell line of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 7(d) is a graph showing the expression level of IL-6 in a human monocyte cell line of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. Figure 7(e) is a graph showing the expression level of IL-13 in a human monocyte cell line of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention.

[0113] Referring to Fig. 7 above, fluorescence staining and confocal microscopy analysis of the nanovesicles were performed to confirm the cell uptake of the stent coating composition. Referring to Fig. 7(a), it was confirmed that uptake by immune cells occurred normally regardless of whether the nanovesicles were freeze-dried or not. Since the freeze-drying process is essential for the clinical application of the stent, all subsequent processes in vitro The experimental group consisted of Fresh-NV (no freeze-drying) and Lyo-NV (freeze-drying). Additionally, referring to Figures 7 (b) to (e), excellent anti-inflammatory effects were confirmed in Fresh-NV and Lyo-NV through rt-qPCR analysis.

[0115] <실험예 7: THP-1 macrophage에서의 oxidized LDL (oxLDL) 흡수 저해 분석>

[0116] Prepare media containing 100 nM PMA, and 2.4 X 10 6 Mix cells with media supplemented with PMA and seed into 24 wells (1 x 10⁶ 5 Differentiation was carried out for 24 hours after cell / well. Afterward, a resting period of 24 hours was conducted in media not containing PMA.

[0117] Subsequently, a stent coating composition containing LPS (1 μg / mL), interferon-γ (20 ng / mL), and mesenchymal stem cell-derived nanovesicles was applied to the cells for 24 hours. Then, DiI-labeled oxLDL was mixed into serum-free media and applied to the cells for 24 hours.

[0118] Afterward, the cells were washed with DPBS, fixed with 4% paraformaldehyde for 5 minutes, and then washed again with DPBS.

[0119] 0.5% Tween-20 solution was diluted in media and cells were permeabilized for 30 minutes.

[0120] After performing F-actin (ReadyProbes™ Reagent F-Actin Phalloidin Conjugates, R37110) staining, the cells were washed with DPBS, and the nuclei were stained with DAPI solution and analyzed using a fluorescence microscope.

[0121] FIG. 8(a) is an image obtained by fluorescent staining and confocal microscopy analysis to confirm oxidized LDL uptake in THP-1 macrophages of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 8(b) is a graph showing the amount of oxidized LDL uptake in THP-1 macrophages of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention.

[0122] Referring to Figure 8 above, THP-1 was treated with phorbol 12-myristate 13-acetate (PMA) to differentiate into macrophages, and then the uptake of oxLDL was evaluated. The inhibitory effect on oxLDL uptake was confirmed in the experimental group using nanovesicles.

[0124] <실험예 8: 인간 관상동맥 내피세포 hCAEC에서의 uptake 및 항염증 효과 확인>

[0125] Uptake by human coronary artery endothelial cells (hCAECs) was confirmed by the following procedure.

[0126] 1 X 10 4 Human coronary artery endothelial cells (hCAECs) were seeded into 24 wells at cells / well and cultured until 60-70% confluency.

[0127] Cultured cells were treated for 4 hours with a mixture of LPS (100 ng / mL) and DiO-labeled mesenchymal stem cell-derived nanovesicles (DiO-labeled NV) in media.

[0128] Afterward, the cells were washed with DPBS, fixed with 4% paraformaldehyde for 5 minutes, and then washed again with DPBS.

[0129] 0.5% Tween-20 solution was diluted in media and cells were permeabilized for 30 minutes.

[0130] After performing F-actin (ReadyProbes™ Reagent F-Actin Phalloidin Conjugates, R37112) staining, the cells were washed with DPBS, and the nuclei were stained with DAPI solution and analyzed using a fluorescence microscope.

[0131] To confirm the anti-inflammatory effect on human coronary artery endothelial cells (hCAECs), qPCR was performed using the following procedure.

[0132] 1 X 10 4 Human coronary artery endothelial cells (hCAECs) were seeded into 24 wells at cells / well and cultured until 60-70% confluency.

[0133] Inflammation was induced in cultured cells by treating them with LPS (100 ng / mL) for 6 hours, and a stent coating composition containing mesenchymal stem cell-derived nanovesicles was treated simultaneously with LPS.

[0134] Six hours after treatment, RNA prep was performed using the RNeasy mini kit, and cDNA was synthesized to proceed with qPCR.

[0135] FIG. 9(a) is an image obtained by fluorescence staining and confocal microscopy analysis to confirm uptake in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 9(b) is a graph showing the expression level of TNF-α in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 9(c) is a graph showing the expression level of IL-1β in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 9(d) is a graph showing the expression level of IL-6 in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 9(e) is a graph showing the expression level of ICAM-1 in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 9(f) is a graph showing the expression level of VCAM-1 in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 9(g) is a graph showing the expression level of E-selectin in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention.

[0136] Referring to Figure 9 above, rt-qPCR analysis was performed on pro-inflammatory markers and endothelial cell dysfunction markers in LPS-stimulated hCAECs, and both the stent coating composition without freeze-drying (Fresh-NV) and the stent coating composition with freeze-drying (Lyo-NV) inhibited the expression of pro-inflammation markers and endothelial cell dysfunction markers.

[0138] <실험예 9: hCAEC VCAM-1 발현 분석>

[0139] To analyze VCAM-1 expression in human coronary artery endothelial cells (hCAECs), the following procedure was performed.

[0140] 1 X 10 4 Human coronary artery endothelial cells (hCAECs) were seeded into 24 wells at cells / well and cultured until 60-70% confluency.

[0141] Cultured cells were treated with LPS (100 ng / mL) for 24 hours, and a stent coating composition containing mesenchymal stem cell-derived nanovesicles was treated simultaneously with LPS.

[0142] After treatment and 24 hours, the cells were washed with DPBS, fixed with 4% paraformaldehyde for 5 minutes, and then washed again with DPBS.

[0143] A blocking solution mixed with 250 mg BSA (bovine serum albumin), 5 mL DPBS, and 30 μL Tween-20 was treated at 200 μL / well and incubated at 4 ℃ for 1 hour.

[0144] Subsequently, the VCAM-1 primary antibody was diluted in blocking solution and treated, followed by incubation at room temperature for 1 hour. Afterward, it was incubated overnight at 4°C.

[0145] Cells were washed with DPBS, and the secondary antibody was diluted in DPBS and incubated at room temperature for 2 hours.

[0146] Subsequently, the cells were washed with DPBS, and the nuclei were stained with DAPI solution before being analyzed by confocal microscopy.

[0147] FIG. 10(a) is an image showing the expression of VCAM-1 in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention, obtained by fluorescent staining and confocal microscopy analysis. FIG. 10(b) is a graph showing the expression amount of VCAM-1 in human coronary artery endothelial cells (hCAECs) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention.

[0148] Referring to Figure 10 above, VCAM-1 protein expression in LPS-stimulated hCAECs was analyzed using a confocal microscope, and as a result, it was confirmed that VCAM-1 expression was inhibited in both the stent coating composition that was not freeze-dried (Fresh-NV) and the stent coating composition that was freeze-dried.

[0150] <실험예 10: hCAEC migration assay 분석>

[0151] 1 X 10 4 Human coronary artery endothelial cells (hCAECs) were seeded into 24 wells at cells / well and cultured until 100% confluency.

[0152] For cultured cells, the center of the well was cut using a 200 μL tip and the cells were washed with DPBS. Then, a stent coating composition containing LPS (100 ng / mL) and mesenchymal stem cell-derived nanovesicles was treated with LPS for 6 hours, and analyzed by microscope immediately after treatment and after 10 hours.

[0153] Figure 11(a) is a photograph showing migration over time when a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention is stimulated with LPS in human coronary artery endothelial cells (hCAECs). Figure 11(b) is a graph showing the relative amount of migration when a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention is stimulated with LPS in human coronary artery endothelial cells (hCAECs).

[0154] Referring to Figure 11 above, a scratch assay was performed on LPS-stimulated hCAECs. In this experiment, it was confirmed that nanovesicles can promote the reendothelialization process after stent implantation in patients with atherosclerosis.

[0156] <실험예 11: Tube formation assay 분석>

[0157] Matrigel (Corning, 354234) was evenly distributed over 24 wells and then gelled.

[0158] 5 ~ 7 X 10 4 Human umbilical cord blood-derived vascular endothelial cells (HUVECs) were evenly seeded into each well, and after 2 hours, a stent coating composition containing CyA and mesenchymal stem cell-derived nanovesicles was simultaneously treated.

[0159] After 12 hours, it was stained with Calcein-AM and analyzed using a fluorescence microscope.

[0160] FIG. 12(a) is a photograph showing the formation of blood vessels in human umbilical cord blood-derived endothelial cells (HUVECs) using a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 12(b) is a graph showing the number of junctions of blood vessels formed in human umbilical cord blood-derived endothelial cells (HUVECs) using a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 12(c) is a graph showing the number of master junctions of blood vessels formed in human umbilical cord blood-derived endothelial cells (HUVECs) using a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 12(d) is a graph showing the number of master segments of blood vessels formed in human umbilical cord blood-derived endothelial cells (HUVECs) using a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. Figure 12 (e) is a graph showing the total length of a blood vessel formed in human umbilical cord blood-derived vascular endothelial cells (HUVEC) using a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention.

[0161] Referring to Figure 12 above, a tube formation assay was performed using human umbilical cord blood-derived vascular endothelial cells (HUVEC). No difference in tube formation was observed depending on whether freeze-drying was performed, and an excellent angiogenesis-promoting effect was observed.

[0163] <실험예 12: 인간 대동맥 평활근 세포(hAoSMC)에서의 uptake 및 항염증 효과 분석>

[0164] Uptake of human aortic smooth muscle cells (hAoSMC) was confirmed by the following procedure.

[0165] 1 X 10 4Human aortic smooth muscle cells (hAoSMC) were seeded into 24 wells at cells / well and cultured until 60-70% confluency.

[0166] Cultured cells were treated for 4 hours with a mixture of TNF-α (10 ng / mL), interferon-γ (10 ng / mL), and DiO-labeled mesenchymal stem cell-derived nanovesicles (DiO-labeled NV) in media.

[0167] Afterward, the cells were washed with DPBS, fixed with 4% paraformaldehyde for 5 minutes, and then washed again with DPBS.

[0168] 0.5% Tween-20 solution was diluted in media and cells were permeabilized for 30 minutes.

[0169] After performing F-actin (ReadyProbes™ Reagent F-Actin Phalloidin Conjugates, R37112) staining, the cells were washed with DPBS, and the nuclei were stained with DAPI solution and analyzed using a fluorescence microscope.

[0170] To confirm the anti-inflammatory effect on human aortic smooth muscle cells (hAoSMC), qPCR was performed using the following procedure.

[0171] 1 X 10 4 Human aortic smooth muscle cells (hAoSMC) were seeded into 24 wells at cells / well and cultured until 60-70% confluency.

[0172] Inflammation was induced in cultured cells by treating them with TNF-α (10 ng / mL) and interferon-γ (10 ng / mL) for 6 hours. A stent coating composition containing mesenchymal stem cell-derived nanovesicles was co-treated with LPS.

[0173] Six hours after treatment, RNA prep was performed using the RNeasy mini kit, and cDNA was synthesized to proceed with qPCR.

[0174] FIG. 13(a) is an image obtained by fluorescence staining and confocal microscopy analysis to confirm uptake in human aortic smooth muscle cells (hAoSMC) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 13(b) is a graph showing the expression level of TNF-α in human aortic smooth muscle cells (hAoSMC) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 13(c) is a graph showing the expression level of IL-1β in human aortic smooth muscle cells (hAoSMC) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 13(d) is a graph showing the expression level of IL-6 in human aortic smooth muscle cells (hAoSMC) of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 13(e) is an image confirming that nanovesicles are absorbed into the intima (vascular endothelial cells) and delivered to smooth muscle cells in the media through an organ-on-a-chip of a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 13(f) is a schematic diagram of an organ-on-a-chip.

[0175] Referring to Figure 13 above, nanovesicle uptake and pro-inflammatory markers in hAoSMCs were confirmed via rt-qPCR. Additionally, using an organ-on-a-chip, it was confirmed that nanovesicles uptaken by endothelial cells (ECs) were transendothelial transported to smooth muscle cells (SMCs).

[0177] <실험예 13: hAoSMC migration assay 분석>

[0178] 1 X 10 4 Human aortic smooth muscle cells (hAoSMC) were seeded into 24 wells at cells / well and cultured until 100% confluency.

[0179] For cultured cells, the center of the well was cut using a 200 μL tip, and after washing the cells with DPBS, TNF-α (10 ng / mL) and interferon-γ (10 ng / mL) were added. A stent coating composition containing mesenchymal stem cell-derived nanovesicles was co-treated with LPS, and analyzed under a microscope immediately after treatment and after 10 hours.

[0180] FIG. 14(a) is a photograph showing migration over time when a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention is stimulated with PDGF-BB, TNF-α, and INF-γ in human aortic smooth muscle cells (hAoSMC). FIG. 14(b) is a graph showing the relative amount of migration when a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention is stimulated with PDGF-BB, TNF-α, and INF-γ in human aortic smooth muscle cells (hAoSMC).

[0181] Referring to Figure 14 above, a scratch assay was performed using human aortic smooth muscle cells (hAoSMC). hAoSMCs stimulated with PDGF-BB, TNF-α, and interferon-γ showed rapid migration and proliferation rates. The results of the assay confirmed that both the stent coating composition without freeze-drying (Fresh-NV) and the stent coating composition with freeze-drying (Lyo-NV) inhibited the abnormal migration and proliferation of LPS-stimulated hAoSMCs.

[0183] <실험예 14: hAoSMC에서 α-smooth muscle actin의 단백질 발현 분석>

[0184] 1 X 10 4 Human aortic smooth muscle cells (hAoSMC) were seeded into 24 wells at cells / well and cultured until 60-70% confluency.

[0185] A composition for stent coating containing PDGF-BB (20 ng / mL), TNF-α (10 ng / mL), interferon-γ (10 ng / mL), and mesenchymal stem cell-derived nanovesicles was mixed in media and treated with cells for 48 hours.

[0186] After treatment and 48 hours, the cells were washed with DPBS, fixed with 4% paraformaldehyde for 5 minutes, and then washed again with DPBS.

[0187] A blocking solution mixed with 250 mg BSA (bovine serum albumin), 5 mL DPBS, and 30 μL Tween-20 was treated at 200 μL / well and incubated at 4 ℃ for 1 hour.

[0188] Subsequently, the α-smooth muscle actin primary antibody was diluted in a blocking solution and treated, followed by incubation at room temperature for 1 hour. Afterward, it was incubated overnight at 4°C.

[0189] Cells were washed with DPBS, and the secondary antibody was diluted in DPBS and incubated at room temperature for 2 hours.

[0190] Subsequently, the cells were washed with DPBS, and the nuclei were stained with DAPI solution and analyzed using a fluorescence microscope.

[0192] FIG. 15(a) is an image obtained by fluorescence staining and confocal microscopy analysis to confirm α-smooth muscle actin expression in human aortic smooth muscle cells (hAoSMC) after stimulating them with PDGF-BB, TNF-α, and INF-γ using a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention. FIG. 15(b) is a graph showing the amount of α-smooth muscle actin expression in human aortic smooth muscle cells (hAoSMC) after stimulating them with PDGF-BB, TNF-α, and INF-γ using a stent coating composition containing mesenchymal stem cell-derived nanovesicles according to one embodiment of the present invention.

[0193] Referring to Figure 15 above, synthetic phenotype switching was induced in hAoSMCs by treating them with PDGF-BB, TNF-α, and INF-γ, followed by immunohistochemistry and confocal microscopy analysis. When differentiation into the synthetic phenotype occurs, the expression of α-smooth muscle actin decreases. As a result, it was confirmed that the stent coating composition without freeze-drying (Fresh-NV) and the stent coating composition with freeze-drying (Lyo-NV) effectively inhibited differentiation into the synthetic phenotype.

[0195] <실험예 15: PLLA 기재인 스텐트의 스텐트 코팅용 조성물의 코팅 및 release test>

[0196] The stent was coated by dip-coating with a stent coating composition (DiD-labeled NV) containing DiD-labeled mesenchymal stem cell-derived nanovesicles at a concentration of 500 μg / mL, and after coating, it was rapidly frozen over gaseous liquid nitrogen. It was stored at -80 ℃ until freeze-drying was performed. The coated stent was placed in a 1.5 mL tube containing 500 μL of DPBS and incubated at 37 ℃, and the amount of released nanovesicles (NV) was measured every 12 hours using Nanoparticle Tracking Analysis (NTA).

[0197] FIG. 16 (a) is a photograph showing the release of mesenchymal stem cell-derived nanovesicles over time from a stent according to one embodiment of the present invention. FIG. 16 (b) is a graph showing the release amount of mesenchymal stem cell-derived nanovesicles over time from a stent according to one embodiment of the present invention.

[0198] Referring to Figure 16 above, the PLLA-based stent used in the experiment has very low hydrophilicity due to the material properties. Accordingly, poly(2-methacryloyloxyethyl phosphorylcholine co-n-butyl methacrylate), an amphiphilic material having a phosphocholine group, was coated to make the PLLA surface hydrophilic. In the present invention, it was proven that the release of nanovesicles on the stent coated with poly(2-methacryloyloxyethyl phosphorylcholine co-n-butyl methacrylate is superior, and it was confirmed that this occurred because poly(2-methacryloyloxyethyl phosphorylcholine co-n-butyl methacrylate increased the hydrophilicity of the stent surface. Explanation of the symbols

[0199] S10: Step of passing through the filter S20: Step of mixing the mixture S30: Step of forming nanovesicles S50: Freeze-drying step

Claims

Claim 1 A stent comprising: a substrate including a biodegradable polymer material; and a mesenchymal stem cell-derived nanovesicle coating layer provided on the substrate; wherein the mesenchymal stem cell-derived nanovesicle coating layer is a stent coating composition including the mesenchymal stem cell-derived nanovesicle coated on the substrate by freeze-drying. Claim 2 The stent of claim 1, wherein the mesenchymal stem cell-derived nanovesicle is formed from an extract obtained by applying pressure to mesenchymal stem cells. Claim 3 A stent according to claim 2, wherein the extract is passed through a filter having an average pore size of 0.1 μm or more and 20 μm or less. Claim 4 A stent according to claim 3, wherein the extract is sequentially passed through a first filter having an average pore size of 9 μm or more and 11 μm or less, a second filter having an average pore size of 4 μm or more and 6 μm or less, and a third filter having an average pore size of 0.1 μm or more and 1.0 μm or less. Claim 5 A stent according to claim 1, wherein the concentration of the protein derived from the mesenchymal stem cells is 0.5 mg / ml or more and 2.0 mg / ml or less. Claim 6 In claim 1, the number of particles of the mesenchymal stem cell-derived nanovesicle is 1 x 10 10 Pieces / ml or more 1 X 10 11 Stents with a count of 1 / ml or less. Claim 7 A stent according to claim 1, wherein the stent coating composition further comprises trehalose. Claim 8 A stent according to claim 7, wherein the concentration of trehalose is 3 (w / v)% or more and 5 (w / v)% or less. Claim 9 A stent according to claim 1, wherein the stent coating composition further comprises phosphate-buffered physiological saline. Claim 10 A stent according to claim 1, wherein the freeze-drying is performed by freezing at -70 to -90°C and then freeze-drying for 1 to 100 hours. Claim 11 A stent comprising: a substrate including a biodegradable polymer material; a mesenchymal stem cell-derived nanovesicle coating layer provided on the substrate; and a hydrophilic coating layer provided between the substrate and the mesenchymal stem cell-derived nanovesicle coating layer, wherein the hydrophilic coating layer comprises a hydrophilic polymer including at least a phosphocholine group. Claim 12 delete Claim 13 A stent according to claim 1 or claim 11, wherein the biodegradable polymer material comprises at least poly(L-lactic acid, PLLA) and further comprises one or more selected from the group consisting of polyglycolide, poly p-dioxanone, polycaprolactone, trimethylene carbonate, polyhydroxyalkanoates, polypropylene fumarate, polyortho esters, other polyesters, polyanhydride, polyphosphazenes, polyalkyl cyanoacrylates, poloxamers, polyamino L-tyrosine, oxidized cellulose, gelatin, and collagen.

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