Biodegradable polymer scaffold comprising drug and / or extracellular vesicles and method for preparing same

Abstract

One aspect provides a biodegradable polymer scaffold for kidney regeneration comprising basic ceramic particles, an extracellular matrix, zinc particles, a material for inducing kidney regeneration, and a biodegradable polymer. The biodegradable polymer scaffold for kidney regeneration according to one aspect comprises a material for inducing kidney regeneration and / or extracellular vesicles that secrete a stem cell recruitment inducing factor, thereby guiding stem cells to a tissue damage site and enhancing kidney regeneration ability so as to effectively induce regeneration of kidney tissue. Accordingly, the biodegradable polymer scaffold can contribute to the medical device industry including the biomedical implant market.

Description

Biodegradable polymer scaffold containing drug and / or extracellular vesicle and method for preparing the same

[0001] The present invention relates to a biodegradable polymer scaffold containing extracellular vesicles secreting a drug for inducing renal tissue regeneration and / or a stem cell recruitment inducing factor, and a method for producing the same.

[0002] The kidneys are two bean-shaped organs that filter the blood and are involved in regulating the volume of various body fluids, osmotic pressure, acid-base balance, electrolyte concentration, and waste excretion. The nephron is the functional unit of the kidney and is composed of a glomerulus and a tubule. The glomerulus is a structure where small capillaries are tangled like a thread and functions to filter waste from the blood. The tubule is a thin tube about 4 to 7 cm long and functions to reabsorb the filtered substances and concentrate waste to form urine. The kidneys are essential organs for maintaining homeostasis in the body, and if their function is impaired, it can have serious effects on various parts of the body depending on the degree of the impairment.

[0003] Tissue engineering, a new field that emerged alongside scientific advancements, is a multidisciplinary discipline that integrates and applies fundamental concepts from life sciences, engineering, and medicine with scientific technology. Tissue engineering is an applied discipline that aims to understand the correlation between the structure and function of biological tissues and, furthermore, to create artificial tissues that can be transplanted into the body to replace or regenerate damaged tissues or organs, thereby maintaining, improving, or restoring bodily functions.

[0004] A summary of representative tissue engineering techniques is as follows. First, the required tissue is harvested from the patient's body, cells are isolated from the tissue fragment, and then the isolated cells are cultured to expand to the required volume. The expanded cells are then seeded on a polymer scaffold and cultured in vitro for a certain period of time, resulting in a hybrid cell / polymer structure that is then transplanted back into the body. The transplanted cells receive oxygen and nutrients through the diffusion of body fluids until new blood vessels form in most tissues and organs. Once blood vessels grow and a blood supply is established, the cells proliferate and differentiate to form new tissues and organs, while the polymer scaffold degrades and is lost.

[0005] Therefore, polymer scaffolds must possess sufficient requirements and functions for human tissue regeneration. Specifically, a material for tissue regeneration must possess cytocompatibility, enabling tissue cells to adhere to the material surface and form new blood vessels, enabling the formation of three-dimensional tissue structures, and function as an intermediate barrier between transplanted cells and host cells. This also means that the scaffold must be non-toxic and biocompatible, preventing blood clotting or inflammatory reactions after transplantation. Furthermore, during the regeneration process, it is desirable for a large number of cells to be recruited and mixed into the scaffold or defect site. Therefore, chemotactic signaling molecules, commonly referred to as chemokines, are used to enhance initial cell recruitment during the mixing process of cells into the scaffold.

[0006] The chemokine Stromal Derived Factor-1α (SDF-1α) has attracted attention because it is chemotactic for mesenchymal stem cells (MSCs) and promotes the expression of genes associated with cell motility. It also recruits MSCs to the injury site, enhancing cell regeneration and guiding cells into the scaffold. Therefore, scaffolds capable of recruiting cells after transplantation must be designed to release signaling molecules over a period of time. In addition to providing an appropriate environment for kidney regeneration, other regenerative factors are also required.

[0007] Therefore, there is a need to develop a multifunctional composite scaffold that can effectively induce regeneration of kidney tissue by inducing stem cells to the site of tissue damage and enhancing kidney regenerative capacity.

[0008] One aspect provides a biodegradable polymer scaffold for kidney regeneration comprising basic ceramic particles, an extracellular matrix, zinc particles, a substance for inducing kidney regeneration, and a biodegradable polymer.

[0009] Another aspect provides a bioimplant for renal regeneration comprising the biodegradable polymer scaffold.

[0010] Another aspect is a step of preparing a first polymer solution by mixing basic ceramic particles, extracellular matrix, zinc particles and a biodegradable polymer;

[0011] A step of preparing a second polymer solution by mixing the first polymer solution and a pore inducer; and

[0012] A method for producing a biodegradable polymer support for kidney regeneration, comprising the step of drying the second polymer solution to produce a porous support,

[0013] The method comprises mixing a substance for inducing renal regeneration together in the step of preparing the first polymer solution;

[0014] A manufacturing method is provided, which further includes a step of loading a material for inducing kidney regeneration after the step of manufacturing the porous support.

[0015] One aspect relates to a biodegradable polymer scaffold for renal regeneration comprising basic ceramic particles, an extracellular matrix, zinc particles, a substance for inducing renal regeneration, and a biodegradable polymer.

[0016] The biodegradable polymer scaffold can control the pore size, density, porosity, etc. of the porous polymer scaffold, and can also control the shape and size of the polymer scaffold including the extracellular matrix and a renal regeneration-inducing substance (e.g., extracellular vesicles or a renal regeneration-inducing drug). In addition, by containing the basic ceramic particles, extracellular matrix, zinc particles, and renal regeneration-inducing substance (e.g., extracellular vesicles or a renal regeneration-inducing drug) at various concentrations, the tissue regeneration ability and hydrophilicity of the biodegradable polymer scaffold are enhanced, and the mechanical strength and degradation period can be controlled.

[0017] According to one aspect, the basic ceramic particles may be at least one selected from the group consisting of alkali metals, alkali metal oxides, alkali metal hydroxides, alkaline earth metals, alkaline earth metal oxides, and alkaline earth metal hydroxides. The alkali metal or alkaline earth metal may be, for example, lithium (Li), beryllium (Be), sodium (Na), magnesium (Mg), potassium (K), calcium (Ca), rubidium (Rb), strontium (Sr), barium (Ba), cesium (Cs), francium (Fr), radium (Ra), and the like.

[0018] In addition, in one aspect, the oxide of the alkali metal, the hydroxide of the alkali metal, the oxide of the alkaline earth metal, or the hydroxide of the alkaline earth metal may be selected from the group consisting of lithium hydroxide, beryllium hydroxide, sodium hydroxide, magnesium hydroxide, potassium hydroxide, calcium hydroxide, rubidium hydroxide, strontium hydroxide, barium hydroxide, cesium hydroxide, francium hydroxide, radium hydroxide, magnesium oxide, sodium oxide, lithium oxide, sodium oxide, manganese oxide, potassium oxide, calcium oxide, barium oxide, cesium oxide, and radium oxide.

[0019] In one aspect, the basic ceramic particles may have their surface modified with a fatty acid, a polymer material, or a mixture thereof.

[0020] The fatty acids may be, for example, caprylic acid, capric acid, lauric acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, ricinoleic acid, linoleic acid, α-linoleic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, stearic acid, DHA, octatecanoic acid, coconut oil, palm oil, cottonseed oil, horsetail oil, soybean oil, olive oil, corn oil, sunflower oil, safflower oil, hemp oil, canola oil, etc.

[0021] In addition, the polymer material is at least one monomer selected from the group consisting of, for example, L-lactide, D-lactide, D,L-lactide, glycolide, caprolactone, dioxanone, trimethylene carbonate, hydroxyalkanoate, peptide, cyanoacrylate, lactic acid, glycolic acid, hydroxycaproic acid, maleic acid, phosphazene, amino acid, hydroxybutyric acid, sebacic acid, hydroxyethoxyacetic acid and trimethylene glycol, or poly-L-lactide, poly-D-lactide, poly-D,L-lactide, polyglycolide, polycaprolactone, poly-L-lactide-co-glycolide, poly-D-lactide-co-glycolide, poly-D,L-lactide-co-glycolide, poly-L-lactide-co-caprolactone, It may be poly-D-lactide-co-caprolactone, poly-D,L-lactide-co-caprolactone, polyglycolide-co-caprolactone, polydioxanone, polytrimethylene carbonate, polyglycolide-co-dioxanone, polyamide ester, polypeptide, polyorthoester, polymaleic acid, polyphosphazene, polyanhydride, polysebacinhydride, polyhydroxyalkanoate, polyhydroxybutyrate, polycyanoacrylate, etc.

[0022] The above term "surface modification" means changing the chemical structure and physical structure of the particle surface, for example, by introducing various functional groups to the surface of basic ceramic particles using a biodegradable polymer, thereby causing a structural change.

[0023] A biodegradable polymer support comprising basic ceramic particles whose surface is modified with fatty acids or polymeric substances according to one aspect not only has improved dispersion stability in an organic solvent, but also can improve mechanical properties, and can improve inflammatory responses and cytotoxicity in the body by neutralizing acidic substances with the basic ceramic particles.

[0024] In addition, according to one aspect, the diameter (size) of the basic ceramic particles or the basic ceramic particles with modified surfaces may be 1 nm to 1 mm. If the diameter of the basic ceramic particles exceeds the above range, there is a problem in that precipitation occurs due to the weight of the basic ceramic particles, resulting in phase separation in the organic solvent.

[0025] In addition, according to one aspect, the basic ceramic particles may be included in an amount of 1 to 20 wt% based on the total weight of the biodegradable polymer support. The basic ceramic particles may be included in an amount of, for example, 1 to 20 wt%, 1 to 16 wt%, 1 to 12 wt%, 5 to 20 wt%, 5 to 16 wt%, 5 to 12 wt%, 10 to 20 wt%, 10 to 16 wt%, or 10 to 12 wt% based on the total weight of the biodegradable polymer support. In this case, when the content of the basic ceramic particles is less than 1 wt%, there is a problem that an acidic substance, which is a decomposition product of the polymer support, cannot be sufficiently neutralized, and when it exceeds 20 wt%, there is a problem that alkalization of the environment around the polymer support may be induced.

[0026] Additionally, in one aspect, the extracellular matrix may be isolated from a human or animal.

[0027] Specifically, the extracellular matrix is ​​a matrix protein derived from tissues or cells of a human or animal, which may be in a complexly mixed state or an artificially separated single molecule state, and the protein may have a denatured structure. The extracellular matrix may be derived from vertebrates such as a human, pig, cow, rat, sheep, horse, dog, or cat, and may be isolated from bone, kidney, amniotic membrane, skin, brain, small intestinal submucosa, fascia, or spinal cord meninges, depending on the purpose. The extracellular matrix may be appropriately selected according to structure or function. In one aspect, the extracellular matrix may be a group having a fibrous structure, a group related to bone differentiation and osteogenesis, a glucosaminoglycan group, a proteoglycan group, etc. The group having a fibrous structure may be, for example, collagen fibers, elastin fibers, laminin, fibrinogen, fibronectin, gelatin, etc. At this time, the collagen may be I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIV, XV, XVI, XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, XXVIII collagen, etc. according to type. In addition, the group related to the bone differentiation and bone formation may be, for example, osteonectin, osteopontin, vitronectin, vimentin, etc. In addition, the glucosaminoglycan group may be, for example, heparan sulfate, keratan sulfate, chondroitin sulfate, dermatan sulfate, heparin, low molecular weight heparin, hyaluronic acid, etc. Additionally, the proteoglycan group may be, for example, decorin, biclycan, versican, tertican, perlecan, bicuin, neurocan, aggrecan, fibromodulin, lumican, etc.

[0028] In one aspect, the extracellular matrix may be included in an amount of 10 to 50 wt% relative to the total weight of the biodegradable polymer scaffold. The extracellular matrix may be included in an amount of, for example, 10 to 50 wt%, 10 to 40 wt%, 10 to 35 wt%, 20 to 50 wt%, 20 to 40 wt%, 20 to 35 wt%, 25 to 50 wt%, 25 to 40 wt%, or 25 to 35 wt% relative to the total weight of the biodegradable polymer scaffold. In this case, when the content of the extracellular matrix is ​​less than 10 wt%, there is a problem in that the cytocompatibility improvement effect cannot be sufficiently exhibited, and when it exceeds 50 wt%, there is a problem in that the mechanical properties of the polymer scaffold are reduced.

[0029] In another aspect, the extracellular matrix may be decellularized, and may be decellularized by a physical or chemical method after culturing tissues or cells. Physical decellularization methods include, for example, freeze-thawing, sonication, physical stirring, etc., and chemical decellularization methods include, for example, treating powder of animal-derived tissue with a storage solution containing water, anionic surfactant, nonionic surfactant, cationic surfactant, DNase, RNase, or trypsin. In the chemical decellularization method, a Tris-HCl (pH 8.0) solution may be used as the storage solution, and the anionic surfactant may be sodium dodecyl sulfate (SDS), sodium deoxycholate, Triton X-200, etc. In addition, as the nonionic surfactant, Triton X-100, Tween 20, or Tween 80 may be used, and as the cationic surfactant, CHAPS, Sulfobetaine-10 (SB-10), Sulfobetaine-16 (SB-16), Tri-n-butyl phosphate, N-lauroyl-sarcosinate, IGEPAL CA-630, etc. may be used. In addition, the decellularization may be performed before or after performing the powdering process after collecting animal tissue, for example, kidney tissue, or simultaneously with the powdering process.

[0030] In addition, in one aspect, the zinc particles may be at least one selected from the group consisting of zinc oxide, zinc sulfide, zinc nitrate, zinc selenide, zinc telluride, zinc nitride, zinc phosphide, zinc arsenide, zinc antimonide, zinc peroxide, zinc hydride, zinc oxalate dihydrate, zinc chloride, zinc bromide, zinc iodide, zinc hydroxide, zinc chlorate, zinc sulfate, zinc phosphate, zinc molybdate, zinc cyanide, zinc metaarsenite, zinc arsenate octahydrate, zinc chromate, zinc pyrithione, and zinc acetate.

[0031] In one aspect, the zinc particles may be surface-modified with a fatty acid, a polymeric material, or a mixture thereof.

[0032] The fatty acids may be, for example, caprylic acid, capric acid, lauric acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, ricinoleic acid, linoleic acid, α-linoleic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, stearic acid, DHA, octatecanoic acid, coconut oil, palm oil, cottonseed oil, horsetail oil, soybean oil, olive oil, corn oil, sunflower oil, safflower oil, hemp oil, canola oil, etc.

[0033] In addition, the polymer material is at least one monomer selected from the group consisting of, for example, L-lactide, D-lactide, D,L-lactide, glycolide, caprolactone, dioxanone, trimethylene carbonate, hydroxyalkanoate, peptide, cyanoacrylate, lactic acid, glycolic acid, hydroxycaproic acid, maleic acid, phosphazene, amino acid, hydroxybutyric acid, sebacic acid, hydroxyethoxyacetic acid and trimethylene glycol, or poly-L-lactide, poly-D-lactide, poly-D,L-lactide, polyglycolide, polycaprolactone, poly-L-lactide-co-glycolide, poly-D-lactide-co-glycolide, poly-D,L-lactide-co-glycolide, poly-L-lactide-co-caprolactone, It may be poly-D-lactide-co-caprolactone, poly-D,L-lactide-co-caprolactone, polyglycolide-co-caprolactone, polydioxanone, polytrimethylene carbonate, polyglycolide-co-dioxanone, polyamide ester, polypeptide, polyorthoester, polymaleic acid, polyphosphazene, polyanhydride, polysebacinhydride, polyhydroxyalkanoate, polyhydroxybutyrate, polycyanoacrylate, etc.

[0034] In one aspect, the diameter (size) of the zinc particles or surface-modified zinc particles may be 10 to 1000 nm. Specifically, the diameter of the zinc particles or surface-modified zinc particles may be, for example, 10 to 1000 nm, 10 to 800 nm, or 50 to 400 nm.

[0035] In addition, in one aspect, the zinc particles may be included in an amount of 1 to 10 wt% relative to the total weight of the biodegradable polymer support. The zinc particles may be included in an amount of, for example, 1 to 10 wt%, 1 to 8 wt%, 1 to 7 wt%, 3 to 10 wt%, 3 to 8 wt%, 3 to 7 wt%, 4 to 10 wt%, 4 to 8 wt%, or 4 to 7 wt% relative to the total weight of the biodegradable polymer support.

[0036] Additionally, in one aspect, the substance for inducing renal regeneration may be edaravone (EDV), extracellular vesicles, or a mixture thereof.

[0037] Edaravone (chemical name: 3-methyl-1-phenyl-2-pyrazolin-5-one), a compound represented by the following chemical formula 1, is a free radical scavenger and a commercially available brain neuroprotective agent (Reference: Yakugaku Zasshi.2004, 124(3): 99-111):

[0038] [Chemical Formula 1]

[0039] .

[0040] Previous studies have shown that edaravone possesses antioxidant activity, significantly improving neurological deficits in animals undergoing cerebral ischemia-reperfusion, including reducing infarct size, the extent of brain damage, alleviating cerebral edema, and inhibiting lipid peroxidation in damaged brain tissue. Recent studies have shown that edaravone can protect against ischemia-reperfusion-induced renal injury, inhibit apoptosis, and improve mitochondrial damage through JAK / STAT signaling (Zhao. 2020, 53(1):28). In addition, it was confirmed that edaravone has a therapeutic effect on renal damage by inhibiting acute mitochondrial DNA damage, renal epithelial cell death, and chronic renal damage induced by cisplatin, which rapidly damages mitochondrial DNA in the renal proximal tubules and induces apoptosis of tubular epithelial cells (Reference: lguchi.2004, 38(4):333-341). Therefore, it was confirmed that edaravone has a preventive or therapeutic effect on renal damage and renal regeneration.

[0041] The above term “prevention” may mean any action that inhibits or delays kidney-related diseases, including kidney damage, in an individual by administration.

[0042] The above term “treatment” may mean any action that improves or beneficially changes the symptoms of a kidney-related disease, including kidney damage, in an individual by administration.

[0043] The term "administration" refers to introducing a given substance into a subject in an appropriate manner, and "subject" refers to any living organism, including rats, mice, and livestock, including humans, that may have a kidney-related disease, including kidney damage. A specific example may be a mammal, including humans.

[0044] The above-mentioned extracellular vesicles are nano-sized vesicles secreted by all cells into the external environment for the exchange of information between cells, and contain various substances exhibiting biological activity, such as proteins, lipids, nucleic acids, and metabolites. The above-mentioned extracellular vesicles may include exosomes and microvesicles. The above-mentioned extracellular vesicles may be isolated from stem cells derived from one or more selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, skin, amniotic membrane, and placenta, for example.

[0045] Additionally, according to one aspect, the stem cells may be genetically engineered to overexpress a stem cell recruitment inducing factor compared to the parent cells, and the extracellular vesicles may secrete the stem cell recruitment inducing factor. In this case, the stem cell recruitment inducing factor may be, for example, SDF-1α (Stromal Derived Factor-1α).

[0046] The above “parent cell” refers to a cell that has not been artificially manipulated to reduce the expression or activity of a gene encoding a stem cell recruitment inducing factor or a stem cell recruitment inducing factor protein according to one aspect, and refers to a cell freshly isolated from a human body and a cell cultured therefrom.

[0047] Additionally, the terms "genetic engineering" or "genetically engineered" refer to the act of introducing one or more genetic modifications into a cell or a cell produced thereby. The genetic modification may be induced by a modification in the nucleic acid sequence of a gene encoding a stem cell recruitment inducing factor, and the modification in the nucleic acid sequence may be artificially introduced, for example, by the CRISPR / Cas9 system.

[0048] Meanwhile, the SDF-1α is a non-glycosylated protein containing 93 amino acids and having a molecular weight of 10.7 kDa, and is a chemokine that plays a role in moving various immune cells to the inflammatory synovium, and is a substance produced in vascular endothelial cells and plays a role in recruiting vascular progenitor cells. In other words, it is known to be involved in angiogenesis. The SDF-1α may be of human origin, and according to one aspect, by transforming mesenchymal stem cells (MSCs), SDF-1α secretory extracellular vesicles (SDF-1α EVs), which are stem cell recruitment inducers capable of continuously expressing SDF-1α, can be produced.

[0049] In addition, in one aspect, the biodegradable polymer may be at least one selected from the group consisting of polylactide, polyglycolide, polycaprolactone, polylactide-co-glycolide, polylactide-co-caprolactone, polyglycolide-co-caprolactone, polydioxanone, polydioxanone, polytrimethylene carbonate, polyglycolide-co-dioxanone, polyamide ester, polypeptide, polyolthoester, polymaleic acid, polyanhydride, polysebacinhydride, polyhydroxyalkanoate, polyhydroxybutyrate, and polycyanoacrylate.

[0050] In addition, according to one aspect, the biodegradable polymer may be included in an amount of 25 to 85 wt% based on the total weight of the biodegradable polymer support. The biodegradable polymer may be included in an amount of, for example, 25 to 85 wt%, 25 to 75 wt%, 25 to 65 wt%, 35 to 85 wt%, 35 to 75 wt%, 35 to 65 wt%, 45 to 85 wt%, 45 to 75 wt%, or 45 to 65 wt% based on the total weight of the biodegradable polymer support.

[0051] According to one aspect, the basic ceramic particles may be 1 to 20 wt%, the extracellular matrix may be 10 to 50 wt%, the zinc particles may be 1 to 10 wt%, and the biodegradable polymer may be 25 to 85 wt%, based on the total weight of the biodegradable polymer support.

[0052] In addition, in one aspect, when the material for inducing renal regeneration includes edaravone, the edaravone may be included in an amount of 0.1 to 1 wt% relative to the total weight of the biodegradable polymer support. When the material for inducing renal regeneration includes edaravone, the material for inducing renal regeneration may be included in an amount of, for example, 0.1 to 1 wt%, 0.1 to 0.8 wt%, 0.1 to 0.6 wt%, 0.2 to 1 wt%, 0.2 to 0.8 wt%, 0.2 to 0.6 wt%, 0.4 to 1 wt%, 0.4 to 0.8 wt%, or 0.4 to 0.6 wt% relative to the total weight of the biodegradable polymer support.

[0053] In addition, in one aspect, when the material for inducing renal regeneration includes extracellular vesicles, the extracellular vesicles in the biodegradable polymer support are 1 x 10 6 1 x 10 12 It can be included as a dog. If the material for inducing renal regeneration includes extracellular vesicles, the material for inducing renal regeneration in the biodegradable polymer support may be, for example, 1 x 10 6 1 x 10 12 Dog, 1 x 10 6 1 x 10 11 Dog, 1 x 10 6 1 x 10 10 Dog, 1 x 10 7 1 x 10 12 Dog, 1 x 10 7 1 x 10 11 Dog, 1 x 10 7 1 x 10 10Dog, 1 x 10 8 1 x 10 12 Dog, 1 x 10 8 1 x 10 11 dog or 1 x 10 8 1 x 10 10 It can be included as a dog.

[0054]

[0055] Another aspect relates to a bioimplant for renal regeneration comprising the biodegradable polymer scaffold.

[0056] The above “biodegradable polymer support” may be within the above-mentioned range.

[0057] In one aspect, the bioimplant can be utilized as a stent, surgical suture, scaffold for tissue regeneration, bio-nano fiber, hydrogel, bio-sponge, pin, screw, rod, implant, other cardiovascular related materials, other dental materials and other biomaterials for neuro / orthopedic / plastic surgery.

[0058]

[0059] Another aspect is a step of preparing a first polymer solution by mixing basic ceramic particles, extracellular matrix, zinc particles and a biodegradable polymer;

[0060] A step of preparing a second polymer solution by mixing the first polymer solution and a pore inducer; and

[0061] A method for producing a biodegradable polymer support for kidney regeneration, comprising the step of drying the second polymer solution to produce a porous support,

[0062] The method comprises mixing a substance for inducing renal regeneration together in the step of preparing the first polymer solution;

[0063] The present invention relates to a manufacturing method, which further includes a step of loading a material for inducing renal regeneration after the step of manufacturing the porous support.

[0064] The above “basic ceramic particles”, “extracellular matrix”, “zinc particles”, “biodegradable polymer”, “material for inducing kidney regeneration”, “biodegradable polymer scaffold”, “extracellular vesicles”, etc. may be within the above-mentioned range.

[0065] In one aspect, the step of preparing the first polymer solution may be mixing the basic ceramic particles, extracellular matrix, zinc particles, and biodegradable polymer in an organic solvent phase.

[0066] In one aspect, the organic solvent may be, for example, alcohols such as methanol, ethanol, propanol, and butanol; aldehydes such as ammonia, dimethyl sulfoxide, dimethylformamide, acetronitrile, tetrahydrofuran, formaldehyde, glutaraldehyde, and acetaldehyde; alkanes such as dioxane, chloroform, heptane, hexane, pentane, octane, nonane, and decane; benzene ring-type solvents such as benzene, toluene, and xylene; ethers such as ether, di-propyl ether, petroleum ether, and methyl-t-butyl ether; ketones such as propanone, butanone, pentanone, hexanone, and heptanone; and common organic solvents such as methylene chloride, tetrafluoroisopropane, and carbon tetrachloride.

[0067] In addition, in one aspect, the pore inducer may be ice particles, and the pore size and porosity of the porous polymer support may be controlled by controlling the size and content of the ice particles. The diameter (size) of the ice particles may be 10 to 500 μm. The diameter of the ice particles may be, for example, 10 to 500 μm, 10 to 450 μm, 10 to 400 μm, 10 to 300 μm, 30 to 500 μm, 30 to 300 μm, 30 to 250 μm, 50 to 500 μm, 50 to 400 μm, 50 to 300 μm, 50 to 200 μm, 100 to 500 μm, 100 to 300 μm, or 150 to 200 μm. At this time, if the diameter of the ice particles is less than 10 ㎛, there is a problem that the degree of cell invasion is low and the angiogenesis effect cannot be sufficiently exerted, and if it exceeds 500 ㎛, there is a problem that the mechanical properties of the biodegradable polymer support are reduced.

[0068] In addition, in the step of preparing the second polymer solution, the pore inducer may be mixed in an amount of 100 to 2000 wt% relative to the total weight of the first polymer solution. Specifically, the pore inducer may be mixed in an amount of 100 to 2000 wt%, 100 to 1500 wt%, 100 to 1300 wt%, 100 to 1000 wt%, 100 to 500 wt%, 500 to 2000 wt%, 500 to 1500 wt%, 500 to 1000 wt%, 1000 to 2000 wt%, or 1500 to 2000 wt% relative to the total weight of the first polymer solution. At this time, if the content of the mixed pore inducer is less than 100 wt% based on the total weight of the first polymer solution, there is a problem that pores are not sufficiently formed inside the polymer support, and if it exceeds 2000 wt% based on the total weight of the first polymer solution, there is a problem that the mechanical properties of the polymer support are reduced.

[0069] In addition, in one aspect, the drying may be freeze-drying, and by manufacturing a polymer support by the freeze-drying method, the detachment of basic ceramic particles and extracellular matrix that may occur in a conventional salt forming method can be prevented.

[0070] In addition, specifically, when the material for inducing renal regeneration used in the method is edaravone, the method may be such that the material for inducing renal regeneration is mixed together in the step of preparing the first polymer solution.

[0071] When the substance for inducing renal regeneration used in the above method is an extracellular vesicle, the method may further include a step of loading the substance for inducing renal regeneration after the step of manufacturing the porous support.

[0072] Furthermore, when the renal regeneration-inducing substance used in the method is a mixture of edaravone and extracellular vesicles, the method may further include a step of mixing edaravone and the renal regeneration-inducing substance together in the step of preparing the first polymer solution, and a step of loading extracellular vesicles after the step of preparing the porous support.

[0073] A biodegradable polymer scaffold for renal regeneration according to one aspect comprises an extracellular vesicle secreting a renal regeneration-inducing agent and / or a stem cell recruitment-inducing factor, thereby effectively inducing renal tissue regeneration by inducing stem cells to the site of tissue damage and enhancing renal regenerative capacity. Accordingly, the biodegradable polymer scaffold can contribute to the medical device industry, including the biotransplant market.

[0074] Figure 1 is a schematic diagram of a polymer support according to one aspect.

[0075] Figure 2 is a schematic diagram of expression vectors that can be used for the production of CRISPR-mediated SDF-1a secretory extracellular vesicles (SDF-1a EVs).

[0076] Figure 3 is a diagram showing the immunoblotting results showing the expression level of SDF-1a conjugated His in cell lysates.

[0077] Figure 4 is a graph showing the degree of SDF-1a secretion from extracellular vesicles determined by ELISA.

[0078] Figure 5 is a photograph showing the pore size and structure of a biodegradable polymer support according to a daily aspect, analyzed using a scanning electron microscope.

[0079] Figure 6 is a diagram showing the effect of a polymer support on the in vitro wound healing ability according to the daily aspect.

[0080] Figure 7 is a diagram showing the effect of a polymer scaffold on in vitro blood vessel formation according to a daily aspect.

[0081] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0082]

[0083] Manufacturing example

[0084] Manufacturing Example 1. Manufacturing of basic ceramic particles

[0085] 1-1. Manufacturing of magnesium hydroxide particles

[0086] A sodium hydroxide solution was prepared by dissolving 10.8 g of sodium hydroxide in 300 ml of distilled water. Then, a magnesium nitrate solution, in which 20 g of magnesium nitrate was dissolved in 150 ml of distilled water, was added dropwise at a rate of 40 drops per minute to the sodium hydroxide solution using a dropping funnel. The nano-magnesium hydroxide particles precipitated in the reaction solution were purified by flowing distilled water, and then filtered to obtain the precipitated magnesium hydroxide particles. The obtained magnesium hydroxide particles were vacuum-dried and stored.

[0087] 1-2. Manufacturing of magnesium oxide particles

[0088] Magnesium hydroxide particles manufactured in the above Manufacturing Example 1-1 were calcined at a temperature of 500 to 1500°C using an electric furnace to manufacture magnesium oxide particles.

[0089]

[0090] 1-3. Preparation of magnesium hydroxide particles surface-modified with polymers

[0091] The basic ceramic particles manufactured in the above Manufacturing Example 1-1 were surface-modified with L-lactide. Specifically, 80 wt% of magnesium hydroxide and 20 wt% of L-lactide of Manufacturing Example 1-1 were mixed based on the total weight of the entire mixture. Thereafter, 0.05 wt% of tin octoate (catalyst) was diluted in toluene and added based on the total weight of the reactants (magnesium hydroxide and L-lactide). The glass reactor containing the reactants was maintained in a vacuum at 70°C for 6 hours while stirring to completely remove toluene and moisture. The sealed glass reactor was stirred in an oil bath adjusted to 150°C while performing a ring-opening polymerization reaction for 48 hours. The recovered polymer was placed in a sufficient amount of chloroform to remove homopolymer and unreacted residues for more than 1 hour, thereby manufacturing basic ceramic particles modified with a polymer.

[0092]

[0093] 1-4. Preparation of magnesium oxide particles surface-modified with polymers

[0094] Basic ceramic particles modified with a polymer were manufactured in the same manner as in Manufacturing Example 1-3, except that the magnesium oxide particles manufactured in Manufacturing Example 1-2 were used instead of the magnesium hydroxide particles.

[0095]

[0096] Manufacturing Example 2. Preparation of decellularized and powdered extracellular matrix

[0097] 2-1. Preparation of human adipose-derived extracellular matrix

[0098] After collecting human adipose tissue, it was washed three times with saline solution for 10 minutes each. The washed tissue was dehydrated with ethanol, and the adipocytes and genetic components present in the tissue were removed, and pure extracellular matrix was obtained through a decellularization process. Specifically, the dehydrated tissue was placed in 1 L of 0.1% sodium dodecyl sulfate (SDS) solution per 10 g and stirred at 100 rpm for 24 hours. Afterwards, it was washed five times with distilled water for 30 minutes each at 100 rpm, and 200 mL of 200 U / mL DNase was added and stirred at 100 rpm for 24 hours at 37°C. Afterwards, it was washed five times with distilled water for 30 minutes each at 100 rpm and dried. The decellularized extracellular matrix was freeze-pulverized into powder with a size of approximately 50 μm using a freeze-pulverizer.

[0099]

[0100] 2-2. Preparation of extracellular matrix derived from human skin

[0101] An extracellular matrix was prepared in the same manner as in Manufacturing Example 2-1, except that skin tissue from an individual was used instead of human adipose tissue.

[0102]

[0103] 2-3. Preparation of extracellular matrix derived from pig kidney

[0104] The extracellular matrix was prepared in the same manner as in Manufacturing Example 2-1, except that pig kidney tissue was used instead of human adipose tissue.

[0105]

[0106] 2-4. Preparation of extracellular matrix derived from rat kidney

[0107] The extracellular matrix was prepared in the same manner as in Manufacturing Example 2-1, except that rat kidney tissue was used instead of human adipose tissue.

[0108]

[0109] Manufacturing Example 3. Zinc oxide particles surface-modified with lactide

[0110] Zinc oxide (ZnO) nanoparticles surface-modified with lactide were prepared. Specifically, 80 wt% zinc oxide and 20 wt% lactide were mixed, placed in an organic reactor at 150°C under vacuum, and stirred for 16 hours. The stirred solution was then dispersed in an organic solvent and filtered to produce zinc oxide particles having a particle size of 80 nm and stably dispersed for 3 weeks.

[0111]

[0112] Manufacturing Example 4. Kidney regeneration-inducing drug for manufacturing biodegradable polymer scaffold

[0113] In the present invention, edaravone having a molecular weight of 174.2 kDa was used as a renal regeneration-inducing drug for use in the manufacture of a biodegradable support for renal regeneration.

[0114]

[0115] Manufacturing Example 5. Production and isolation of exosomes secreting stem cell recruitment inducing factors.

[0116] To generate SDF-1a tonsil-derived mesenchymal stem cells (TMSCs), mRNA targeting the safe harbor sites of AAVS1 (adeno-associated virus integration site 1) CRISPR / Cas9 (Cosmogene) and the AAVS1 target region of SDF-1a: 5'-CTCCACCCCACAGTGGGGCCACTAGGGGCAGGA-3' (SEQ ID NO: 1) were transfected into AAVS1. Nucleofection was performed using the SDF-1a sequence (used as a donor vector in Fig. 2) and transfection substrates under the following conditions. The tonsil-derived mesenchymal stem cells were seeded on culture dishes and then stabilized in a 5% CO2 incubator at 37°C. Afterwards, tonsil-derived mesenchymal stem cells were cultured, and the supernatant was collected four times every 24 hours. The culture medium was filtered through a 0.2 μm filter to remove impurities, and exosomes were selectively isolated and concentrated using a MWCO 500 kDa filter and tangential flow filtration (TFF).

[0117]

[0118] Example

[0119] Example 1. Polymer scaffold containing extracellular vesicles secreting renal regeneration-inducing drugs and stem cell recruitment-inducing factors (1)

[0120] A polymer scaffold containing a renal regeneration-inducing drug and stem cell recruitment-inducing factor-secreting extracellular vesicles was prepared. Specifically, 55 wt% of polylactide-co-glycolide (PLGA; 50:50, molecular weight: 40,000 Da) based on the total weight, 11 wt% of the surface-modified magnesium hydroxide particles of Preparation Example 1-3, 28 wt% of the human adipose-derived extracellular matrix of Preparation Example 2-1, 5.5 wt% of the zinc particles of Preparation Example 3, and 0.5 wt% of edaravone (EDV) of Preparation Example 4 were mixed in an organic solvent to prepare a polymer solution. Thereafter, ice particles of 100 to 200 μm in size were uniformly mixed into the polymer solution, and then freeze-dried for 48 hours using a Teflon mold to prepare a porous scaffold. The porous support was sterilized by immersing it in 70% ethanol, washed with sterilized distilled water to remove ethanol, and hydrated by immersing it in physiological saline solution. The amount of exosomes isolated in the above manufacturing example 5 was measured by nanoparticle tracking analysis (NTA), and 1 x 10 exosomes 9 A polymer scaffold containing a renal regeneration-inducing drug was manufactured by loading the particles of the dog into the porous polymer scaffold using a simple loading method.

[0121]

[0122] Example 2. Polymer scaffold containing extracellular vesicles secreting renal regeneration-inducing drugs and stem cell recruitment-inducing factors (2)

[0123] A polymer scaffold for bone regeneration was prepared in the same manner as in Example 1, except that polylactide-co-glycolide (PLGA; 50:50, molecular weight: 110,000 Da) was used instead of polylactide-co-glycolide (PLGA; 50:50, molecular weight: 40,000 Da).

[0124]

[0125] Example 3. Polymer scaffold containing extracellular vesicles secreting renal regeneration-inducing drugs and stem cell recruitment-inducing factors (3)

[0126] A polymer scaffold for bone regeneration was manufactured in the same manner as in Example 1, except that polylactide-co-glycolide (PLGA; 75:25, molecular weight: 110,000 Da) was used instead of polylactide-co-glycolide (PLGA; 50:50, molecular weight: 40,000 Da).

[0127]

[0128] Comparative example

[0129] Comparative Example 1. Polymer Support

[0130] A polymer scaffold was prepared in the same manner as in Example 1, except that only 40K polylactide-co-glycolide (50:50) biodegradable polymer was used, without using surface-modified magnesium hydroxide particles, extracellular matrix, zinc particles, and edaravone (EDV).

[0131]

[0132] Comparative Example 2. Polymer scaffold without edaravone as a kidney regeneration-inducing drug.

[0133] A polymer scaffold was prepared in the same manner as in Example 1, except that edaravone (EDV) was not used, and 55 wt% of polylactide-co-glycolide (PLGA; 50:50, molecular weight: 40,000 Da), 11 wt% of the surface-modified magnesium hydroxide particles of Manufacturing Example 1-3, 28 wt% of the human adipose-derived extracellular matrix of Manufacturing Example 2-1, and 5.5 wt% of the zinc particles of Manufacturing Example 3 were used.

[0134]

[0135] Comparative Example 3. Polymer scaffold containing edaravone as a kidney regeneration-inducing drug (3)

[0136] A polymer scaffold was prepared in the same manner as in Example 1, except that 55 wt% of polylactide-co-glycolide (PLGA; 50:50, molecular weight: 40,000 Da), 11 wt% of the surface-modified magnesium hydroxide particles of Manufacturing Example 1-3, 28 wt% of the human adipose-derived extracellular matrix of Manufacturing Example 2-1, 5.5 wt% of the zinc particles of Manufacturing Example 3, and 0.5 wt% of edaravone (EDV) of Manufacturing Example 4 were used without using (loading) the stem cell recruitment inducing factor secreting extracellular vesicles (exosomes).

[0137]

[0138] Experimental example

[0139] Experimental Example 1. Characterization of SDF-1a-MSC

[0140] The characteristics of SDF-1a-secreting mesenchymal stem cells (SDF-1a-MSC) produced in the above manufacturing example 5 were analyzed.

[0141] First, as previously described, the CRISPR / Cas9 system was used to induce mesenchymal stem cells to secrete SDF-1a, and SDF-1a was used after being labeled with His (see Figure 2). The expression (presence) of SDF-1a loaded within the cells and exosomes of SDF-1a-MSCs was confirmed by immunoblotting and ELISA, and the results are shown in Figures 3 and 4.

[0142] Figure 3 shows the immunoblotting results showing the expression level of SDF-1a-His conjugated His in the cell lysate of SDF-1a-MSCs, confirming the expression of SDF-1a. Figure 4 shows the results of analyzing the amount of SDF-1a secreted from SDF-1a-MSCs and exosomes of MSCs by ELISA, confirming that the amount of SDF-1a secreted from exosomes of SDF-1a-MSCs was 2.5 times higher than the amount secreted from exosomes of MSCs.

[0143]

[0144] Experimental Example 2. Confirmation of pore size and structure of polymer support

[0145] The pore size and structure of the polymer supports manufactured in Example 1 and Comparative Examples 2 to 3 were analyzed using a scanning electron microscope.

[0146] Figure 5 is a photograph showing the pore size and structure of a polymer support according to one specific example, analyzed using a scanning electron microscope. As a result, as shown in Figure 5, in the case of Example 1, the average size of the pores within the support was measured to be 50 μm.

[0147] That is, in the case of a polymer support according to a daily aspect, it was found that the size and structure of the pores can be controlled by using the size and content of the pore inducer.

[0148]

[0149] Experimental Example 3. In Vitro Wound Healing Migration Analysis

[0150] To confirm the in vitro wound healing ability of biodegradable polymer scaffolds, the migration-enhancing effect of human kidney-2 (HK-2) cells was confirmed. Specifically, the polymer scaffolds prepared in Example 1 and Comparative Examples 2 and 3 were cultured with HK-2 cells using a transwell for 24 hours, and the open wound gap was measured.

[0151] Figure 6 shows the effect of a polymer support on in vitro wound healing according to a daily aspect, specifically, the results of measuring the wound distance using ImageJ software.

[0152] As a result, as shown in Table 1 below, it was confirmed that the wound healing ability of Example 1 was superior to that of Comparative Examples 2 and 3.

[0153]

[0154] Control group Comparative Example 2 Comparative Example 3 Example 1 Wound healing ability 21.8±1.96% 40.6±2.07% 60.0±0.21% 77.5±2.37%

[0155]

[0156] That is, it was found that the polymer scaffold according to the daily aspect can promote wound healing in vitro by edaravone and stem cell recruitment-induced secreted extracellular vesicles.

[0157]

[0158] Experimental Example 4. Confirmation of in vitro blood vessel formation

[0159] To confirm the in vitro vascularization of the polymer scaffold, the degree of vascularization of human umbilical vein endothelial cells (HUVECs) was examined.

[0160] Specifically, HUVECs were dispensed into wells coated with matrigel, and the polymer supports prepared in Example 1 and Comparative Examples 2 to 3 were cultured for 24 hours using a transwell, and then the degree of blood vessel formation was measured.

[0161] Figure 7 illustrates the effect of polymer scaffolds on in vivo blood vessel formation according to various aspects, specifically, the results of observing the number of branch points and tubule length after staining with Calcein-AM. As a result, as shown in Figure 7, it was confirmed that Example 1 had superior in vitro blood vessel formation ability compared to Comparative Examples 2 and 3.

[0162] That is, it was found that the polymer scaffold according to the daily aspect could promote in vitro angiogenesis by edaravone and stem cell recruitment inducing factor secreted extracellular vesicles.

Claims

1. A biodegradable polymer support for renal regeneration comprising basic ceramic particles, extracellular matrix, zinc particles, a substance for inducing renal regeneration, and a biodegradable polymer.

2. A biodegradable polymer support according to claim 1, wherein the basic ceramic particles are at least one selected from the group consisting of an alkali metal, an oxide of an alkali metal, a hydroxide of an alkali metal, an alkaline earth metal, an oxide of an alkaline earth metal, and a hydroxide of an alkaline earth metal.

3. A biodegradable polymer support according to claim 2, wherein the alkali metal oxide, alkali metal hydroxide, alkaline earth metal oxide or alkaline earth metal hydroxide is selected from the group consisting of lithium hydroxide, beryllium hydroxide, sodium hydroxide, magnesium hydroxide, potassium hydroxide, calcium hydroxide, rubidium hydroxide, strontium hydroxide, barium hydroxide, cesium hydroxide, francium hydroxide, radium hydroxide, magnesium oxide, sodium oxide, lithium oxide, sodium oxide, manganese oxide, potassium oxide, calcium oxide, barium oxide, cesium oxide and radium oxide.

4. A biodegradable polymer support according to claim 1, wherein the basic ceramic particles have their surface modified with a fatty acid, a polymer material, or a mixture thereof.

5. A biodegradable polymer support according to claim 1, wherein the basic ceramic particles have a diameter of 1 nm to 1 mm.

6. A biodegradable polymer support according to claim 1, wherein the extracellular matrix is ​​isolated from a human body or an animal.

7. A biodegradable polymer support according to claim 1, wherein the zinc particles are at least one selected from the group consisting of zinc oxide, zinc sulfide, zinc nitrate, zinc selenide, zinc telluride, zinc nitride, zinc phosphide, zinc arsenide, zinc antimonide, zinc peroxide, zinc hydride, zinc oxalate dihydrate, zinc chloride, zinc bromide, zinc iodide, zinc hydroxide, zinc chlorate, zinc sulfate, zinc phosphate, zinc molybdate, zinc cyanide, zinc metaarsenite, zinc arsenate octahydrate, zinc chromate, zinc pyrithione, and zinc acetate.

8. A biodegradable polymer support according to claim 1, wherein the zinc particles are surface-modified with a fatty acid, a polymer material, or a mixture thereof.

9. A biodegradable polymer support according to claim 1, wherein the diameter of the zinc particles is 10 nm to 1 mm.

10. A biodegradable polymer support according to claim 1, wherein the substance for inducing renal regeneration is edaravone (EDV), extracellular vesicles or a mixture thereof.

11. A biodegradable polymer support according to claim 10, wherein the extracellular vesicles secrete a stem cell recruitment inducing factor.

12. A biodegradable polymer support according to claim 10, wherein the extracellular vesicles are isolated from stem cells derived from at least one selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, skin, amniotic membrane, and placenta.

13. A biodegradable polymer support according to claim 12, wherein the stem cells are cells genetically engineered to overexpress a stem cell recruitment inducing factor compared to parent cells.

14. A biodegradable polymer support according to claim 10 or 13, wherein the stem cell recruitment inducing factor is SDF-1α (Stromal Derived Factor-1α).

15. A biodegradable polymer support according to claim 1, wherein the biodegradable polymer is at least one selected from the group consisting of polylactide, polyglycolide, polycaprolactone, polylactide-co-glycolide, polylactide-co-caprolactone, polyglycolide-co-caprolactone, polydioxanone, polydioxanone, polytrimethylene carbonate, polyglycolide-co-dioxanone, polyamide ester, polypeptide, polyolthoester, polymaleic acid, polyanhydride, polysebacinhydride, polyhydroxyalkanoate, polyhydroxybutyrate, and polycyanoacrylate.

16. A biodegradable polymer support according to claim 1, wherein the basic ceramic particles are present in an amount of 1 to 20 wt%, the extracellular matrix is ​​present in an amount of 10 to 50 wt%, the zinc particles are present in an amount of 1 to 10 wt%, and the biodegradable polymer is present in an amount of 25 to 85 wt%, based on the total weight of the biodegradable polymer support.

17. A biodegradable polymer support according to claim 16, wherein, when the material for inducing renal regeneration includes edaravone, the amount of edaravone is 0.1 to 1 wt% based on the total weight of the biodegradable polymer support.

18. In claim 16, if the material for inducing renal regeneration includes extracellular vesicles, the extracellular vesicles in the biodegradable polymer support are 1 x 10 6 Inside 1 x 10 12 Personal, biodegradable polymer support.

19. A bioimplant for renal regeneration comprising the biodegradable polymer support of claim 1.

20. A step of preparing a first polymer solution by mixing basic ceramic particles, extracellular matrix, zinc particles, and biodegradable polymer; A step of preparing a second polymer solution by mixing the first polymer solution and a pore inducer; and A method for producing a biodegradable polymer support for kidney regeneration, comprising the step of drying the second polymer solution to produce a porous support, The method comprises mixing a substance for inducing renal regeneration together in the step of preparing the first polymer solution; A manufacturing method further comprising a step of loading a material for inducing renal regeneration after the step of manufacturing the porous support.

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