CN116492448A - PEG-EPO and mesenchymal stem cell-loaded composition, medicament and preparation method thereof - Google Patents
PEG-EPO and mesenchymal stem cell-loaded composition, medicament and preparation method thereof Download PDFInfo
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- CN116492448A CN116492448A CN202310404410.1A CN202310404410A CN116492448A CN 116492448 A CN116492448 A CN 116492448A CN 202310404410 A CN202310404410 A CN 202310404410A CN 116492448 A CN116492448 A CN 116492448A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 19
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- 229940105423 erythropoietin Drugs 0.000 description 20
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 20
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 10
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
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- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/505—Erythropoietin [EPO]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/37—Digestive system
- A61K35/39—Pancreas; Islets of Langerhans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1816—Erythropoietin [EPO]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5026—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Engineering & Computer Science (AREA)
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- Cell Biology (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Diabetes (AREA)
- Organic Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Developmental Biology & Embryology (AREA)
- Virology (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Nutrition Science (AREA)
- Emergency Medicine (AREA)
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- Endocrinology (AREA)
- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention belongs to the field of biological pharmacy, and in particular relates to a composition and a medicament for loading PEG-EPO and mesenchymal stem cells and a preparation method thereof. According to the invention, the PEG-EPO is obtained by modifying the EPO molecule with the terminal modified PEG, and the PEG modification can not only reduce the immunogenicity of the protein molecule EPO, but also reduce the sensitization and increase the water solubility and stability of the protein molecule EPO. More importantly, PEG modification reduces the hydrolyzability of EPO and prolongs the in vivo half-life. Meanwhile, when PEG-EPO, mesenchymal stem cells and pancreatic islets are loaded together for pancreatic islet transplantation, the biological curative effect of EPO can be greatly improved, and the reconstruction of blood transport after transplantation is promoted.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and in particular relates to a composition and a medicament for loading PEG-EPO and mesenchymal stem cells and a preparation method thereof.
Background
Diabetes is one of the major diseases that severely threatens human health. According to the international diabetes mellitus consortium (IDF) report, it was shown that adults 20-79 years old, about 4.63 million worldwide, had diabetes. Islet transplantation is the only therapeutic approach that can stably control blood glucose by minimally invasive means. Erythropoietin (EPO) is the primary regulator of erythropoiesis and has been shown to promote angiogenesis. The main precondition for the survival of transplanted islets is rapid revascularization after transplantation; thus, EPO improves the revascularization of transplanted islets.
Disclosure of Invention
A first object of the present invention is to provide a cell complex and its use in the preparation of a medicament.
A cell complex contains protein linear PEG-EPO, islet cells and mesenchymal stem cells as effective components.
The preparation method of the protein linear PEG-EPO in the cell complex comprises the following steps: the linear PEG is combined with the EPON terminal amino group and the lysine epsilon amino group through stable covalent bonds; the monosubstituted PEG glycoprotein is obtained after purification; by synthesis, hydrophobic, desalting, ion exchange, ultrafiltration; obtaining the protein linear PEG-EPO.
More specifically, the preparation method of the protein linear PEG-EPO comprises the following steps:
the linear PEG is combined with the EPON terminal amino group and the lysine epsilon amino group through stable covalent bonds; taking an EPO stock solution, adding a proper amount of PBS solution with pH of 7.0+/-0.5 to prepare an EPO solution, and shaking uniformly;
weighing PEG according to the mass ratio of EPO to PEG of 0.07+/-0.03:1, adding PBS solution with pH of 3.0+/-0.5 to prepare PEG solution, and shaking uniformly; adding PEG solution into EPO solution for reaction, and sampling SEC-HPLC detection after reaction, wherein the single substitution rate is more than or equal to 40%;
obtaining a target peak after gradient elution by using Buffer A and Buffer B with the pH value of 7.0+/-1.0, desalting and eluting by using Buffer C with the pH value of 3.5+/-1.0, collecting, finally obtaining target protein by gradient elution by using Buffer D with the pH value of 3.5+/-1.0, and obtaining the protein linear PEG-EPO by intercepting the molecular weight of 10 kd.
The second object of the invention is a water-soluble drug sustained release microsphere and application thereof in preparing drugs.
A water-soluble drug sustained release microsphere comprising:
an inner core region, the inner core region consisting of an isolated oil phase encapsulating a water-soluble drug; the water-soluble medicine is protein linear PEG-EPO, islet cells and mesenchymal stem cells;
and the external diffusion area is composed of medically acceptable high polymer auxiliary materials.
The preparation method of the water-soluble medicine sustained-release microsphere comprises the steps that islet cells, mesenchymal stem cells and protein linear PEG-EPO are dissolved in a solution containing polyvinyl alcohol/carbomer/Arabic/polyvinylpyrrolidone to form an inner water phase W1;
adding W1 into an acetone/ethyl acetate/dichloromethane organic solvent in which PLGA and span are dissolved to form an oil phase/dispersion medium O;
carrying out micro-jet homogenization treatment to form W1/O primary emulsion;
then dispersing the primary emulsion in a polyvinyl alcohol solution containing tween to form an external water phase W2, and carrying out micro-jet homogenization treatment to obtain W1/O/W2 compound emulsion;
injecting the complex emulsion into a polyvinyl alcohol solution, stirring, and volatilizing an organic solvent to form microspheres; centrifuging the suspension with the dispersed microspheres, filtering, washing, drying and collecting the microspheres.
According to the invention, the PEG-EPO is obtained by modifying the EPO molecule with the terminal modified PEG, and the PEG modification can not only reduce the immunogenicity of the protein molecule EPO, but also reduce the sensitization and increase the water solubility and stability of the protein molecule EPO. More importantly, PEG modification reduces the hydrolyzability of EPO and prolongs the in vivo half-life. Meanwhile, when PEG-EPO, mesenchymal stem cells and pancreatic islets are loaded together for pancreatic islet transplantation, the biological curative effect of EPO can be greatly improved, and the reconstruction of blood transport after transplantation is promoted.
The microsphere has the advantages of stable and feasible preparation process, round shape, smooth surface, good fluidity and uniform particle size distribution.
The slow release microspheres have the advantages of prolonging the acting time of the medicine, avoiding frequent injection and improving the compliance of patients; the blood concentration is stable, the peak-valley fluctuation is small, and the adverse reaction is reduced; the first pass effect of the medicine is avoided; has good treatment effect, reduces the workload of clinical preparation distribution, drug administration and monitoring, has low total treatment cost, and can degrade microsphere encapsulation materials without toxicity or harm to human bodies.
Drawings
The invention will be further described with reference to the drawings and examples.
FIG. 1 shows the results of staining islet cells of mice to identify their activity.
Fig. 2 shows islet cell results from insulin release experiments.
Fig. 3 is a diagram of a process of implantation of a kidney capsule in a mouse.
FIG. 4 is a comparison of normoglycemia maintenance in diabetic mice in the control group versus the experimental group (MSCs+PEG-EPO).
FIG. 5 is a liquid chromatogram of the prepared protein linear PEG-EPO.
Fig. 6 and 7 are electron microscope pictures of the prepared water-soluble drug sustained-release microspheres.
FIG. 8 shows the average erythrocyte HB content of mice after administration of the prepared water-soluble drug sustained release microsphere, EPO and PEG-EPO.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
In specific embodiments, steps, material selections, numerical parameters that are not described in detail are all routine selections in the art, or any prior art that is presently disclosed.
Example 1 animal experiments with cell complexes
(1) Isolation, purification and digestion of mouse islets
1) The pancreas was digested with BALB/c mice (donor) and 2.0mL collagenase solution was aspirated with a 2.5mL syringe.
1) Putting into a water bath kettle at 37 ℃ for digestion until pancreas becomes creamy or quicksand, and stopping digestion;
2) Filtering to a 50mL centrifuge tube by using a 450 μm screen of a glass funnel;
3) Islet cells were purified with density 1.119, 1.083, 1.077Histopaque solution and HBSS solution.
(2) Islet identification, islet equivalent and purity calculation
1) The islets were stained with DTZ at room temperature for 3min and counted.
2) Islet Equivalent (IEQ) was calculated with reference to the method proposed by Lembert et al.
(3) Establishment of mouse-mouse islet allograft model and sample collection
1) The isolated mouse islet cell mass suspension was dropped to a counting dish, counted under a microscope and islet equivalents were counted.
2) 300 islets isolated from BALB/C mice (donor) were prepared for transplantation into the kidney capsule of one C57BL/6 diabetic mouse (recipient).
3) The transplanted kidney was replaced and the wound was sutured.
(4) Preparation of PEG-EPO
The PEG-EPO injection is prepared from PEG-EPO obtained by PEG synthesis reaction and high purification of EPO.
The PEG-EPO is formed by combining linear PEG with EPO N-terminal amino and lysine epsilon amino through stable covalent bonds, wherein the main combining sites are N-terminal amino acid, 52-position lysine and 45-position lysine, and the mono-substituted PEG glycoprotein is obtained after high purification, and the target protein linear PEG-EPO is obtained through synthesis, hydrophobicity, desalination, ion exchange and ultrafiltration.
The method comprises the following steps: the linear PEG is combined with the N-terminal amino group of EPO and the epsilon amino group of lysine through stable covalent bonds; taking an EPO stock solution, adding a proper amount of PBS solution with pH of 7.0+/-0.5 to prepare an EPO solution, and shaking uniformly;
weighing PEG, adding PBS solution with pH of 3.0+ -0.5 to prepare PEG solution, and shaking; adding PEG solution into EPO solution for reaction, and sampling SEC-HPLC detection after reaction, wherein the single substitution rate is more than or equal to 40%;
obtaining a target peak after gradient elution by using Buffer A and Buffer B with pH value of 7.0+/-1.0, desalting and eluting by using Buffer C with pH value of 3.5+/-1.0, collecting, finally obtaining target protein by gradient elution by using Buffer D with pH value of 3.5+/-1.0, and obtaining the protein linear PEG-EPO with cut-off molecular weight of 10 kd.
As shown in FIG. 5, a liquid chromatogram of the protein linear PEG-EPO is shown.
(5) Construction and Performance of PEG-EPO and mesenchymal Stem cell-loaded islet transplantation Complex
Based on the optimized formula process and after PEG-EPO is introduced, finally, the islets and the mesenchymal stem cells are wrapped in the PEG-EPO, and the islets are transplanted after being cultured for 4 hours at 37 ℃.
(6) Islet cell culture and insulin release assay
1) Placing the encapsulated islet cells in RPMI 1640 medium, and culturing for 2h under stimulation of low sugar (concentration of 2.8 mmol/L) and high sugar (concentration of 16.7 mmol/L); and detecting the insulin content in the supernatant by using an insulin enzyme-linked immunosorbent assay kit.
(7) Islet cell activity assay
Placing the encapsulated islet cells in 10mLRPMI 1640 medium at 37deg.C CO 2 Culturing in an incubator for 8 hours, and detecting the activity of islet cells by using a Calcein-AM/PI method.
(8) Experimental results
(1) After isolation and purification of the islets from mice (shown in FIG. 1, part A, 50-fold magnification), they were identified by DTZ (dithizone) staining (shown in FIG. 1, part B, islet cells appear scarlet, oval or circular, 100-fold magnification after DTZ staining). Islet activity was measured by Calcein-AM/PI staining, and more than about 90% of islets exhibited green fluorescence, indicating that islets had good activity (as shown in section C, D of fig. 1, the Calcein-AM/PI staining measured islet activity, and the green islet cells demonstrated activity, 100-fold magnification).
(2) Isolated mouse islet cell insulin release experiments demonstrate that islet cells are capable of secreting insulin, are sensitive to high sugar stimuli, and can secrete more insulin. Insulin stimulation index reached 2.5 times on average (fig. 2, n=5).
(3) The islet transplantation compound loaded with PEG-EPO and mesenchymal stem cells is used for carrying out islet transplantation of the kidney capsule membrane of the mouse, as shown in figure 3;
(4) Comparison of the hypoglycemic function of the control group with the experimental group (PEG-EPO and mesenchymal stem cell-loaded mouse islet graft).
The results show that the PEG-EPO and mesenchymal stem cell loaded allogenic mouse islet grafts were able to significantly lower blood glucose to the normal range in diabetic C57BL/6 receptor mice and maintained euglycemic for significantly longer time than the control group (the islets of mice not loaded with PEG-EPO and mesenchymal stem cells were used as grafts) (as shown in fig. 4).
Example 2 preparation and experiment of Water-soluble drug sustained-Release microspheres
The protein of example 1, straight-chain PEG-EPO
(1) Preparation of the inner aqueous phase
Islet cells, mesenchymal stem cells and protein linear PEG-EPO are dissolved in a solution containing polyvinyl alcohol/carbomer/acacia/polyvinylpyrrolidone to form an inner water phase W1;
(2) Preparation of the oil phase
Adding W1 into an acetone/ethyl acetate/dichloromethane organic solvent in which PLGA and span are dissolved to form an oil phase/dispersion medium O;
(3) Preparation of the Primary emulsion
Carrying out micro-jet homogenization treatment to form W1/O primary emulsion;
(4) Preparation of multiple emulsion
Dispersing the W1/O primary emulsion in a polyvinyl alcohol solution containing tween to form an external water phase W2, and carrying out micro-jet homogenization treatment to obtain W1/O/W2 compound emulsion;
(5) Preparation of water-soluble medicine slow-release microsphere
Injecting the complex emulsion into a polyvinyl alcohol solution, stirring, and volatilizing an organic solvent to form microspheres; centrifuging the suspension with the dispersed microspheres, filtering, washing, drying and collecting the microspheres.
The prepared water-soluble medicine slow-release microsphere electron microscope pictures are shown in fig. 6 and 7, and the microsphere has round shape, smooth surface, good fluidity and uniform particle size distribution.
In order to compare the sustained release effect of the water-soluble drug sustained release microsphere, the detection of the average red blood cell HB content of the mice was performed in the same experimental manner as in example 1, and the results are shown in FIG. 8.
In fig. 8, EPO is administered continuously, once a week; PEG-EPO is administered continuously, once every 2 weeks; the sustained release PEG-EPO (i.e. water-soluble drug sustained release microsphere) is continuously administered once every 2 weeks. It can be seen that the water-soluble drug sustained release microspheres can better maintain the average erythrocyte hemoglobin level in the case of the same number of administrations as PEG-EPO.
The foregoing detailed description is directed to one of the possible embodiments of the present invention, which is not intended to limit the scope of the invention, but is to be accorded the full scope of all such equivalents and modifications so as not to depart from the scope of the invention.
Claims (7)
1. The cell complex is characterized in that the active ingredients are protein linear PEG-EPO, islet cells and mesenchymal stem cells.
2. The cell complex of claim 1, wherein the protein linear PEG-EPO is prepared by the process of: the linear PEG is combined with the EPON terminal amino group and the lysine epsilon amino group through stable covalent bonds; the monosubstituted PEG glycoprotein is obtained after purification; by synthesis, hydrophobic, desalting, ion exchange, ultrafiltration; obtaining the protein linear PEG-EPO.
3. The preparation method of the protein linear PEG-EPO is characterized by comprising the following steps:
the linear PEG is combined with the EPON terminal amino group and the lysine epsilon amino group through stable covalent bonds; taking an EPO stock solution, adding a proper amount of PBS solution with pH of 7.0+/-0.5 to prepare an EPO solution, and shaking uniformly;
weighing PEG according to the mass ratio of EPO to PEG of 0.07+/-0.03:1, adding PBS solution with pH of 3.0+/-0.5 to prepare PEG solution, and shaking uniformly; adding PEG solution into EPO solution for reaction, and sampling SEC-HPLC detection after reaction, wherein the single substitution rate is more than or equal to 40%;
obtaining a target peak after the gradient elution of BufferA and BufferB with the pH value of 7.0+/-1.0, desalting and eluting with BufferC with the pH value of 3.5+/-1.0, collecting, finally obtaining target protein by the gradient elution of BufferD with the pH value of 3.5+/-1.0, and obtaining the protein linear PEG-EPO with the cut-off molecular weight of 10 kd.
4. Use of the cell complex of claim 1 or 2 in the manufacture of a medicament.
5. A water-soluble drug sustained release microsphere, comprising:
an inner core region, the inner core region consisting of an isolated oil phase encapsulating a water-soluble drug; the water-soluble medicine is protein linear PEG-EPO, islet cells and mesenchymal stem cells;
and the external diffusion area is composed of medically acceptable high polymer auxiliary materials.
6. The method for preparing the water-soluble medicine sustained-release microsphere according to claim 5, wherein islet cells, mesenchymal stem cells and protein linear PEG-EPO are dissolved in a solution containing polyvinyl alcohol/carbomer/acacia/polyvinylpyrrolidone to form an inner water phase W1;
adding W1 into an acetone/ethyl acetate/dichloromethane organic solvent in which PLGA and span are dissolved to form an oil phase/dispersion medium O;
carrying out micro-jet homogenization treatment to form W1/O primary emulsion;
then dispersing the primary emulsion in a polyvinyl alcohol solution containing tween to form an external water phase W2, and carrying out micro-jet homogenization treatment to obtain W1/O/W2 compound emulsion;
injecting the complex emulsion into a polyvinyl alcohol solution, stirring, and volatilizing an organic solvent to form microspheres; centrifuging the suspension with the dispersed microspheres, filtering, washing, drying and collecting the microspheres.
7. The use of the water-soluble drug-eluting microsphere as defined in claim 5 in the preparation of a medicament.
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