WO2018131890A1 - Microsphères d'acide poly (lactique-co-glycolique) revêtues de polydopamine, et méthode de modification de la surface cellulaire utilisant ces dernières - Google Patents

Microsphères d'acide poly (lactique-co-glycolique) revêtues de polydopamine, et méthode de modification de la surface cellulaire utilisant ces dernières Download PDF

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WO2018131890A1
WO2018131890A1 PCT/KR2018/000488 KR2018000488W WO2018131890A1 WO 2018131890 A1 WO2018131890 A1 WO 2018131890A1 KR 2018000488 W KR2018000488 W KR 2018000488W WO 2018131890 A1 WO2018131890 A1 WO 2018131890A1
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microspheres
cells
pancreatic islet
drug
plga
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Korean (ko)
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정지헌
응웬티엔티엡
팜탄텅
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영남대학교 산학협력단
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Priority claimed from KR1020180003155A external-priority patent/KR102080689B1/ko
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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
    • A61K35/33—Fibroblasts
    • 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
    • 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
    • A61K35/407—Liver; Hepatocytes
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

Definitions

  • the present invention relates to poly (lactic-co-glycolic acid) microspheres coated with polydopamine, a method of cell surface modification using the microspheres, and a diabetes treatment using cells surface-modified with the microspheres.
  • Diabetes diabetes mellitus, DM is a disease characterized by hyperglycemic symptoms and complications caused by abnormal insulin secretion of pancreatic ⁇ -cells or abnormal receptors on insulin action organs or organs.
  • exercise therapy and diet are mainly performed along with insulin injection therapy, but there are limitations in the treatment such as inability to cure and the risk of complications still exists.
  • pancreas transplantation has the advantage that in vitro manipulation such as in vitro culture of isolated pancreatic islet cells is possible.
  • in vitro manipulation such as in vitro culture of isolated pancreatic islet cells is possible.
  • infinite pancreatic islet cells can be supplied using pigs, etc., and transplantation can be easily performed without complications due to a relatively simple procedure.
  • pancreatic islet cell transplantation requires minimizing pancreatic islet cell damage during the development and isolation of effective pancreatic islet cells, and cell damage caused by nonspecific inflammatory processes during transplantation and engraftment failure. Problems such as minimizing, overcoming cellular damage due to immune responses occurring after transplantation, and securing pancreatic islet cell sources should be addressed.
  • the initial inflammatory response after pancreatic islet cell transplantation causes functional incompatibility or destruction of pancreatic islet cells due to cell necrosis and apoptosis, and thus, pancreatic islet cells must be transplanted with a larger amount of pancreatic islet cells than are actually necessary. .
  • Macro encapsulation is a concept that uses a hydrogel structure for grafting of transplanted pancreatic islet cells. Although this method is very simple, a large amount of hydrogel causes hypoxia in transplanted cells due to a lack of blood vessel regeneration, and as a result, pancreatic islet cells transplanted subcutaneously exhibit blood glucose fluctuations.
  • pancreatic islet cells are small in size and can be transplanted to various sites such as portal and kidney capsules.
  • pancreatic islet cells are transplanted into the portal vein, the transplanted pancreatic islet cells are directly exposed to the blood, causing blood coagulation around the pancreatic islet cells due to activation of the blood coagulation system such as platelets and complement and rapidly destroying the pancreatic islet cells.
  • An instant blood mediated inflammatory reaction may occur.
  • pancreatic islet cell transplantation an efficient method for surface modification of pancreatic islet cells and a method for suppressing a nonspecific immune response are needed.
  • An object of the present invention is a microsphere coated with polydopamine and made of a poly (lactic-co-glycolic acid) (PLGA) polymer and encapsulating a drug or bioactive substance in the microsphere. It is to provide a microsphere characterized in that.
  • PLGA poly (lactic-co-glycolic acid)
  • Another object of the present invention is to provide a method for producing the microspheres and cell surface modification method using the microspheres.
  • Still another object of the present invention is to provide a pharmaceutical composition for treating diabetes comprising the cells whose surface is modified by the microspheres as an active ingredient.
  • the present invention is microspheres coated with polydopamine and made of poly (lactic-co-glycolic acid) (PLGA) polymer and the drug or physiology in the microspheres It provides a microsphere characterized in that the active material is encapsulated.
  • PLGA poly (lactic-co-glycolic acid)
  • the present invention is a first step of encapsulating a drug or bioactive material in the microspheres by stirring the PLGA microspheres and the drug or bioactive material, the polymer suspension of the PLGA microspheres containing the drug or bioactive material and dopamine It provides a method for producing a microsphere comprising a second step of reacting the solution and a third step of obtaining a PLGA microsphere coated with polydopamine by centrifuging the reacted reactant.
  • the present invention is a first step of encapsulating a drug or bioactive material in the microspheres by stirring the PLGA microspheres and the drug or bioactive material, the polymer suspension of the PLGA microspheres containing the drug or bioactive material and dopamine
  • the second step of reacting the solution the third step of obtaining the PLGA microspheres coated with polydopamine by centrifugation of the reacted reactant and the cells of the polydopamine-coated PLGA microspheres are reacted to modify the surface of the cells. It provides a cell surface modification method comprising the fourth step.
  • the present invention provides a pharmaceutical composition for treating diabetes comprising the cells whose surface is modified by the microsphere as an active ingredient.
  • the present invention relates to a method for modifying a cell surface such as pancreatic islet cells using poly (lactic-co-glycolic acid) microspheres coated with polydopamine, which is simpler than a conventional cell surface modification method.
  • Targeted drug encapsulation is possible to prolong drug release around the microenvironment of the cell and deliver several types of drugs or bioactive agents, such as immunosuppressants or anticoagulants, to achieve multiple effects.
  • the cell surface modification method of the present invention having the above effect has the advantage of improving the transplantation rate of cells, and reduce the non-specific immune response can be utilized as a novel cellular drug delivery system.
  • Figure 1 (A) is a schematic diagram showing a method of surface modification of pancreatic islet cells using poly (lactic-co-glycolic acid) microspheres coated with polydopamine
  • Figure 1 (B) is a micro-dopamin coated micro Schematic representation of the binding mechanism between the quinone group of polydopamine in spear and the amine group of extracellular collagen matrix on the surface of pancreatic islets.
  • Figure 2 schematically shows the effect of pancreatic islet cell modification using PLGA microspheres coated with an immunosuppressant (FK506) and coated with polydopamine.
  • Figure 3 shows the characteristics of PLK microspheres loaded with FK506 (a) scanning electron microscopy (SEM) images, (b) X-ray powder diffraction analysis (XRD) spectra and (c) Fourier transform infrared spectroscopy (FT-IR) It was confirmed by spectrum.
  • SEM scanning electron microscopy
  • XRD X-ray powder diffraction analysis
  • FT-IR Fourier transform infrared spectroscopy
  • FIG. 4 shows the characteristics of polygapamine-coated PLGA microspheres in (ab) SEM images according to dopamine oxidation time (1 hour and 7 hours), and (c) in microspheres coated with polydopamine at 1 hour dopamine oxidation time.
  • FIG. 5 shows the relative fluorescence intensity of unmodified pancreatic islet cells and modified pancreatic islet cells in (a) fluorescence images, (b) confocal images, (c) pancreatic islet cells modified with pD-Cou / M, and (d) It was confirmed by SEM image.
  • Figure 6 shows the stability of microspheres over time on the surface of pancreatic islet cells by (a) SEM image and (b) confocal image.
  • FIG. 7 shows the survival and function of microspheres modified pancreatic islet cells (a) survival / kill assay, (b) CCK-8 assay, (c) western blot, (de) glucose stimulated insulin secretion (GSIS) assay and It is confirmed by the value of the stimulus index (SI).
  • IPGTT intraperitoneal glucose tolerance test
  • the inventors of the present invention have developed a method for surface modification of pancreatic islet cells using poly (lactic-co-glycolic acid) (PLGA) microspheres coated with polydopamine. It was confirmed that the quinone group interacted with the amine group of the extracellular collagen matrix surrounding the pancreatic islet cells, and that the pancreatic islet cells modified with the polygapamine coated PLGA microspheres showed no human toxicity.
  • the present invention has been completed.
  • the present invention is coated with polydopamine (polydopamine) and encapsulated microspheres made of poly (lactic-co-glycolic acid) (PLGA) polymer and the drug or bioactive material in the microspheres It provides a microsphere characterized in that.
  • polydopamine polydopamine
  • encapsulated microspheres made of poly (lactic-co-glycolic acid) (PLGA) polymer and the drug or bioactive material in the microspheres It provides a microsphere characterized in that.
  • the average diameter of the microspheres may be 1 to 1000 ⁇ m, but is not limited thereto.
  • the drug or bioactive material may be one or more selected from the group consisting of immunosuppressants, anticoagulants, anti-inflammatory agents, antioxidants and hormones, but is not limited thereto.
  • the drug or bioactive material may be provided in a form selected from the group consisting of chemicals, proteins, peptides, antibodies, genes, siRNA and microRNA, but is not limited thereto.
  • the immunosuppressive agent is Tacrolimus, Cyclosporin, Sirolimus, Everolimus, Ridaforolimus, Temsirolimus, Temsirolimus, Thysirolimus Umirolimus, Zotarolimus, Leflunomide, Methotrexate, Rituximab, Ruplizumab, Daclizumab, Daclizumab, Abatacept and It may be one or more selected from the group consisting of Bellatacept, but is not limited thereto.
  • the anticoagulant may include argatroban, coumarin, heparin, low molecular weight heparin, hirudin, dabigatran, Melagatran, Clopidogrel, Ticlopidine and Abxisimab (Abciximab) may be one or more selected from the group consisting of, but not limited to.
  • the anti-inflammatory agent is acetoaminephene, aspirin, ibuprofen, dicrofenac, indomethacin, pyroxicam, phenopropene, flubiprofen, ketoprofen, naproxen, supropene, roxofero It may be, but is not limited to, one or more selected from the group consisting of pens, synoxycamps and tenoxycamps.
  • the present invention is a first step of encapsulating a drug or bioactive material in the microspheres by stirring the PLGA microspheres and the drug or bioactive material, the polymer suspension of the PLGA microspheres containing the drug or bioactive material and dopamine It provides a method for producing a microsphere comprising a second step of reacting the solution and a third step of obtaining a PLGA microsphere coated with polydopamine by centrifuging the reacted reactant.
  • the second step may include, but is not limited to, adding the polymer suspension of the PLGA microspheres and the dopamine solution in a volume ratio of 1: 1.5 to 3.
  • the second step may be carried out for 30 minutes to 2 hours at a condition of 15 to 40 °C, pH 8 to 9, but is not limited thereto.
  • the present invention is a first step of encapsulating a drug or bioactive material in the microspheres by stirring the PLGA microspheres and the drug or bioactive material, the polymer suspension of the PLGA microspheres containing the drug or bioactive material and dopamine A second step of reacting the solution, a third step of obtaining the PLGA microspheres coated with polydopamine by centrifugation of the reacted reactants, and a reaction of the cells with the PLGA microspheres coated with polydopamine to modify the cell surface.
  • a cell surface modification method comprising the fourth step.
  • the fourth step of cells is not limited to specific cells, pancreatic islet cells, stem cells, hepatocytes, fibroblasts, lymphocytes, vascular endothelial cells, neurons, enamel cells, keratinocytes, adipocytes, osteoblasts
  • the fourth step may be repeated 2 to 5 times at 5 to 15 minutes at 15 to 40 °C, but is not limited thereto.
  • the present invention provides a pharmaceutical composition for treating diabetes comprising the cells whose surface is modified by the microsphere as an active ingredient.
  • the pharmaceutical composition may be transplanted in various ways depending on the site of transplantation, and may be transplanted to any site in the living body such as liver, kidney, stomach, abdominal cavity, testes, eye, and bone marrow subcutaneous.
  • An effective amount of the active ingredient of the pharmaceutical composition means an amount required for the treatment of the disease.
  • the type of disease, the severity of the disease, the type and amount of the active and other ingredients contained in the composition, the type of formulation and the age, weight, general health, sex and diet, sex and diet, time of administration, route of administration and composition of the patient may be adjusted according to various factors including, but not limited to, the rate of secretion, the duration of treatment, and the drug used concurrently.
  • Collagenase P was purchased from Roche (Roche diagnostic GmBH, Mainheim, Germany) and dichloromethane (DCM) was purchased from Junsei, Korea.
  • Fetal bovine albumin (FBS) and phosphate buffered saline (PBS) were purchased from Hyclone, and the live / dead cell viability / cytotoxicity assay kit Life Technologies, Oregon, USA and CCK-8 (cell counting kit-8) were purchased from Dojindo Laboratories, Japan.
  • PLGA microspheres were prepared using solvent-evaporation. Briefly, 40 mg PLGA was dissolved in 0.5 ml of dichloromethane, then 5 ml of 1% PVA was added and at 17,400 rpm using a homogenizer (homogenizer, T25 digital ULTRA-TURRAX ® , IKA-Werke GmbH & Co. KG). Homogenized for 5 minutes. Next, 10 ml of 1% PVA was added to the obtained emulsion to stabilize, and stirred for 4 hours to completely evaporate the solvent. Thereafter, microspheres were obtained by washing and centrifuging five times with deionized water, and the obtained microspheres were freeze-dried and stored at -20 ° C.
  • homogenizer homogenizer, T25 digital ULTRA-TURRAX ® , IKA-Werke GmbH & Co. KG. Homogenized for 5 minutes.
  • 10 ml of 1% PVA was added to the obtained emulsion to stabilize, and stir
  • FK506-encapsulated microspheres FK506-M
  • FK506-M FK506-encapsulated microspheres
  • FK506 was encapsulated in various dosages using various types of PLGA.
  • DLG 4A LA: GA 50:50, acid-terminated, Evonik
  • M (F3) and M (F4) were prepared for the purpose of extending the release time of FK506 using PLGA polymers with similar drug loading capacity as M (F1) (3.36 ⁇ 0.05% and 3.31 ⁇ 0.07%).
  • M (F1) 3.36 ⁇ 0.05% and 3.31 ⁇ 0.07%
  • PLGA microspheres were filled with 1.5% of coumarine-6, a fluorescent substance (Cou-M), and a confocal laser scanning microscope. It was analyzed using.
  • the PLGA microspheres were analyzed by a scanning electron microscope, and the average diameter was 1 to 8 ⁇ m, and the size of the microspheres was found to be well controlled when FK506 was enclosed. However, as can be seen from M (F2), it was observed that the high drug loading content darkens the particle surface and changes the surface roughness.
  • the characteristics of the polygapamine-coated PLGA microspheres were analyzed, and the dopamine reaction time was important among the variables affecting the properties.
  • the experiment was conducted for 1 to 7 hours while maintaining the final concentration of dopamine at 1 mg / ml. Was performed.
  • the increase in the reaction time increased the amount of unconjugated polydopamine nanoparticles, it was confirmed to have a particle size of 100 to 200 ⁇ m.
  • the nanoparticles are thought to interfere with the reaction between microspheres and pancreatic islet cells. Therefore, the problem can be solved by applying the reaction time to 1 hour.
  • FT-IR Fourier transform infrared
  • Nicolet Nexus 670 FT-IR spectrometer Thermo Fisher Scientific Inc., Waltham, Mass., USA
  • X-ray powder diffraction analysis (X-ray powder) using an X'Pert PRO MPD diffractometer (PANalytical, Almelo, the Netherlands) to analyze the phase state of the FK506 enclosed in PLGA microspheres diffraction, XRD).
  • the FK506 has a highly crystallized structure with sharp peaks from 10 ° to 30 °, whereas once enclosed into the microspheres it is molecularly dispersed without observing any peaks in the spectrum. Confirmed.
  • FT-IR confirmed the presence of polydopamine conjugated to the cell surface in the polysphere coated with polydopamine.
  • the polydopamine spectrum showed a broad peak in the range of 3500 to 3000 cm ⁇ 1 .
  • the stretching vibration of the hydroxyl group (-OH) was observed, whereas the spectrum of the microspheres coated with polydopamine confirmed that the stretching vibration of the hydroxyl group was not observed.
  • the spectra of polydopamine and polydopamine coated microspheres showed peaks in the 2400 to 2250 cm ⁇ 1 range. From the above results, it was confirmed that the polydopamine was physically bonded to the microspheres.
  • the tube was replaced with another 50 ml tube containing 5 ml of release medium to allow liquid to pass from both sides of the membrane, and was shaken using a shake incubator (SI-64, 150; Hanyang Scientific Equipment Co., Ltd, Republic of Korea) 37 Incubated at °C. After some time, the effluent solution in the ultracentrifuge tube was centrifuged to separate from the particles and concentrated to analyze the FK506 concentration. Experiments were performed by filling fresh tubes with tubes to maintain sink conditions for drug release.
  • pancreatic islet cells were isolated using collagenase type P.
  • Pancreatic islet cells were composed of 10% fetal bovine serum (FBS, Gibco), 2 mM sodium bicarbonate, 11 mM glucose (Sigma), 6 mM HEPES and 1% penicillin / streptomycin, Invitrogen Co., Carlsbad, Calif.) was cultured in RPMI-1640 medium. Animal experiments were approved by the Institutional Review Board of Yeungnam University.
  • pancreatic islet cells were obtained by washing twice with HBSS (pH 8.0) and centrifuging for 1 minute at 1,000 rpm.
  • HBSS pH 8.0
  • the coating efficiency was evaluated by two culture methods. The first method is to incubate cells with microspheres three times continuously for 1 hour or 10 minutes in succession, and the second method is to remove unconjugated microspheres by washing with HBSS before the next incubation.
  • microsphere-conjugated pancreatic islet cells were cultured for 24 days and observed by scanning electron microscopy and confocal laser scanning microscopy.
  • the optimal conditions for conjugating microspheres to the pancreatic islet cell surface are as follows: (1) Microsphere coating step: 1 mg / ml dopamine, 1 hour oxidation time; (2) Conjugation of pancreatic islet cells: microsphere concentration 2 mg / ml, incubation time 10 minutes, repeated 3 times.
  • microspheres were observed on the surface of pancreatic islets until day 7. As observed in the SEM image, the microspheres disappeared from the surface of the pancreatic islets after 14 days, but were detected with high density in the confocal microscope laser scanning image.
  • pancreatic islet cells 150 IEQ
  • pD-M polydopamine coated microspheres
  • the instrument used an Agilent 1260 Infinity HPLC system (Agilent Technologies, Santa Clara, Calif.) And an API-400 mass spectrometer (AB SCIEX, Framingham, Mass.).
  • Chromatographic separations consisted of an Atlatis® dC18 column (2.1 x 150 mm, 3 ⁇ m; Water Corporations, Milford, Mass.) And a mobile phase consisting of acetonitrile (A) and an aqueous buffer containing 2 mM ammonium acetate and 0.1% formic acid (B). It was performed using. The gradient started at 90% A and changed from 2.5 minutes to 10.5 minutes to 5% A and from 10.5 to 16.0 minutes to 90%. The flow rate was set to 250 ⁇ l / min and the column temperature to 60 ° C. Electrospray ionization was performed in cation mode. Nitrogen was used as the sheath gas and auxiliary gas in the elecrospray source. Tandem mass spectrometry was performed in mulitple reaction monitoring (MRM) mode. Decelerating potential (DP), collision energy (CE) and collision cell exit potential (CXP) were 81, 29 and 22, respectively.
  • MRM mulitple reaction monitoring
  • pancreatic islet cells were analyzed using CCK-8 analysis. Briefly, 100 ⁇ l of pancreatic islet cells suspended in RPMI medium were placed in a 96 well plate (20 cells / well) and WST-8 [2- (2-methoxy-4-nitrophenyl) -3- ( 10 ⁇ l of 4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-etrazoium, monosodium salt] was treated. After 4 hours reaction, absorbance was measured at 450 nm and final data was analyzed by normalizing to the dsDNA content of each sample.
  • pancreatic islet cell viability was analyzed using a live / dead assay kit. Briefly, pancreatic islet cells are collected from the culture medium and washed, containing 0.67 ⁇ M of acridine orange (AO, Sigma) and 75 ⁇ M of propidium iodide PI (Sigma) in shaded condition. Reacted with HBSS for 10 minutes. Stained pancreatic islet cells were then observed under a fluorescence microscope. Acridine orange has cell permeability, so all nucleated cells are stained to give green fluorescence. PI only enters cells with dysfunctional cell membranes and stains dead and necrotic cells to show red fluorescence.
  • AO acridine orange
  • PI propidium iodide
  • FK506 is known to cause severe toxicity to many organs and cell types, including the pancreas.
  • conjugation processes that cause chemical and physical modifications can cause pancreatic islet cell dysfunction. Therefore, it was confirmed whether the FK506 delivery system affects the survival and function of pancreatic islet cells.
  • Experiments were performed using unmodified pancreatic islet cells (control), pancreatic islet cells modified with pD-M, pancreatic islet cells modified with pD-M (F1), and pancreatic islet cells modified with pD-M (F2). was performed.
  • pancreatic islet cells are alive in all groups, and referring to Figure 7b, the control group (100 ⁇ 11.55%) in CCK-8 analysis ),
  • the survival rate of pancreatic islet cells modified with pD-M, pancreatic islet cells modified with pD-M (F1) and pancreatic islet cells modified with pD-M (F2) were 103.22 ⁇ 7.32%, respectively. It was confirmed that it is 96.78 ⁇ 8.09% and 113.15 ⁇ 7.75%.
  • Bcl-2 and Bax expression levels of pancreatic islet cells did not show a significant difference between groups.
  • the modification of pancreatic islet cells using FK506-encapsulated, polydopamine-coated microspheres did not affect cell viability.
  • pancreatic islet cells modified with microspheres The function of pancreatic islet cells modified with microspheres was determined by the amount of insulin secreted from pancreatic islet cells using low glucose and high glucose Krebs-Ringer bicarbonate buffer (KRBB, pH 7.4) solution (Sigma, St. Louis, MO). Measured and evaluated. Briefly, pancreatic islet cells were washed twice and then pretreated with 1 ml of a low glucose KRBB solution at 2.8 mM concentration for 1 hour, followed by incubation with 1 ml of a new 2.8 mM low glucose KRBB solution for 2 hours, followed by 28 mM Incubated further with 1 ml of high glucose KRBB solution at 37 ° C for 2 hours.
  • KRBB low glucose and high glucose Krebs-Ringer bicarbonate buffer
  • Type 1 diabetes was induced by C57BL / 6 mice with a single intraperitoneal injection of streptozotocin (STZ) 200 mg / kg. After 3 days, mice with blood glucose levels of 350 mg / dl or more for 2 consecutive days were selected as diabetic mice. Mice were anesthetized by intraperitoneal injection of ketamine 80 mg / kg and xylazine 16 mg / kg, exposing the left kidney of the mouse to the lumbar incision and then using a 31 gauge needle to the bottom of the kidney. Made a small wound. The curved capillary was then inserted into the capsule and gently moved in all directions to create a pouch under the capsule containing the implant.
  • STZ streptozotocin
  • Microsphere-modified pancreatic islet cells or unmodified pancreatic islet cells (400 IEQ) included in cutdown tubing were transferred to Hamilton syringes (Hamilton company, Nevada, USA). It was injected into the bag. The wound was then cauterized carefully with low heat, then the kidneys were returned to the peritoneum and the incisions closed. Mice were allowed to consume water and feed autonomously during the experiment.
  • pancreatic islet cell transplantation After pancreatic islet cell transplantation, non-fasting blood glucose levels were measured in mouse tail veins using a handheld blood glucose meter (Contour TS, Bayer Healthcare LLC, IN, USA). Experiments were considered successful if blood glucose levels dropped below 200 mg / dl for two consecutive days, and transplanted pancreatic islet cells were considered to have rejected for two consecutive days above 200 mg / dl.
  • pD-M (F1) not only improved the graft survival rate, but also regulated blood glucose much better than pD-M (F2)
  • the content of FK506 in pD-M (F1) is pD- Only half of the FK506 content in M (F2) was 3.33 ⁇ 0.07% and 6.71 ⁇ 0.11%, respectively.
  • the level of FK506 released in pD-M (F2) was about three times higher than the FK506 level released in pD-M (F1).
  • the results indicate that high levels of FK506 released from pD-M (F2) may interfere with the survival and function of transplanted pancreatic islets.
  • FK506 released from pD-M (F1) was confirmed to be sufficient to protect the pancreatic islets cells from immune rejection without adversely affecting the pancreatic islets cells.
  • mice were allowed to ingest water freely, but fasted for 12 hours prior to intraperitoneal injection of glucose solution (2.0 g / kg) dissolved in saline. Blood glucose levels were measured and recorded at 0, 5, 10, 15, 20, 30, 45, 60, 90 and 120 minutes after injection. Normal mice and diabetic mice were used as controls.
  • IPGTT intraperitoneal glucose tolerance test
  • the blood glucose level of diabetic mice rapidly increased to 600 mg / dL or more after 10 minutes of high glucose administration, and maintained at 400 mg / dL or more at 120 minutes.
  • the pattern of change in blood glucose levels of normal mice and pancreatic islet cell recipients modified with pD-M (F1) was similar and increased to 15 minutes and gradually decreased to normal levels within 120 minutes.
  • pancreatic islet cells modified with pD-M (F1) and unmodified pancreatic islet cells do not show significant differences in function in vivo.
  • pancreatic islet cells modified with pD-M showed normal development by gaining weight, demonstrating the effect of the drug delivery system.

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Abstract

La présente invention concerne une méthode de modification de la surface de cellules telles que les cellules des îlots pancréatiques à l'aide de microsphères d'acide poly (lactique-co-glycolique) revêtues de polydopamine. La présente invention offre un processus plus simple que celui des méthodes classiques de modification de la surface cellulaire, permet l'encapsulation d'une grande quantité d'un médicament cible afin de prolonger la libération du médicament dans le microenvironnement des cellules, et permet l'administration simultanée de divers types de médicaments, tels que des immunosuppresseurs ou des anticoagulants, ou des substances bioactives afin d'obtenir des effets multiples. Du fait que la méthode de modification de la surface cellulaire de la présente invention possède les effets ci-dessus, elle présente l'avantage d'améliorer les taux de réussite des transplantations cellulaires et de diminuer les réponses immunitaires non spécifiques, ce qui la rend utilisable en tant que nouveau système cellulaire d'administration de médicament.
PCT/KR2018/000488 2017-01-11 2018-01-10 Microsphères d'acide poly (lactique-co-glycolique) revêtues de polydopamine, et méthode de modification de la surface cellulaire utilisant ces dernières WO2018131890A1 (fr)

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KR20170004145 2017-01-11
KR10-2017-0004145 2017-01-11
KR1020180003155A KR102080689B1 (ko) 2017-01-11 2018-01-10 폴리도파민으로 코팅된 폴리(락틱-코-글리콜산) 마이크로스피어 및 이를 이용한 세포 표면 개질 방법
KR10-2018-0003155 2018-01-10

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US20220105047A1 (en) * 2019-01-16 2022-04-07 Research Cooperation Foundation Of Yeungnam University Microcapsule composition using alginate gel, and method for producing same
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CN115025723A (zh) * 2022-05-10 2022-09-09 吉林大学 经神经生长因子及多巴胺改良的peg-plga微球
CN116983269A (zh) * 2023-07-26 2023-11-03 上海交通大学医学院附属第九人民医院 一种载细胞多孔微球及其制备方法和应用

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