WO2018131890A1 - Poly(lactic-co-glycolic acid) microspheres coated with polydopamine and cell surface modification method using same - Google Patents

Abstract

The present invention relates to a method for modifying the surface of cells such as pancreatic islet cells by using poly(lactic-co-glycolic acid) microspheres coated with polydopamine. The present invention has a process simpler than that of conventional cell surface modification methods, enables the encapsulation of a large amount of a target drug so as to prolong drug release around the microenvironment of cells, and enables the simultaneous delivery of various types of drugs, such as immunosuppressants or anti-coagulants, or bioactive substances in order to achieve multiple effects. Therefore, the cell surface modification method, of the present invention, having the above effects, has advantages of improving cell transplantation rates and reducing nonspecific immune responses, thereby being usable as a novel cell pharmaceutical delivery system.

Description

Poly (lactic-co-glycolic acid) microspheres coated with polydopamine and cell surface modification method using the same

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 β-cells or abnormal receptors on insulin action organs or organs. In order to treat diabetes, 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.

Recently, diabetes treatment through pancreas transplantation and pancreatic islet cell transplantation has been carried out. In the case of pancreas transplantation, there are problems such as absolute shortage of donors, high surgical complications, and difficulty in post-transplantation management including continuous administration of immunosuppressive agents. In contrast, pancreatic islet cell transplantation has the advantage that in vitro manipulation such as in vitro culture of isolated pancreatic islet cells is possible. In addition, when the immune response of the xenotransplantation is overcome, infinite pancreatic islet cells can be supplied using pigs, etc., and transplantation can be easily performed without complications due to a relatively simple procedure.

However, successful 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. In particular, 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.

Recently, superiority of surface modification of pancreatic islets cells has been reported. Compared to macro- and micro-encapsulation, the transport of oxygen and nutrients in surface-modified pancreatic islet cells has dramatically increased, and pancreatic islet cells are small in size and can be transplanted to various sites such as portal and kidney capsules. . However, when the 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 (IBMIR) may occur.

Therefore, for successful 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.

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.

In order to achieve the above object, 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.

In addition, 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.

In addition, 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.

In addition, 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.

Therefore, 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.

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. TEM images of the extracted polydopamine capsules, (d) FT-IR spectra and (e) in vitro release of FK506.

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.

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).

8 shows in vivo effects of transplanted pancreatic islet cells (a) pancreatic islet cells (n = 5), (b) pancreatic islet cells (n = 7) modified with pD-M (506 unpacked), ( c) equivalent amounts of unmodified pancreatic islet cells and pD-M transplanted under contralateral renal capsule (n = 4), (d) pancreatic islet cells modified with pD-M (F1) (n = 12), (e ) pancreatic islet cells modified with pD-M (F2) (n = 6), (f) pancreatic islet cells modified with pD-M (F3) (n = 3) and (g) with pD-M (F4) Non-fasting glucose levels in diabetic C57BL / 6 mice transplanted with modified pancreatic islet cells (n = 3), (h) graft survival in each group, (i) diabetic mice (▲, red line, n = 2), normal Confirmed by intraperitoneal glucose tolerance test (IPGTT) 20 days after transplantation of pancreatic islet cell recipients modified with mice (●, black line, n = 1) and pD-M (F1) (◆, blue line, n = 4) will be.

FIG. 9 shows (a) control pancreatic islet cells (n = 5), (b) pancreatic islet cells modified with pD-M (unfilled FK506) (n = 7), (c) non transplanted under contralateral kidney capsule Equivalent amount of modified pancreatic islet cells and pD-M (n = 4), (d) pancreatic islet cells modified with pD-M (F1) (n = 12), (e) modified with pD-M (F2) Pancreatic islet cells (n = 6), (f) Pancreatic islet cells modified with pD-M (F3) (n = 3) and (g) Pancreatic islet cells modified with pD-M (F4) (n = 3) The percentage change in body weight of mice transplanted with.

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.

Preferably, the average diameter of the microspheres may be 1 to 1000 μm, but is not limited thereto.

Preferably, 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.

Preferably, 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.

Preferably, 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.

Preferably, 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.

Preferably, 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.

In addition, 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.

Preferably, 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.

Preferably, the second step may be carried out for 30 minutes to 2 hours at a condition of 15 to 40 ℃, pH 8 to 9, but is not limited thereto.

In addition, 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. Provided is a cell surface modification method comprising the fourth step.

Preferably, 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 You can choose from a variety of cells, including ciliated cells, macrophages, dendritic cells, brain neurons, egg cells, and neomyocytes.

Preferably, the fourth step may be repeated 2 to 5 times at 5 to 15 minutes at 15 to 40 ℃, but is not limited thereto.

In addition, 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. Thus, 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. It is noted that it 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.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1: Reagent Preparation

Poly (lactic-co-glycolic acid), PLGA, MW = 38-54 kDa, 50:50 LA: GA, is available from Evonik Industries AG, Darmstadt, Germany Hank's balanced salt solution (HBSS), RPMI 1640 medium, Histopaque-1077, poly (vinyl alcohol), PVA, MW = 31-50 kDa) and dopamine hydrochloride (dopamine hydrochloride) were sigma-aldrich ( Sigma-Aldrich, Korea). 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. FK506, an immunosuppressant, was provided by Hanmi Pharmaceutical (Seoul, Republic of Korea).

Example 2 Preparation of poly (lactic-co-glycolic acid, PLGA) microspheres

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.

To prepare FK506-encapsulated microspheres (FK506-M), FK506 was added to a dichloromethane solution in which PLGA microspheres were dissolved, and formulated by mixing thoroughly before the emulsion step. The theoretical inclusion content of FK506 in the formulation was 5% and 10%.

To assess whether drug release and drug release time influence the protection of transplanted pancreatic islet cells from in vivo immune rejection responses, FK506 was encapsulated in various dosages using various types of PLGA. Referring to Formulations M (F1) and M (F2) in Table 1 below, DLG 4A (LA: GA 50:50, acid-terminated, Evonik) improved the encapsulation capacity for FK506 at M (F2) ( 3.33 ± 0.07% and 6.71 ± 0.11%). Formulations 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%). Interestingly, as shown in Table 1, it was confirmed that the FK506 encapsulation efficiency is slightly different in all formulations.

Formulation	PLGA type	Initial ratio between FK506 and PLGA (w / w)	LC (%)	EE (%)
M (F1)	DLG 4A (50:50, acid terminated)	5:95	3.33 ± 0.07	66.55 ± 1.30
M (F2)	DLG 4A (50:50, acid terminated)	10:90	6.71 ± 0.11	67.06 ± 1.12
M (F3)	PLGA 756S (75:25, 76-115kDa, ester terminated)	5:95	3.36 ± 0.05	67.14 ± 1.04
M (F4)	PLGA 858S (85:15, 190-240kDa, ester terminated)	5:95	3.31 ± 0.07	66.27 ± 1.49

In addition, in order to analyze the splicing efficiency of microspheres on the surface of pancreatic islets, 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.

Example 3 Preparation of PLGA Microspheres Coated with Polydopamine

PLGA microspheres coated with polydopamine (polydopamine coated microspheres, pD-Ms) were prepared at room temperature using alkaline buffer (bicarbonate, pH = 8.5, 10 mM). Briefly, dopamine hydrochloride was dissolved in 10 mM bicarbonate buffer (pH 8.5), PLGA microsphere suspension was added and stirred at constant speed for a period of time. The concentration of dopamine hydrochloride in the reaction mixture was 1 mg / ml and the concentration of microspheres was 0.5 mg / ml. In the process, the dopamine is oxidized and polymerized to form a polydopamine coating layer on the microsphere surface. Thereafter, washed and centrifuged five times with deionized water to obtain a microsphere coated with polydopamine, and the obtained microsphere was lyophilized and stored at -20 ° C.

Example 4 Characterization of PLGA Microspheres

In order to observe the size and surface topography of the microspheres according to the particle fixation and platinum coating steps, a scanning electron microscope (SEM; S-4100, Hitachi, Japan) was used. In addition, after removing the PLGA polymer from the core of the polyspheres coated with polydopamine using acetonitrile, a transmission electron microscope system (TEM 120kV, H-7600; Hitachi, Japan) was used to analyze the polydopamine capsules. .

As a result, referring to FIG. 3A, the PLGA microspheres were analyzed by a scanning electron microscope, and the average diameter was 1 to 8 μm, 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.

Next, 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.

As a result, referring to Figures 4a and 4b, 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 μm. 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.

In order to confirm whether a polydopamine coating layer was formed on the surface of the microspheres, after removing the PLGA core with acetonitrile, transmission electron microscopic analysis was performed.

As a result, referring to FIG. 4C, a flexible polydopamine capsule composed of two parts, a very thin sealing membrane and small particles bonded to the surface thereof, was identified.

Fourier transform infrared (FT-IR) spectroscopy, Nicolet Nexus 670 FT-IR spectrometer, Thermo Fisher Scientific Inc., Waltham, Mass., USA, was performed to analyze the functional groups of the molecules from the samples. In addition, 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). To analyze PLGA microspheres or PLGA microspheres (control) with FK506 encapsulation, copper anodes (Cu Kα radiation) at a voltage of 40 kV and a current of 30 mA and 2θ detector resolution between 10 and 70 degrees at ambient temperature. Was used.

As a result, referring to FIG. 3B, 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.

In addition, referring to FIG. 3C, when compared to PLGA microspheres in the FT-IR spectral results, PLK microspheres containing FK506 did not show formation of new peaks. The results indicate that there is no chemical interaction between FK506 and PLGA.

In addition, FT-IR confirmed the presence of polydopamine conjugated to the cell surface in the polysphere coated with polydopamine. Referring to FIG. 4D, 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.

In order to determine the loading capacity (LC) and encapsulation efficiency (EE) of the various PLGA microsphere formulations for FK506, a high performance liquid chromatography (HPLC) system was used in the formulation. Drug concentration was quantified. Briefly, PLK microspheres loaded with FK506 were dissolved in acetonitrile and the supernatant was taken after centrifugation. An analytical column was used with an Inertsil column (4.6 x 150 mm, 5 μm), a mobile phase consisting of acetonitrile and 0.1% phosphoric acid in an 85:15 ratio, flow rate of 1 ml / min and 210 nm. The experiment was carried out under the condition of wavelength, column temperature of 60 ℃. Encapsulation capacity and capsule efficiency were calculated using Formula 1 and Formula 2 below.

[Calculation 1]

Encapsulation Capacity (%)

= Total volume of FK506 in microspheres / actual injection volume of FK506 X 100%

[Calculation 2]

Capsule efficiency (%)

= Total amount of FK506 in microspheres / amount of microspheres X 100%

In vitro release of FK506 from PLGA microspheres was also analyzed using phosphate buffered saline (PBS) containing 1% Tween20 (Sigma-Aldrich) as a surfactant to prevent precipitation of FK506 during the release process. Briefly, an appropriate amount of polydopamine-coated FK506-encapsulated PLGA microspheres are suspended in 5 ml of release medium and enclosed in ultracentrifuge tubes equipped with a 300,000 Da MWCO membrane (Sartorius Stedim Lab Ltd, Stonehouse, Gloucestershire, UK). It was. 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 ℃. 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.

As a result, referring to FIG. 4E, it was confirmed that the release kinetics of FK506 was not clearly observed at the initial time of the four formulations (F1 to F4), but the release kinetics were affected by the type of PLGA used. In particular, pD-M (F1) and pD-M (F2) release the entire encapsulated drug within 40 days, while pD-M (F3) and pD-M (F4) continue to release the drug after 40 days. It was confirmed that.

Example 5 Pancreatic Islet Cell Isolation

At 8 weeks of age, 250-300 g of normal male Sprague-Dawley (SD) rat 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.

Example 6 Conjugation of Microspheres Coated with Polydopamine on Pancreatic Islet Cell Surface

Three days after isolation, pancreatic islet cells were obtained by washing twice with HBSS (pH 8.0) and centrifuging for 1 minute at 1,000 rpm. In order to conjugate microspheres to the pancreatic islet cell surface, a suspension of pD-Ms added at 2 mg / ml concentration to HBSS (pH = 8.0, 10 mM) was added to pancreatic islet cells and mixed at 37 ° C. for 2 minutes. . Cells were then collected and resuspended in culture medium.

For maximum conjugation of microspheres on the surface of pancreatic islet cells, 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.

To analyze the coating effect of polydopamine on pancreatic islet cell surface, nonfunctionalized microspheres were used as controls. To demonstrate the stability of the conjugation, microsphere-conjugated pancreatic islet cells were cultured for 24 days and observed by scanning electron microscopy and confocal laser scanning microscopy.

Referring to FIG. 5A, it was confirmed that the non-functionalized microspheres did not react with the pancreatic islets, whereas the microspheres functionalized with polydopamine were uniformly coated on the surface of the pancreatic islets.

However, referring to Figures 5b and 5d, it was confirmed that the increase in dopamine oxidation time for microsphere coating has a negative effect on conjugation by inhibiting the interaction between microspheres and pancreatic islet cells by polydopamine nanoparticles. It was. Therefore, it is important to maintain the dopamine oxidation time at 1 hour in order to prepare uniform microspheres coated with polydopamine. In addition, it was confirmed that when the microspheres and pancreatic islet cells were reacted three times for 10 minutes, the density of the microspheres on the surface of the pancreatic islets was increased for 1 hour.

Thus, 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.

In addition, referring to FIG. 6, 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.

Example 7: Quantification of FK506 Encapsulated in Modified Pancreatic Islet Cells

Briefly, pancreatic islet cells (150 IEQ) modified with polydopamine coated microspheres (pD-M (F1)) were incubated in 1 ml of acetonitrile, sonicated, and centrifuged at 17,000 rpm for supernatant. Was taken and used for LC / MS / MS analysis. 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 μm; 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 μl / 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.

Example 8 Analysis of Viability of Pancreatic Islet Cells

Survival rates of unmodified pancreatic islet cells and encapsulated pancreatic islet cells were analyzed using CCK-8 analysis. Briefly, 100 μl 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 μl 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 μM of acridine orange (AO, Sigma) and 75 μM 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.

FK506 is known to cause severe toxicity to many organs and cell types, including the pancreas. In addition, 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.

As a result, referring to Figure 7a, as can be seen in the survival / killing analysis, it was confirmed that the 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%. In addition, referring to Figure 7c, Bcl-2 and Bax expression levels of pancreatic islet cells did not show a significant difference between groups. Thus, it was confirmed that the modification of pancreatic islet cells using FK506-encapsulated, polydopamine-coated microspheres did not affect cell viability.

Example 9 Glucose Stimulating Insulin Secretion

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. Supernatants were taken under each condition and the amount of insulin secreted from pancreatic islet cells was measured using a Rat / Mouse Insulin ELISA kit (Millipore, MA). Final data were analyzed by normalizing to the dsDNA content of each sample. The stimulation index (SI) value was calculated by dividing the insulin secretion under high glucose condition by the insulin secretion under low glucose condition.

As a result, referring to Figures 7d and 7e, it was confirmed that the insulin secretion of the modified pancreatic islet cells under low glucose (2.8 MM) and high glucose (28 MM) conditions slightly decreased. However, there was no statistically significant difference in stimulus index between the four groups. From the above results, it was confirmed that the cell surface modification process did not affect the function of the pancreatic islets.

Example 10 Xenograft of Rat Pancreatic Islet Cells Using Kidney Capsule of Diabetic Mice

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. Microsphere-modified pancreatic islet cells or unmodified pancreatic islet cells (400 IEQ) included in cutdown tubing (JMS Co., Ltd, South Korea) 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.

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.

As a result, referring to FIGS. 8A and 8B, the pancreatic islet cell beneficiary (n = 5) and the pancreatic islet cell beneficiary (n = 7) modified with pD-M were 12 days of median survival. normal blood sugar was maintained for a short time at time;

On the other hand, referring to Figures 8d and 8e, when the FK506 is sealed, and the polyspheres coated with polydopamine is conjugated to pancreatic islet cells, it was confirmed that the graft survival rate is greatly improved. The MST of pancreatic islet cell beneficiaries modified with pD-M (F1) (n = 12) and pancreatic islet cell beneficiaries modified with pD-M (F2) (n = 6) was 2.8- and 2.2-fold higher than the control group. (MST = 34 days and 26.5 days).

In addition, referring to Figure 8h, 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. In addition, 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.

Example 11 Intraperitoneal Sugar Loading Test

In order to evaluate the function of transplanted pancreatic islet cells in lowering blood glucose levels, an intraperitoneal glucose tolerance test (IPGTT) was performed 20 days after transplantation. Briefly, 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.

As a result, referring to FIG. 8I, 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. In contrast, 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. The results indicate that pancreatic islet cells modified with pD-M (F1) and unmodified pancreatic islet cells do not show significant differences in function in vivo.

In addition, referring to Figure 9, in the change in body weight of the transplanted mice, only pancreatic islet cells modified with pD-M (F1) showed normal development by gaining weight, demonstrating the effect of the drug delivery system. .

As described above in detail certain parts of the present invention, it is apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

The scope of the present invention is represented by the following claims, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention.

Claims (11)

1. It is coated with a polydopamine (polydopamine) and the microspheres made of poly (lactic-co-glycolic acid) (PLGA) polymer and the drug or bioactive material in the microspheres Microspheres.
2. The microsphere of claim 1, wherein the microspheres have an average diameter of 1 to 1000 μm.
3. The microsphere of claim 1, wherein the drug or bioactive substance is at least one selected from the group consisting of immunosuppressants, anticoagulants, anti-inflammatory agents, antioxidants, and hormones.
4. The microsphere of claim 3, wherein the drug or bioactive material is provided in a form selected from the group consisting of chemicals, proteins, peptides, antibodies, genes, siRNAs and microRNAs.
5. A first step of encapsulating the drug or bioactive material in the microspheres by stirring the PLGA microspheres with the drug or the bioactive material;

   A second step of reacting the dopamine solution with the polymer suspension of the PLGA microspheres containing the drug or the bioactive material; And

   Centrifuging the reacted reactants to obtain a PLGA microsphere coated with polydopamine;

   Microsphere manufacturing method comprising a.
6. 6. The method of claim 5, wherein the second step is a method for producing a microsphere, characterized in that the polymer suspension of the PLGA microspheres and the dopamine solution is added in a volume ratio of 1: 1.5 to 3.
7. The method of claim 5, wherein the second step is performed at 15 to 40 ° C. and pH 8 to 9 for 30 minutes to 2 hours.
8. A first step of encapsulating the drug or bioactive material in the microspheres by stirring the PLGA microspheres with the drug or the bioactive material;

   A second step of reacting the dopamine solution with the polymer suspension of the PLGA microspheres containing the drug or the bioactive material;

   Centrifuging the reacted reactants to obtain a PLGA microsphere coated with polydopamine; And

   A fourth step of modifying a cell surface by reacting the polygapamine-coated PLGA microspheres with cells;

   Cell surface modification method comprising a.
9. The method of claim 8, wherein the fourth stage cells are pancreatic islet cells, stem cells, hepatocytes, fibroblasts, lymphocytes, vascular endothelial cells, neurons, enamel cells, keratinocytes, adipocytes, osteoblasts, ciliated cells, macrophages Cell surface modification method, characterized in that selected from the group consisting of dendritic cells, brain neurons, egg cells and neomyocytes.
10. 9. The method of claim 8, wherein the fourth step is repeated 2 to 5 times at 5 to 15 minutes at 15 to 40 ℃.
11. A pharmaceutical composition for treating diabetes, comprising a cell whose surface is modified with a microsphere according to claim 1 as an active ingredient.

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