KR101721977B1 - Sustained drug-release nanoparticle and pharmaceutical composition for treating diabetes comprising pancreatic islets having modified surface by the nanoparticle - Google Patents

Abstract

The present invention relates to a biodegradable polymer encapsulating a drug; A linear polymer having one end of a linear polymer attached to the surface of the biodegradable polymer; And an adhesive substance bonded to the other end of the linear polymer, and a pancreatic islet cell modified with a drug sustained release nanoparticle according to the present invention, It is about.
Since the nanoparticles according to the present invention can release the encapsulated immunosuppressant or anticoagulant to regulate cytokine release or blood coagulation system, modification of the pancreatic islet cell surface with the nanoparticles can improve survival after transplantation of pancreatic islet cells have.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for the treatment of diabetes, comprising a sustained-release drug nanoparticle and a nanoparticle-modified pancreatic islet cell,

The present invention relates to a pharmaceutical composition for treating diabetes comprising nanoparticles for suppressing cytokine release or blood coagulation system and pancreatic islet cells surface-modified with said nanoparticles in pancreatic islet cell transplantation for treating diabetes .

Diabetes Mellitus is a disease characterized by hyperglycemia due to insulin secretion abnormality in the pancreatic β-cell or insulin action or organ receptor organs and complications thereof. In diabetes therapy, exercise therapy and dietary therapy are mainly performed with insulin injection therapy, but there is a limitation that the cure is impossible and the risk of complication still exists.

Recently, diabetes treatment through pancreas transplantation and pancreatic islet cell transplantation has been performed. In the case of pancreas transplantation, there are problems such as absolute donor shortage, high surgical complication, persistent immunosuppressive drug administration and difficulty in post-transplant management. In contrast, pancreatic islet cell transplantation has the advantage of being able to perform in vitro processing such as in vitro culture of the isolated pancreatic islet cells and immunity control. In addition, when the immune response of xenotransplantation is overcome, infinite pancreatic islet cells can be supplied using pigs and relatively easy procedure can be easily performed without complications.

However, successful transplantation of pancreatic islet cells requires minimization of pancreatic islet cell damage resulting from the development and isolation of effective pancreatic islet cells, cell damage due to nonspecific inflammation during transplantation, Minimization, overcoming of cell damage caused by immune response after transplantation, and securing supply of pancreatic islet cell. In particular, the initial inflammatory reaction after transplantation of pancreatic islet cells results in a functional incompatibility or destruction of pancreatic islet cells due to cell necrosis and apoptosis, so that there is a problem in that a larger amount of pancreatic islet cells than the actually required pancreatic islet cells must be transplanted .

These nonspecific inflammatory responses are caused by various proinflammatory factors such as tumor necrosis factor-alpha, TNF-alpha, interleukin-1 beta, IL-1 beta and interferon- The cytokine induces the production of nitric oxide (NO) by inducible NO synthase (iNOS) in pancreatic islet cells, which is induced by the Krebs cycle (Aconitase) in the Krebs cycle, reducing glucose oxidation, ATP production and insulin production.

In addition, prostaglandin E2 (Prostaglandin E2, PGE2) has been reported to inhibit glucose-induced insulin secretion in pancreatic islet cells or? -Cells and is known to be stimulated by IL-1?. A monoclonal antibody to the tumor necrosis factor-alpha receptor (infliximab) has been developed to prevent cellular damage caused by nonspecific inflammatory processes occurring during the above-described transplantation, and has been used for pancreatic islet cell transplantation. N-monomethyl arginine and 15-deoxypergualin, which regulate the function of macrophages, have also been tried, but they have not been fully applied to clinical practice.

Therefore, if the inflammatory cytokine can be controlled, the maximum pancreatic islet cell transplantation effect can be expected by inducing the stabilization of the pancreatic islet cell in the early stage of transplantation, so that the transplantation success rate can be improved.

In addition, when pancreatic islet cells are transplanted into the portal vein and the pancreatic islet cells are directly exposed to the blood, blood coagulation system such as platelets and complement activates, resulting in blood coagulation around the pancreatic islet cells, resulting in rapid breakdown of pancreatic islet cells. It is also a problem that an instant blood mediated inflammatory reaction (IBMIR) occurs.

Thus, for successful pancreatic islet cell transplantation, there is a need for a method that can regulate the cytokine release or blood coagulation system as described above.

1. Korean Patent No. 10-0505954.

Accordingly, it is an object of the present invention to provide nanoparticles capable of regulating cytokine release or blood coagulation system upon pancreatic islet cell transplantation.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of diabetes comprising pancreatic islet cells whose surface is modified with the nanoparticles.

In order to achieve the above object, the present invention provides a biodegradable polymer encapsulating a drug; A linear polymer having one end of a linear polymer attached to the surface of the biodegradable polymer; And an adhesive material bonded to the other end of the linear polymer.

In order to accomplish still another object, the present invention provides a pharmaceutical composition for the treatment of diabetes comprising pancreatic islet cells whose surface is modified with the drug sustained release nanoparticles.

The present invention relates to a biodegradable polymer encapsulating a drug; A linear polymer having one end of a linear polymer attached to the surface of the biodegradable polymer; And an adhesive material bonded to the other end of the linear polymer, and a pancreatic islet cell modified with the nanoparticles, wherein the pharmaceutical composition for the treatment of diabetes mellitus The nanoparticles can release the encapsulated immunosuppressant or anticoagulant to regulate cytokine release or blood coagulation system, and modifying the surface of the pancreatic islet cell with the nanoparticles can improve survival after transplantation of pancreatic islet cells.

In FIG. 1, A is the drug sustained release nanoparticle according to the present invention, and B in FIG. 1 is an illustration of the effect of the immunoprecipitated nanoparticles on the pancreatic islet cell surface. In FIG. 1, Fig. 2 shows the effect of nanoparticles on the pancreatic islet cell surface.
2 is a result of thin layer chromatography (TLC) analysis of PLGA-PEG-dopamine.
FIG. 3 shows Fourier Transform Infrared Spectroscopy (FT-IR) analysis results of PLGA-PEG-dopamine.
4 and 5 are H-nuclear magnetic resonance (H-NMR) analysis results of PLGA-PEG-dopamine.
6 shows the results of H-NMR analysis of PLGA-PEG-NHS copolymer (PLGA-PEG-NHS co-polymer).
FIG. 7 shows the UV-Visible spectrophotometer (UV-VIS) analysis results of PLGA-PEG-NHS and PLGA-PEG-dopamine.
Figure 8 shows the results of analysis of the dopamine content of PLGA-PEG-dopamine.
Fig. 9 shows the results of confirming the influence of the organic solvent on PLGA-PEG-dopamine nanoparticles.
FIG. 10 shows the results of examining the influence of the drug / polymer weight ratio on the size and polydispersity index (PDI) of the nanoparticles.
Fig. 11 shows the results of confirming the influence of the addition of the cryoprotectant on the stability of the nanoparticles.
Fig. 12 shows the result of confirming the distribution of the optimized nanoparticles.
FIG. 13 shows the result of checking the size of the optimized nanoparticles.
Fig. 14 shows the result of confirming the binding of PLGA-PEG-dopamine nanoparticles in a collagen-coated culture dish.
FIG. 15 shows the intensity distribution of PLGA-PEG-dopamine nanoparticles in a collagen-coated culture dish.
FIGS. 16 and 17 show the results of confirming the binding of PLGA-PEG-dopamine nanoparticles to the surface of pancreatic islet cells.
18 shows the results of confirming cell survival after binding of the surface of pancreatic islet cells with PLGA-PEG-dopamine nanoparticles.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention have been studying a method for regulating cytokine release or blood coagulation system for successful pancreatic islet cell transplantation, including a biodegradable polymer encapsulating a drug; A linear polymer having one end of a linear polymer attached to the surface of the biodegradable polymer; And an adhesive material bonded to the other end of the linear polymer, and modifying the surface of the pancreatic islet cell with the nanoparticles to complete the present invention.

Accordingly, the present invention provides a biodegradable polymer encapsulating a drug, such as A in FIG. 1; A linear polymer having one end of a linear polymer attached to the surface of the biodegradable polymer; And an adhesive material bonded to the other end of the linear polymer.

The adhesive material may be any one selected from the group consisting of dopamine, 3,4-dihydroxyhydrocinnamic acid (DOHA), and derivatives thereof, and more preferably dopamine.

The linear polymer is any one selected from the group consisting of polyethylene glycol (PEG), heparin, heparin analogue, chitosan, hyaluronic acid and derivatives thereof. Preferably, it is not limited to PEG.

The biodegradable polymer may be selected from the group consisting of poly (lactide-co-glycolide), polylactic acid (PLA), polylactide, polylactic-co- glycolic acid, polylactide-co- glycolide (PLGA) , Polyiminocarbonates, polyphosphoesters, polyanhydrides, polyorthoesters, copolymers of lactic acid and caprolactone, polycaprolactone, polyhydroxyvalerate, polyhydroxybutyrate, polyamino acid, lactic acid and amino acid A copolymer, a polyethylene glycol derivative, a chitosan derivative, a heparin derivative, and a mixture thereof, more preferably PLGA, but is not limited thereto.

The medicament may be selected from the group consisting of tacrolimus, Cyclosporin, Sirolimus, Everolimus, Ridaforolimus, Temsirolimus, Umirolimus, But are not limited to, Zotarolimus, Leflunomide, Methotrexate, Rituximab, Ruplizumab, Daclizumab, Abatacept and Belatacept. ) Or an immunosuppression drug selected from the group consisting of argatroban, cumarin, heparin, low molecular weight heparin, Hirudin, Is an anti-coagulant drug selected from the group consisting of Dabigatran, Melagatran, Clopidogrel, Ticlopidine and Abciximab.

The immunosuppressant or anticoagulant may be encapsulated according to an encapsulation method well known to those skilled in the art.

The nanoparticles according to the present invention can be attached between the collagen matrix or the nanoparticles via dopamine attached to the PEG.

As shown in FIG. 1B, immunoprecipitated nanoparticles encapsulate pancreatic islets to form an immune system (complement, macrophage, polymorphonuclear leukocyte ) And K cells) and inhibit the secretion of cytokines by the release of tacrolimus, an immunosuppressive agent.

In addition, as shown in C of Fig. 1, nanoparticles encapsulating pancreatic islets encapsulate the immune system (complement, macrophage, polymorphonuclear leukocyte, K cells, etc.), and the release of argatroban, an anticoagulant, inhibits an intrinsic blood-mediated inflammatory reaction (IBMIR), thereby preventing the damage of pancreatic islets.

The nanoparticles according to the present invention are characterized by having an average diameter of 50 nm to 1000 nm. If the average diameter of the nanoparticles is too small beyond the above range, the drug release time may be reduced, If the average diameter of the particles is too large, it may be difficult to uniformly bind to the surface of the islet cell.

In addition, the present invention provides a pharmaceutical composition for the treatment of diabetes comprising pancreatic islet cells whose surface is modified with the drug sustained release nanoparticles.

In addition, the dose of the surface-modified pancreatic islet cell modified with the drug sustained release nanoparticle according to the present invention may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All methods of transplantation can be expected, such as portal vein, eye, kidney, muscle, subcutaneous, gastrointestinal, etc.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

< Reference example > Experimental material

(Lactide-co-glycolide) -b-poly (ethylene glycol) -N-hydroxysuccinamide, MW ~ 17000 Da: 3000 Da, 50:50 LA: GA) and PLGA-FITC (poly (lactide-co-glycolide) -FITC, MW ~ 10000 Da, 50:50 LA: GA) were purchased from Polyscitech (Indiana, USA) Respectively. RPMI 1640 medium, Hank's balanced salt solution (HBSS), Histopaque-1077 and dopamine hydrochloride were purchased from Sigma-aldrich, Korea. Acetone, dimethylformamide (DMF), dichloromethane (DCM), and diethylether were purchased from Junsei, Korea. Fetal bovine serum (FBS) and phosphate buffer saline (PBS) were purchased from Hyclone. Collagenase P was purchased from Roche Diagnostics GmBH (Roche diagnostic GmBH, Mainheim, Germany). Live / dead cell viability / cytotoxicity assay kit was purchased from Life Technologies (Oregon, USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Dojindo, Japan). Amicon centrifugation tubes (Pore size: 100,000 Da) were purchased from Millipore Co. (Billerica, MA, USA). The Quant-iT ™ PicoGreen® dsDNA reagent (Quant-iT ™ PicoGreen® dsDNA reagent) was purchased from Invitrogen (Invitrogen, Carlsbad, Calif., USA). The collagen coated culture dish was purchased from MatTech Corporation, Homer Ave, Ashland, USA.

< Example  1> PLGA -PEG-Dopamine Complex

100 mg of PLGA-PEG-NHS and 2.83 mg of dopamine hydrochloride were dissolved in 1 ml of DMF and the mixture was stirred overnight at 4 캜. Then, 20 ml of methylene chloride was added and the reaction was stirred vigorously. Since dopamine is not mixed with methylene chloride, unreacted dopamine is removed by centrifugation to precipitate. The supernatant containing the product, PLGA-PEG-dopamine, was collected and the purity was checked using thin layer chromatography (TLC). If unreacted dopamine was not found, the solution was evaporated under reduced pressure at 40 &lt; 0 &gt; C to collect the viscous product and purified by precipitation in cold ether. Finally, the product was lyophilized at -20 &lt; 0 &gt; C for 4 hours and then lyophilized at -70 &lt; 0 &gt; C for 12 hours to obtain a powdered form.

< Example  2> PLGA -PEG-dopamine complexes

2.1) Thin layer  Thin layer chromatography (TLC)

A solution of dopamine hydrochloride, PLGA-PEG-NHS in DMF, and the reaction material were dropped onto a TLC plate (4 cm x 15 cm) after free dopamine was removed.

The TLC plate was stored at room temperature for 30 minutes and then introduced into a glass tank containing mobile phase (DMF: methanol = 1: 1) for 30 minutes and dried at 60 ° C for 1 hour.

As a result, as shown in FIG. 2, no chemical trace of dopamine was found in the reaction product, and almost all of the free dopamine was removed from the final product.

2.2) Fourier transform Infrared spectroscopy (Fourier Transform Infrared Spectroscopy, FT-IR)

The FT-IR spectra were measured using a Thermo Scientific Nicolet Nexus 670 FT-IR Spectrometer, a Smart iTR and a diamond window (Thermo Fisher Scientific Inc., Waltham, MA) .

The chemical structure of the PLGA-PEG-dopamine complex was confirmed by FT-IR. As shown in FIG. 3, the characteristic peak for the alcohol stretch (3200-3550 cm ^-1 , OH stretch) The spectra of the final product were significantly increased when compared to the same peak of the PLGA spectra.

In addition, amide (amide) group (~ 1600 ^cm-1) carbonyl peak (carbonyl peak) corresponding to the dopamine residue (moiety) and co-demonstrated coupling between the block polymer (co-block polymer).

2.3) H-nuclear magnetic resonance (H-NMR)

The spectra of PLGA and PLAG-PEG-DOPA were registered in DMSO-d6 and an Agilent-NMR-VNMRS600 instrument operating at 600 MHz and chemical shifts were reported in ppm. The spectrum of PLGA-PEG-NHS was obtained from Polysytec.

As a result, the prevalence of the polymer signal was observed in the 1 H-NMR spectrum of the PLGA-AL bond as shown in Fig.

However, the presence of dopamine was confirmed at two small peaks at 2.57 ppm and 3.28 ppm (peak area: 0.16) belonging to the hydrogen of dopamine.

The hydrogen atom corresponding to the lactic acid unit provided a peak from 4.829 ppm to 4.945 ppm with a peak area of 7.72.

In addition, as shown in Fig. 5, the absorbance of the peak corresponding to NHS (4.46 ppm) of PLGA-PEG-dopamine spectra confirmed the interaction between PLGA-PEG-NHS and dopamine, in which the NHS residue was rearranged by dopamine.

The H-NHR results of the PLGA-PEG-NHS copolymer (PLGA-PEG-NHS co-polymer) supplied by Polycattech showed that the relative ratio between the peak areas of PLGA and dopamine The base was calculated and the binding efficiency reached almost 60%.

2.4) Gel permeation chromatography &lt; RTI ID = 0.0 &gt;

The Varian system was used with 1 ml / min DCM flow cross. The molecular weight of PLGA-PEG-dopamine was measured using two Phenogel 5 μm columns (DCM flow across two Phenogel 5 μm columns) and one PLgelResipore column (Agilent) Were measured. These results were corrected based on polystyrene standards and the number average molecular weight (Mn), molecular weight and polydispersity index (PDI) were calculated and shown in Table 1 below (PLGA-PEG-NHS and HO- The results of PEG-COOH were obtained from Polysytec).

Polymer M _n
(from GPC ) M _w
(from GPC ) PDI PLGA-PEG-NHS 16,048 24,882 1.55 HO-PEG-COOH Average M _n by GPC ~ 3300 PLGA-PEG-Dopa

2.5) UV-Visible Spectrophotometer (UV- VIS )

UV-VIS profiles of PLGA-PEG-NHS and PLGA-PEG-dopamine were analyzed using a U-2800 spectrophotometer (Hitachi, Japan) to confirm the chemical structure of the polymer. The reaction was confirmed by the change of the NHS peak to the dopamine peak (PLGA-PEG-NHS solution of DMF (black line in Figure 7) and PLGA-PEG-dopamine solution of DMF (blue line in Figure 7)).

In FIG. 7, it was confirmed that the NHS moiety was rearranged by dopamine through the shift of the peak from 260 nm to 280 nm.

2.6) Determination of the dopamine content of the conjugate

The dopamine content of the conjugate was confirmed using a spectrophotometer. The absorbance of the PLGA-PEG-dopamine solution in DMF at different concentrations was measured at 280 nm on a calibration graph of a serial solution of dopamine (1.40625 μg / ml, 2.8125 μg / ml, 5.625 μg / ml and 11.25 μg / ml , 22.5 μg / ml, 45 μg / ml). This was repeated three times.

As a result, as shown in Fig. 8, the content of dopamine in the conjugate was measured to be 6.22 μg in 1 mg of the binding substance. Assuming that the molecular weight of the PLGA-PEG moiety is 20,000 Da, the binding efficiency of this reaction can be calculated to be about 80%.

< Example  3> PLGA -PEG-dopamine nanoparticles ( Nanoparticles , NPs )

PLGA-PEG-dopamine NPs were prepared by the precipitation method. That is, 10 mg polymer was dissolved in 1 ml acetone and slowly injected into 10 ml with stirring of the oil layer. After stirring at room temperature for 2 hours, the solution was dialyzed against 1 L of distilled water to completely remove the organic solvent.

To evaluate the distribution of NPs on the surface of pancreatic islet cells, PLGA-FITC and PLGA-PEG-dopamine were dissolved in acetone at a ratio of 20:80 (w / w) to prepare fluorescently labeled nanoparticles.

< Example  4> PLGA -PEG-dopamine Of NPs  Character rating

4.1) Assess the effect of organic solvents

To evaluate the effect of organic solvents, two organic solvents (DMF or acetone) mixed with water were used in nanoparticle production.

Tacrolimus was added to different concentrations of polymer solution (1%, 2% or 5%) to confirm the effect of drug content on the PDI and size of the nanoparticles.

In particular, nanoparticles prepared without surfactant (cryoprotectant) are known to be unstable after lyophilization. Therefore, the nanoparticles with or without surfactant are lyophilized and placed at room temperature. The size and PDI of each nanoparticle are compared The effect of surfactant on the stability of nanoparticles during freeze drying process was investigated.

The hydrodynamic particle size, zeta-potential and PDI were measured by dynamic light scattering (DLS) of a Nano-S90 ZetaSizer (Malvern Instruments, Worcestershire, UK) , At a fixed angle of 90 [deg.].

The hydrodynamic size was determined using the Stokes-Einstein equation. The ζ-potential and PDI were determined using manufacturer's instructions using nano-DTS software (version 6.34). All measurements were performed at 25 ° C and were performed at least three times per 10 cycles.

As a result, using DMF to produce nanoparticles, as shown in Figure 9, provided better PDI and smaller size of nanoparticles. However, if the size of the NPs is too small, the drug release time may be reduced. Acetone was chosen because it has a low boiling point and is easy to remove from the suspension.

In addition, the effect of drug content and weight ratio of drug and polymer on the PDI and size of nanoparticles was confirmed, and the results are shown in Table 2 and FIG.

Drug content
(Drug content) Size (nm) SD PDI SD Blank 80.48 1.316 0.174 0.009 One% 82.56 3.149 0.184 0.013 2% 81.3 0.9379 0.172 0.052 5% 172.9 4.234 0.276 0.02

From the above results, it was confirmed that tacrolimus should not be filled in an amount of 2% or more.

In addition, the effect of the surfactant (cryoprotectant) on the stability of the nanoparticles during the freeze-drying process was confirmed, and the results are shown in Table 3 and FIG. When PLGA-PEG-dopamine conjugate was prepared as in Example 1, 10% sucrose was added to the dialysis solution as a cryoprotectant after dialyzing with distilled water, followed by lyophilization.

Size (nm) SD PDI SD Control group 71.94 0.4179 0.125 0.011 After adding sucrose, it is lyophilized. 79.83 0.4409 0.181 0.03 After adding sucrose, freeze-dried. 1334 33.71 0.637 0.072

From the above results, it was confirmed that PDI and size of NPs after lyophilization did not change significantly even in the presence of 10% sucrose.

Therefore, a protocol for preparing an optimized preparation was established through the results of Tables 2 and 3 and FIGS. 9 and 10.

Briefly, 0.1 mg of tacrolimus and 9.9 mg of PLGA-PEG-dopamine were dissolved in 1 ml of acetone and the solution was slowly poured into 10 ml of water. After stirring at room temperature for 2 hours, the suspension was dialyzed against 1 L of distilled water for 4 hours to completely remove the organic solvent and the free drug.

The dialysate was then collected and lyophilized under reduced pressure in the presence of 10% sucrose, an antifreeze. Finally, the product was sterilized via n-irradiation.

4.2) TEM  And DLS NPs  Measure size and distribution

TEM images of NPs were obtained using a JEOL 2011 instrument at an accelerated voltage of 80 kV.

The samples were prepared by depositing 10 μL of NP suspension (0.1 mg / mL) on a 300-mesh carbon-coated copper grid.

It was incubated for 2 minutes, then stained, air-dried and further dried in vacuo.

The grid was counterstained with 1% uranyl formate to enhance the difference. This process was repeated three times to obtain a representative image.

As a result, as shown in FIGS. 12 and 13, the size of the optimized NPs was about 80 nm, and DLS and TEM images confirmed that they were distributed in a narrow size.

< Example  5> Of NPs  Encapsulation

A polymer of a different weight ratio than tacrolimus was used to obtain an optimized formulation.

To completely remove the organic solvent and the free drug, the final suspension was dialyzed against 1 L of distilled water, collected, added with sucrose (5%) and lyophilized. And then stabilized by n-irradiation.

5.1) Of pancreas islets  detach

Pancreatic islet cells of sprague-Dawley (SD) rats (8 weeks of age, male, Korea) were isolated by collagenase type IV (Sigma) digestion method. The cells were treated with 10% FBS, 2mN sodium bicarbonate, 11mM glucose, 6mM HEPES (4- hydroxyethyl-1-piperazineethanesulfonic acid) and 1% penicillin / streptomycin Lt; RTI ID = 0.0 &gt; RPMI-1640 &lt; / RTI &gt; All the mouse experiments were approved by the Ethics Committee of Yeungnam University (South Korea) and conducted according to national guidelines.

5.2) Analysis of collagen binding

A suspension of NPs labeled with FITC in HBSS at pH 8.0 (1 mg / ml) was added to the collagen-coated culture dish and incubated at 37 ° C for 1, 2 or 4 hours. The suspension was then removed and washed 10 times with HBSS. Fluorescence images were obtained using a fluorescence microscope. The intensity profile was analyzed using a 3D-surface plotting graph, and the physical form of the grafted culture dish was analyzed using an atomic force microscope (AFM) The results are shown in FIG.

Optical images (Optical images) of a culture dish coated with untreated collagen (1-A) in FIG. 14, fluorescence images of a culture dish coated with untreated collagen (1-B) C) 3D-surface plotting of the fluorescence intensity of the uncoated collagen-coated culture dish surface, (2-A) incubation with PLGA-PEG-dopamine NPs labeled with FITC for 2 hours, (2-C) FITC-labeled PLGA-PEG-dopamine NPs labeled with (2-B) FITC and incubated for 2 hours with fluorescence images of collagen- 3-surface plotting of the fluorescence intensity of the surface of the coated dish after culturing for 2 hours with NPs.

In addition, the intensity distribution in the range of 100 μm on the surface was confirmed and shown in FIG.

(B) a culture plate coated with collagen after incubation of PLGA-PEG-dopamine NPs labeled with FITC for 2 hours, (C) labeled with FITC of distilled water, Gt; PLGA-PEG-dopamine NPs &lt; / RTI &gt; suspension.

14 and 15, PLGA-PEG-dopamine NPs exhibited a high covering ratio and were found to bind to the surface of the collagen-coated culture dish.

5.3) Pancreatic islet cell  Surface PLGA -PEG-dopamine Of NPs  join

After isolation of pancreatic islet cells, cells were washed twice with HBSS pH 8.0 and collected by centrifugation at 1800 rpm. For the attachment of NPs to the pancreatic islet cell surface, a suspension of PLGA-PEG-dopamine NPs (1 mg / ml) was added to the cell pellet and allowed to react for 1 hour at 37 ° C with slight shaking every 10 minutes. The surface modified pancreatic islet cells were washed twice with HBSS pH 8.0 and resuspended in culture medium. To evaluate the distribution of NPs, FITC-labeled NPs were used for conjugation and observed over time with a fluorescence microscope and a confocal laser scanning microscope.

As a result, as shown in FIGS. 16 and 17, PLGA-PEG-dopamine NPs covered all the surfaces of pancreatic islets.

5.4) Pancreatic islet cell  Viability assessment

The viability of NPs-conjugated pancreatic islet cells was quantitatively assessed by CCK-8 analysis using a cell counting kit-8 (Dojindo molecular technologies Inc., Rockville, Md.).

Unbound pancreatic islet cells or conjugated pancreatic islet cells in 100 [mu] l of RPMI-1640 were dispensed into 96-well plates and cultured for 24 hours. After the incubation, a solution of WST-8 (2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4- disulfophenyl) -2H- tetrazoium, monosodium salt) The cells were added to each well, followed by further incubation for 4 hours. The amount of formazan produced was determined by reading the absorbance at 450 nm and the final data were normalized to the DNA content of each chamber.

In addition, the viability of pancreatic islet cells was quantitatively analyzed using a viability / cytotoxicity kit (Live / dead assay viability / toxicity kit).

Since the activity of the intracellular esterase causes AM to be the intense fluorescence of non-fluorescent cell-permeant calcein, viable pancreatic islet cells produce uniform green. Ethidium-1 (Ethidium homodimer-1) enters the damaged pancreatic islet cell membrane and binds to nucleic acid, producing red fluorescence from dead cells.

As a result, as shown in FIG. 18, 90% of the surface-modified pancreatic islet cells and unmodified pancreatic islet cells showed green fluorescence, and morphology was also observed in pancreatic islet cells and heparin-DOPA And preserved after incubation. Thus, these results confirmed that PLGA-PEG-dopamine NPs conjugated to the surface of pancreatic islet cells did not affect cell survival.

Claims (7)

delete delete delete A pancreatic islet cell whose surface has been modified with drug sustained release nanoparticles,
The drug sustained release nanoparticles include PLGA (poly (lactide-co-glycolide)), a drug-encapsulating biodegradable polymer; Polyethylene glycol (PEG), a linear polymer having one end of a linear polymer adhered on the surface of the biodegradable polymer; And dopamine coupled to the other end of the linear polymer,
The medicament may be selected from the group consisting of tacrolimus, Cyclosporin, Sirolimus, Everolimus, Ridaforolimus, Temsirolimus, Umirolimus, Or an immunosuppression drug selected from the group consisting of Zotarolimus, Leflunomide and Methotrexate or any one selected from argatroban or Cumarin. Wherein the anti-coagulant drug is an anti-coagulant drug. delete 5. The method of claim 4,
Wherein the drug sustained release nanoparticles have an average diameter of 50 nm to 1000 nm.


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