KR101436466B1 - Method of modifying surface of pancreatic islets using heterofunctional-high order structural PEG with bioactive complex and pancreatic islets having modified surface using the method - Google Patents

Abstract

The method for surface modification of pancreatic islets according to the present invention is characterized in that the surface of pancreatic islets is first coated with a high-order structure PEG having a bifunctionality to reduce the immune response more effectively, The anti-thrombotic effect is induced by tea coating, and the survival time of pancreatic islets can be increased by effectively inhibiting the intrinsic blood-mediated inflammatory reaction (IBMIR) that occurs when transplanting pancreatic islets into the portal vein , And may be useful for the pretreatment of pancreatic islet transplants.

Description

[0001] The present invention relates to a method for modifying a surface of a pancreatic islet using a bi-functional higher order structure PEG and a bioactive complex, and a surface modified pancreatic islet using the same. the method}

The present invention relates to a method for surface modification of a pancreatic islet using a bi-functional higher order PEG and a bioactive complex.

Polyethylene glycol (PEG) is known as a hydrophilic polymer that is effective for hydrogen bonding with water molecules. It has the property of melting not only in aqueous solution but also in many kinds of organic solvents, and has chemical stability.

In addition, a conjugate of a drug having a biological activity such as a protein, a peptide, or a gene exhibits a half-life in the blood due to a high hydrodynamic volume in an aqueous solution of PEG, and the recognition by the bio-immune system is inhibited.

In order to show a difference in the hydrodynamic volume to form a more effective conjugate, PEG has a higher order structure in the form of linear, double branch, triple branch, dendron, dendrimer in terms of structural structure of PEG and bioactive substance and conjugate The research on biotechnology has been carried out steadily until now.

In Dongkun Han et al., Bioactive and Compatible Polymers, 2009, 24, 316-328, the surface of a stent made of titanium-nickel alloy was surface treated with linear isocyanate PEG (2 kDa) Lt; RTI ID = 0.0 > non-specific < / RTI > protein binding and platelet adhesion.

Republic of Korea Patent Publication No. 2008-0072960 In succinimidyl carbonate in a linear mono having the active group, the ethoxy -PEG (5 ~ 30 kDa) L - by reacting the lysine L - and a (core) center of the lysine carboxyl group (20-120 kDa) containing the dendrimer-like structure PEG (20-120 kDa) were synthesized and purified by chromatography to test a combination of a protein having pharmacological activity such as interferon alpha-2b and a dendrimer-like structure PEG in vitro or in vivo To increase in vivo stability.

However, in recent medical and biotechnological fields, complex requirements have arisen that a high-order structure substance having a functional group of Heterofunctional or more is required. For example, there is a growing demand for dual functionality as a result of attempting to deliver several types of bioactive materials and imaging materials for diagnosis at once.

Three-generation bow-tie dendrimers were conjugated to linear PEG (5 kDa) at the ends and doxorubicin, the anticancer agent, in the core at Frechet, Szoka et al., Molecular Pharmaceutics, 2005, Discloses an anticancer effect of a bi-functional dendron-type polyester.

In Dirksen et al., Chemical Communications, 15, 2006, 1667-1669, it has been disclosed that two dendritic polylysines can be attached to one side to introduce a fluorophore on one side and a peptide to the other side for therapeutic purposes .

In order to overcome the above-mentioned problems, the inventors of the present invention have found that by first coating the surface of a pancreas canal with a high-order structure of PEG having a dual functionality, the immune reaction is more effectively reduced and a bioactive complex Secondary coating induces an antithrombotic effect, thereby effectively reducing the intrinsic blood-mediated inflammatory reaction (IBMIR) that occurs during transplantation of the pancreatic islet into the portal vein, thereby prolonging the survival time of the pancreatic islet And the present invention has been completed.

It is an object of the present invention to provide a method for modifying the surface of pancreatic islets in order to reduce the hyper-acute blood-mediated inflammatory reaction that occurs when transplanting pancreatic islets into the portal vein.

Another object of the present invention is to provide a surface modified pancreatic islet by the above method.

In order to achieve the above object, the present invention provides a method for activating PEG or a derivative thereof, comprising the steps of: (1) activating a PEG or derivative thereof with a bifunctional higher-order structure to bind to a surface of a pancreatic islet;

(Step 2) of binding the activated PEG or derivative thereof to the amine group on the surface of the pancreatic islet surface to effect primary coating; And

(Step 3) of binding a bioactive complex to a terminal functional group of PEG or a derivative thereof bound to the surface of the pancreas canthus (step 3) in step 2, thereby providing a method for surface modification of pancreatic islets.

The present invention also relates to a coating layer comprising a bi-functional high-order structure PEG or derivative thereof on a pancreatic islet; And

And a bioactive complex coating layer on the surface of the PEG or derivatives thereof. The present invention also provides a surface modified pancreatic islet.

The method for surface modification of a pancreatic islet according to the present invention is a method for surface modification of a pancreatic islet by first coating a surface of a pancreatic islet with a bi-functional high-order structure PEG, thereby effectively reducing the immune response, The anti-thrombotic effect is induced by coating, and the survival time of pancreatic islets can be increased by more effectively inhibiting the rapid blood mediated inflammatory reaction (IBMIR) that occurs when pancreatic islets are transplanted into the portal vein.

Brief Description of the Drawings Fig. 1 is a cross-sectional view of an 8-branched PEG and a heparin catechol coated according to one embodiment of the present invention, It is an image representing pancreatic islet.
2 is a ¹ H-NMR peak of dendritic PEG of each generation (G0-G3) prepared in Preparation Example 2. Fig.
3 is a GPC peak for confirming the molecular weight of the dendritic PEG for each generation (G0-G3) prepared in Production Example 2. FIG.
4 is a ¹ H-NMR peak for confirming the introduction of a carboxyl group at the center of the dendritic PEG prepared in Preparation Example 2. FIG.
FIG. 5 is a ¹ H-NMR peak for confirming the introduction of an amine group at the terminal of the dendritic PEG prepared in Production Example 2. FIG.
FIG. 6 is a graph showing that dopamine is introduced into the central carboxyl group of the dendritic PEG prepared in Preparation Example 2 with an ultraviolet detector.
7 is a ¹ H-NMR peak for confirming the 4-branched PEG prepared in Production Example 3. Fig.
8 is a GPC peak for confirming that the 8-branch terrestrial PEG prepared in Production Example 3 is synthesized.
9 is a ¹ H-NMR peak for confirming the introduction of an amine group at the terminal of the 8-branch terrestrial PEG prepared in Production Example 3. FIG.
10 is a graph obtained by using an ultraviolet detector to confirm that the terminal amine group of the 8-branched PEG prepared in Production Example 3 is bound to the bioactive substance.
Figure 11 is a graph showing the results of a < RTI ID = 0.0 > It is an image taken by an optical microscope of the pancreas sodomy.
Figure 12 shows that 8-branched PEG is coated and surface modified A fluorescent image taken with a confocal scanning microscope based on the central compartment of the pancreatic islet.
Figure 13 shows that 8-branched PEG is coated and surface modified It is a fluorescence image taken with a confocal scanning microscope overlaying multiple compartment images of pancreatic islets.
14 is an image of a normal pancreatic islet taken by fluorescence microscope.
Figure 15 is a graph showing the results of a < RTI ID = 0.0 > It is an image of a pancreatic islet taken by fluorescence microscope.
Figure 16 is a graph showing the results of a surface modified PEG- FIG. 2 is a graph showing the results of cell viability using a cell quantification kit for pancreatic islets. FIG.
Figure 17 is a graphical representation of the surface modified < RTI ID = 0.0 > FIG. 2 is a graph showing changes in blood glucose level in a mouse transplanted with a pancreatic islet. FIG.
Figure 18 shows the results of a < RTI ID = 0.0 > It is an image taken by an optical microscope of the pancreas sodomy.
Figure 19 is a graph showing the results of a < RTI ID = 0.0 > A fluorescent image taken with a confocal scanning microscope based on the central compartment of the pancreatic islet.
Figure 20 shows the results of a < RTI ID = 0.0 > It is a fluorescence image taken with a confocal scanning microscope overlaying multiple compartment images of pancreatic islets.
Figure 21 is a graph showing the results of a < RTI ID = 0.0 > It is an image of a pancreatic islet taken by fluorescence microscope.
Figure 22 is a graph showing the effect of the surface modified < RTI ID = 0.0 > FIG. 2 is a graph showing the results of cell viability using a cell quantification kit for pancreatic islets. FIG.
Figure 23 shows the results of a < RTI ID = 0.0 > 2 is a graph showing the results of insulin secretion ability of pancreatic islets.
Figure 24 shows that when not injected with the immunosuppressant, it is coated with a normal pancreatic islet and a 6-branch terra-PEG, FIG. 2 is a graph showing blood glucose changes of each mouse transplanted with pancreatic islets. FIG.
Figure 25 shows that when injected with FK506, it was coated with normal pancreatic islets and 6-branched PEG, FIG. 2 is a graph showing blood glucose changes of each mouse transplanted with pancreatic islets. FIG.
FIG. 26 shows the results of immunohistochemical staining for anti-CD154 monoclonal antibody injected into normal pancreatic islets and 6-branched PEG- FIG. 2 is a graph showing blood glucose changes of each mouse transplanted with pancreatic islets. FIG.
Figure 27 shows that when injected with both FK506 and anti-CD154 monoclonal antibodies, the surface was modified by coating with normal pancreatic islets and 6-branched PEG FIG. 2 is a graph showing blood glucose changes of each mouse transplanted with pancreatic islets. FIG.
28 is a graph showing the results of a < RTI ID = 0.0 > It is an image taken by an optical microscope of the pancreas sodomy.
29 is a graph showing the results of a heparin- A fluorescent image taken with a confocal scanning microscope based on the central compartment of the pancreatic islet.
Figure 30 shows the results of a heparin- It is a fluorescence image taken with a confocal scanning microscope overlaying multiple compartment images of pancreatic islets.
Figure 31 is a graph showing the results of normal pancreatic isoderm and heparin- It is an image of a pancreatic islet taken by fluorescence microscope.
32 is a graph showing the results of a comparison between normal pancreatic islets and heparin-waveguide coated surface modified FIG. 2 is a graph showing the results of cell viability using a cell quantification kit for pancreatic islets. FIG.
33 is a graph showing the results of a typical pancreatic islet and heparin-waveguide coated surface modified 2 is a graph showing the results of insulin secretion ability of pancreatic islets.
FIG. 34 shows the results of immunohistochemical analysis of normal pancreatic islets and heparin-waveguide-coated, surface-modified, anti-CD154 monoclonal antibody- FIG. 2 is a graph showing blood glucose changes of each mouse transplanted with pancreatic islets. FIG.
Figure 35 shows that 8-branched PEG and heparin catechol were coated in a multi-layered, A fluorescent image taken with a confocal scanning microscope based on the central compartment of the pancreatic islet.
Figure 36 shows that 8-branched PEG and heparin catechol were coated in a multi-layered, It is a fluorescence image taken with a confocal scanning microscope overlaying multiple compartment images of pancreatic islets.
Figure 37 shows that 8-branched terpene PEG and heparin catechol were coated in a multi- It is an image of a pancreatic islet taken by fluorescence microscope.
Figure 38 is a graph showing the results of a comparison between normal pancreatic islets, 8-branched PEG and heparin catechol FIG. 2 is a graph showing the results of cell viability using a cell quantification kit for pancreatic islets. FIG.

The term "heterofunctional" as used herein means two or more functional groups having different functions.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for activating PEG or a derivative thereof, comprising the steps of: (a) activating a bi-functional higher order structure PEG or derivative thereof to bind to the surface of a pancreatic islet;

(Step 2) of binding the activated PEG or derivative thereof to the amine group on the surface of the pancreatic islet surface to effect primary coating; And

(Step 3) of binding a bioactive complex to a terminal functional group of PEG or a derivative thereof bound to the surface of the pancreas canthus (step 3) in step 2, thereby providing a method for surface modification of pancreatic islets.

Hereinafter, the present invention will be described in more detail by step.

In the method for surface modification of pancreatic islets according to the present invention, step 1 is a step of preparing a bi-functional high-order structure PEG or derivative thereof used for primary coating of pancreatic islets, To activate it.

Since polyethylene glycol (PEG) has a very high hydrodynamic capacity and is highly capable of collecting water molecules, polyethylene glycol bound to the surface of the pancreatic islets plays a role in preventing the immune cells from being recognized as antigens by forming water molecules .

The above-mentioned dual functional higher order PEG derivative serves to prolong the survival time of the pancreatic islet by decreasing the immune response upon transplantation of the pancreatic islet into the portal vein. PEG derivatives such as PEG-DOPA, monomethoxy polyethylene glycol (PEG), succinimide of PEG propionic acid, succinimide of PEG butanoic acid, Succinimide of carboxymethylated PEG, succinimide of PEG butanoic acid, branched PEG-NHS, PEG succinimidyl succinate, succinimide of carboxymethylated PEG, Benzotriazole carbonate of PEG, PEG-glycidyl ether, PEG-oxycarbonylimidazole, PEG nitrophenyl carbonates, PEG-aldehyde (PEG-aldehyde), PEG succinimidyl carboxymethyl ester, PEG succinimidyl ester, and the like.

The above-mentioned dual functional higher order structure PEG or its derivative has two functional groups attached to the central part and / or the terminal part to have a difunctional property. At this time, the functional group may be introduced with a catechol group, an amine group, a hydroxyl group, a carboxyl group, a sulfone group, or the like.

A biologically active substance is introduced into one functional group out of two different functional groups introduced into the central part and / or the terminal of the bi-functional high-order structure PEG or its derivative. When the bioactive substance binds to an amine group on the surface of the pancreatic islet amine group), and the other one functional group binds to the biologically active complex coated with the secondary. At this time, the bioactive substance can use catechol derivatives, and it is preferable to use dopamine or 3,4-dihydroxyhydrocinnamic acid (DOHA).

Next, the biologically active substance introduced into the end of the above-prepared bi-functional high-order structure PEG or its derivative is reacted with pancreatic islets in a HBSS medium of pH 8 to bind to the pancreatic islet surface.

In the method for surface modification of pancreatic islets according to the present invention, step 2 is a step of first coating PEG or its derivative activated in step 1 with an amine group on the surface of pancreatic islets.

Specifically, various functional groups at the ends of a bioactive substance capable of reacting with an amine group on the surface of the pancreas canal are covalently bonded to each other and grafted.

In the method for surface modification of pancreatic islets according to the present invention, the step 3 is a step of binding the bioactive complex to the functional group of the double functional higher order structure PEG or derivatives thereof grafted onto the surface of the pancreatic islet in the step 2, Coating.

The bioactive complexes play a role in reducing the production of thrombosis caused by transplantation of pancreatic islets into the portal vein. As the bioactive complex, heparin or a derivative thereof and a bioactive substance may be used.

The bioactive substance may be a catechol derivative. It is preferable to use dopamine or 3,4-dihydroxyhydrocinnamic acid (DOHA). Examples of the heparin derivative include recombinant heparin, heparin analogue ) May be used, but it is preferable to use heparin.

Specifically, a method for binding a bioactive complex is a method in which functional groups of a functional functional group of a bi-functional high-order structure grafted onto the surface of a pancreatic islet are conjugated with various functional groups at the end of the bioactive substance of the bioactive complex, .

The method of grafting the biologically active complex on the surface of the PEG or its derivatives coated with the double-functional high-order structure coated on the pancreatic islet can be carried out by using an amine group, A thiol group, and an azide group.

The present invention also provides a surface-modified pancreatic islet in which a pancreatic islet is first coated with a bi-functional higher-order structure of PEG (Polyethylene glycol) or a derivative thereof, and a bioactive complex is secondarily coated on the surface of the PEG or derivative thereof do.

The surface-modified pancreatic islets according to the present invention can be obtained by first coating a surface of a pancreatic islet with a high-order structure PEG having a bifunctionality to effectively reduce the immune response, and applying a bioactive complex to the surface of the primary coating The antithrombotic effect is induced and the survival time of pancreatic islets can be increased by more effectively inhibiting the intrinsic blood-mediated inflammatory reaction (IBMIR) that occurs when pancreatic islets are transplanted into the portal vein.

In the present invention, a dual-function higher order structure PEG or derivative thereof is used in the primary coating of the pancreatic islet, and the higher order PEG reduces the immune response upon transplantation of the pancreatic islet into the portal vein, thereby prolonging the survival time of the pancreatic islet .

At this time, examples of the PEG derivatives having a bifunctional higher-order structure include PEG-DOPA, monomethoxy polyethylene glycol (PEG), succinimide of PEG propionic acid, Succinimide of carboxymethylated PEG, succinimide of PEG butanoic acid, branched PEG-NHS, PEG succinimidyl succinate, succinimide of carboxymethylated PEG, PEG Benzotriazole carbonate of PEG, PEG-glycidyl ether, PEG-oxycarbonylimidazole, PEG nitrophenyl carbonates, PEG-oxycarbonylimidazole, PEG-aldehyde, PEG succinimidyl carboxymethyl ester, PEG succinimidyl ester, and the like can be used.

The above-mentioned dual functional higher order structure PEG or its derivative has two functional groups attached to the central part and / or the terminal part to have a difunctional property. The functional group may be a catechol group, an amine group, a hydroxyl group, a carboxyl group, a sulfone group, or the like.

A biologically active substance is introduced into one functional group out of two different functional groups introduced into the central part and / or the terminal of the bi-functional high-order structure PEG or its derivative. When the bioactive substance binds to an amine group on the surface of the pancreatic islet amine group), and the other one functional group binds to the biologically active complex coated with the secondary. At this time, the bioactive substance can use catechol derivatives, and it is preferable to use dopamine or 3,4-dihydroxyhydrocinnamic acid (DOHA).

In the present invention, a bioactive complex is used as a secondary coating material which is additionally coated on the primary coating surface of the pancreas cantho. The bioactive complex serves to reduce the thrombosis that occurs upon transplantation of the pancreatic islet into the portal vein.

At this time, the bioactive complex may be a heparin or a derivative thereof combined with a bioactive substance. The bioactive substance is as described above. As the heparin derivative, recombinant heparin, heparin analogue or the like may be used, but heparin is preferably used.

In order to minimize the immune rejection reaction and prevent the formation of thrombosis, the method for coating the pancreas small intestine according to the present invention comprises coating the surface of the pancreatic islet with a bi-functional high order structure PEG or a derivative thereof several times, , The biologically active complex is grafted on the surface of the primary coating by secondary coating to maximize the density of the bi-functional high-order structure PEG or derivatives thereof and the bioactive complex grafted onto the surface of the pancreas canal.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited thereto.

< Manufacturing example  1> Heparin- Waveguide  synthesis

As shown in the structural formula shown below, heparin is reacted with NHS (N-hydroxysuccinimide) under weakly acidic conditions to activate the -COOH group of heparin, and then reacted with dopamine to finally react with heparin-dopa Complex.

[Heparin]

[Activated heparin]

[Dopamine]

[Heparin-dopa]

< Manufacturing example  2> Dual functionality Dendritic PEG Synthesis of

Step 1: Each generation PEG  synthesis

) 0.1 g과 동일 몰 수의 DPMK(Diphenylmethane potassium)을 무수 THF 조건에서 30분 동안 반응시키고 모노머로 에틸렌옥사이드(ethylene oxide)를 넣은 후 3일 동안 실온에서 반응시켰다. 모든 중합 과정은 고진공하에서 실시하였다.To polymerize the linear PEG, isopropylidene-1,1-bis (hydroxymethyl) -1-hydroxymethylpropane (

) Was reacted for 30 minutes under the condition of anhydrous THF, ethylene oxide was added as a monomer, and the reaction was carried out at room temperature for 3 days. All polymerization processes were carried out under high vacuum.

Then, the polymerization of the next generations was carried out under a solvent condition in which the ratio of anhydrous DMSO to THF was 1/4. Specifically, 0.1 g of PEG used as an initiator was added thereto, DPMK having a molar ratio of 1/2 of the hydroxyl group of the PEG was added, and the mixture was reacted at 60 ° C. for 30 minutes. Then, ethylene oxide was added as a monomer and reacted at room temperature for 3 days .

Step 2: PEG on Branches ( branched point Introduction

The branch was introduced through the reaction of the allyl group introduction and the dihydroxylation of the allyl group in the PEG prepared in the step 1 above.

In the allyl group introduction step, PEG produced under nitrogen was dissolved in THF, and a saturated NaOH solution was added in the same amount as that of THF. Then, tert-butylammonium bromide was added in an amount of 1/10 times as much as the prepared PEG hydroxyl group, Lt; 0 &gt; C. Thereafter, allyl bromide was added by 5 times the number of hydroxyl groups of PEG, followed by reaction for 24 hours to introduce allyl into the first generation PEG.

[First-generation PEG with allyl group introduced]

In the step of converting the allyl group into a diacid group, the allyl-introduced PEG is dissolved in the same amount of acetone and water, and then the mixture is purged with nitrogen for 1 hour. Then, the molar number of NMO (N-methylmorpholine N-oxide) 0.05 ml of osmium tetroxide stabilized in tert-butanol was added, and the reaction was carried out for 24 hours to carry out a diacid conversion reaction. The structural formula after the first-generation PEG is subjected to the diacid conversion reaction is shown in the following formula (3).

(3)

Step 3: Dendritic PEG Polymerization of

The above steps 1 and 2 were repeated to polymerize the dendritic PEG having the third-generation higher-order structure. The number of generations can be adjusted by the number of repetitions of steps 1 and 2 above.

Step 4: Dendritic PEG Introduction of a carboxyl group at the center of

The dendritic PEG prepared in step 3 was dissolved in the same amount of THF and methanol, and 0.1 M hydrochloric acid was added thereto, followed by reaction at room temperature for 24 hours, followed by purification. Then, DPMK was added to the center of the dendritic PEG at an equivalent amount of DPMK, and the reaction was carried out for 30 minutes. Then, 4-bromobutanoic acid was added thereto and reacted for 2 days to introduce a carboxyl group into the center of the dendritic PEG .

Step 5: Dendritic PEG At the end of Amine group  Introduction

3-Aminopropane-1-thiol was added to the PEG prepared in the step 4 and irradiated with ultraviolet rays having a wavelength of 356 nm for 24 hours to synthesize a dendritic PEG having a bifunctional group introduced therein.

[Chemical Formula 4]

Step 6: Bioactivity  Materials (dopamine and heparin- Waveguide ) Introduction

Dendritic PEG having a carboxyl group at the center and an amine group introduced at the center through the above steps 4 and 5 was doped at the center through dopamine EDC / NHS and heparin-dopa prepared in Preparation Example 1 at the end.

Identification of compounds

In order to confirm the structure of the third generation dendritic PEG prepared in the above steps 1 to 3, each characteristic ¹ H-peak was identified by ¹ H-NMR in each generation (G0-G3) Is shown in Fig.

In order to confirm the molecular weight of the dendritic PEG for each generation (G1-G3), peaks of each generation were confirmed by gel permeation chromatography (GPC). At this time, it was confirmed that the peak position shifts as the number of generations increases, and the generation of dendritic PEG was confirmed by confirming the molecular weight and the molecular weight distribution for each generation using the PEG control group (G0). The results are shown in Fig.

Further, in order to confirm that the carboxyl group was introduced at the center of the dendritic PEG in the step 4, the peak corresponding to the acetal disappeared at about 0.8-1.4 ppm using ¹ H-NMR, confirming that the carboxyl group was introduced , And the results are shown in Fig.

Further, in order to confirm that an amine group was introduced at the end of the dendritic PEG in the step 5, it was confirmed by ¹ H-NMR that a peak corresponding to cysteamine appeared near 2.7-2.9 ppm An amine group was introduced. The results are shown in Fig.

Furthermore, in order to confirm that dopamine, which is a bioactive substance, was introduced into the central carboxyl group of the dendritic PEG in step 6, it was confirmed by using an ultraviolet detector (UV detector) that the absorption wavelength was emitted at 280 nm, . The results are shown in Fig.

< Manufacturing example  3> 6 minutes topography PEG Synthesis of

Step 1: Synthesis of 6-minute terrestrial PEG

)을 무수 DMSO에 녹인 후 쏘비톨 말단의 히드록실기 몰 수의 30 %에 해당하는 DPMK(Diphenylmethylpotassium)를 넣고 히드록실기를 활성화시킨 다음 모노머로 에틸렌옥사이드를 넣은 후 3일 동안 40 ^oC에서 반응시켜 하기와 같은 6분지형 PEG를 중합하였다. 이때, 모든 중합은 고진공하에서 실시하였다. In order to polymerize the 6-branched terpolymer, sorbitol (Sorbitol,

) Was dissolved in anhydrous DMSO, DPMK (Diphenylmethylpotassium) corresponding to 30% of the number of hydroxyl groups of the terminal of the covalvol was added, the hydroxyl group was activated, ethylene oxide was added as a monomer, and the reaction was carried out at 40 ^° C for 3 days. 6-branched terpene PEG was polymerized. At this time, all the polymerization was carried out under high vacuum.

[6-minute terrain PEG]

Step 2: Six-minute terrain PEG  At the end Allyl group  Introduction

To the saturated NaOH solution under nitrogen was added TBAB (tert-butylammonium bromide) 1/10 times the mole number of the PEG hydroxyl group, and the 6-branched PEG prepared in the above step 1 was dissolved in THF and injected for 30 minutes at 50 ^° C Lt; / RTI &gt; Then, allylbromide was added 5 times the number of moles of PEG hydroxyl group, followed by reaction for 24 hours to introduce an allyl group at the 6-branch terrestrial PEG end.

[6-terpoled PEG with allyl group introduced]

Step 3: Six-minute terrain PEG  At the end Amine group  Introduction

An amine group was introduced through a chain transfer reaction in order to introduce an amine group into the 6-branched terpolymer PEG prepared in Step 2 above. Under nitrogen, 2,2-dimethoxy-2-phenylacetophenone, which is a photoinitiator, was added 0.05 times the molar number of allyl groups, cysteamine hydrochloride was added in triplicate to the mole number of allyl groups, and ultraviolet light of 365 nm wavelength was irradiated for 24 hours To introduce an amine group at the terminal of the 6-branch terrestrial PEG.

[6-branch terpolymer PEG with amine group introduced]

Step 4: Bioactivity  Introduction of substance (catechol group)

In order to introduce a catechol group capable of adsorbing on the cell surface into 6-branch type PEG having an amine group introduced at the terminal prepared in the step 3, 3,4-dihydroxyhydrocinnamic acid (DOHA) composed of a carboxyl group in the catechol group acid was activated with NHS (N-hydroxysuccinimide) and EDC (1-ethyl-3- (3-dimethylaminopropyl) carboimide hydrochloride) and reacted with an amine group of PEG at pH 3.5-4.0 for 24 hours. The reaction solution was injected into a membrane tube, dialyzed for 3 days, and the unreacted DOHA was removed. The freeze-dried PEG was added to the terminal catechol group.

[6-branch terrain PEG with catechol group introduced]

< Manufacturing example  4> 8 minutes terrain PEG Synthesis of

Step 1: Four-minute terrain PEG Synthesis of

)을 무수 DMSO에 녹인 후 펜타에리쓰리톨 말단의 히드록실기 몰 수의 30%에 해당하는 DPMK(Diphenylmethylpotassium)를 넣고 히드록실기를 활성화시킨 다음 모노머로 에틸렌옥사이드를 넣은 후 3일 동안 40 ℃에서 반응시켜 하기와 같은 4분지형 PEG를 중합하였다. 이때, 모든 중합은 고진공하에서 실시하였다.In order to polymerize the tetrahedral PEG, pentaerythritol,

) Was dissolved in anhydrous DMSO, DPMK (Diphenylmethylpotassium) corresponding to 30% of the number of hydroxyl groups at the end of pentaerythritol was added, the hydroxyl group was activated, ethylene oxide was added as a monomer, Followed by the polymerization of the 4-branched terpene PEG as follows. At this time, all the polymerization was carried out under high vacuum.

[4-minute terrain PEG]

Step 2: Four-minute terrain PEG At the end of Point  Introduction

In step 1, the branch was introduced through the introduction of allyl group and the dihydroxylation of the allyl group to the polymerized PEG end.

In the allyl group introduction step, TBAB ( tert- butylammonium bromide) was added to the saturated NaOH solution at 1/10 times the number of moles of the PEG hydroxyl group, and the 4-branched PEG prepared in the above step 1 was dissolved in THF And reacted at 50 캜 for 30 minutes. Then, allylbromide was added 5 times the number of moles of the PEG hydroxyl group, and reacted for 24 hours to introduce an allyl group at the 4-branched terpolymer PEG end.

[4-Tetrahedron PEG with allyl group introduced]

In the diisocyanate conversion step of the allyl group, the 4-branched PEG having the allyl group introduced therein was dissolved in the same amount of acetone and water, followed by nitrogen substitution for 1 hour. Next, NMO (N-methylmorpholine N-oxide) and OsO ₄ (osmium tertroxide) stabilized in tert -butanol were added in the same amount as the moles of allyl introduced, and the reaction was carried out for 24 hours, Hydroxyl groups were introduced at the ends of the terrain PEG.

[4-terpoled PEG with hydroxyl group incorporated]

Step 3: 8-minute terrain PEG Manufacturing

The 4-branched terpolymer PEG having hydroxyl groups at the terminal prepared in the step 2 was dissolved in dioxane anhydride and lyophilized to remove water. Then, the 8-branched terpolymer PEG was polymerized in the same manner as in step 1.

[8-minute Terrain PEG]

Step 4: 8-minute terrain PEG At the end of Amine group  Introduction

In order to introduce an amine group into the terminal of the 8-branched terpolymer prepared in the step 3, an amine group was introduced through an introduction of an allyl group at the terminal of the PEG and a chain transfer reaction. Specifically, after the allyl group was introduced in the same manner as in the step 2, 2,2-dimethoxy-2-phenylacetophenone as a photoinitiator was added in an amount of 0.05 times the mole number of the allyl group under nitrogen, and then cysteamine hydrochloride The amine group was introduced at the terminal of the 8-minute terrestrial PEG by irradiating ultraviolet rays having a wavelength of 365 nm for 24 hours by adding 3 times of the number of moles.

[Eight-minute topographic PEG with amine group introduced at the terminal]

Step 5: Bioactivity  Introduction of substance (catechol group)

In order to introduce a catechol group capable of adsorbing on the cell surface into the 8-branched PEG having an amine group introduced at the terminal prepared in the above step 4, 3,4-dihydroxyhydrocinnamic acid (DOHA) composed of a carboxyl group in the catechol group acid was activated with NHS (N-hydroxysuccinimide) and EDC (1-ethyl-3- (3-dimethylaminopropyl) carboimide hydrochloride) and reacted with an amine group of PEG at pH 3.5-4.0 for 24 hours. The reaction solution was injected into a membrane tube, and dialyzed for 3 days. Unreacted DOHA was removed and lyophilized to prepare an 8-branched terpene PEG having a catechol group introduced at the terminal.

[8-terrain PEG with catechol group introduced]

Identification of compounds

In order to identify the tetrabranched PEG with the branches prepared in Step 2, the hydrogen peak corresponding to the allyl group was confirmed at 5.2-5.4 ppm and 5.8-6.0 ppm by ¹ H-NMR, Later, it was found that all allyl groups were all converted to hydroxyl groups by elimination of all allyl groups. The results are shown in Fig.

In order to confirm whether or not the 4-branched PEG having branched branches prepared in the step 2 obtained by the ring-opening polymerization of ethylene oxide had a desired molecular weight, 4-branched terpolymer PEG and 8-branched terpolymer were obtained by GPC (permeation chromatography) The molecular weight of PEG was measured. As a result of the measurement, it was confirmed that the 8-minute terrestrial PEG was synthesized by confirming that the peak shifts completely and the desired molecular weight was obtained in the 4-minute terrestrial PEG. The results are shown in Fig.

Further, in order to confirm that the amine group was introduced at the end of the 8-branch terrestrial PEG prepared in the step 3, the ¹ H-NMR result of the compound introduced with allyl before the chain transfer reaction and the ¹ H- ¹ H-NMR results were confirmed. Allyl was introduced at 5.2-5.4 ppm and 5.8-6.0 ppm, and hydrogen peak corresponding to allyl group appeared. After the chain transfer reaction, all of the allyl peak disappeared, and at 2.7 ppm and 2.9 ppm, cysteamine And the amine group was introduced. The results are shown in Fig.

In order to confirm that the carboxyl group of the catechol group was bonded to the terminal amine group of the 8-branched terpolymer prepared in Step 4 and the bioactive substance, a UV detector was used to measure the absorption wavelength of 280 nm , It was found that the catechol group was bonded to the PEG terminal. The results are shown in Fig.

< Example  1> Pancreatic islet  8 minute terrain on the cell PEG  coating

Preparation phase: Pancreatic  detach

In the capsule for enclosing the pancreas small intestine with the surface modified with the biocompatible polymer according to the present invention, the pancreatic islet was obtained by the following method.

First, SD rats (Korean Experimental Animal Center, Korea) weighing 250-300 g fasted from day before isolation of pancreatic islets were anesthetized by subcutaneous injection with a mixed solution of ketamine and xylazine. After the abdominal cavity was opened, collagenase (1.5 mg collagenase / HBSS 1 mL, 12.5 mL / SD rat) was infused through bile ducts to inflate the pancreas, and the pancreas immediately inflated Lt; / RTI &gt; The extracted pancreas was shredded with scissors, shaken while shaking in a 37 ° C water bath, and cold HBSS was added to prevent further degradation. The degraded pancreatic tissue was sieved and washed several times. The supernatant was removed by centrifugation at about 1400 rpm for 3 minutes, centrifuged at about 2400 rpm for 24 minutes after a gradient of ficoll concentration of 27, 23, 20 and 11%. Islets present between 20% and 23% layers and between 11% and 20% layers were collected and washed, and cultured in RPMI-1640 medium (Sigma, 11.1 mM glucose, 2 mM sodium pyruvate, 6 mM HEPES, 10% FBS, 1% penicillin / streptomycin), and cultured at 37 ° C with 5% CO ₂ . The culture medium was replaced with a new one every two days, and the pancreatic islets were separated therefrom.

Step 1: 8-minute terrain PEG Activation of the terminal catechol group

The 8-branched PEG with catechol group introduced in Preparation Example 4 was activated as follows to react with an amine group present on the surface of pancreatic islets. First, an 8-minute topographic PEG was added to a two-necked flask, and methylene chloride was poured and dissolved. Next, NHS (N-hydrosuccinimide) was added to completely dissolve and 1,3-dicyclohexylcarbodiimide was added in an ice water bath, followed by reaction for one day and filtration. The methylene chloride was evaporated, and about 50 to 100 ml of benzene was added thereto, and the solution was maintained for about 6 hours and filtered again. The filtrate was precipitated in ether and filtered again to obtain an 8-minute topographic PEG with terminal DOHA activated.

Step 2: Pancreatic islet  Active 8-minute terrain on the surface PEG Combination of

The pancreatic islets isolated in the preparation step were cultured in RPMI-1640 medium for 3 days, and then bound to the activated 8-branched PEG of step 1. Specifically, pancreatic islets were washed with HBSS so that pancreatic islets could bind well to PEG, and 10 ml of HBSS buffer solution was added to the pancreatic islets with 1% (w / v) ₂ , and 37 ℃ in the cell incubator. After pegylation, the pancreatic islets were washed with culture medium to remove any 8-minute topographic PEG that did not react with the surface of the pancreatic islets in order to prevent further reaction.

< Example  2> Pancreatic islet  6 minute topography on cells PEG Coating

In the same manner as in Example 1 except that the 6-branched PEG in which the catechol group prepared in Preparation Example 3 was used was used instead of the 8-branched PEG in which the catechol group was introduced, Topographic PEG was coated.

< Example  3> Pancreatic islet  Heparin- Waveguide  coating

The heparin-dopa prepared in Preparation Example 1 was coated on pancreatic islet cells obtained in the preparation step of Example 1 as follows.

10 mg of the heparin-dopa prepared in Preparation Example 1 was dissolved in 10 ml of HBSS (pH 8) buffer, added to the pancreatic islets prepared in the preparation step of Example 1, and incubated in a cell incubator under the conditions of 37 ° C and 5% , And the pancreatic islets were washed with fresh HBSS buffer to prevent further reaction, and cultured in RPMI-1640 medium until use.

< Example  4> Pancreatic islet  8 minute terrain on the cell PEG  And a dual layer coating of heparin catechol

First, 8-minute topographic PEG was coated on the surface of pancreatic islet cells in the same manner as in Example 1,

Heparin catechol of 1% by weight (w / v%) was dissolved in HBSS (pH 8) buffer and added to the 8-minute topical PEG-coated pancreatic islets. The cells were incubated in a cell incubator at 37 ° C under 5% After reaction, pancreatic islets were washed with fresh HBSS buffer to prevent further reaction, and then cultured in RPMI-1640 medium until use. FIG. 1 is a graph showing the relationship between the 8-branched terpene PEG and the heparin catechol prepared in this Example Images of pancreatic islets were shown.

< Experimental Example  1> Pancreatic islet  8-minute terrain on the surface PEG Confirmation of coupling

8-minute topographic FITC-PEG was coated on the surface of the pancreas canal by performing the same procedure as in Example 1 except that the fluorescent substance-labeled FITC-PEG was used to confirm the binding of PEG to the surface of the pancreatic islet. FIG. 11 shows a photograph of the FITC-PEG fused to the surface of the small intestine, and FIG. 12 shows a photograph of the fluorescence image taken by the confocal laser scanning microscope FIG. 13 shows a photograph in which multiple compartment images are overlaid.

FIG. 11 is an optical micrograph of an 8-branched PEG-coated pancreatic islet. FIG.

Figure 12 is a fluorescence image taken with a confocal scanning microscope based on the central compartment of the 8-branched PEG-coated pancreatic islets.

Figure 13 is a fluorescence image taken with a confocal scanning microscope overlaying multiple compartment images of an 8-branched PEG-coated pancreatic islet.

As shown in Figs. 11 to 13, it was confirmed that the heparin-dopa, which is the terminal group of the 8-branch terrestrial PEG, was well bound to the surface of the pancreatic islet.

Therefore, since the 8-branched PEG according to the present invention binds well to the surface of the pancreatic islet, it can be usefully used in the pretreatment process of pancreatic islet transplantation.

< Experimental Example  2> 8 min terrain PEG Coated Pancreatic Survivability  Measure

Example 1 in vitro for 8 bun prepared to investigate the viability of pancreatic islets coated with a branched PEG in the (in In vitro) conditions, the following experiment was performed.

First, pancreatic islets coated with normal pancreatic islets and 8-well topical PEG were added separately to each culture plate. For the culture, RPMI-1640 containing 10% FBS was used and cultured in a cell incubator for two days. Each of the cultured pancreatic islets was washed with HBSS buffer, and reacted with a live / dead cell kit for 15 minutes. Cell viability was confirmed by fluorescence microscopy. At this time, dead cells are reddish, and living cells are dyed green. An image of a normal pancreas is shown in FIG. 14, and an image of a pancreatic islet coated with an 8-minute topical PEG is shown in FIG.

14 is an image of a normal pancreatic islet taken by fluorescence microscope.

15 is an image obtained by photographing a pancreatic islet coated with an 8-branch type PEG using a fluorescence microscope.

Next, 8-minute topical PEG-coated pancreatic islets were transferred to a 96-well plate to which RPMI-1640 medium was added, and cultured at 37 ° C and 5% CO ₂ for 24 hours. After 4 hours from the induction of the secretion of the enzyme in living cells, the absorbance was measured at 450 nm by adding a solution of cell counting kit-8 (manufactured by Dojindo Molecular Technologies Inc., Rockville, MD) The viability of the cells was confirmed by this, and the results compared with the normal pancreatic islet are shown in FIG. 16 as a graph.

16 is a graph showing cell viability results of pancreatic islets coated with general pancreatic islets and 8-branched PEGs using a cell quantitation kit.

As shown in Figs. 14 to 16, it was confirmed that binding of the 8-branched terpene PEG to the surface of the pancreatic islet does not affect the viability of the pancreatic islet.

Therefore, the 8-branched PEG according to the present invention does not affect the viability of the pancreatic islet, and thus can be usefully used in the pretreatment process of pancreatic islet transplantation.

< Experimental Example  3> 8 min terrain PEG Coated Pancreatic  transplantation

The experiment was conducted as follows to examine the change of blood glucose level by transplanting the 8-branch type PEG-coated pancreatic islets prepared in Example 1 into experimental rats.

First, about 20 g of C57BL / 6 mice were injected with streptozotocin 180 mg / kg streptozotocin to induce diabetes. Among them, mice having a blood glucose concentration of 350 mg / dl or more were selected as diabetes induced, and were selected as recipients of the 8-minute topical PEG-coated pancreas small intestine transplant prepared in Example 1, transplanted through the portal vein, -CD154 monoclonal antibody was injected on days 0, 2, 4 and 6 to confirm the regulation of blood concentration. At this time, in order to compare the amount of blood glucose change in the 8-branch type PEG-coated pancreatic islet transplantation group, the following general pancreatic islet was set as a control group, and the result is shown in FIG.

FIG. 17 is a graph showing blood glucose changes in mice transplanted with 8-branched PEG-coated pancreatic islets and normal pancreatic islet cells.

As shown in FIG. 17, in the case of the control group, blood glucose levels of all the mice transplanted with the pancreatic islet cells were not regulated to the normal range, but it was confirmed that the blood glucose control was more stable after the 8-minute topographic PEG-coated pancreatic islet transplantation Respectively.

Therefore, the 8-branched PEG-coated pancreatic islets according to the present invention can be usefully used in the pretreatment process of pancreatic islet transplantation because blood glucose control can be performed in a normal range.

< Experimental Example  4> Pancreatic islet  Six-minute terrain on the surface PEG Confirmation of coupling

It was confirmed that the 6-minute topographic PEG was bound to the surface of the pancreatic islet by the same method as Experimental Example 1 except that the 6-branched PEG-coated pancreatic islet prepared in Example 2 was used. A photograph of the FITC-PEG fused to the surface of the pancreas isoform is shown in Fig. 18, and a photograph of the fluorescence image of the pancreas canal centered on confocal laser scanning microscopy is shown in Fig. 19 20, and a photograph of a plurality of compartment images superimposed on each other is shown in FIG.

18 is an optical microscope image of pancreatic islets coated with a 6-minute topographic PEG.

Figure 19 is a fluorescence image taken with a confocal scanning microscope based on the central compartment of the pancreatic islets coated with 6-branched PEG.

20 is a fluorescence image taken with a confocal scanning microscope overlaying multi-folded compartment images of a 6-branched PEG-coated pancreatic islet.

As shown in FIGS. 18 to 20, it was confirmed that the heparin-dopa, which is the terminal group of the 6-minute terrestrial PEG, was well bound to the surface of the pancreatic islet.

Therefore, since the 6-branch type PEG according to the present invention binds well to the surface of the pancreatic islet, it can be usefully used in the pretreatment process of pancreatic islet transplantation.

< Experimental Example  5> 6-minute terrain PEG Coated Pancreatic Survivability  Measure

The viability of the pancreatic islets coated with the 6-branch type PEG prepared in Example 2 was examined in the same manner as in Experimental Example 2.

An image of pancreatic islets coated with a 6-minute topographic PEG is shown in FIG.

FIG. 21 is an image obtained by photographing a pancreatic islet coated with a 6-branch type PEG using a fluorescence microscope.

22 is a graph showing the results of cell viability using pancreatic islets coated with a normal pancreatic islet and a 6-branch type PEG using a cell quantitation kit.

As shown in FIGS. 21 and 22, it was confirmed that binding of the 6-branched PEG to the surface of the pancreatic islet does not affect the viability of the pancreatic islet.

Therefore, the 6-branch type PEG according to the present invention does not affect the viability of the pancreatic islet, and thus can be usefully used for the pretreatment of pancreatic islet transplantation.

< Experimental Example  6> 6 minutes topography PEG Coated Pancreatic  Determination of insulin secretion by glucose concentration

In order to measure the insulin secreted according to the glucose (glucose) concentration in the 6-branched PEG-coated pancreatic islets prepared in Example 2, in vitro vitro ) conditions.

First, into groups 6 minutes insert culturing the pancreatic islets coated with a branched PEG plate KRBB culture solution (3.469 g NaCl, 0.175 g KCl , 0.160 g MgSO 4 7H 2 O, 0.0816 g KH 2 PO4, 0.1054 g CaCl 2, 1.05 g NaHCO ₃ , 1.19 g HEPES, 0.025 g BSA, 1 L water) and incubated at 37 ° C with 5% CO ₂ . The cells were preincubated in a culture medium having a sugar concentration of 2.8 mM for 1 hour and cultured for 2 hours at a concentration of 2.8 mM for 2 hours in a medium having a glucose concentration of 28 mM. At this time, the inserter was shaken every 10 minutes. After 2 hours of incubation, samples were collected from 2.8 mM and 28 mM culture medium, and the insulin concentration was measured with an ELISA kit (manufacturer: Millipore Corp.). The results are shown in FIG.

23 is a graph showing the results of insulin secretion ability of pancreatic islets coated with 6-branch type PEG.

As shown in FIG. 23, significant changes were observed in the insulin concentration measured two days after the culture in the sugar solution with different glucose concentrations, indicating that the sensitivity to the sugar concentration of the 6-minute topical PEG-coated pancreatic islets was significantly .

Therefore, the 6-branched PEG-coated pancreatic islets according to the present invention can be effectively used for the pretreatment of pancreatic islet transplantation since the sensitivity to the sugar concentration is not influenced and does not affect the function of pancreatic islet cells .

< Experimental Example  7> 6 minutes Terrain PEG Coated Pancreatic  transplantation

The experiment was carried out as follows to examine the change of blood glucose by transplanting the 6-branch type PEG-coated pancreatic islets prepared in Example 2 into experimental rats.

First, about 20 g of C57BL / 6 mice were injected with streptozotocin 180 mg / kg streptozotocin to induce diabetes. Among them, mice having a blood glucose concentration of 350 mg / dl or more were selected as having been induced with diabetes, and each recipient of a pancreatic islet (control group) coated with a 6-minute topical PEG-coated pancreatic islet prepared in Example 2 ). The results are shown in Fig. 24, in which glucose was transplanted through the kidney capsule portion and the injection of immunosuppressant was not carried out. The results are shown in Fig. 24. In the case of intraperitoneal injection of Tacrolimus (FK506) FIG. 25 shows the comparison result of the blood concentration control of the anti-CD154 monoclonal antibody, and FIG. 26 shows the comparison result of the glucose concentration control when the anti-CD154 monoclonal antibody was intraperitoneally injected on days 0, 2, 4 and 6, FIG. 27 shows the comparison result of the glucose concentration control in the case of treatment with various kinds of drugs.

24 is a graph showing blood glucose changes in each mouse transplanted with pancreatic islets coated with normal pancreatic islets and 6-branched PEGs when no immunosuppressant was injected.

25 is a graph showing changes in blood glucose of each mouse transplanted with pancreatic islets coated with normal pancreatic islets and 6-branched PEG when FK506 was injected.

FIG. 26 is a graph showing blood glucose changes of each mouse transplanted with pancreatic islets coated with a general pancreatic islet and a 6-branch type PEG when anti-CD154 monoclonal antibody was injected.

FIG. 27 is a graph showing blood glucose changes in each mouse transplanted with pancreatic islets coated with a common pancreatic islet and a 6-branch-type PEG when FK506 and anti-CD154 monoclonal antibody were simultaneously injected.

As shown in FIGS. 24 to 27, it was confirmed that pancreatic islets coated with 6-branch terra-PEG had a more stable concentration of clucose in the blood and increased survival rate compared to the normal pancreatic islet without modification of the surface of the pancreatic islet I could.

Therefore, the 6-branched PEG-coated pancreatic islets according to the present invention can be usefully used in the pretreatment process of pancreatic islet transplantation since the survival rate is increased and the blood glucose concentration can be controlled stably for a long time in the body.

< Experimental Example  8> Pancreatic islet  On the surface, heparin- Waveguide  Confirm binding

The heparin-dopa-coated pancreatic islets prepared in Example 3 were used in the same manner as Experimental Example 1 to confirm that heparin-dopa was bound to the surface of the pancreatic islets. A photograph of the FITC-heparin-dopa bound to the surface of the pancreatic islet is shown in FIG. 28, and a fluorescence image of the pancreas small intestine is shown by Confocal Laser Scanning Microscopy 29, and a photograph in which multiple compartment images are overlaid is shown in Fig.

28 is an optical microscope image of heparin-dopa coated pancreatic islets.

Figure 29 is a fluorescence image taken with a confocal scanning microscope based on the central compartment of heparin-dopa coated pancreatic islets.

Figure 30 is a fluorescence image taken with a confocal scanning microscope overlaying multiple compartment images of heparin-dopa coated pancreatic islets.

As shown in FIGS. 28 to 30, it was confirmed that heparin-dopa was well bound to the surface of the pancreatic islet.

Therefore, since heparin-dopa according to the present invention binds well to the surface of the pancreatic islet, it can be usefully used in the pretreatment process of pancreatic islet transplantation.

< Experimental Example  9> Heparin- Waveguide  Coated Pancreatic Survivability  Measure

The viability of the heparin-waveguide-coated pancreatic islets prepared in Example 1 was examined in the same manner as in Experimental Example 2.

An image of a normal pancreas canal and a heparin-waveguide coated pancreas canal is shown in Fig.

31 is an image obtained by photographing a normal pancreas canal and heparin-waveguide coated pancreatic islets with a fluorescence microscope.

32 is a graph showing the results of cell viability using a cell quantitation kit for a normal pancreatic islet and a heparin-waveguide coated pancreatic islet.

As shown in FIGS. 31 and 32, it was confirmed that the binding of heparin-dopa to the surface of the pancreatic islet does not affect the viability of the pancreatic islet.

Therefore, the 6-branch type PEG according to the present invention does not affect the viability of the pancreatic islet, and thus can be usefully used for the pretreatment of pancreatic islet transplantation.

< Experimental Example  10> Heparin- Waveguide  Coated Pancreatic  Determination of insulin secretion by glucose concentration

In the heparin-waveguide-coated pancreatic islets prepared in Example 3, in order to measure insulin secreted according to the glucose (glucose) concentration, the pancreatic islets coated with 6-branch type PEG prepared in Example 2 were used The same procedure as in Experimental Example 6 was carried out except that heparin-waveguide-coated pancreatic islets prepared in Example 3 were used, and the result of measuring insulin secreted according to the glucose concentration is shown in FIG. 33

33 is a graph showing the results of insulin secretion ability of a normal pancreatic islet and a heparin-waveguide coated pancreatic islet.

As shown in FIG. 33, significant changes were observed in the insulin concentration measured two days after the culture with different sugar concentrations, indicating that the sensitivity to the sugar concentration of heparin-waveguide coated pancreatic islets was excellent.

Therefore, the heparin-waveguide-coated pancreatic islets according to the present invention have excellent sensitivity in response to glucose concentration, and thus can be usefully used in the pretreatment process of pancreatic islet transplantation.

< Experimental Example  11> Heparin- Waveguide  Coated Pancreatic  transplantation

The heparin-waveguide-coated pancreatic islets were transplanted into experimental rats to examine changes in blood glucose levels, and the same procedure as in Experimental Example 3 was carried out except that heparin-waveguide-coated pancreatic islets prepared in Example 3 were used .

FIG. 34 is a graph showing blood glucose changes of each mouse transplanted with a normal pancreas islet and a heparin-waveguide coated pancreatic islet when an anti-CD154 monoclonal antibody was injected when no immunosuppressor was injected.

As shown in FIG. 34, heparin-waveguide-coated pancreatic islets more stable than normal pancreatic islets without modifying the surface of the pancreatic islets, and it was confirmed that the survival rate was increased and the concentration of the clucose was more stable.

Therefore, the heparin-waveguide-coated pancreatic islets according to the present invention can be used for the pretreatment of the pancreas transplantation because the survival rate is increased and the blood glucose concentration can be controlled stably for a long time in the body.

< Experimental Example  12> Pancreatic islet  8-minute terrain on the surface PEG  And double bond binding of heparin catechol

The 8-minute topographic PEG and heparin catechol prepared in Example 4 were treated in the same manner as in Experimental Example 1 except that pancreatic islets coated with a multilayer were used, . &Lt; / RTI &gt; FIG. 35 shows a fluorescence image of FITC-PEG fused to the surface of a pancreas isoform based on the central compartment of the pancreas small intestine using Confocal Laser Scanning Microscopy. FIG. 35 shows a photograph of multiple compartment images Is shown in Fig.

35 is a fluorescence image taken with a confocal scanning microscope based on the central compartment of a pancreatic islet in which the 8-branched PEG and heparin catechol are coated in multiple layers.

Figure 36 is a fluorescence image taken with a confocal scanning microscope overlaying multiple compartment images of a pancreatic islet in which the 8-branched PEG and heparin catechol are coated in multiple layers.

As shown in FIGS. 35 and 36, it was confirmed that the 8-minute topographic PEG and the heparin catechol bilayer were well bonded to the surface of the pancreatic islet.

Therefore, the 8-branched PEG and heparin catechol according to the present invention can be effectively used in the pretreatment of pancreas transplantation because the bilayer is well bonded to the surface of the pancreas.

< Experimental Example  13> 8 minutes Terrain PEG  And heparin catechol in a multi-layer coated Pancreatic Survivability  Measure

The viability of the 8-branched PEG and heparin catechol-coated pancreatic islets prepared in Example 4 was tested in the same manner as in Experimental Example 2.

Fig. 37 shows images of pancreatic islets coated with a multilayer of 8-branched terpene PEG and heparin catechol.

FIG. 37 is an image obtained by photographing a pancreatic islet coated with a multilayer of 8-branch type PEG and heparin catechol by fluorescence microscope.

FIG. 38 is a graph showing cell viability results of pancreatic islets coated with a double layer of a general pancreatic islet, 8-branched PEG and heparin catechol using a cell quantification kit.

As shown in FIGS. 37 and 38, it was confirmed that binding of the 8-branched terpene PEG and the heparin catechol bapule layer to the surface of the pancreatic islet does not affect the viability of the pancreatic islet.

Therefore, the 8-branched PEG and heparin catechol bilayer coating according to the present invention does not affect the viability of the pancreatic islets, and thus can be usefully used in the pretreatment of pancreas transplantation.

Claims (17)

A bi-functional high-order structure having an amine group, a hydroxyl group, a carboxyl group, or a sulfone group introduced into some terminals of the branched ends of each branch of polyethylene glycol (PEG) or derivative thereof of 6-branched to 32- Of PEG is treated with N-hydroxysuccinimide (NHS) and 1,3-dichlorocarbodiimide so that the catechol group introduced at the branch terminus of the above-mentioned dual functional higher order structure PEG can be combined with the amine group on the surface of the pancreatic islet (Step 1);
(Step 2) of binding the activated dual functional high-order structure PEG to the amine group on the surface of the pancreatic islet; And
(Step 3) of binding a biologically active complex to an amine group, a hydroxyl group, a carboxyl group or a sulfone group introduced at a certain end of a PEG having a bifunctional higher-order structure bound to the surface of the pancreas canthus in the step 2, Including,
The derivatives of PEG-6 to PEG-DOPA, monomethoxy PEG, succinimide of PEG propylic acid, succinimide of PEG butanoic acid, branched PEG-NHS, PEG Succinimide of carboxymethylated PEG, benzotriazole carbonate of PEG, PEG-glycidyl ether, PEG-oxycarbonylimidazole, PEG nitrophenyl carbonate, PEG-aldehyde, PEG succinimidyl Carboxymethyl ester, PEG succinimidyl ester, and the like.
Wherein the bioactive complex is a conjugate of dopamine or 3,4-dihydroxyhydrocinnamic acid (DOHA) to the carboxyl group of heparin, wherein the heparin is heparin or recombinant heparin. / RTI &gt;
delete delete delete delete delete delete delete The method according to claim 1, wherein grafting the biologically active complex to the branched terminal amine group, hydroxyl group, carboxyl group or sulfone group of the double functional high order structure PEG coated on the pancreatic islet of step 3 is a chemical covalent bond A method for surface modification of pancreatic islets.
Pancreatic islet;
A bi-functional high-order structure having an amine group, a hydroxyl group, a carboxyl group, or a sulfone group introduced into some terminals of the branched ends of each branch of polyethylene glycol (PEG) or derivative thereof of 6-branched to 32- The first coating layer formed by combining the catechol group and the amine group on the surface of the pancreatic islet in the PEG of the first coating layer; And
A second coating layer formed by binding a biologically active complex to a branch terminal amine group, a hydroxyl group, a carboxyl group or a sulfone group of the bi-functional high-order structure PEG,
The derivatives of PEG-6 to PEG-DOPA, monomethoxy PEG, succinimide of PEG propylic acid, succinimide of PEG butanoic acid, branched PEG-NHS, PEG Succinimide of carboxymethylated PEG, benzotriazole carbonate of PEG, PEG-glycidyl ether, PEG-oxycarbonylimidazole, PEG nitrophenyl carbonate, PEG-aldehyde, PEG succinimidyl Carboxymethyl ester, PEG succinimidyl ester, and the like.
Wherein the bioactive complex is a conjugate of dopamine or 3,4-dihydroxyhydrocinnamic acid (DOHA) to the carboxyl group of heparin, wherein the heparin is heparin or recombinant heparin. delete delete delete delete delete delete delete

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