KR20100083872A - Islet microcapsule of alginate-chitosan bilayer for treating diabetes mellitus and process for preparing the same - Google Patents

Abstract

PURPOSE: A method for an islet microcapsule of alginate-chitosan bilayer is provided to suppress deposition of inflammatory immunocytes and to treat diabetes. CONSTITUTION: An islet microcapsule of alginate-chitosan bilayer is used for treating diabetes. The islet microcapsule forms immune barrier by capsulating different kind islet with sodium alginate and coating with chitosan. The different kind islet is an islet isolated from adult pig. A method for preparing islet microcapsule of alginate-chitosan for treating diabetes comprises: a step of isolating islet from islet source; a step of diluting isolated islet in sodium alginate solution; a step of dropping in BaCl_2 to form a capsule; and a step of coating outer membrane of the capsule with chitosan at pH 4.5.

Description

Islet microcapsule of alginate-chitosan bilayer for treating diabetes mellitus and process for preparing the same}

The present invention relates to a pancreatic islet microcapsules of alginate-chitosan bilayer for diabetic treatment and a method for preparing the same.

Diabetes is a disease caused by the lack or absence of insulin, a hormone secreted from the pancreatic islets, and can be divided into insulin-dependent type 1 diabetes and insulin-independent type 2 diabetes.

Type 1 diabetes usually occurs suddenly in young or young age groups before the age of 30 to 40 years, with severe weight loss and consciousness problems. Most patients have a thin body and are susceptible to diabetic ketoacidosis, a type of diabetic acute complication. In addition, type 1 diabetes has little risk of making insulin in the pancreas, so if you do not use insulin causes a risk of life, must be treated with the use of insulin, proper diet and exercise.

Type 2 diabetes occurs mainly after age 40, and the insulin secretion ability of the pancreas is less than normal or insulin secretion is normal, but the action of insulin appears due to obesity and the like. Type 2 diabetes is often characterized by inconspicuous clinical symptoms, and metabolic disorders appear due to an increase in insulin resistance prior to a decrease in insulin secretion, and do not cause diabetic ketoacidosis except in special cases. Therefore, in the early stages of weight loss by diet and exercise therapy, diabetes is often improved.

Type 1 diabetes is a chronic disease that cannot be cured by current methods of treatment. Once it occurs, it requires thorough self-management for life. Only near-normal blood sugar control can prevent diabetic chronic complications. However, there are limitations such as exercise therapy and diet, together with the current treatment of insulin injection therapy, which cannot be cured and there is still a risk of complications. In addition, in some patients with type 1 diabetes, thorough hypoglycemia and insufficiency of hypoglycemic cognitive impairment are impossible. Type 2 diabetes also needs to be administered insulin injections in the same way as patients with type 1 diabetes. Therefore, the most basic and ideal treatment for maintaining blood sugar near normal in diabetic patients is pancreatic or pancreatic islet transplantation in which insulin secretion is physiologically controlled.

Therefore, recently, a lot of diabetes treatments through pancreas transplantation and pancreatic islet transplantation have been performed. However, in the case of pancreas transplantation, there are problems such as an absolute shortage of donors, high surgical complications, and difficulty in management after transplantation including continuous administration of immunosuppressive agents. It can be expected to reduce side effects of using immunosuppressive agents by inducing immune tolerance through preoperative immunomodulation of islet cells and maintaining the isolated islet cells in vitro for the most appropriate transplantation. There is an advantage to it. However, pancreatic islet transplantation, as well as pancreatic transplantation, has difficulty in treating diabetes through transplantation due to the absolute shortage of donors.

Therefore, in vitro proliferation of beta cells, induction of differentiation of pancreatic ducts as adult stem cells, induction of differentiation of embryonic stem cells, and proliferation of fetal pancreatic islets in order to solve the problem of absolute shortage of donors in the treatment of diabetes through pancreatic islet transplantation. In addition to the proliferation of various human pancreatic islet cells, xenotransplantation using animal tissues other than humans has been sought.

The best known animal source for xenotransplantation is pigs. This is because pig's insulin has been used in humans for a long time without side effects, and because pig's insulin metabolism is similar to human and widely used for food, it has less rejection, relatively easy to handle, and has a large amount of islet cells. This is because there is an advantage. Therefore, studies are being conducted around the world that can be used as a source of xenograft islet cells of pigs having beta cells with human-like insulin metabolism (Wang, T., et al., Successful allotransplantation of encapsulated islets). in pancreatectomized canines for diabetic management without the use of immunosuppression.Transplantation, 2008. 85 (3): p. 331-7; Elliott, RB, et al., Live encapsulated porcine islets from a type 1 diabetic patient 9.5 yr after xenotransplantation. Xenotransplantation, 2007. 14 (2): p. 157-61; Lijun Bai, Kathrin Maedler, Marc Donath and Bernard E. Tuch, Expression of Fas but not Fas ligand on fetal pig β cells.Xenotransplantation, 2004. 11: p. 426435).

However, despite the above advantages of xenograft transplantation using porcine islet cells, rapid immune rejection reactions may occur after transplanting pig islet cells, which may adversely affect the health of the recipient pig islets as well. In addition, even if the immunosuppressive agent is used, the immunorejection reaction may be caused by the inconsistency of the fundamental histocompatibility reaction, and the side effects of the immunosuppressive agent may also be serious.

In order to overcome such immunosuppression, a method of isolating pig islets from the recipient's immune system has been envisioned, and specific examples thereof include the formation of an immune barrier, and representative examples thereof include alginic acid capsules.

Alginate capsules were first attempted in 1981 to encapsulate the islets with sodium alginate. Sodium alginate is a material extracted from brown algae and is a polymer composed of β-1,4-D-mannuronic acid and β-1,4-L-glucuronic acid. The refined processing material extracted from brown algae is used to increase the purity. However, the purified pure sodium alginate has an endogenous toxin present, which causes inflammatory effects. Pure sodium alginate has the property of solidifying by strongly bonding with divalent cations (Ca ²⁺ , Ba ²⁺ ) except Al ²⁺ . Representatively, Ca ²⁺ is used a lot. Since Ca ²⁺ is less robust, a method of coating poly-L-lysine on the outside is also proposed and used (Aileen King, Evaluation of Alginate Microcapsules for Use in Transplantation Of Islets of Langerhans.ACTA Universitatis Upsaliensis, 2001.). However, since poly-L-lysine has a side effect of promoting cytokine secretion, the islets within the capsule are eventually destroyed by cytokines (Juste, S., et al., Effect of poly-L-lysine coating on macrophage). activation by alginate-based microcapsules: assessment using a new in vitro method.J Biomed Mater Res A, 2005. 72 (4): p. 389-98; Ronn, SG, et al., Suppressor of cytokine signaling-3 expression inhibits cytokine-mediated destruction of primary mouse and rat pancreatic islets and delays allograft rejection.Diabetologia, 2008. 51 (10): p. 1873-82; Aiba, S. and TL Creazzo, Comparison of the number of dihydropyridine receptors with the number of functional L-type calcium channels in embryonic heart.Circ Res, 1993. 72 (2): p. 396-402.). In addition, sodium alginate capsules with Ba ²⁺ do not require poly-L-lysine because they are robust. However, Ba ²⁺ is more toxic than Ca ²⁺ , so care must be taken in its use.

When the immune barrier is formed, that is, the encapsulation of the pancreatic islet with sodium alginate, small molecules such as insulin, oxygen, and glucose are easily passed, but polymers such as immunoglobulins are not passed, which greatly reduces the possibility of attack from immunization. Although good, sodium alginate capsules also cause the deposition of immune cells, such as macrophage and monocytes, due to the endogenous toxin of the material itself. In addition, poly-L-lysine is coated on the outer surface of the sodium alginate capsule, and macrophages, helper T cells (CD4 + T cells), and monocytes are deposited on the capsule outer membrane when they are present in vivo for a long period of more than 6 months. The secretion of caffeine and complement, or thick deposits, blocks the smooth transfer of oxygen and nutrients between the inside and outside of the capsule, causing severe damage to the pancreatic islets inside the capsule. In addition, cytokines, etc., can move freely inside the capsule as small molecules, which may induce death of islet cells in the capsule (Kobayashi, T., et al., Immune mechanisms associated with the rejection of encapsulated neonatal porcine islet xenografts Xenotransplantation, 2006. 13 (6): p. 547-59; de Groot, M., et al., Macrophage overgrowth affects neighboring nonovergrown encapsulated islets.J Surg Res, 2003. 115 (2): p. 235-41. Lacy, PE, et al., Maintenance of normoglycemia in diabetic mice by subcutaneous xenografts of encapsulated islets.Science, 1991. 254 (5039): p. 1782-4.).

In order to solve the above problems, a variety of methods have been proposed, such as forming a capsule using a variety of materials or coating another film on the existing outer film of sodium alginate (Chandy, T., DL Mooradian, and GH Rao). , Evaluation of modified alginate-chitosan-polyethylene glycol microcapsules for cell encapsulation.Artif Organs, 1999. 23 (10): p. 894-903; Calafiore, R., et al., Standard technical procedures for microencapsulation of human islets for graft into nonimmunosuppressed patients with type 1 diabetes mellitus.Transplant Proc, 2006. 38 (4): p. 1156-7; Safley, SA, et al., Inhibition of cellular immune responses to encapsulated porcine islet xenografts by simultaneous blockade of two different costimulatory pathways.Transplantation, 2005. 79 (4): p. 409-18.). However, it has not yet solved the deposition of inflammatory immune cells caused by sodium alginate.

On the other hand, chitosan is a polysaccharide extracted from crustaceans, which contains nitrogen, and exists for the first time as chitin insoluble in water. When reacted with acid to make chitin soluble, it becomes soluble chitosan. Chitosan contains about 20% chitin and is extremely difficult to purify and isolate. In addition, chitosan is dissolved at a pH of 4.5 or less, has a number of positive charges, and has a characteristic of strongly binding to a material having a negative charge, and has an action of alleviating inflammatory effects. Therefore, it is thought that coating the sodium alginate capsule outer membrane with chitosan having an anti-inflammatory action and a strong binding property with a negatively charged substance can suppress the deposition of inflammatory immune cells caused by sodium alginate.

However, there have been no studies on the method for inhibiting the deposition of inflammatory immune cells by coating chitosan on the outer membrane of sodium alginate capsule.

Therefore, after encapsulating the heterologous islets with sodium alginate, chitosan is coated on the capsule outer membrane to prepare a capsule, and xenografting using the capsule is expected to be effective in treating diabetes. Therefore, there is a need for development and research on islet microcapsules of alginate-chitosan bilayers.

The present inventors have been studying to inhibit the deposition of inflammatory immune cells such as macrophages and monocytes on the alginate islet microcapsule outer membrane, and then, after encapsulating the heterologous islets with sodium alginate, chitosan having anti-inflammatory action on the capsule outer membrane The coating was prepared for the islet isotonic microcapsules of alginate-chitosan bilayer, and it was confirmed that the deposition of inflammatory immune cells on the outer membrane of the prepared islet microcapsules was remarkably reduced, thereby completing the present invention.

The present invention is to provide a pancreatic islet microcapsules of alginate-chitosan bilayer for the treatment of diabetes mellitus and a method for preparing the same.

The present invention provides islet microcapsules of alginate-chitosan bilayer for treating diabetes.

In addition, the present invention

1) separating the islets from the islet source,

2) diluting the isolated islets in sodium alginate solution and dropping them in BaCl ₂ solution to form capsules, and

3) It provides a method for producing a pancreatic islet microcapsules of the alginate-chitosan double membrane for treating diabetes, comprising coating the chitosan at pH 4.5 on the formed capsule outer membrane.

Hereinafter, the present invention will be described in detail.

The islets of the alginate-chitosan diaphragm for diabetic treatment according to the present invention are characterized in that the islets of the heterogeneous islets are encapsulated with sodium alginate and the outer membrane is coated with chitosan having anti-inflammatory action to form an immune barrier.

The heterologous islets are preferably islets isolated from adult pigs.

The method for preparing the islet microcapsules of the alginate-chitosan bilayer according to the present invention will be described in detail step by step.

Step 1) is a step of separating the pancreatic islets from the source of pancreatic islets, the pancreatic source is preferably an adult pig. This is because the insulin metabolism of pigs is similar to that of humans, and is widely used for food, so it has little objection, is relatively easy to handle, and has a large amount of islets. The islets isolated from adult pigs are incubated for one day in RPMI 1640 medium containing 10% FBS.

Step 2) is to encapsulate the isolated pancreatic islets with sodium alginate. The pancreatic islets isolated in step 1) are diluted to 1000 to 100,000 per 1 ml of sodium alginate solution and placed in a 5cc syringe. Attach a 5cc syringe to the syringe pump and connect it to the microdroplet maker. The carbon dioxide injection tube is connected to the prepared air inlet on the side of the micro drop maker, and the carbon dioxide is injected at a pressure of 5 to 10 kgf / cm 2 while pushing the syringe pump at a speed of 15 to 25 ml / hr. Sodium alginate and diluted islet cells are dropped into 10 mM BaCl ₂ solution to form a spherical capsule. The formed capsules are washed with HBSS solution and then incubated in D-MEM culture medium containing 10% FBS for one day at 37 ° C. and 5% CO ₂ .

In step 3), the chitosan is coated on the capsule outer membrane. The sodium alginate capsule including the pancreatic islet is placed in a 1% chitosan solution and coated at pH 4.5 to prepare a chitosan-coated sodium alginate capsule. The pancreatic islets of the alginate-chitosan bilayer prepared above were washed with HBSS solution, and then cultured in RPMI 1640 culture medium containing 10% FBS for 1 week.

In the case of the pancreatic islets of the alginate-chitosan bilayer prepared by the above method, the islet viability is not abnormal in the capsule and secretes insulin normally by external stimulation. In addition, after implanting the pancreatic islet microcapsules of the sodium alginate-chitosan bilayer prepared above into the abdominal cavity of a diabetic mouse, the blood glucose change was measured to maintain normal blood glucose, and the islets of the alginate-chitosan bilayer of the transplanted islet microcapsules As a result of collecting and observing, the deposition of inflammatory immune cells on the capsule envelope is markedly reduced.

Therefore, the islet-capsular microcapsules of the alginate-chitosan bilayer according to the present invention effectively inhibit the deposition of inflammatory immune cells on the microcapsule outer membrane to protect the heterologous islet islets in immune rejection reactions resulting from xenografts, thereby making them very useful in treating diabetes. Can be used.

The concentration of pancreatic islets in the islets of the alginate-chitosan bilayer according to the present invention is preferably 1000 to 100,000 IEq / ml.

The pancreatic islet capsule of the alginate-chitosan bilayer according to the present invention can be implanted into a patient in need of diabetes treatment. Spinal fluid or lymph fluid is preferred.

In addition, the pancreatic islet microcapsules of the alginate-chitosan bilayer according to the present invention may be administered to patients in need of diabetes treatment in combination with immunosuppressants or anti-inflammatory agents. The immunosuppressive agent may be selected from the group consisting of cyclosporine, sirolimus, rapamycin and ortacrolimus, but is not limited thereto. The anti-inflammatory agent may be selected from the group consisting of aspirin, ibuprofen, steroids, and non-steroidal anti-inflammatory agents, but is not limited thereto. The immunosuppressive agent or anti-inflammatory agent is preferably administered for 6 months, preferably 1 month after transplantation of the islet microcapsules of the alginate-chitosan bilayer.

In order to implant the islets of the alginate-chitosan bilayer according to the present invention into recipients, the amount of islets to be transplanted is preferably 5,000 to 20,000 IEq / kg (weight of the recipient), and the donor's gender and pancreas. The range varies depending on the condition, weight of the recipient, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the disease.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example 1  : Rat islet microcapsules of sodium alginate-chitosan bilayer

1. Isolation of Pancreatic Islet Cells from Rats

An average of 200 g SD-rat males were anesthetized using a lump of ketamine mixture. The abdominal cavity of the rat was opened to bind the pancreatic ducts connected to the duodenum, and the incision was made between the pancreatic ducts connected to the liver and the polyethylene-50 (PE-50) tube was connected to the incision. 8-9 ml of a solution in which 1 mg of collagenase P was dissolved per 1 ml of prepared Media 199 was put into the pancreas through PE-50. The pancreas was separated and digested by shaking at 37 rpm for 60 minutes at 60 rpm. After washing three times with Media 199 containing 10% FBS (fetal bovine serum), it was filtered through a sieve having a pore size of 500 ㎛ to remove pancreas tissues of a predetermined size or more. In order to increase the purity of the pancreatic islets, the pancreatic islets were purely separated using a separation medium (Histopaque 1077). Isolated pancreatic islets were washed with Media 199 containing 10% FBS, and the number of pancreatic islets was measured. It was then incubated for one day in RPMI 1640 medium containing 10% FBS.

2. Encapsulating Pancreatic Islets with Sodium Alginate

Sodium alginate was dissolved in KRH (krebs-ringer-hepes) buffer at a concentration of 2% and filtered through a Hepa filter. The pancreatic islet cells isolated in 1 were diluted to 1000 per 1 ml of sodium alginate solution and placed in a 5cc syringe. A 5 cc syringe was mounted on the syringe pump and then connected to the microdroplet maker. The carbon dioxide injection tube was connected to the prepared air inlet on the side of the micro drop maker. Carbon dioxide was injected at 6.0 kgf / cm 2 pressure while pushing the syringe pump at 20 ml / hr. Sodium alginate and diluted pancreatic islet cells were added to a 10 mM BaCl ₂ solution placed on a magnetic stirrer and stirred for 10 minutes to solidify rapidly and form spherical capsules. The formed capsules were washed 2-3 times with Hanks' balanced salt solution (HBSS) solution, and then cultured in D-MEM culture medium containing 10% FBS for ₁ day under 37 ° C. and 5% CO ₂ .

3. Chitosan Coating on Sodium Alginate Capsule Outer Membrane

The sodium alginate capsule including the islets prepared in 2 was put in a 1% chitosan solution at pH 4.5 and incubated with stirring for 10 minutes. After 10 minutes, chitosan-coated sodium alginate capsules were washed with HBSS solution and incubated for one week in RPMI 1640 medium containing 10% FBS.

Example 2  : Adult Porcine Islet Microcapsules of Sodium Alginate-chitosan Double Layer

1. Isolation of Islet Cells from Adult Pigs

Adult pig organs extracted from the slaughterhouses were washed with saline. The mesentery surrounding the pancreas was cut with scissors to make the pancreas come out. After finishing the peripancreatic care, the pancreas was excised to about 80g. A catheter was placed in the catheter of the resected pancreas, followed by 20-40 ml long-term preservation solution (HTK solution; histidine-tryptophan-ketoglutarate solution) and stored at 4 ° C. The pancreas transferred to the laboratory was put into betadine solution and antibiotic solution, respectively, and washed with D-PBS (dulbecco's phosphate buffer solution). 100 g of 2 g / ml collagenase P was injected through a catheter connected to the pancreatic catheter. After injection, the pancreas was divided into 4 to 8 portions, placed in a ricordi chamber, and slowly shaken while maintaining the 35 ° C. After digestion for 5 to 15 minutes, M199 containing 4% BSA (Bovine serum albumin) is injected into the chamber, and the digested pancreatic cells are collected in 250 ml conical tube while maintaining the inside of the chamber at 4 to 10 ° C. Washed three times with M199 containing BSA. The washed pancreatic cells were placed in 200 ml of Ficoll solution having a specific gravity of 1.108 and then placed in a cobe bag. While rotating the cobe bag at 1200 rpm, specific gravity 1.100 and 1.060 were slowly mixed into the gradient maker beaker. Finally, 100 ml of HBSS solution was added and then rotated at 2200 rpm for 5 minutes. After 5 minutes, each solution was received in a 50 ml conical tube by 35 ml in a cobe bag, and cells were extracted from each tube and stained with DTZ (dithizone). After confirming the distribution of pancreatic islets for each tube cell sample, only 30% or more of the tubes were collected and washed, and cultured in RPMI 1640 medium containing 10% FBS for 1 day.

2. Encapsulating Pancreatic Islets with Sodium Alginate

Sodium alginate capsules were prepared in the same manner as in Example 1, 2 except that the pancreatic islet cells isolated in 1 were diluted to a concentration of 10000 IEq per 1 ml of sodium alginate solution.

3. Chitosan Coating on Sodium Alginate Capsule Outer Membrane

The pancreatic islet microcapsules of sodium alginate-chitosan double membranes were prepared in the same manner as in Example 3 using the sodium alginate capsule including the islets prepared in 2 above.

The production process of the islet isotonic microcapsules of the sodium alginate-chitosan bilayers prepared in Examples 1 and 2 is shown in Figure 1, the physical shape and characteristics of these microcapsules are shown in Figure 2 by scanning electron microscopy, The pore size of these microcapsules is shown in FIG. 3 under a confocal microscope.

As shown in Figure 2, it was confirmed that the pancreatic islet microcapsules of the sodium alginate-chitosan bilayer according to the present invention was coated with chitosan.

In addition, as shown in Fig. 3, the islet isotonic microcapsules of the sodium alginate-chitosan bilayer according to the present invention was confirmed that there is no movement of the FITC-bound dextran with a molecular weight of 70kDa, 150kDa, 250kDa into the capsule. This suggests that immunoglobulin G, a molecule central to immunization, is not transported into the capsule at 150 kDa.

Comparative Example 1  : Sodium alginate microcapsules including rat islets

In Example 1, the chitosan coating process was not performed, but the same procedure was performed only to separate the islets from the rats and to encapsulate the separated islets with sodium alginate to prepare sodium alginate islets.

Comparative Example 2  : Sodium alginate microcapsules including adult pig islets

In Example 2, the chitosan coating process was not performed, but the same procedure was performed until the islets were separated from adult pigs and the isolated islets were encapsulated with sodium alginate to prepare sodium alginate pancreatic microcapsules.

Experimental Example 1  : Measurement of pancreatic islet viability in islet microcapsules of sodium alginate-chitosan bilayer according to the present invention (in vitro)

In order to confirm the islet survival rate in the islet microcapsules of the sodium alginate-chitosan bilayer according to the present invention, the following experiment was performed.

Some of the islet microcapsules prepared in Example 1 and Comparative Example 1 were collected for 14 days from the day of preparation, and treated with AO / PI (acridine orange / propidium iodide) to confirm the islet survival rate in the microcapsules. Unencapsulated rat islets were used as a control.

The results are shown in FIG.

As shown in FIG. 4, it was confirmed that the islet viability in the islet microcapsules of the sodium alginate islet microcapsules and the sodium alginate-chitosan bilayer was significantly reduced by 7 days compared to the rat uncapsulated islets. However, on day 14, there was almost no difference from the unencapsulated rat pancreas.

Experimental Example 2  : Measurement of insulin secretion in pancreatic islet microcapsules of sodium alginate-chitosan bilayer according to the present invention (in vitro)

In order to confirm the insulin secretion ability in the pancreatic islet microcapsules of the sodium alginate-chitosan bilayer according to the present invention, the following experiment was performed.

1. Measurement of insulin secretion in rat islet microcapsules

The pancreatic islet microcapsules prepared in Example 1 and Comparative Example 1 were treated with 2.8 mM and 16.8 mM of glucose in 20 IEqs, and then insulin secretion ability was measured. Unencapsulated rat islets were used as a control.

The results are shown in FIG.

As shown in FIG. 5, unencapsulated rat pancreatic islets, sodium alginate islet microcapsules and pancreatic microcapsules of sodium alginate-chitosan bilayer were all normally secreted by external stimuli.

2. Determination of insulin secretion in adult pig microcapsules

Insulin secretion ability of the islet microcapsules prepared in Example 2 and Comparative Example 2 was measured by the same method as described above.

The results are shown in FIG.

As shown in FIG. 6, it was confirmed that both of sodium alginate islet microcapsules and pancreatic islet of sodium alginate-chitosan bilayer normally secrete insulin by external stimulation.

Experimental Example 3  : Effect of Islet Alginate-chitosan Double Membrane Microcapsules on Blood Glucose Changes in the Present Invention

In order to determine the effect of pancreatic islet microcapsules of sodium alginate-chitosan bilayer according to the present invention on the blood glucose change, the following experiment was performed.

Balb / c male mice were intraperitoneally injected with 180 mg / kg of streptozotocin to induce diabetes. Diabetic Balb / c mice were anesthetized, and the abdominal cavity was cut into 1 cm in size. Pancreatic islet microcapsules of sodium alginate-chitosan bilayers prepared in Examples 1 and 2 and Comparative Examples 1 and 2 were respectively injected at 500 IEq, and the incision site was closed. After transplantation, peripheral blood was collected from the tail of the transplanted mouse between 9 and 10 am and blood glucose was measured using a blood glucose meter. As a control, unencapsulated rat islets or adult porcine islets were transplanted into the kidney and blood glucose levels were measured.

Glucose change after transplantation of rat pancreatic islets of sodium alginate-chitosan bilayer into the abdominal cavity of diabetic-induced Balb / c mice is shown in FIG. 7. Blood glucose changes after transplantation of the abdominal cavity of Balb / c mice are shown in FIG. 8.

As shown in FIG. 7, when the non-encapsulated rat pancreatic islets were transplanted, blood glucose rapidly increased from the 3rd day after transplantation, but when the alginate islet microcapsules and the pancreatic microcapsules of sodium alginate-chitosan bilayer were transplanted, the normal blood glucose was long. It was observed to keep.

In addition, as shown in FIG. 8, when the unencapsulated adult porcine islets were transplanted, blood glucose rapidly increased from 2 days after transplantation, but when the alginate islet microcapsules and the pancreatic islet of alginate-chitosan bilayer were transplanted for a long time, Maintaining normal blood sugar was observed.

Experimental Example 4  : Pancreatic islet analysis by collecting islet microcapsules from transplanted sodium alginate-chitosan bilayer: DTZ staining

In order to confirm the presence of beta cells in the capsule and the degree of inflammatory immune cell deposition in the collected capsules by collecting the islets of the alginate-chitosan bilayer transplanted into the abdominal cavity of diabetes-induced mice, the following experiments were performed. It was.

After implanting the islets of the alginate-chitosan bilayers prepared in Examples 1 and 2 and Comparative Examples 1 and 2 into the abdominal cavity of Balb / c male mice, 28 days, 56 days, 189 days, or blood glucose levels may rise again. Intraperitoneal section of the mouse was inoculated, and the physiological saline at 37 ° C. was added again, and the microcapsules were collected by repeating the method of subtraction again. The collected islet microcapsules were stained with DTZ solution, which is specifically stained only on beta cells, followed by observation, thereby primarily confirming the presence of insulin secreting beta cells. In addition, the degree of deposition of inflammatory immune cells in the collected islet microcapsules was confirmed by the number of capsules, respectively.

The rat pancreatic islet microcapsules of the collected sodium alginate-chitosan bilayers were stained with DTZ solution to observe the staining of inflammatory immune cells deposited on the microcapsule outer membrane and the islets of the microcapsules. The yield of clean microcapsules not deposited was shown in FIG. 10. In addition, the adult pig pancreatic islet microcapsules of the collected sodium alginate-chitosan bilayer were stained with DTZ solution, and the results of observing staining of inflammatory immune cells deposited on the microcapsule outer membrane and the islets of the microcapsules were shown in FIG. 11. , The quantity of clear microcapsules in which inflammatory immune cells were not deposited is shown in FIG. 12.

As shown in FIG. 9, rat pancreatic islet microcapsules of the collected sodium alginate-chitosan bilayers were observed with and without deposition of inflammatory cells (9-A). In addition, some of the pancreatic islets inside the capsule where no inflammatory cells were deposited were observed to die after prolonged transplantation (9-B), and only a part of the pancreatic islets inside the capsule where the inflammatory cells were deposited were stained with DTZ (9-B). -C). In addition, as shown in Figure 10, the inflammatory cell deposition rate of the rat pancreatic islet microcapsules of the collected sodium alginate-chitosan double membrane was confirmed that there is a significant difference (P <0.05) compared to the sodium alginate rat islet microcapsules, but all Inflammatory cells were deposited only in capsules up to 20%.

As shown in FIG. 11, it was observed that adult pig pancreatic islet microcapsules of the collected sodium alginate-chitosan bilayers had less inflammatory cells and most of the pancreatic islets stained with DTZ (11-A and 11-C). . On the other hand, the collected sodium alginate adult porcine islet microcapsules were mostly deposited with inflammatory cells, and the islets inside the microcapsules were mostly stained with DTZ (11-B and 11-D). In addition, as shown in Figure 12, the inflammatory cell deposition rate of adult porcine islet islets of the collected sodium alginate-chitosan double membrane was confirmed to be significantly different (P <0.01) compared to the sodium alginate adult porcine islet microcapsules. Adult algal pancreatic microcapsules of sodium alginate-chitosan bilayer were not deposited more than 60%, whereas sodium alginate adult porcine islet microcapsules were not deposited less than 5%.

Experimental Example 5  : Determination of Inflammatory Cell Deposition in Pancreatic Islet Microcapsules of Collected Sodium Alginate-Citosan Bilayer

To confirm the degree of deposition of inflammatory cells in adult porcine islet microcapsules of the collected sodium alginate-chitosan bilayer, the following experiment was performed.

1. Insulin Fluorescent Immunostaining

Adult pig pancreatic islet microcapsules of the collected sodium alginate-chitosan bilayers were fixed with 4% paraformaldehyde (PFA) solution, paraffin embedded, and cut into 5-10 μm sizes. After removing paraffin from the sections, water was added again, and then a solution of 1: 100 diluted guinea pig host insulin antibody was sprinkled onto the tissue sections and incubated at room temperature for 1 hour. After sufficient washing with 1 × PBS, anti-guinea pig antibodies with rhodamine were stained with secondary antibodies.

2. Microwave Fluorescent Immunostaining

After removing paraffin from the sections, water was added again, and a solution of the rat-digested microphage antibody at a ratio of 1: 100 was sprayed onto the tissue sections, and then incubated at 4 ° C. for 10 hours. After sufficient washing with 1 × PBS, the cells were stained using a secondary antibody with Alexa 526 (yellow) fluorescence.

3. Masson Trichrome Dyeing

After removing paraffin from the sections, water was added again, and then Weigert's iron hematoxylin was attached and incubated at room temperature for 10 minutes. After washing sufficiently with water, the sections were put in Biebrich scarlet acid fuchsin solution and incubated at room temperature for 20-30 minutes. After washing with water was incubated for 3 minutes at room temperature in 5% aqueous phosphotungstic acid, then incubated for 3 minutes at 1% glacial acetic acid. It was then stained by incubating for 10 minutes at room temperature in a light green solution.

The results are shown in FIG.

As shown in FIG. 13, both the collected adult sodium pancreatic islet microcapsules (13-A) and adult porcine pancreatic islet microcapsules (13-E) of the sodium alginate-chitosan bilayer (13-E) were obtained by insulin fluorescence immunostaining. It was confirmed that the cells were stained (red). However, it was confirmed that inflammatory cells were deposited on the outer membrane of the adult sodium alginate porcine islet microcapsules (blue) (13-A), but no inflammatory cells were deposited on the adult porcine islet microcapsules of the sodium alginate-chitosan bilayer (13-E). ).

Microphage fluorescence immunostaining results also confirmed that the inflammatory cells deposited on the sodium alginate adult porcine islet microcapsule outer membrane were stained with the microphage antibody (yellow) (13-B).

In addition, Masson Trichrome staining results confirmed that the inflammatory cells deposited on sodium alginate adult porcine islet microcapsule outer membrane were stained blue (collagen) (13-C and 13-D).

Therefore, the islet isotonic microcapsules of the sodium alginate-chitosan bilayer according to the present invention can effectively inhibit the deposition of inflammatory immune cells to protect the heterologous islet cells in the immune rejection reaction in xenografts.

Islet microcapsules according to the present invention encapsulates heterogeneous islets with sodium alginate and then coats the outer membrane with chitosan to form an immune barrier, thereby suppressing the deposition of inflammatory immune cells caused by sodium alginate, resulting in immune rejection reactions in xenografts. Can protect heterogeneous islet cells. Therefore, the islet microcapsules of the alginate-chitosan bilayer according to the present invention can be very useful for treating diabetes.

1 is a view showing the manufacturing process of the islet microcapsules of sodium alginate-chitosan bilayer according to the present invention.

2 is a diagram illustrating islet microcapsules of the sodium alginate-chitosan bilayer according to the present invention with a scanning electron microscope.

Figure 3 is a view of the islet microcapsules microcapsules of sodium alginate-chitosan bilayer according to the present invention by confocal microscopy.

Figure 4 is a diagram showing the survival rate of pancreatic islets in the islet microcapsules of sodium alginate-chitosan bilayer according to the present invention.

Figure 5 is a diagram showing the insulin secretion ability in the rat pancreatic islet microcapsules of the sodium alginate-chitosan bilayer according to the present invention.

Figure 6 is a diagram showing the insulin secretion ability in adult porcine pancreatic islet microcapsules of sodium alginate-chitosan bilayer according to the present invention.

Figure 7 is a diagram showing the change in blood glucose after transplanting the rat pancreatic islet microcapsules of sodium alginate-chitosan bilayer according to the present invention in the abdominal cavity of diabetes-induced Balb / c mice.

Figure 8 is a diagram showing the change in blood glucose after transplanting adult porcine islet islet microcapsules of sodium alginate-chitosan bilayer according to the present invention in the abdominal cavity of diabetes-induced Balb / c mice.

FIG. 9 is a diagram illustrating that the inflammatory immune cells deposited on the microcapsule outer membrane and the islets of the microcapsules were stained by staining the rat pancreatic islets of the collected sodium alginate-chitosan bilayer with DTZ solution.

FIG. 10 is a diagram showing the yield of clean microcapsules in which inflammatory immune cells are not deposited by staining rat pancreatic islets of the collected sodium alginate-chitosan bilayer with DTZ solution.

FIG. 11 is a diagram showing that adult porcine islet pancreatic microcapsules of the collected sodium alginate-chitosan bilayers were stained with DTZ solution to stain inflammatory immune cells deposited on the microcapsule outer membrane and the islets inside the microcapsules.

FIG. 12 is a diagram showing the yield of clean microcapsules in which inflammatory immune cells are not deposited by staining adult porcine islets of the collected alginate-chitosan bilayer with DTZ solution.

FIG. 13 is a diagram illustrating fluorescence immunostaining of inflammatory cell deposition in adult porcine islet microcapsules of collected sodium alginate-chitosan bilayers.

Claims (6)

Pancreatic islet microcapsules of alginate-chitosan bilayer for diabetes treatment. The method of claim 1, wherein the islet microcapsules are encapsulated with heterogenous islets and sodium alginate, and then the outer membrane is coated with chitosan having anti-inflammatory action to form an immune barrier. The islet microcapsules according to claim 2, wherein the heterologous islets are isolated islets from adult pigs. 1) separating the islets from the islet source, 2) diluting the isolated islets in sodium alginate solution and dropping them in BaCl ₂ solution to form capsules, and 3) A method for preparing the islet microcapsules of the alginate-chitosan bilayer for treating diabetes of claim 1, comprising coating the formed capsule outer membrane at pH 4.5. 5. The method of claim 4, wherein the pancreatic islet source in step 1) is an adult pig. [5] The method of claim 4, wherein the pancreatic islets in step 2) are diluted 1000 to 100,000 per 1 ml of sodium alginate solution.

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