Anti-fibrosis artificial pancreas islet with spherical core-shell structure as well as preparation method and application of anti-fibrosis artificial pancreas islet

Abstract

The invention discloses an anti-fibrosis artificial islet with a spherical core-shell structure as well as a preparation method and application of the anti-fibrosis artificial islet. The preparation method comprises the following steps: step 1, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into a sodium alginate solution; adding 4-iodobenzylamine after fully dissolving, fully stirring, dialyzing, and freeze-drying to obtain 4-iodobenzylamine modified alginate; 2, resuspending the islet cells in matrigel to obtain an internal phase solution; step 3, adopting the 4-iodobenzylamine modified alginate obtained in the step 1 as an external phase solution and the internal phase solution obtained in the step 2 to obtain core-shell liquid drops of which the internal phase is wrapped by the external phase; 4, dropwise adding the core-shell liquid drops obtained in the step 3 into a calcium ion solution to obtain an artificial pancreas islet with a cross-linked structure; the obtained artificial pancreas islet is high in anti-fibrosis degree, and the survival condition of pancreas islet cells is better.

Description

Technical Field

[0001] The invention relates to the technical field of polymer materials, and in particular to an anti-fibrosis spherical core-shell structure artificial pancreatic islet, a preparation method and application thereof. Background Art

[0002] Cell therapy has already had an important impact in the healthcare field, providing new treatments for some intractable diseases. Such as mesenchymal stem cells for tissue regeneration, islet transplantation for diabetes treatment, and T cell delivery for cancer immunotherapy. As a carrier that provides 3D tissue and supports cell viability and function, biomaterials have the potential to expand the therapeutic effects of cell therapy. One of the biggest challenges of cell therapy at present is that after the explant is implanted into the body, a dense collagen fiber capsule will be generated around it, blocking the delivery of external nutrients to the internal cells, leading to the death of the encapsulated cells. Therefore, how to reduce the degree of fibrosis caused by the implantation of explants into the body is the most urgent problem to be solved. Summary of the invention

[0003] In view of the problems existing in the prior art, the present invention provides an anti-fibrosis spherical core-shell structure artificial pancreatic islet, a preparation method and application thereof.

[0004] The technical solution adopted by the present invention is: a method for preparing an anti-fibrotic spherical core-shell structure artificial pancreatic islet, comprising the following steps:

[0005] Step 1: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the sodium alginate solution; add 4-iodobenzylamine after fully dissolving, stir well, dialyze, and freeze-dry to obtain 4-iodobenzylamine-modified alginate;

[0006] Step 2: resuspend the islet cells in the matrix gel to obtain the inner phase solution;

[0007] Step 3: using the 4-iodobenzylamine-modified alginate obtained in step 1 as the external phase solution and the internal phase solution obtained in step 2 to obtain core-shell droplets in which the external phase envelops the internal phase;

[0008] Step 4: The core-shell liquid droplets obtained in step 3 are added with a calcium ion solution to obtain an artificial pancreatic islet with a cross-linked structure.

[0009] Furthermore, in the step 1, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and 4-iodobenzylamine is 1:1:1.

[0010] Furthermore, in step 1, the molar ratio of sodium alginate to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:100.

[0011] Furthermore, in step 2, the matrix gel contains L-alanine, L-aspartic acid, L-arginine, L-cysteine, L-glutamic acid, glycine, histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-ascorbic acid, 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxynucleoside, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleoside sodium phosphate, calcium chloride, magnesium sulfate, potassium chloride, sodium acetate, sodium chloride, disodium hydrogen phosphate, D-glucose, phenol red-sodium, glutathione, D-glucuronic acid, sodium bicarbonate, glucose, and fetal bovine serum.

[0012] Furthermore, in step 3, the pushing speed of the inner phase is 1 ml / min, and the pushing speed of the outer phase is 0.5 ml / min to 2.5 ml / min; the voltage is 5 kV.

[0013] Furthermore, the concentration of calcium ions in the calcium ion solution in step 4 is 20 mM.

[0014] An artificial pancreatic islet with an anti-fibrosis spherical core-shell structure, wherein the shell thickness of the artificial pancreatic islet is 0.1-0.5 mm.

[0015] The invention discloses an application of an artificial pancreatic islet with an anti-fibrosis spherical core-shell structure, and the artificial pancreatic islet is used in the preparation of a diabetes drug.

[0016] Furthermore, the diabetes drug is a type I diabetes drug.

[0017] The beneficial effects of the present invention are:

[0018] (1) The present invention obtains artificial pancreatic islets with a core-shell structure, and uses alginate modified with 4-iodobenzylamine as the shell layer, which has a significant anti-fibrosis effect and can isolate the recognition of immune cells and protect the encapsulated cells, so that the cells can survive for a long time inside the shell layer without being recognized and attacked by external immune cells;

[0019] (2) The core-shell structure artificial pancreatic islets obtained by the present invention use a new type of matrix gel as the nuclear layer, which can make the encapsulated pancreatic islet cells survive longer than the pancreatic islet cells encapsulated by ordinary alginate, and have a better function of releasing insulin. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The diagram is a diagram of the interaction between alginate and FnⅢ9-10 fibronectin in the artificial pancreatic islets obtained by the present invention.

[0021] Figure 2 The figure is the Gibbs free energy diagram of alginate and FnⅢ9-10 fibronectin in the artificial pancreatic islet obtained by the present invention.

[0022] Figure 3 This is a schematic diagram of the process of preparing artificial pancreatic islets and implanting them into mice for treatment.

[0023] Figure 4 It is a schematic diagram of the protein adsorption effect of the artificial pancreatic islets obtained in Example 1 of the present invention and the artificial pancreatic islets obtained in Comparative Examples 1 and 2.

[0024] Figure 5 Schematic diagram of the results of cell adhesion experiments on the artificial islets obtained in Example 1 of the present invention and the artificial islets obtained in Comparative Examples 1 and 2.

[0025] Figure 6 Schematic diagram of the shape and structure of the artificial pancreatic islet obtained in Example 1 of the present invention and the artificial pancreatic islet obtained in Comparative Examples 3, 4, and 5.

[0026] Figure 7 It is a schematic diagram of the fibrosis effect of the artificial islets obtained in Example 1 of the present invention and the artificial islets obtained in Comparative Examples 3, 4, and 5 in mice.

[0027] Figure 8 This is a schematic diagram of the fibrosis effects of the artificial pancreatic islets obtained in Example 1 of the present invention and the artificial pancreatic islets obtained in Comparative Examples 1 and 2 in mice.

[0028] Fig. 9 Schematic diagram of the fluorescent staining effect of the artificial pancreatic islets obtained in Examples 1 to 5 of the present invention.

[0029] Fig.10 This is a schematic diagram of the experimental results of the artificial pancreatic islets obtained in Example 1 of the present invention and the artificial pancreatic islets obtained in Comparative Examples 1 and 2.

[0030] Fig.11 The statistical results of the survival rates of the artificial islets obtained in Example 1 of the present invention and the artificial islets obtained in Comparative Examples 1 and 2 are shown.

[0031] Fig.12 This is a schematic diagram of the change in blood glucose concentration after the artificial pancreatic islets obtained in Example 1 of the present invention were implanted into mice. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0033] A method for preparing an anti-fibrotic spherical core-shell structure artificial pancreatic islet comprises the following steps:

[0034] Step 1: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the sodium alginate solution; add 4-iodobenzylamine after fully dissolving, stir fully, dialyze, and freeze-dry to obtain 4-iodobenzylamine-modified alginate; the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and 4-iodobenzylamine is 1:1:1. The molar ratio of sodium alginate and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:100.

[0035] Step 2: resuspend the islet cells in the matrix gel to obtain the inner phase solution;

[0036] Matrigel contains L-alanine, L-aspartic acid, L-arginine, L-cysteine, L-glutamic acid, glycine, histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-ascorbic acid, 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxyribonucleoside, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleoside sodium phosphate, calcium chloride, magnesium sulfate, potassium chloride, sodium acetate, sodium chloride, disodium hydrogen phosphate, D-glucose, phenol red-sodium, glutathione, D-glucuronic acid, sodium bicarbonate, glucose, and fetal bovine serum.

[0037] Step 3: Use the 4-iodobenzylamine-modified alginate obtained in step 1 as the external phase solution and the internal phase solution obtained in step 2 to obtain core-shell droplets in which the external phase envelops the internal phase; the pushing speed of the internal phase is 1 ml / min, and the pushing speed of the external phase is 0.5 ml / min to 2.5 ml / min; the voltage is 5 kV.

[0038] Step 4: Calcium ion solution is added to the core-shell liquid droplets obtained in step 3 to obtain artificial pancreatic islets with a cross-linked structure.

[0039] The concentration of calcium ions in the ion solution is 20 mM. The thickness of the shell layer of the artificial islet is 0.1 to 0.5 mm.

[0040] Example 1

[0041] A method for preparing an anti-fibrotic spherical core-shell structure artificial pancreatic islet comprises the following steps:

[0042] Step 1: Weigh 100 mg of sodium alginate and dissolve it in 10 ml of RO water, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC (10 eq.) and N-hydroxysuccinimide NHS (10 eq.) to completely dissolve. After stirring at room temperature for 12 hours, add 4-iodobenzylamine (10 eq.) dissolved in DMSO, stir overnight, transfer the reaction solution to a dialysis bag (molecular weight cutoff 1000 Da), dialyze in pure water for 24 hours, collect the dialysate and freeze-dry to obtain the novel alginate used in the present invention.

[0043] Step 2: resuspend the islet cells in the matrix gel to obtain the inner phase solution;

[0044] The formulation of the matrix gel is as follows: 500 mL of the matrix gel (commonly used matrix gel at present), to which are added 0.01 g of L-alanine, 0.02 g of L-aspartic acid, 0.03 g of L-arginine, 0.01 g of L-cysteine, 0.05 g of L-glutamic acid, 0.04 g of glycine, 0.01 g of histidine, 0.01 g of L-isoleucine, 0.03 g of L-leucine, 0.04 g of L-lysine, 0.01 g of L-methionine, 0.02 g of L-phenylalanine, 0.02 g of L-proline, 0.015 g of L-serine, 0.015 g of L-threonine, 0.01 g of L-tryptophan, and 0.05 g of L-tyrosine. .02 g, L-valine 0.02 g, L-ascorbic acid 0.025 g, 2'-deoxyadenosine 0.01 g, 2'-deoxyguanosine 0.01 g, 2'-deoxyribonucleoside 0.01 g, nicotinamide adenine dinucleotide 0.01 g, nicotinamide adenine dinucleoside sodium phosphate 0.001 g, calcium chloride 0.1 g, magnesium sulfate 0.01 g, potassium chloride 0.2 g, sodium acetate 0.03 g, sodium chloride 3 g, disodium hydrogen phosphate 0.1 g, D-glucose 0.5 g, phenol red-sodium 0.01 g, glutathione 0.01 g, D-glucuronic acid 0.01 g, sodium bicarbonate 1 g, glucose 3 g, 5 ml fetal bovine serum.

[0045] After the matrix gel is prepared, the obtained matrix gel is filtered and sterilized through a 0.2 μm filter. The whole process is carried out in a type II A2 class biosafety cabinet to maintain the sterility of the encapsulated islet cell material. The islet cells are suspended in it at a density of 0.85 IEQ / ul. At this time, the islet density is most conducive to the survival and function of the islet cells. Next, the artificial islets with a core-shell structure are made.

[0046] Step 3: If Figure 3As shown, two microfluidic pumps are prepared, one for pushing the 4-iodobenzylamine-modified alginate obtained in step 1 of the shell layer of the outer phase, and the other for pushing the new matrix gel for encapsulating islet cells in the inner phase, and two soft rubber tubes with replaceable needles are prepared, and the needle of the soft rubber tube connected to the inner phase is inserted into the soft rubber tube connected to the outer phase, perpendicular to the tube wall of the soft rubber tube of the outer phase, so that the matrix gel for encapsulating islet cells in the inner phase can be cut off by the 4-iodobenzylamine-modified alginate in the outer phase with sufficient shear force, forming core-shell droplets of the outer phase encapsulating the inner phase, and an electrostatic generator is connected to the needle of the outer phase.

[0047] The distance between the receiver and the needle was controlled at 30 cm; the pushing speed of the inner phase was 1 ml / min, and the pushing speed of the outer phase was 1.5 ml / min; the voltage of the electrostatic generator was 5 kV.

[0048] Step 4: A 5% mannitol solution with a calcium ion concentration of 20 mM is placed on the receiving platform below the external phase needle. When the droplets are dropped into the solution, they are instantly cross-linked into artificial pancreatic islets with a spherical core-shell structure. After cross-linking, they are immediately washed 4 times with 50 ml of CMRLM culture medium. The shell thickness of the core-shell structure obtained by testing is 0.3 mm.

[0049] The artificial pancreatic islets obtained by the present invention use alginate molecules modified with 4-iodobenzylamine. The material used has been found through analysis and kinetic simulation to reduce the adhesion of fibronectin and further reduce the adhesion of fibroblasts to the material. The degree of fibrosis caused by implantation in the body is lighter than that of ordinary alginate molecules.

[0050] Since alginate molecules have good biocompatibility, and the calcium ion solution used during cross-linking is non-toxic and harmless to the encapsulated cells, and the cell scaffold made after the material is cross-linked into a gel is also non-toxic and harmless to the surrounding tissues after being implanted in the body, and has no immunogenicity, and will not cause immune rejection in the body. After cross-linking, a large number of pores will be formed between the colloids, which can enable the proteins released by the encapsulated cells to be released through the pores to the outside of the material to play their corresponding functions. At the same time, it can also enable the nutrients in the tissues, such as glucose, vitamins, serum, oxygen, etc., to reach the encapsulated cells through the hydrogel aperture channel, giving the encapsulated cells nutrition and oxygen, and also allowing the substances produced by the encapsulated cells to be released into the tissues around the material for material transfer and exchange. In addition, the pore size of the hydrogel scaffold is smaller than the size of the immune cells, which plays a role in blocking external immune cells, preventing the immune cells from entering and identifying the encapsulated cells inside, thereby preventing the immune cells from attacking them.

[0051] Firstly, by using Gauss View molecular modeling to model different modified alginate molecules and docking them with fibronectin, the binding energy of different types of alginate with fibronectin was investigated. The 4-iodobenzylamine alginate molecules with weak binding energy were analyzed with ordinary alginate molecules by gromacs dynamic simulation. The hydrogen bonds and Gibbs free energy formed between the two alginates and fibronectin were statistically compared. The types of alginate with anti-protein adhesion were screened through computational simulation. Secondly, the protein adsorption effect of 4-iodobenzylamine alginate molecules and ordinary alginate molecules was verified by protein adsorption experiment. Then the adhesion effect at the cell level was investigated. The degree of adhesion of different materials to fibroblasts was statistically analyzed by co-culturing the materials with l929 fibroblasts and performing fluorescent staining.

[0052] After the explant obtained in Example 1 is implanted into the body, the material will adsorb the protein, and the fibronectin FnⅢ9-10 is mainly recognized by fibroblasts. Reducing the adsorption of fibronectin on the material can further reduce the adhesion of fibroblasts to the material. The kinetic simulation results are shown in Figure 1 As shown in the figure, it can be seen that the alginate obtained by the present invention has a higher binding energy with fibronectin FnⅢ9-10, and the number of dynamic hydrogen bonds is also the least, indicating that the interaction effect between the new alginate and fibronectin FnⅢ9-10 is the weakest and the binding ability is the worst.

[0053] Gibbs free energy diagram Figure 2 As shown in the figure, it can be seen that the binding energy value between the alginate obtained by the present invention and fibronectin is high and the binding is unstable. From the results, it can be inferred that the core-shell material used as an artificial pancreatic islet can reduce the adsorption effect of fibronectin FnⅢ9-10, thereby reducing the degree of fibrosis after implantation in the body.

[0054] Example 2

[0055] The other steps of this embodiment are the same as those of embodiment 1, except that in step 3, the pushing speed of the inner phase is 1 ml / min, and the pushing speed of the outer phase is 0.5 ml / min; the voltage of the electrostatic generating device is 5 kV.

[0056] The shell thickness of the core-shell structure obtained in this embodiment was tested to be 0.1 mm.

[0057] Example 3

[0058] The other steps of this embodiment are the same as those of embodiment 1, except that in step 3, the pushing speed of the inner phase is 1 ml / min, the pushing speed of the outer phase is 1 ml / min, and the voltage of the electrostatic generating device is 5 kV.

[0059] The shell thickness of the core-shell structure obtained in this embodiment was tested to be 0.2 mm.

[0060] Example 4

[0061] The other steps of this embodiment are the same as those of embodiment 1, except that in step 3, the pushing speed of the inner phase is 1 ml / min, and the pushing speed of the outer phase is 2.0 ml / min; the voltage of the electrostatic generating device is 5 kV.

[0062] The shell thickness of the core-shell structure obtained in this embodiment was tested to be 0.4 mm.

[0063] Example 5

[0064] The other steps of this embodiment are the same as those of embodiment 1, except that in step 3, the pushing speed of the inner phase is 1 ml / min, and the pushing speed of the outer phase is 2.5 ml / min; the voltage of the electrostatic generating device is 5 kV.

[0065] The shell thickness of the core-shell structure obtained in this embodiment was tested to be 0.5 mm.

[0066] Comparative Example 1

[0067] The other steps of this comparative example are the same as those of Example 1, except that ordinary alginate is used in step 1.

[0068] Comparative Example 2

[0069] The other steps of this comparative example are the same as those of Example 1, except that the zwitterionic material commonly used as an implantation material is used in step 1.

[0070] The artificial pancreatic islets obtained in Example 1, Comparative Example 1 and Comparative Example 2 of the present invention were used to perform protein adsorption experiments. The results are as follows: Figure 4 As shown, the artificial islets obtained by the present invention have a lower protein adsorption effect than the artificial islets obtained from the currently available common alginate and zwitterionic materials, and can reduce fibrosis; reduce the recognition and adhesion of subsequent fibroblasts, and reduce the progress of fibrosis.

[0071] The artificial pancreatic islets obtained in Example 1, Comparative Example 1 and Comparative Example 2 of the present invention were used to perform a cell adhesion experiment. The results are as follows: Figure 5 As shown in the figure, it can be seen that the artificial islets obtained by the present invention have weaker cell adhesion effect than the artificial islets obtained by currently available ordinary alginate and zwitterionic materials, reduce the recognition and adhesion of fibroblasts, and reduce the number of fibroblasts around the material during the fibrosis process.

[0072] Comparative Example 3

[0073] The other steps of this comparative example are the same as those of Example 1, except that the shape of the artificial pancreatic islet prepared by the template method in step 4 is a cubic structure.

[0074] Comparative Example 4

[0075] The other steps of this comparative example are the same as those of Example 1, except that the shape of the artificial pancreatic islet prepared by the template method in step 4 is a cylindrical structure.

[0076] Comparative Example 5

[0077] The other steps of this comparative example are the same as those of Example 1, except that the shape of the artificial pancreatic islet prepared by the template method in step 4 is a hemispherical structure.

[0078] The shape models of the artificial pancreatic islets obtained in Example 1 and Comparative Examples 3, 4 and 5 of the present invention are as follows: Figure 6 As shown. A template was made using 3D modeling software to form a solid template with holes of different shapes, and 4-iodobenzylamine alginate was filled into the holes of different shapes, and then cross-linked and solidified to form and take out the hydrogels in four shapes: cube, cylinder, hemisphere, and sphere. These four materials were modeled using abqus software and mechanically analyzed to calculate the degree of mechanical receptors of materials of different shapes, and the degree of fibrosis caused by implantation into the body was verified to show that the degree of fibrosis caused by the cube is greater than that of the cylinder, greater than that of the hemisphere, and greater than that of the sphere.

[0079] The artificial pancreatic islets obtained in Example 1, Comparative Examples 3, 4, and 5 were implanted into mice. Figure 3 The fibrosis results are shown in Figure 7 The artificial pancreatic islets obtained in Example 1, Comparative Examples 2 and 3 were implanted into mice. The results were as follows: Figure 8 As shown. Figure 7 It can be seen that the artificial islets obtained in the present invention have a lower degree of fibrosis than the artificial islets obtained in Comparative Examples 3, 4, and 5.

[0080] from Figure 8 It can be seen that the artificial pancreatic islets obtained by the present invention have a lower degree of fibrosis.

[0081] Choosing appropriate propulsion speed and voltage can obtain artificial pancreatic islets with appropriate shell thickness. If the flow rate of the external phase microfluidic pump exceeds the range of the present invention, the liquid of the internal phase will be squeezed, so that the core-shell structure sphere cannot be obtained.

[0082] The fluorescence staining results of the core-shell structure artificial islets obtained in Examples 1 to 5 are as follows: Fig. 9 As shown in the figure, it can be seen that the outer layer is the shell layer, the inner layer is the encapsulated islet cells, and the shell layer thickness from left to right is 0.1mm, 0.2mm, 0.3mm, 0.4mm, and 0.5mm. The new alginate with different shell layer thicknesses can not only well protect the encapsulated islet cells inside, but also have an anti-fibrosis effect.

[0083] The fluorescent staining results of the artificial islet cell survival experiment using the artificial islet obtained in Example 1 of the present invention and the artificial islet obtained in Comparative Examples 1 and 2 are as follows: Fig.10 As shown. It can be seen from the figure that compared with the current group of islet cells encapsulated directly with ordinary alginate and encapsulated with zwitterionic anti-fibrotic materials, the survival rate of islet cells in this core-shell structure artificial islet with a new matrix glue is higher. The higher the green intensity and the greater the number, the higher the islet survival rate. It can be seen that the use of the prepared new matrix glue as the nuclear layer material for encapsulating the islets has a stronger effect on the survival of islet cells than directly using ordinary alginate and zwitterionic materials.

[0084] The statistical results are as follows Fig.11 As shown in the figure, it can be seen that the survival rate of islet cells in the artificial islets obtained in Example 1 of the present invention is the highest.

[0085] The artificial pancreatic islets obtained in Example 1 of the present invention were implanted into type Ⅰ diabetic mice for treatment, and the therapeutic effect was investigated. Fig.12 The artificial islets with a core-shell structure that reduces fibrosis and improves islet survival rate can be treated continuously for more than 120 days.

[0086] Compared with the alginate and zwitterion fibrosis-reducing materials commonly used to encapsulate islet cells, the new alginate made of the outer shell structure of the artificial islet obtained by the present invention has a stronger effect of reducing fibrosis and is more conducive to the supply of nutrients and survival of the encapsulated islets. In addition, the new matrix gel prepared by encapsulating islet cells in the inner nuclear layer improves the survival rate of islet cells. After the artificial islet was implanted into mice, the blood sugar concentration of type Ⅰ diabetic mice was successfully regulated within the normal range for more than 120 days.

[0087] The present invention uses 4-iodobenzylamine modified alginate as a shell layer, which can well isolate the recognition of immune cells and protect the encapsulated cells, so that the cells can survive for a long time inside the shell layer and are not attacked by the recognition of external immune cells, play the function of the cells themselves, and have a better anti-fibrosis effect. Its good biocompatibility also ensures that the material has a wide range of application prospects in the medical field. And the spherical shape produced produces less fibrosis compared to other shapes with sharp edges. And a new type of matrix glue is prepared for the material for encapsulating pancreatic islets in the nuclear layer, which can make the encapsulated pancreatic islet cells survive longer than the pancreatic islet cells encapsulated by ordinary alginate, and the function of releasing insulin is better. The artificial pancreatic islet is made into a spherical core-shell structure with a strong anti-fibrosis effect. The anti-fibrotic new alginate shell layer and the preparation of a new matrix glue make the encapsulated pancreatic islet survive longer and release insulin better. Compared with the artificial pancreatic islet made by directly encapsulating pancreatic islet cells with ordinary alginate encapsulation, the effect of treating type Ⅰ diabetes is better.

Claims

1. A method for preparing an anti-fibrotic spherical core-shell structure artificial pancreatic islet, characterized in that: The following steps are involved: Step 1: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the sodium alginate solution; add 4-iodobenzylamine after fully dissolving, stir well, dialyze, and freeze-dry to obtain 4-iodobenzylamine-modified alginate; Step 2: resuspend the islet cells in the matrix gel to obtain the inner phase solution; Step 3: using the 4-iodobenzylamine-modified alginate obtained in step 1 as the external phase solution and the internal phase solution obtained in step 2 to obtain core-shell droplets in which the external phase envelops the internal phase; Step 4: The core-shell liquid droplets obtained in step 3 are added with a calcium ion solution to obtain an artificial pancreatic islet with a cross-linked structure.

2. The method for preparing an anti-fibrotic spherical core-shell structured artificial islet according to claim 1, characterized in that: In the step 1, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and 4-iodobenzylamine is 1:1:

1.

3. The method for preparing an anti-fibrotic spherical core-shell structured artificial islet according to claim 1, characterized in that: In the step 1, the molar ratio of sodium alginate to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:

100.

4. The method for preparing an anti-fibrotic spherical core-shell structured artificial islet according to claim 1, characterized in that: The matrix gel in step 2 contains L-alanine, L-aspartic acid, L-arginine, L-cysteine, L-glutamic acid, glycine, histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-ascorbic acid, 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxynucleoside, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleoside sodium phosphate, calcium chloride, magnesium sulfate, potassium chloride, sodium acetate, sodium chloride, disodium hydrogen phosphate, D-glucose, phenol red-sodium, glutathione, D-glucuronic acid, sodium bicarbonate, glucose, and fetal bovine serum.

5. The method for preparing an anti-fibrotic spherical core-shell structured artificial islet according to claim 1, characterized in that: In step 3, the pushing speed of the inner phase is 1 ml / min, and the pushing speed of the outer phase is 0.5 ml / min to 2.5 ml / min; the voltage is 5 kV.

6. The method for preparing an anti-fibrotic spherical core-shell structured artificial islet according to claim 1, characterized in that: The concentration of calcium ions in the calcium ion solution in step 4 is 20 mM.

7. The anti-fibrotic spherical core-shell structured artificial pancreatic islet obtained by any one of the preparation methods of claims 1 to 6, characterized in that: The shell thickness of the artificial pancreatic islet is 0.1-0.5 mm.

8. The use of an anti-fibrotic spherical core-shell structured artificial islet according to claim 7, characterized in that: The artificial pancreatic islet is used in the preparation of diabetes medicine.

9. The use of an anti-fibrotic spherical core-shell structured artificial islet according to claim 8, characterized in that: The diabetes drug is a type I diabetes drug.

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