Heparinized gelatin / sodium alginate pancreas islet packaging material with anti-fibrosis function as well as preparation method and application of heparinized gelatin / sodium alginate pancreas islet packaging material

Abstract

The invention relates to a heparinized gelatin / sodium alginate pancreas islet packaging material with an anti-fibrosis function as well as a preparation method and application of the heparinized gelatin / sodium alginate pancreas islet packaging material. The preparation method comprises the following steps: adding gelatin into normal saline, soaking, performing autoclaved sterilization to obtain a gelatin solution, then adding epsilon-polylysine hydrochloride into the gelatin solution, and finally adding glutamine transaminase for reaction to obtain an epsilon-polylysine grafted gelatin solution; adding heparin into the solution, and reacting to obtain a heparinized gelatin solution; adding sodium alginate and glutamine transaminase, reacting, diluting, preparing hydrogel microspheres by adopting an electrostatic spraying method, and cross-linking to obtain the hydrogel microspheres. According to the invention, heparin molecules are incorporated into a pancreas islet packaging system, so that fibrosis at the periphery of the microspheres can be relieved, the pancreas islet cells are packaged by using the pancreas islet packaging material to prepare the biological artificial pancreas, the pancreas islet cell proliferation and insulin secretion can be promoted, and the pancreas islet packaging material has a good diabetes treatment effect.

Description

Technical Field

[0001] The present invention relates to a heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function, a preparation method and application thereof, and belongs to the field of biomedical material preparation. Background Art

[0002] Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by the immune system attacking and destroying insulin-producing beta cells in the pancreas. Currently, the main treatment for T1DM, exogenous insulin injections, is unable to halt the progression of diabetic complications. Pancreas and islet transplants can be immune damaged by autoimmune and allogeneic immune rejection mechanisms. While immunosuppressive therapy can effectively delay or prevent transplant rejection in the host, it can also adversely affect other tissues and organs, and even increase the risk of infection and cancer.

[0003] The development of islet encapsulation technology has provided a possible solution to the problem of immune rejection in islet transplantation. Hydrogel microspheres are used to encapsulate islets to prepare a bio-artificial pancreas, replacing traditional islet transplantation. Hydrogel microspheres act as a physical barrier between the transplant and the recipient, isolating the islet cells from contact with immune cells, thereby protecting the islet cells. During islet encapsulation, the loading of islet cells and the preparation of hydrogel microspheres are often carried out simultaneously, which requires that the hydrogel prepolymer has good biocompatibility, and the cross-linking and post-processing methods also need to be relatively mild. Therefore, natural polymer materials sodium alginate (SA) and gelatin (Gel) with excellent biocompatibility have become the first choice. The disadvantage of poor mechanical strength of natural biomaterials can also be improved by forming an interpenetrating network between sodium alginate and gelatin in a double cross-linking manner. The mechanical strength and toughness of the double cross-linked interpenetrating network hydrogel are significantly improved compared to the structure formed by 3D cross-linking of a single polymer.

[0004] After islet transplantation, the revascularization process typically takes more than 10 days, and complete vascular remodeling may take up to 3 months. At this stage, the survival of the islets depends primarily on the passive diffusion of nutrients and oxygen. However, the islets are affected by the host immune response, which activates the foreign body reaction, leading to the formation of fibrosis around the implant, preventing the entry of nutrients and oxygen, and ultimately leading to islet cell loss due to beta cell starvation and hypoxia. In addition, fibrosis also prevents the diffusion of insulin from the implant. Fibrosis is the main cause of failure of bioartificial pancreas transplantation. Heparin (Hep) is a highly sulfated glycosaminoglycan present in the extracellular matrix that has anticoagulant properties, anti-inflammatory effects, and a high affinity for vascular endothelial growth factor.

[0005] Therefore, the preparation of a new islet encapsulation material with anti-fibrosis function can be used to encapsulate islet cells to create a bioartificial pancreas, which is of great significance for islet transplantation and the treatment of type 1 diabetes. Summary of the Invention

[0006] The present invention aims to solve the difficult problem of low survival rate after islet transplantation. Aiming at the two main aspects of the use of immunosuppressants and peripheral fibrosis of islets, the present invention provides a heparinized gelatin / sodium alginate islet encapsulation material with anti-fibrosis function, as well as its preparation method and application. The heparinized gelatin / sodium alginate islet encapsulation material of the present invention is used to encapsulate islet cells, thereby preparing a bioartificial pancreas (BAP). The present invention first grafts polylysine (PLL) onto gelatin using transglutaminase (TG) to increase its surface positive charge, and then utilizes electrostatic assembly to prepare heparinized gelatin; secondly, after mixing with sodium alginate, heparinized gelatin / sodium alginate hydrogel microspheres are prepared by electrostatic spraying to obtain heparinized gelatin / sodium alginate islet encapsulation material. The present invention incorporates heparin molecules into the islet encapsulation system, which can reduce fibrosis around the microspheres. Using this islet encapsulation material to encapsulate islet cells to prepare a bioartificial pancreas can promote islet cell proliferation and insulin secretion, and has a good effect on the treatment of diabetes.

[0007] The technical solutions of the present invention are as follows:

[0008] A method for preparing a heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function comprises the following steps:

[0009] (1) adding gelatin (Gel) into physiological saline, soaking and then autoclaving to obtain a gelatin (Gel) solution, then adding ε-polylysine hydrochloride (ε-PLL) into the gelatin (Gel) solution, and finally adding transglutaminase (TG) to react to obtain an ε-polylysine grafted gelatin solution;

[0010] (2) adding heparin to the ε-polylysine grafted gelatin solution to react and obtain a heparinized gelatin solution; then adding sodium alginate and transglutaminase to react and obtain a heparinized gelatin / sodium alginate solution;

[0011] (3) After diluting the heparinized gelatin / sodium alginate solution obtained in step (2), hydrogel microspheres are prepared by electrostatic spraying; the obtained hydrogel microspheres are cross-linked to obtain a heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function.

[0012] Preferably, according to the present invention, the ratio of the volume of the physiological saline to the mass of the gelatin in step (1) is 40-60 mL:1 g.

[0013] Preferably, according to the present invention, the gel strength of the gelatin in step (1) is 150-300 g Bloom.

[0014] According to the preferred embodiment of the present invention, the soaking time in step (1) is 5 to 15 minutes; the high-pressure sterilization conditions are: pressure of 103 to 137 kPa, temperature of 120 to 122° C., and time of 15 to 25 minutes.

[0015] According to the present invention, preferably, the mass ratio of the ε-polylysine hydrochloride (ε-PLL) to gelatin in step (1) is 0.05-0.15:1; the average molecular weight of the ε-polylysine hydrochloride is not specifically limited, and preferably the ε-polylysine hydrochloride has a molecular weight of less than 5000.

[0016] Preferably, according to the present invention, the mass ratio of the transglutaminase (TG) to gelatin in step (1) is 0.0002-0.0003:1; and the enzyme activity of the transglutaminase (TG) is 150-200 U / g.

[0017] According to the preferred embodiment of the present invention, the reaction temperature in step (1) is 40-50° C., and the reaction time is 2-3 h.

[0018] According to the preferred embodiment of the present invention, in step (1), after the reaction is completed, a post-treatment step is further included, specifically as follows: using an ultrafiltration tube with a molecular weight cutoff of 30 kDa, ultrafiltration centrifugation at a speed of 3500-4000 r / min for 20-30 min, then resuspending with physiological saline, and centrifuging and replacing 4-6 times using the ultrafiltration tube to remove TG and PLL that have not participated in the reaction to obtain a purified product, and dispersing the purified product with physiological saline to obtain an ε-polylysine grafted gelatin solution; the ratio of the volume of physiological saline used for resuspension to the mass of gelatin is 50-100 mL:1 g; the volume of the obtained ε-polylysine grafted gelatin solution is the same as the volume of physiological saline in the gelatin solution in step (1).

[0019] Preferably, according to the present invention, the mass ratio of heparin to ε-polylysine hydrochloride (ε-PLL) in step (2) is 2 to 3:1.

[0020] Preferably, according to the present invention, in step (2), heparin is added to the ε-polylysine grafted gelatin solution, the reaction temperature is 37° C., and the reaction time is 0.5 to 1 h.

[0021] Preferably, according to the present invention, the mass of the sodium alginate in step (2) is equal to the mass of the gelatin, and the amount of the added glutamine transaminase is the same as that in step (1).

[0022] Preferably, according to the present invention, in step (2), sodium alginate and transglutaminase are added, the reaction temperature is 40-50° C., and the reaction time is 2-4 h.

[0023] According to the preferred embodiment of the present invention, in step (3), the dilution is performed by adding physiological saline to the obtained heparinized gelatin / sodium alginate solution for dilution, and the volume ratio of the added physiological saline to the volume of the heparinized gelatin / sodium alginate solution is 0.3-0.4:1.

[0024] According to the preferred embodiment of the present invention, in step (3), the parameters of the electrostatic spray are: an operating voltage of 6 to 12 kV, a propulsion speed of 12 to 16 mL / h, a receiving solution of 0.1 mol / L CaCl2 solution, and a receiving distance of 8 to 12 cm.

[0025] According to the preferred embodiment of the present invention, the cross-linking step in step (3) is as follows: first, soaking in a 0.1 mol / L CaCl2 solution for 20 to 40 minutes to promote gelation of sodium alginate, then removing excess CaCl2 solution, rinsing with ultrapure water three times, and incubating in a 37°C water bath for 0.5 to 1.5 hours to promote further cross-linking of gelatin to form interpenetrating network hydrogel microspheres.

[0026] The present invention also provides a heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function, which is prepared by the above-mentioned preparation method.

[0027] According to the present invention, the above-mentioned heparinized gelatin / sodium alginate islet encapsulation material with anti-fibrosis function is used to encapsulate islet cells and prepare a bio-artificial pancreas.

[0028] According to the application of the present invention, a method for encapsulating pancreatic islet cells to prepare a bioartificial pancreas comprises the following steps:

[0029] The pancreatic islet secretory cells MIN6 were prepared into a cell suspension, which was then mixed with a heparinized gelatin / sodium alginate solution. Then, an electrostatic spray method was used to prepare hydrogel microspheres encapsulating the islet cells. After cross-linking, washing, and culturing, the islet cells were encapsulated to obtain a bioartificial pancreas.

[0030] According to a preferred embodiment of the present invention, the cell suspension is prepared by culturing pancreatic secretory cells MIN6 to the logarithmic growth phase, digesting them with trypsin solution, counting them, and dispersing them in a culture medium to prepare a cell suspension with a concentration of 1×10 6 -5×10 6The cell suspension is 100 mL / mL; the culture medium is DMEM complete medium containing 10% fetal bovine serum and 1% double-resistance penicillin-streptomycin. 100 mL of DMEM complete medium includes 10 mL of fetal bovine serum, 1 mL of penicillin-streptomycin mixture (100X) and 89 mL of DMEM high-glucose medium; the concentration of the trypsin solution is 0.25%, which is a common commercially available product.

[0031] Preferably, according to the present invention, the volume ratio of the cell suspension to the heparinized gelatin / sodium alginate solution is 0.3-0.4:1.

[0032] According to the application of the present invention, the preparation method of the heparinized gelatin / sodium alginate solution is as described in steps (1) to (2) of the preparation method of the heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function.

[0033] According to the application of the present invention, the parameters of the electrostatic spray are the same as those in the method for preparing the heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function.

[0034] According to the application of the present invention, the cross-linking step is: soaking the hydrogel microspheres encapsulating pancreatic islet cells in a 0.1 mol / L CaCl2 solution for 20 to 40 minutes, and then removing the CaCl2 solution; the washing step is washing three times with DMEM complete culture medium containing 10% fetal bovine serum and 1% double-streptomycin; and the culturing step is inoculating the washed sample into a 6-well plate and culturing it in a cell culture incubator at 37°C for 24 to 72 hours to obtain a bioartificial pancreas.

[0035] The present invention adopts the principle of glutamine transaminase catalyzing gelatin self-crosslinking and gelatin-g-polylysine grafting reaction Figure 1 shown.

[0036] Anything not described in detail in the present invention is based on conventional techniques in the art.

[0037] The technical features and beneficial effects of the present invention are as follows:

[0038] 1. The present invention utilizes an enzymatic method to graft polylysine onto gelatin molecules to prepare gelatin-g-polylysine. Heparin molecules are then fixed to the positively charged polylysine through electrostatic assembly to form heparinized gelatin. The electrostatically adsorbed heparin not only achieves a good controlled-release effect but also maintains its natural conformation, maximally retaining its activity.

[0039] 2. This invention uses a combination of enzymatic and ionic crosslinking to prepare (heparinized) gelatin / alginate interpenetrating network hydrogel microspheres. Not only are the microspheres of suitable size for cell encapsulation, but their porous internal structure also provides an extracellular matrix-like environment for cell adhesion and proliferation.

[0040] 3. In view of the fact that islet transplants are prone to fibrosis, the present invention incorporates heparin molecules into the islet encapsulation system. It can inhibit IBMIR by utilizing its anticoagulant properties and prevent excessive growth of peripheral fibrosis of the microspheres by reducing the adsorption of fibrinogen, thereby effectively improving the islet encapsulation efficiency and long-term transplant function.

[0041] 4. Heparinized gelatin / sodium alginate islet encapsulation material did not affect the activity and function of MIN6 cells. Instead, it promoted the proliferation of MIN6 cells, upregulated the expression of the Insulin gene, increased the amount of insulin released, and prolonged the residence time of MIN6 cells in the mouse peritoneal cavity. It also showed a better hypoglycemic effect in the treatment of T1DM mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The figure is a schematic diagram of the principle of gelatin self-crosslinking and gelatin-g-polylysine grafting reaction catalyzed by transglutaminase of the present invention.

[0043] Figure 2 These are the ε-polylysine grafted gelatin sample prepared in Example 1, the gelatin self-crosslinking product prepared in Comparative Example 1, and infrared spectra of ε-polylysine and gelatin.

[0044] Figure 3 These are atomic force microscope photos of the hydrogel microspheres prepared in Example 2 and Comparative Examples 2-3.

[0045] Figure 4 This is a bar graph showing the adsorption results of fibrinogen on the surface of hydrogel microspheres in Experimental Example 1.

[0046] Figure 5 Fluorescence photograph (top) and cell density statistics (bottom) of cells adhered to the surface of hydrogel microspheres after co-culture of RAW264.7 cells in Experimental Example 1.

[0047] Figure 6 The fluorescence image of CD86 staining (left) and semi-quantitative analysis of CD86 (right) of RAW264.7 cells induced to activate into M1 macrophages in Experimental Example 1 are shown.

[0048] Figure 7 This is a live / dead cell staining photograph of MIN6 cells in the bioartificial pancreas of Example 3 in Experimental Example 2.

[0049] Figure 8 These are three-dimensional distribution photos of MIN6 cells in the bioartificial pancreas in Experimental Example 2 and scanning electron microscope photos of the cross sections of the bioartificial pancreas in Example 3 and Comparative Examples 4-5.

[0050] Figure 9This is a bar graph showing the proliferation results of MIN6 cells in the bioartificial pancreas in Experimental Example 2.

[0051] Figure 10 This is the insulin gene expression and secretion capacity of MIN6 cells in the bioartificial pancreas in Experimental Example 2.

[0052] Figure 11 The figures show the changes in average body weight (left) and blood glucose levels (right) of diabetic mice after bioartificial pancreas transplantation in Experimental Example 2.

[0053] Figure 12 This is the fluorescence distribution diagram of the bioartificial pancreas in diabetic mice in Experimental Example 2.

[0054] Figure 13 This is a diagram of the total radiation efficiency of the bioartificial pancreas in diabetic mice in Experimental Example 2. DETAILED DESCRIPTION

[0055] In order to further illustrate the technical solution of the present invention, the following is a detailed description with reference to the following embodiments. It should be understood that the present invention is not limited to the following embodiments.

[0056] All the raw materials in the examples are conventional raw materials and commercially available products.

[0057] Gelatin: Sigma, USA, gel strength 175g Bloom;

[0058] Glutamine transaminase: Shanghai Shifeng Biotechnology Co., Ltd., enzyme activity is 200 U / g;

[0059] Heparin: Solebao Biotechnology Co., Ltd., potency >140 units / mg;

[0060] Sodium alginate: Shanghai MacLean Biochemical Technology Co., Ltd., M / G = 1:2;

[0061] Polylysine hydrochloride: Shanghai MacLean Biochemical Technology Co., Ltd., MW less than 5000, product number P832586;

[0062] The culture medium used in the Examples, Comparative Examples and Test Examples, unless otherwise specified, is DMEM complete medium containing 10% fetal bovine serum and 1% double antibody. 100 mL of culture medium includes 10 mL of fetal bovine serum, 1 mL of penicillin-streptomycin mixture (100X) and 89 mL of DMEM high glucose medium, wherein the DMEM high glucose medium was purchased from Wuhan Punosai Life Science Technology Co., Ltd., the fetal bovine serum was purchased from Hangzhou Tianhang Biotechnology Co., Ltd., and the penicillin-streptomycin mixture (100X) was purchased from Genview, USA.

[0063] Example 1: Enzymatic preparation of ε-polylysine grafted gelatin sample

[0064] 200 mg of type A gelatin (Gel) was added to 10 mL of normal saline, soaked for 10 min, and then sterilized under high pressure (137 kPa, 121 ° C, 20 min), and naturally cooled to room temperature to obtain a gelatin (Gel) solution; the gelatin (Gel) solution was heated to 50 ° C, and then 20 mg of ε-polylysine hydrochloride (ε-PLL) was added to the gelatin (Gel) solution, stirred evenly, and finally 0.05 mg of glutamine transaminase (TG) was added to react at 50 ° C for 3 h. After the reaction was completed, , select an ultrafiltration tube with a molecular weight cutoff of 30 kDa, and ultrafiltration centrifuge at a speed of 4000 r / min for 30 minutes; then resuspend with physiological saline (15 mL), use an ultrafiltration tube for centrifugal replacement 5 times (4000 r / min, 30 minutes) to remove TG and PLL that do not participate in the reaction, collect the purified product, add physiological saline to the purified product to a total volume of 10 mL, and disperse evenly to obtain ε-polylysine grafted gelatin (Gel-g-PLL) solution.

[0065] The ε-polylysine grafted gelatin solution prepared in this example was tested using an automatic amino acid analyzer. The lysine content of the gelatin grafted with PLL increased from 3.55% to 7.45%, indicating a significant increase in lysine. This indicates that PLL was successfully grafted onto the gelatin molecule and caused a change in the secondary structure of the protein. The ε-polylysine grafted gelatin solution was freeze-dried and its infrared spectrum was tested. The results are shown in Figure 2. Figure 2 As shown in the figure, the infrared spectra of gelatin and ε-polylysine were tested simultaneously. Compared with the Gel sample, the characteristic peak light absorption in the infrared spectra of the Gel-Gel sample and the Gel-g-PLL sample was significantly weakened, the peak shape became flatter from sharp, and the peak position shifted. This indicates that the protein structure of gelatin changes or transforms under the action of TG.

[0066] Comparative Example 1 Preparation of gelatin self-crosslinking sample by enzymatic method

[0067] 200 mg of type A gelatin (Gel) was added to 10 mL of normal saline, soaked for 10 minutes, and then sterilized under high pressure (137 kPa, 121°C, 20 minutes). The mixture was naturally cooled to room temperature to obtain a gelatin (Gel) solution. The gelatin (Gel) solution was heated to 50°C, and 0.05 mg of TG was added and reacted at 50°C for 3 hours. After the reaction, an ultrafiltration tube with a molecular weight cutoff of 30 kDa was selected and ultrafiltration was performed at a speed of 4000 r / min for 30 minutes. The mixture was then resuspended with normal saline (15 mL) and centrifuged five times using an ultrafiltration tube (4000 r / min, 30 minutes). The purified product was collected, and normal saline was added to the purified product to a total volume of 10 mL. The product was evenly dispersed to obtain a gelatin self-crosslinked product (Gel-Gel) solution.

[0068] After freeze-drying the gelatin self-crosslinked product solution in this comparative example, its infrared spectrum was tested, and the results were as follows: Figure 2 shown.

[0069] Example 2: Preparation of heparinized gelatin / sodium alginate hydrogel microspheres

[0070] An ε-polylysine grafted gelatin (Gel-g-PLL) solution was prepared according to the steps of Example 1, and heated to 37° C. 50 mg of heparin (Hep) was added to the Gel-g-PLL solution and reacted at 37° C. for 1 h to obtain a heparinized gelatin solution, i.e., a Gel-g-PLL / Hep solution. 200 mg of autoclaved (137 kPa, 121° C., 20 min) sodium alginate (SA) was then added to the Gel-g-PLL / Hep solution. The mixture was stirred and dissolved at 37°C on a magnetic stirrer. After thorough mixing for 10 minutes, 0.05 mg of transglutaminase (TG) was added and the mixture was reacted at 50°C for 3 hours to prepare a SA / Gel-g-PLL / Hep hydrogel precursor solution. The hydrogel precursor solution was then diluted 0.75-fold by adding 1 / 3 of the volume of normal saline to the hydrogel precursor solution. The solution was then transferred to a 10 mL syringe with a 21-gauge needle. The syringe was embedded in a microinjection pump. A flat dish containing 0.1 mol / L CaCl2 solution was placed on a copper plate as a receiving dish. The positive and negative electrodes of the electrostatic high-voltage generator were connected to the needle tip and the receiving dish, respectively. The propulsion speed, working voltage, and receiving distance were set to 14 mL / h, 7 kV, and 10 cm, respectively. Under the dual action of voltage and gravity, the microspheres ejected from the syringe fell evenly into the CaCl2 solution. Finally, the hydrogel microspheres were immersed in CaCl2 solution for 30 minutes to promote the gelation of sodium alginate. After removing the excess CaCl2 solution, they were rinsed three times with ultrapure water and incubated in a 37°C water bath for 1 hour to promote further cross-linking of gelatin to form interpenetrating network hydrogel microspheres, which are heparinized gelatin / sodium alginate islet encapsulation materials with anti-fibrosis function, recorded as SA / Gel-g-PLL / Hep HMS.

[0071] Comparative Example 2

[0072] A gelatin self-crosslinking product (Gel-Gel) solution was prepared according to the steps of Comparative Example 1, heated to 37°C, 200 mg of autoclaved (137 kPa, 121°C, 20 min) sodium alginate (SA) was added, and stirred and dissolved under a magnetic stirrer at 37°C. After thorough mixing for 10 min, 0.05 mg of transglutaminase (TG) was added and reacted at 50°C for 3 h to prepare a SA / Gel-Gel hydrogel precursor solution; then, hydrogel microspheres were prepared according to the method of Example 2, recorded as SA / Gel-Gel HMS.

[0073] Comparative Example 3

[0074] A gelatin self-crosslinking product (Gel-Gel) solution was prepared according to the steps of Comparative Example 1, heated to 37°C, 50 mg of heparin (Hep) was added to the above Gel-Gel solution, and the mixture was reacted at 37°C for 1 hour to obtain a Gel-Gel / Hep solution; 200 mg of autoclaved (137 kPa, 121°C, 20 min) sodium alginate (SA) was added to the Gel-Gel / Hep solution, stirred and dissolved under a magnetic stirrer at 37°C, and thoroughly mixed for 10 minutes. Then, 0.05 mg of transglutaminase (TG) was added, and the mixture was reacted at 50°C for 3 hours to prepare a SA / Gel-Gel / Hep hydrogel precursor solution; hydrogel microspheres were then prepared according to the method of Example 2, and recorded as SA / Gel-Gel / Hep HMS.

[0075] Test Example 1

[0076] The hydrogel microspheres prepared in Example 2 and Comparative Examples 2-3 were subjected to the following tests: surface morphology, anti-protein adsorption performance, anti-macrophage adhesion performance, and macrophage polarization of the microspheres were measured.

[0077] (1) Surface morphology of microspheres

[0078] Place a drop of quick-drying glue in the center of a plastic Petri dish. After blotting the surface moisture of the microspheres with filter paper, carefully place them onto the quick-drying glue and gently press them to secure them. Once the glue has dried, slowly add deionized water to the dish until the hydrogel microspheres are submerged. Place the dish on the atomic force microscope stage and prepare for performance testing, with a scan range of 5μm x 5μm.

[0079] (2) Anti-protein adsorption properties of microspheres

[0080] To determine the Fgn standard curve: First, prepare a 1 mg / mL Fgn standard solution in PBS. Then, dilute this solution with PBS to create a series of Fgn test solutions at concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL. Measure the absorbance of Fgn at 280 nm using a microplate reader. Plot an absorbance-concentration curve.

[0081] Fgn protein adsorption assay: First, dilute the 1 mg / mL fibrinogen (Fgn) standard solution to 700 μg / mL with PBS to obtain Fgn solution. Take 0.5 mL of microsphere saline dispersion (concentration is 1000 / mL) and add it to 1 mL of prepared Fgn solution. Place it at 37°C for 2 hours to allow Fgn to fully adsorb on the microspheres, then centrifuge at 1000 rpm for 3 minutes, take the supernatant and measure its absorbance at 280 nm. Calculate the corresponding Fgn concentration based on the standard curve, and repeat each experiment twice. The protein adsorption amount of the microspheres (q, μg / cm 2) is calculated as follows: q = (Co - C) V / S, where Co is the initial Fgn concentration, C is the Fgn concentration after incubation with microspheres, V is the total volume of the solution, and S is the total surface area of ​​the microspheres added to the solution.

[0082] (3) Anti-macrophage adhesion properties of microspheres

[0083] RAW264.7 cells were collected and counted, and dispersed in culture medium to prepare a concentration of 2×10 5 / mL cell suspension; take a 12-well plate, spread 2% w / v agarose gel on the bottom of the well plate to prevent cells from adhering to the wall, add 400μL of microsphere saline dispersion (concentration is 1000 / mL), and then inoculate the cells into the 12-well plate containing microspheres. Add culture medium to 2mL, set up 3 replicates for each group, and place in a 37℃ constant temperature incubator for 24h. In the initial 4h, gently shake the well plate for 5min every 0.5h to allow the cells to fully adhere to the surface of the microspheres. After 24h of cell culture, remove the well plate, wash twice with PBS, add 1mL LDAPI dye to each well, stain in the dark for 10min, wash three times with PBS, observe the cell adhesion on the microsphere surface with LSCM, and then count the cells with the help of Image J software.

[0084] (4) Observation of macrophage polarization by CD86 fluorescence staining

[0085] Sterile 20mm round cell slides were placed in a 12-well plate and the above (3) was added with a concentration of 2×10 5Cell suspension was prepared at 37°C for 24 hours. Different interventions were then given according to the grouping: the blank group received only culture medium, the experimental group received culture medium and 400 μL of the microsphere saline dispersion prepared in Example 2 and Comparative Example 2-3 (at a concentration of 1000 cells / mL), and the positive control group received culture medium containing 1000 ng / mL lipopolysaccharide. Three replicates were set up in each group. After 24 hours of culture at 37°C, the old culture medium and microspheres were discarded and washed twice with PBS. 200 μL of 4% paraformaldehyde was added to each well and fixed at room temperature for 20 minutes, washed twice with PBS, incubated with 1% Triton-X100 solution for 10 minutes, rinsed twice with PBS, and then blocked with 200 μL of 5% bovine serum albumin (BSA) solution at room temperature for 30 minutes. The BSA supernatant was discarded. The primary antibody (diluted at a ratio of 1:20) was incubated at 37°C for 2 hours and washed twice with PBS. Cells were incubated with Alexa Fluor 488 fluorescently labeled secondary antibody (1:100 dilution) at 37°C in the dark for 2 h, washed twice with PBS, and then incubated with rhodamine-labeled phalloidin (1:200 dilution) in the dark for 30 min to visualize the cytoskeleton. The cells were then washed three times with PBS. Finally, cell nuclei were labeled with ready-to-use DAPI in the dark for 5 min at room temperature. After three washes with PBS, cell morphology was observed using LSCM within 1 h. Fluorescence intensity was then quantified and analyzed using Image J software.

[0086] The results are analyzed as follows:

[0087] The surface roughness of materials is one of the important factors affecting cell adhesion, proliferation and maturation. The rough surface structure can expand the contact area between materials and cells, promoting cell adhesion, differentiation and proliferation. Fibrinogen (Fgn) is a sticky protein with high adsorption capacity. It can spontaneously adsorb to the surface of materials, promote the adhesion of cells such as macrophages, epithelial cells and platelets, and ultimately lead to the occurrence of fibrosis and proliferation on the surface of materials. Macrophages are divided into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages. M1 macrophages are the main phenotype in all stages of foreign body response. CD86, as a specific surface marker of M1 macrophages, is often used to detect the polarization state of macrophage M1.

[0088] like Figure 3 As shown, the SA / Gel-Gel group contains a high and low micropattern, the SA / Gel-Gel / Hep group can clearly see the presence of several pits, and the surface of the SA / Gel-g-PLL / Hep group is relatively flat. The flat surface will inhibit cell adhesion and is less likely to cause inflammatory response.

[0089] like Figure 4-6As shown in the results, compared with the SA / Gel-Gel group of microspheres, the two groups of microspheres with heparin added had less Fgn adsorbed on the surface, reduced the amount of RAW264.7 cells adsorbed and the amount of activated M1 macrophages, had better anti-Fgn adhesion effect and anti-macrophage adhesion effect, and is a promising anti-fibrosis material that helps to maintain the function of the graft in the long term.

[0090] Example 3: Preparation of heparinized gelatin / sodium alginate bioartificial pancreas

[0091] MIN6 cells were cultured in DMEM complete medium containing 10% fetal bovine serum and 1% double antibody until the logarithmic growth phase, digested with trypsin solution (0.25%, Biological Industries), counted, and dispersed in the culture medium to prepare a concentration of 1×10 6 The cell suspension was prepared by mixing the SA / Gel-g-PLL / Hep hydrogel precursor solution (prepared according to the method of Example 2) cooled to 37°C by autoclaving (137 kPa, 121°C, 20 min) at a volume ratio of 1:3 and pipetting to mix. The hydrogel and cell suspension were transferred to a 10 mL syringe with a 21-gauge needle, and the syringe was embedded in a microinjection pump. A plate containing 0.1 mol / L CaCl2 solution was placed on a copper plate as a receiving dish. The positive and negative electrodes of the electrostatic high-voltage generator were connected to the needle tip and the receiving dish, respectively. The propulsion speed, operating voltage, and receiving distance were set to 14 mL / h, 7 kV, and 10 cm, respectively. Under the dual action of voltage and gravity, the microspheres ejected from the syringe fell evenly into the CaCl2 solution to form BAP, which was recorded as SA / Gel-g-PLL / Hep BAP. After soaking in CaCl2 solution for 30 min, the CaCl2 solution was removed and the cells were washed three times with culture medium, seeded in a 6-well plate, and cultured in a cell culture incubator at 37°C.

[0092] Comparative Example 4

[0093] As described in Example 3, except that SA / Gel-Gel hydrogel precursor solution was used instead of SA / Gel-g-PLL / Hep hydrogel precursor solution, which was recorded as SA / Gel-Gel BAP.

[0094] Comparative Example 5

[0095] As described in Example 3, except that SA / Gel-Gel / Hep hydrogel precursor solution was used instead of SA / Gel-g-PLL / Hep hydrogel precursor solution, which was recorded as SA / Gel-Gel / Hep BAP.

[0096] Test Example 2

[0097] The BAP prepared in Example 3 and Comparative Examples 4-5 were subjected to the following tests to observe the particle size and pores of the BAP as well as the distribution, proliferation, gene expression and hypoglycemic effect of MIN6 cells in the BAP.

[0098] (1) Particle size and cell viability of the bioartificial pancreas

[0099] Example 3: After 3 days of culture, cells were harvested and washed three times with PBS. A staining solution containing 2 mol / L calcein-AM and 4.5 μmol / L PI was prepared using the animal cell viability / toxicity kit. 5 μL of the staining solution was added to every 100 μL of cell suspension, mixed thoroughly, and incubated for 20 minutes. The cells were washed three times with PBS, and the BAP particle size and cell viability were observed using an inverted fluorescence microscope.

[0100] (2) Observation of cell distribution and pores in the bioartificial pancreas

[0101] BAPs were cultured in 6-well plates for 72 hours, the culture medium was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 3 hours and washed three times with PBS. Cross-sections of the BAPs were cut using a cryostat. The BAPs were collected and washed three times with PBS to remove the OCT gel. The cells were then dehydrated through a gradient of ethanol (50%, 60%, 70%, 80%, 90%, 95%, and 100%), with each gradient dehydration lasting 10 minutes. Finally, the cells were freeze-dried. The microspheres were mounted on a metal sample holder with the cross-section facing upward and vacuum-coated with gold for 240 seconds. The morphology of the cells within the BAPs was then observed using a scanning electron microscope (SEM).

[0102] After culturing BAP in a 6-well plate for 72 hours, the medium was discarded and the cells were washed three times with sterile PBS before being transferred to a 35mm confocal microplate. The cells were fixed with 4% paraformaldehyde for 20 minutes, rinsed three times with PBS for 5 minutes each, incubated with 1% Triton-X100 solution for 10 minutes, and rinsed three times with PBS. The cells were then incubated in the dark with rhodamine-labeled phalloidin (1:200 diluted in PBS) for 30 minutes to visualize the cytoskeleton. The cells were then washed three times with PBS, preferably within 1 hour. The three-dimensional distribution of the cells within the microspheres was observed using LSCM.

[0103] (3) Cell proliferation in the bioartificial pancreas

[0104] The BAP prepared in Example 3 and Comparative Examples 4-5 were inoculated in a 96-well plate, and the control group was added with the same volume of naked cells (1×10 6Cells were cultured in a CO2 incubator for 1, 3, 5, and 7 days. The old culture medium was aspirated and 100 μL of culture medium and 10 μL of CCK-8 reagent were added to each well. After incubation at 37°C for 1 hour, 100 μL of the solution was transferred to an enzyme-labeled strip and the absorbance was measured at 450 nm. The relative cell growth rate was then calculated according to GB / T 16886.5-2017.

[0105] (4) Gene expression and secretion of insulin in the bioartificial pancreas

[0106] First, 2 mg / mL papain and 200 mol / L sodium citrate were dissolved in 5 mL of PBS to prepare microsphere lysis buffer. A certain amount of BAP was then taken, the culture medium removed, and the cells were rinsed three times with PBS. Then, 5 mL of microsphere lysis buffer was added and incubated at 37°C for 5 minutes with gentle shaking until no gel was visible. Lysis was terminated by adding 5 mL of culture medium (containing 10% serum and antibiotics), and the supernatant was discarded after centrifugation at 1000 rpm. The cells were washed once with culture medium, and then centrifuged to collect the cells for RNA extraction, reverse transcription to cDNA, and RT-qPCR analysis.

[0107] Three types of BAP and naked cells were seeded into 24-well plates and cultured for 24 hours. The culture medium was then switched to complete DMEM low-glucose medium and incubated at 37°C for 30 minutes. The culture medium was then switched to complete DMEM high-glucose medium and complete DMEM low-glucose medium for 24 hours, respectively. The supernatants were collected. Absorbance was measured according to the protocol described in the mouse insulin ELISA kit. The insulin content in the supernatant was calculated using a standard curve. The stimulation response index was calculated as the ratio of the insulin content in the high- to low-glucose media.

[0108] (5) Implantation of bioartificial pancreas to treat T1DM mice

[0109] Construction of T1DM mouse model:

[0110] One week before the experiment, Kunming mice were ear-tagged. They were fasted but not watered for 12 hours before modeling and weighed. Streptozotocin was administered intraperitoneally at a standard dose of 50 mg / kg for one week, followed by a 2-hour fast after each injection. Blood glucose levels were measured in the tail vein using a glucometer on an empty stomach. Blood glucose levels were measured continuously for one week after modeling. A successful glycemic model was considered established if the fasting blood glucose level was ≥16.7 mmol / L for three consecutive days. After successful modeling, the mice were weighed.

[0111] Intraperitoneal injection of bioartificial pancreas

[0112] Sixty T1DM model mice were randomly divided into six groups of 10 mice each. Each T1DM mouse was intraperitoneally injected with approximately 2,000 MIN6 cells. Post-injection observation and recording were performed on the general condition, toxicity, and mortality of the mice in both the experimental and control groups.

[0113] The animals are grouped as follows:

[0114] Group A: T1DM model mice with SA / Gel-PLL / Hep BAP transplanted into the peritoneal cavity;

[0115] Group B: T1DM model mice with SA / Gel-Gel / Hep BAP transplantation in the peritoneal cavity;

[0116] Group C: T1DM model mice intraperitoneal SA / Gel-Gel BAP transplantation;

[0117] Group D: T1DM model mice transplanted with MIN6 cells into the peritoneal cavity;

[0118] Group E: T1DM model mice injected with normal saline intraperitoneally;

[0119] Group F: normal mice injected with normal saline intraperitoneally;

[0120] Blood sugar and weight monitoring

[0121] After transplantation, blood was collected from the tail vein of the mice at 10 am every 5 days to measure fasting blood glucose concentration, and the body weight of the mice was recorded and the symptoms of diabetes were observed.

[0122] (6) Residence time of the bioartificial pancreas in the body

[0123] DiR was first prepared in DMSO to a 5 mmol / L DiR stock solution, which was then diluted in PBS to a 5 μmol / L DiR working solution. After 72 hours of culture, the three BAPs and bare cells were harvested, resuspended in the appropriate amount of DiR working solution, and incubated at 37°C for 20 minutes. The cells were washed three times with culture medium and then harvested. Bare cells and the three BAPs were injected intraperitoneally into mice using a 1 mL syringe. Live animal imaging was performed on days 1, 14, and 28.

[0124] The particle size of the bioartificial pancreas prepared in Example 3 is mainly distributed between 200-400 μm, which is in line with the practical principles of cell encapsulation. In addition, most of the MIN6 cells in the BAP are dyed green and maintain a good survival state. The material has good biocompatibility. Figure 7 shown.

[0125] The microspheres obtained after cross-linking are porous micro-nanostructures, which are similar to the nanofiber network structure of natural extracellular matrix, and have preliminarily achieved the goal of structurally simulating the natural tissue cell microenvironment, such as Figure 8 As shown in the mechanical stability test, some microspheres showed damage compared to before the shock, but the integrity of the microspheres remained above 98% after 5 weeks, suggesting potential applications in biomimetic extracellular matrix design for pancreatic islet encapsulation. LSCM 3D images clearly show that MIN6 cells are evenly dispersed within the BAP, with some cells forming clusters. This demonstrates that cells can proliferate, migrate, and form clusters normally within all three HMS.

[0126] like Figure 9 As shown, MIN6 cells within the BAP showed a significant proliferation trend and maintained good survival over 7 days, indicating that HMS caused minimal damage to cells during the cell encapsulation process, enabling relatively efficient and non-destructive encapsulation of pancreatic islet cells and exhibiting good biocompatibility. Furthermore, the SA / Gel-g-PLL / HepBAP group was found to have better cell compatibility than the SA / Gel-Gel / HepBAP group. This is because heparin, with its strong negative charge, electrostatically repels similarly negatively charged cells, hindering cell adhesion. However, the SA / Gel-g-PLL / HepBAP group, upon slow release of heparin, exposes positively charged PLL, which improves cell-matrix interactions and promotes cell adhesion.

[0127] pass Figure 10 It was not difficult to find that compared with bare cells, the expression of the Insulin gene was significantly upregulated in all three BAPs. Furthermore, both the three BAP groups and the bare cell group were able to secrete insulin normally under both high and low glucose conditions, and the amount of insulin released was greater than that of the bare cell group. Encapsulation of MIN6 cells by hydrogel microspheres did not affect their insulin secretion function at either the gene or protein level. Furthermore, the three BAPs had similar stimulation secretion indices, all greater than 1, indicating that all three BAPs secrete insulin in response to changes in glucose.

[0128] The prepared BAP has good injectability and can be smoothly injected into the abdominal cavity of mice through a No. 5 needle. The mice that received BAP transplants grew well and showed no toxic symptoms. Figure 11It can be seen that the average weight of T1DM mice in the normal saline group and MIN6 cell group never returned to the initial weight within 30 days, and the hypoglycemic effect was not ideal. However, the average weight of diabetic mice increased slightly after injection of BAP, and it had a good hypoglycemic effect, especially the SA / Gel-g-PLL / Hep BAP group had a more significant therapeutic effect on T1DM mice, and its blood sugar was closer to that of normal healthy mice. This is because large molecular heparin can reduce the surface fibrosis of BAP and improve the survival rate of pancreatic islet cells. Figure 12-13 From the results of in vivo imaging of small animals, we found that the SA / Gel-g-PLL / Hep BAP group can significantly increase the residence time of MIN6 cells, play an immune shielding effect, and provide a suitable environment for the survival of MIN6 cells, thereby better playing the role of lowering blood sugar.

[0129] The detection and analysis of the above results show that this material not only has good anti-adhesion effect and lower immunogenicity, but also can promote pancreatic cell proliferation, insulin gene expression and has a good blood sugar lowering effect. It has anti-fibrosis properties and does not affect the treatment effect of diabetes. It is a promising new type of pancreatic islet encapsulation material.

Claims

1. A method for preparing a heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function, comprising the following steps: (1) adding gelatin into physiological saline, soaking and then sterilizing under high pressure to obtain a gelatin solution, then adding ε-polylysine hydrochloride into the gelatin solution, and finally adding glutamine transaminase to react to obtain an ε-polylysine grafted gelatin solution; (2) adding heparin to the ε-polylysine grafted gelatin solution to react and obtain a heparinized gelatin solution; Then, sodium alginate and transglutaminase are added to react to obtain a heparinized gelatin / sodium alginate solution; (3) After diluting the heparinized gelatin / sodium alginate solution obtained in step (2), hydrogel microspheres are prepared by electrostatic spraying; the obtained hydrogel microspheres are cross-linked to obtain a heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function.

2. The method for preparing the heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function according to claim 1, characterized in that: The ratio of the volume of the physiological saline to the mass of the gelatin in step (1) is 40-60 mL:1 g; The gel strength of the gelatin is 150-300 g Bloom; the soaking time is 5-15 min; and the high-pressure sterilization conditions are: pressure of 103-137 kPa, temperature of 120-122° C., and time of 15-25 min.

3. The method for preparing the heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function according to claim 1, characterized in that: The mass ratio of the ε-polylysine hydrochloride to gelatin in step (1) is 0.05-0.15:1; the mass ratio of the transglutaminase to gelatin is 0.0002-0.0003:1; and the enzyme activity of the transglutaminase is 150-200 U / g.

4. The method for preparing the heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function according to claim 1, characterized in that The reaction temperature is 40-50°C, and the reaction time is 2-3 hours; In step (1), after the reaction is completed, a post-processing step is further included, which is specifically as follows: using an ultrafiltration tube with a molecular weight cutoff of 30kD, ultrafiltration centrifugation at a speed of 3500-4000r / min for 20-30min, then resuspending with physiological saline, and using the ultrafiltration tube for centrifugation and replacement 4-6 times to obtain a purified product, and dispersing the purified product with physiological saline to obtain an ε-polylysine grafted gelatin solution; the ratio of the volume of physiological saline used for resuspension to the mass of gelatin is 50-100mL:1g; the volume of the obtained ε-polylysine grafted gelatin solution is the same as the volume of physiological saline in the gelatin solution in step (1).

5. The method for preparing the heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function according to claim 1, characterized in that: The mass ratio of heparin to ε-polylysine hydrochloride in step (2) is 2-3:1; heparin is added to the ε-polylysine grafted gelatin solution, the reaction temperature is 37° C., and the reaction time is 0.5-1 h.

6. The method for preparing the heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function according to claim 1, characterized in that: In step (2), the mass of the sodium alginate is equal to the mass of the gelatin, and the amount of the added glutamine transaminase is the same as that in step (1); sodium alginate and glutamine transaminase are added, the reaction temperature is 40-50° C., and the reaction time is 2-4 hours.

7. The method for preparing the heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function according to claim 1, characterized in that: In step (3), the dilution is performed by adding physiological saline to the obtained heparinized gelatin / sodium alginate solution for dilution, and the volume ratio of the added physiological saline to the volume of the heparinized gelatin / sodium alginate solution is 0.3 to 0.4:1; The parameters of electrostatic spraying are: working voltage 6-12 kV, propulsion speed 12-16 mL / h, receiving solution 0.1 mol / L CaCl2 solution, and receiving distance 8-12 cm; The cross-linking step is as follows: first, soaking in a 0.1 mol / L CaCl2 solution for 20 to 40 minutes to promote gelation of sodium alginate, then removing excess CaCl2 solution, rinsing with ultrapure water three times, and incubating in a 37°C water bath for 0.5 to 1.5 hours.

8. A heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function, characterized in that: The preparation method according to claim 1 is used for preparation.

9. The use of the heparinized gelatin / sodium alginate pancreatic islet encapsulation material with anti-fibrosis function according to claim 8, characterized in that: Used to encapsulate pancreatic islet cells and prepare a bio-artificial pancreas.

10. The use according to claim 9, characterized in that: The method for encapsulating pancreatic islet cells to prepare a bioartificial pancreas comprises the following steps: Pancreatic islet secretory cells MIN6 were prepared into a cell suspension, which was then mixed with a heparinized gelatin / sodium alginate solution. Hydrogel microspheres encapsulating the islet cells were then prepared using an electrostatic spray method. After cross-linking, washing, and culturing, the islet cells were encapsulated to obtain a bioartificial pancreas. The cell suspension is prepared by culturing pancreatic islet secretory cells MIN6 to the logarithmic growth phase, digesting them with trypsin solution, counting them, and dispersing them in culture medium to prepare a cell suspension with a concentration of 1×10 6 -5×10 6 The cell suspension is 100 mL / mL; the culture medium is DMEM complete medium containing 10% fetal bovine serum and 1% double-streptomycin. 100 mL of DMEM complete medium includes 10 mL of fetal bovine serum, 1 mL of penicillin-streptomycin mixture (100X) and 89 mL of DMEM high-glucose medium. The volume ratio of the cell suspension to the heparinized gelatin / sodium alginate solution is 0.3-0.4:1; The cross-linking step comprises: soaking the hydrogel microspheres encapsulating pancreatic islet cells in a 0.1 mol / L CaCl2 solution for 20 to 40 minutes, and then removing the CaCl2 solution; washing the microspheres three times using a DMEM complete culture medium containing 10% fetal bovine serum and 1% double-streptomycin; and culturing the washed samples in a 6-well plate and placing them in a cell culture incubator at 37°C for 24 to 72 hours to obtain a bioartificial pancreas.

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