Injectable hierarchical structure composite hydrogel for pancreas islet transplantation and preparation method of injectable hierarchical structure composite hydrogel

Abstract

The invention discloses injectable hierarchical structure composite hydrogel for pancreas islet transplantation and a preparation method of the injectable hierarchical structure composite hydrogel, and belongs to the technical field of biomedical engineering. The preparation method comprises the following steps: introducing bubbles into an inner phase tube in a microfluidic device, introducing a hydrogel cell pre-polymerization solution into an outer phase tube, uniformly distributing the bubbles in the hydrogel cell pre-polymerization solution, then mixing with an ionic crosslinking aqueous phase solution, irradiating with blue light to obtain bubble-loaded hydrogel microfibers, and obtaining a pancreas islet-loaded short fiber stent after the bubbles are broken; mixing the oxidized sodium alginate solution with the carboxymethyl chitosan solution, and wrapping the angiogenesis promoting component to obtain a self-healing hydrogel matrix; and mixing the short fiber scaffold with the self-healing hydrogel matrix to obtain the injectable hierarchical structure composite hydrogel. The composite hydrogel has good biocompatibility, oxidation resistance and immune isolation effect, meanwhile, the hierarchical structure promotes formation of external vascularization, and long-term survival and function maintenance of transplanted plants after pancreas islet transplantation are facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical engineering, and in particular relates to an injectable hierarchical structure composite hydrogel for pancreatic islet transplantation and a preparation method thereof. Background Art

[0002] Islet transplantation is an emerging treatment for diabetes, with current research focused on prolonging survival and enhancing graft function. However, existing techniques typically require open surgical implantation, which causes significant trauma to the recipient. Furthermore, in some poorly vascularized transplant sites, islets can rapidly lose their vitality due to ischemia and hypoxia, leading to transplant failure. Therefore, an injectable hierarchical composite hydrogel for islet transplantation and its preparation method are proposed. Summary of the Invention

[0003] In view of the deficiencies of the prior art, the present invention aims to provide an injectable hierarchical structure composite hydrogel for islet transplantation and a preparation method thereof, thereby solving the problems in the prior art.

[0004] The purpose of the present invention can be achieved through the following technical solutions:

[0005] A method for preparing an injectable hierarchical structure composite hydrogel for pancreatic islet transplantation comprises the following steps:

[0006] In the microfluidic device, air bubbles are introduced into the inner phase tube and the hydrogel cell prepolymer solution is introduced into the outer phase tube, so that the bubbles are evenly distributed in the hydrogel cell prepolymer solution. The solution is then mixed with an ion-crosslinked aqueous solution and irradiated with blue light to obtain bubble-loaded hydrogel microfibers. After the bubbles burst, pancreatic islet-loaded short fiber scaffolds are obtained.

[0007] The oxidized sodium alginate solution was mixed with the carboxymethyl chitosan solution to encapsulate the pro-angiogenic components to obtain a self-healing hydrogel matrix;

[0008] The short fiber scaffold is mixed with the self-healing hydrogel matrix to obtain the result.

[0009] Furthermore, the hydrogel cell prepolymerization solution comprises: deionized water, sodium alginate, methacrylated hyaluronic acid, phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt and primary pancreatic islets;

[0010] The mass fraction of the sodium alginate is 1.5%, the mass fraction of the methacrylated hyaluronic acid is 2%, and the mass fraction of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 0.25%.

[0011] Furthermore, the amount of primary islets added was 10 4 pcs / ml.

[0012] Furthermore, bubble bursting is achieved by vacuum negative pressure suction or needle puncture.

[0013] Furthermore, the ion-crosslinked aqueous solution is a calcium chloride solution, and the mass concentration of the calcium chloride solution is 2-5%.

[0014] Furthermore, the inner phase tube and the outer phase tube are both glass capillaries, the inner diameter of the inner phase tube is 100-200 μm, and the inner diameter of the outer phase tube is 580-750 μm.

[0015] Furthermore, the mass fraction of the oxidized sodium alginate solution is 8%, the mass fraction of the carboxymethyl chitosan solution is 5%, and the mixing volume ratio of the oxidized sodium alginate solution to the carboxymethyl chitosan solution is 1:1.

[0016] Furthermore, the angiogenesis-promoting components are human umbilical vein endothelial cells and vascular endothelial growth factor.

[0017] A composite hydrogel is prepared using the method for preparing the injectable hierarchical structure composite hydrogel for pancreatic islet transplantation.

[0018] Application of the above-mentioned composite hydrogel in the preparation of pancreatic islet transplantation carriers.

[0019] Beneficial effects of the present invention:

[0020] 1. This invention uses microfluidic technology to prepare bubble-laden hydrogel microfibers, which are then broken and cut to obtain short fiber scaffolds. This method is simple to operate, low-cost, and the short fiber size can be adjusted by adjusting microfluidic parameters, allowing for stable mass production.

[0021] 2. The self-healing hydrogel prepared based on the Schiff base reaction of the present invention is injectable so that minimally invasive transplantation can be performed by injection.

[0022] 3. The composite hydrogel system of the present invention has excellent biocompatibility, antioxidant properties and immune isolation. At the same time, its hierarchical structure promotes the formation of external vascularization, which is conducive to the long-term survival and functional maintenance of islet transplantation. It is expected to be used as an ideal islet transplantation model in the fields of cell therapy and tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0024] Figure 1 Schematic diagram of the core-shell structured pancreatic cell three-dimensional culture hydrogel microfiber of the present invention;

[0025] Figure 2 This is a light microscopic image of the bubble-laden microfiber and short fiber scaffold according to an embodiment of the present invention;

[0026] Figure 3 This is a graph showing the relationship between short fiber parameters and solution flow rate according to an embodiment of the present invention;

[0027] Figure 4 This is a scanning electron micrograph of the self-healing hydrogel matrix according to an embodiment of the present invention;

[0028] Figure 5 This is a characterization diagram of the self-healing properties of the self-healing hydrogel according to an embodiment of the present invention;

[0029] Figure 6 This is a characterization diagram of the injectable properties of the composite hydrogel according to an embodiment of the present invention;

[0030] Figure 7 This is a diagram showing the islet function test results after encapsulating the islets according to an embodiment of the present invention;

[0031] Figure 8 This is a diagram showing the in vivo imaging test results after transplantation according to an embodiment of the present invention;

[0032] Figure 9 This is a graph showing the results of in vivo blood glucose monitoring after transplantation according to an embodiment of the present invention;

[0033] In the figure: 1-internal phase air inlet device; 2-external phase liquid inlet device; 3-internal phase capillary; 4-observation tube; 5-external phase capillary; 6-405nm blue light source; 7-collection device; 8-short fiber scaffold; 9-oxidized sodium alginate; 10-carboxymethyl chitosan; 11-self-healing hydrogel matrix; 12-hierarchical structure composite hydrogel. DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0035] A method for preparing an injectable hierarchical structure composite hydrogel for pancreatic islet transplantation comprises the following steps:

[0036] S1, in the microfluidic device, air bubbles are introduced into the inner phase tube and the hydrogel cell prepolymer solution is introduced into the outer phase tube, so that the bubbles are evenly distributed in the hydrogel cell prepolymer solution. The solution is then mixed with the ion-crosslinked aqueous solution and irradiated with blue light to obtain air bubble-loaded hydrogel microfibers. After the bubbles burst, pancreatic islet-loaded short fiber scaffolds can be obtained;

[0037] S2, mixing the two-component aqueous solution that achieves dynamic crosslinking based on Schiff base bonds to encapsulate the pro-angiogenic component to obtain a self-healing hydrogel matrix;

[0038] S3, mixing the short fiber scaffold with the self-healing hydrogel matrix to obtain an injectable hierarchical structure composite hydrogel;

[0039] In S1, the hydrogel cell prepolymerization solution includes: deionized water, sodium alginate, methacryloylated hyaluronic acid, phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and primary pancreatic islets; wherein the mass fraction of sodium alginate is 1.5%, the mass fraction of methacryloylated hyaluronic acid is 2%, and the mass fraction of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 0.25%.

[0040] In S1, bubble rupture can be achieved by vacuum suction or needle puncture.

[0041] In S1, the ion-crosslinked aqueous solution is a calcium chloride solution, and the mass concentration of the calcium chloride solution is 2-5%.

[0042] In S1, the inner phase tube and the outer phase tube of the microfluidic device are both glass capillaries, the inner diameter of the inner phase tube is 100-200 μm, and the inner diameter of the outer phase tube is 580-750 μm.

[0043] In S2, the two-component aqueous solutions for achieving dynamic cross-linking based on Schiff base bonds are: oxidized sodium alginate solution and carboxymethyl chitosan solution, wherein the mass fraction of the oxidized sodium alginate solution is 8%, and the mass fraction of the carboxymethyl chitosan solution is 5%; the mixing volume ratio of the oxidized sodium alginate solution and the carboxymethyl chitosan solution is 1:1.

[0044] In S2, the pro-angiogenic components are human umbilical vein endothelial cells and vascular endothelial growth factor.

[0045] The technical solutions of the present invention are described below through the following embodiments;

[0046] Example 1

[0047] In this embodiment, the construction of a bubble-laden hydrogel microfiber generation device is described. Figure 1 As shown, the steps for constructing the bubble-laden hydrogel microfiber generation device include:

[0048] 1) Fabrication of the inner capillary tube 3, the outer capillary tube 5, and the observation tube 4;

[0049] A glass capillary with an outer diameter of 1000 μm and an inner diameter of 580 μm was stretched using a tube puller until the tip was needle-shaped. The tip was then polished with 1500-grit sandpaper to a smooth finish, resulting in an inner diameter of 100-200 μm. This served as inner capillary tube 3. Another glass capillary with an outer diameter of 1000 μm and an inner diameter of 580 μm was selected, cut to an appropriate length, and polished at both ends to serve as outer capillary tube 5. A glass capillary with an outer diameter of 1500 μm and an inner diameter of 1100 μm was cut and polished at both ends to serve as observation tube 4 in the generation device.

[0050] Finally, all the treated capillaries were immersed in ethanol solution and ultrasonically cleaned for 5-10 minutes. After cleaning, they were blown dry with nitrogen or allowed to dry naturally for later use.

[0051] 2) Assembly of the bubble-laden hydrogel microfiber generation device;

[0052] A glass slide is selected as the carrier of the microfluidic core-shell hydrogel microfiber generation device. First, use quick-drying glue to fix the observation tube 4 in the center of the glass slide. Then, the inner phase capillary 3 is nested in the outer phase capillary 5, ensuring that the tip of the inner phase capillary 3 is inserted into the outer phase capillary 5 and aligned with the central axis of the two. The inner phase capillary 3 and the outer phase capillary 5 are fixed on the glass slide by quick-drying glue, and the opening at the junction of the observation tube 4 and the outer phase capillary 5 is closed. Afterwards, take a flat-head needle, carve a groove at the bottom that fits the outer diameter of the glass capillary, and fix it vertically on the glass slide at the junction of the opening of the inner phase capillary 3 and the observation tube 4. Finally, use quick-drying glue to seal around the base of the needle to ensure that it is stable for subsequent use.

[0053] Example 2

[0054] In this example, a method for preparing an injectable hierarchical composite hydrogel using the bubble hydrogel microfiber generation device constructed in Example 1 is described. Figure 1 As shown, the following steps are included:

[0055] Step 1, solution preparation;

[0056] A 10 ml syringe was filled with air to serve as the internal phase component.

[0057] 0.15 g of sodium alginate (Alg) was added to 10 ml of deionized water and stirred to fully dissolve. 0.2 g of methacryloylated hyaluronic acid (HAMA) and 0.025 g of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) were dissolved in the aforementioned sodium alginate solution and stirred to dissolve in the dark to obtain a mixed solution. The mixed solution was sterilized by filtering with a 0.22 μm filter membrane. The solution was then mixed with the extracted primary pancreatic islets according to 10 4The hydrogel cell prepolymerization solution was prepared by mixing the cells at a ratio of 1:1 to 2:1.

[0058] 1 g of calcium chloride solid was dissolved in 50 ml of pure water, and the solution was sterilized by filtration using a 0.22 μm filter membrane to obtain an ion-crosslinked aqueous phase solution (collected solution).

[0059] Step 2, preparation of short fiber scaffold 8;

[0060] The internal phase bubbles are introduced into the inlet of the internal phase capillary 3 of the microfluidic device through the inward air inlet device 1. The hydrogel cell prepolymer solution is introduced into the opening of the external phase capillary 5 through the external phase liquid inlet device 2. The flow rates of the internal and external phase solutions are adjusted to ensure that the bubbles are evenly distributed throughout the hydrogel cell prepolymer solution. At the outlet of the microfluidic device, the external phase solution contacts the collection liquid in the collection device 7. The sodium alginate in the solution is cross-linked by the calcium ions in the collection liquid, initially forming hydrogel microfibers. Subsequently, blue light irradiation with a 405nm blue light source 6 achieves secondary cross-linking, solidifying the internal phase HAMA and ultimately forming bubble-laden hydrogel microfibers. The collected microfibers are punctured with a needle tip to obtain segmented short fiber scaffolds 8.

[0061] In order to observe the structure of the bubble-laden microfibers and short fiber scaffolds, they were characterized using optical microscopy, e.g. Figure 2 As shown in the figure, it is clearly observed that the collected bubble-laden microfibers maintain continuity and integrity, and the bubbles evenly divide the hydrogel microfibers. By puncturing the microfibers, short fibers of similar length are obtained.

[0062] By adjusting the parameters of the microfluidic system, the inner diameter, outer diameter and shell thickness of the hydrogel microfiber can be precisely controlled, such as Figure 3 The length of the short fiber is proportional to the flow rate of the external phase solution and inversely proportional to the flow rate of the internal phase bubble phase; the diameter of the short fiber is proportional to the flow rate of the external phase solution.

[0063] Step 3, preparation of the self-healing hydrogel matrix 11;

[0064] 0.8g of oxidized sodium alginate 9 (OSA) was added to 10ml of deionized water and stirred to fully dissolve; 0.5g of carboxymethyl chitosan 10 (CMCS) was added to 10ml of deionized water and stirred to fully dissolve. The two solutions were sterilized by filtering with a 0.22μm filter membrane. Afterwards, the two solutions were mixed in a 1:1 volume ratio and the angiogenesis-promoting component, human umbilical vein endothelial cells (HUVECs, cell density: 10 6 / mL) and vascular endothelial growth factor (VEGF, mass concentration: 400ng / mL), and wait for about 2 minutes to cross-link into a self-healing hydrogel matrix11;

[0065] In order to observe the microstructure of the self-healing hydrogel matrix 11, it was freeze-dried and observed using a scanning electron microscope. Figure 4 As shown, it can be seen that it has a loose and porous structure. By marking the hydrogel with different colors, it is cut and reassembled, as shown in Figure 5 As shown, the self-healing hydrogel can be completely repaired.

[0066] Step 4, preparation of hierarchical structure composite hydrogel 12;

[0067] By mixing the short fiber scaffold 8 with the self-healing hydrogel matrix 11, a hierarchical composite hydrogel can be obtained; the hydrogel is injectable. Figure 6 As shown, rheology confirms its shear-thinning behavior, and the word "SEU" can also be extruded using a needle tip.

[0068] Due to the incorporation of vascularized components into the self-healing matrix, the composite hydrogel also improved islet function, e.g. Figure 7 Results of a glucose-stimulated insulin release experiment are shown in the figure. As can be seen in the figure, pancreatic islets wrapped in vascularized composite hydrogels can responsively secrete more insulin under high glucose stimulation.

[0069] Example 3

[0070] In this example, the composite hydrogel prepared in Example 2 was implanted subcutaneously on the back of diabetic mice, and in vivo imaging of the small animals was performed to observe the survival status of the transplanted tissues.

[0071] like Figure 8 As shown, the group transplanted with composite hydrogel-encapsulated islets (v-Gel@islet) showed the best cell survival time compared with the groups directly transplanted with naked islets (Naked) and without added vascularization components (Gel@islet).

[0072] Blood glucose monitoring results such as Figure 9 As shown, similar good blood sugar control effect was also demonstrated (the arrow indicates the v-Gel@islet group).

[0073] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0074] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.

Claims

1. A method for preparing an injectable hierarchical structure composite hydrogel for pancreatic islet transplantation, characterized in that: The following steps are involved: In the microfluidic device, air bubbles are introduced into the inner phase tube and the hydrogel cell prepolymer solution is introduced into the outer phase tube, so that the bubbles are evenly distributed in the hydrogel cell prepolymer solution. The solution is then mixed with an ion-crosslinked aqueous solution and irradiated with blue light to obtain bubble-loaded hydrogel microfibers. After the bubbles burst, pancreatic islet-loaded short fiber scaffolds are obtained. The oxidized sodium alginate solution was mixed with the carboxymethyl chitosan solution to encapsulate the pro-angiogenic components to obtain a self-healing hydrogel matrix; The short fiber scaffold is mixed with the self-healing hydrogel matrix to obtain the result.

2. The method for preparing the injectable hierarchical structure composite hydrogel for pancreatic islet transplantation according to claim 1, characterized in that: The hydrogel cell prepolymerization solution comprises: deionized water, sodium alginate, methacrylated hyaluronic acid, phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt and primary pancreatic islets; The mass fraction of the sodium alginate is 1.5%, the mass fraction of the methacrylated hyaluronic acid is 2%, and the mass fraction of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 0.25%.

3. The method for preparing the injectable hierarchical structure composite hydrogel for pancreatic islet transplantation according to claim 2, characterized in that: The amount of primary islets added was 10 4 pcs / ml.

4. The method for preparing the injectable hierarchical structure composite hydrogel for pancreatic islet transplantation according to claim 1, characterized in that: Bubble bursting is achieved by vacuum aspiration or needle puncture.

5. The method for preparing the injectable hierarchical structure composite hydrogel for pancreatic islet transplantation according to claim 1, characterized in that: The ion-crosslinked aqueous solution is a calcium chloride solution, and the mass concentration of the calcium chloride solution is 2-5%.

6. The method for preparing the injectable hierarchical structure composite hydrogel for pancreatic islet transplantation according to claim 1, characterized in that: The inner phase tube and the outer phase tube are both glass capillaries. The inner diameter of the inner phase tube is 100-200 μm, and the inner diameter of the outer phase tube is 580-750 μm.

7. The method for preparing the injectable hierarchical structure composite hydrogel for pancreatic islet transplantation according to claim 1, characterized in that: The mass fraction of the oxidized sodium alginate solution is 8%, and the mass fraction of the carboxymethyl chitosan solution is 5%; the mixing volume ratio of the oxidized sodium alginate solution to the carboxymethyl chitosan solution is 1:

1.

8. The method for preparing the injectable hierarchical structure composite hydrogel for pancreatic islet transplantation according to claim 1, characterized in that: The angiogenesis-promoting components are human umbilical vein endothelial cells and vascular endothelial growth factor.

9. A composite hydrogel, characterized in that The injectable hierarchical structure composite hydrogel for pancreatic islet transplantation is prepared using the preparation method of any one of claims 1 to 8.

10. Use of the composite hydrogel according to claim 9 in preparing a pancreatic islet transplantation carrier.

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