Pancreatic islet-like sphere injectable hydrogel based on double-sequence packaging as well as preparation and application of pancreatic islet-like sphere injectable hydrogel

Abstract

The invention discloses pancreas islet-like sphere injectable hydrogel based on double-sequence packaging as well as preparation and application of the pancreas islet-like sphere injectable hydrogel, and belongs to the technical field of bioengineering medical materials. According to the preparation method, a high polymer material with positive and negative charges is self-assembled layer by layer in an electrostatic adsorption manner to form a pancreas islet beta cell immune protection film, and then the pancreas islet beta cell immune protection film is prepared by using the characteristic of temperature sensitivity of a spleen extracellular matrix, namely the characteristic of self-assembling into a nanofiber gel structure at an in-vitro liquid state and an in-vivo physiological temperature. The injectable pancreatic islet-like sphere hydrogel is prepared by using a nano-fiber as a template, so that the stability of the hydrogel structure is ensured, the hydrogel is assembled into a nano-fiber three-dimensional gel structure in vivo, a natural specific microenvironment is provided for self-renewal and differentiation of cells, beta cells are promoted to be gathered into pancreatic islet-like spheres, and the insulin secretion and vascularization capability are enhanced. The gelatin and the alginate are self-assembled layer by layer to form an immune protection film on the surface of the pancreatic beta cell, so that beta cell immune protection encapsulation is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering medical materials, and in particular relates to an injectable hydrogel of islet-like spheres based on double sequential encapsulation, and the preparation and application thereof. Background Art

[0002] As of 2021, diabetes has affected over 537 million people worldwide, leading to serious health complications and death. Patients with type 2 diabetes (T2D) experience multiple impairments in blood glucose regulation, including: (1) decreased insulin secretion; (2) insulin resistance in muscle, liver, and adipocytes; and (3) glucose uptake problems in visceral areas.

[0003] Islet transplantation is typically focused on treating type 1 diabetes. This approach is also applicable to patients with advanced type 2 diabetes, whose beta-cell function is failing, as opposed to those in the earlier stages who present with insulin resistance. Late stage type 2 diabetes is characterized by insulin deficiency. Exogenous pancreatic beta-cell transplantation is an attractive strategy to restore glycemic control and prevent the progression of diabetes and its associated complications. However, the potential risks of long-term immunosuppression and the long-term survival of the transplanted cells remain hot issues that need to be addressed.

[0004] Recent advances in microencapsulation technology for cell delivery have shown promising results. This technique can mitigate rejection of immune cell transplants without the use of immunosuppressive drugs with side effects. Alginate is an optimal material for microencapsulation, and its performance has been validated in multiple animal models. To leverage the immunoprotective properties of alginate while mitigating the immune response caused by structural disruption of the encapsulation, the applicant's laboratory developed a method for immunoprotective encapsulation of β cells via layer-by-layer (LbL) self-assembly of gelatin and alginate onto the cell surface. This coating does not interfere with cell migration and exhibits excellent immunogenicity. However, LbL coatings alone cannot provide a favorable three-dimensional environment to promote β cell growth and self-assembly. On surfaces, cell movement is two-dimensional, with nearly every layer being planar rather than a true three-dimensional shape. A 3D environment, such as a scaffold or hydrogel, provides cells with opportunities for movement and multidimensional interactions; in this way, it more effectively replicates the tissue environment than a two-dimensional environment. Typically, cells function best under the realistic environmental conditions of their life cycle, which resemble the natural "three-dimensional" cellular tissue structure and thus allow for diverse directional interactions. In LbL systems, the layers may not form structures that allow the passage of nutrients, gases, and other factors that are essential for the survival and proliferation of living cells. In natural tissues, cells can communicate with each other and with the extracellular matrix (ECM) within a three-dimensional spatial geometry. The traditional extracellular matrix microenvironment can only provide limited oxygen and nutrients in vivo, resulting in limited vascularization of β cells after transplantation. This ultimately hinders their long-term survival and function.

[0005] The inherently two-dimensional nature of LBL self-assembly makes it difficult to provide the comprehensive conditions that a true three-dimensional environment can provide to promote optimal cell growth and function. The extracellular matrix within the cell encapsulation can provide a natural, specific microenvironment for cell self-renewal and differentiation. Decellularized extracellular matrix, composed of collagen, elastin, glycosaminoglycans, and growth factors, has demonstrated numerous benefits in tissue repair services and healing, such as promoting cardiac regeneration (zebrafish cardiac extracellular matrix), reducing oxidative stress (liver extracellular matrix), and improving the survival and integration of transplanted grafts. Current research indicates that the extracellular matrix influences β-cell growth, differentiation, and function through interactions with integrins, cell adhesion receptors. Furthermore, impairment of islet-extracellular matrix interactions throughout the islet isolation process can lead to significant impairment of islet function.

[0006] Related technologies disclose the use of silk fibroin to prepare hydrogels. However, this type of silk fibroin requires a relatively long gelation time (4 days) in aqueous media, making it unsuitable for preparing injectable hydrogels and exhibiting extremely poor degradation properties. Related technologies utilize phenylboronic acid in combination with silk fibroin to promote its gelation, thereby ensuring the degradation properties of the hydrogel. These technologies primarily focus on the degradation properties of the hydrogels and do not address how to effectively maintain long-term blood sugar control and alleviate related complications in the treatment of diabetes and its complications. Summary of the Invention

[0007] The first purpose of the present invention is to provide a preparation method of an injectable islet-like spheroid hydrogel based on double sequential encapsulation. The preparation method utilizes polymer materials with positive and negative charges, and self-assembles layer by layer through electrostatic adsorption to form an immune protective membrane for pancreatic beta cells. The temperature-sensitive characteristics of the spleen extracellular matrix, that is, the liquid state in vitro and the self-assembly into a nanofiber gel structure at physiological temperature in vivo, are then utilized to prepare an injectable islet-like spheroid hydrogel, thereby ensuring the stability of the hydrogel structure.

[0008] The second object of the present invention is to provide an islet-like spheroid injectable hydrogel based on double sequential encapsulation, which is prepared by mixing LbL-coated β cells with spleen hydrogel (SpGel) to prepare an islet-like spheroid injectable hydrogel, and transplanting it into the body. By applying LbL coating without the use of immunosuppressants, initial immune protection of β cells is achieved. At the same time, the spleen hydrogel SpGel can self-assemble into nanofiber hydrogel under physiological temperature conditions and can promote immunomodulation and angiogenesis properties. Through the combination of these two methods, β cells transplanted into the kidney tissue of diabetic rats can effectively maintain long-term blood sugar level control, thereby effectively alleviating the in vivo complications caused by T2D.

[0009] The present invention is achieved through the following technical solutions:

[0010] A method for preparing an injectable hydrogel of islet-like spheroids based on double sequential encapsulation comprises the following steps:

[0011] S1. Using positively charged polymer materials and negatively charged polymer materials, a layer-by-layer self-assembly technique is used to form an immune protective membrane on the surface of pancreatic β cells.

[0012] The specific process of the layer-by-layer self-assembly technology is as follows:

[0013] Add a solution of positively charged polymer materials to pancreatic β cells, shake, centrifuge, and discard the supernatant;

[0014] Pancreatic beta cells are incubated in a negatively charged polymer solution, repeatedly incubated to obtain a cell coating with different interlayer structures as an immune protective membrane;

[0015] The ratio of the pancreatic beta cells, the positively charged polymer material solution, and the negatively charged polymer material solution is 1:1-10ml:1-10ml;

[0016] The concentration of pancreatic β cells is 1×10 6 ~5×10 6 , the mass concentration of the positively charged polymer solution is 0.1% to 2%, and the mass concentration of the negatively charged polymer solution is 0.1% to 2%;

[0017] S2. The cells encapsulated by the immune protective membrane are combined with spleen decellularized extracellular matrix hydrogel of the same or xenogeneic origin to form an injectable three-dimensional islet-like spheroid hydrogel.

[0018] Preferably, in S1, the positively charged polymer material includes one or more of gelatin and gelatin derivatives, chitosan and chitosan derivatives, and polylysine and polylysine derivatives;

[0019] The negatively charged polymer material includes one or more of sodium alginate and its derivatives, heparin and its derivatives, hyaluronic acid and its derivatives;

[0020] The positively charged polymer material solution is added to the pancreatic β cells, shaken for 5 to 20 minutes, and centrifuged at 2000 rpm for 5 to 8 minutes;

[0021] The pancreatic islet β cells are incubated in the negatively charged polymer material solution for 10 to 60 minutes;

[0022] The number of repetitions is 1 to 7 times.

[0023] Preferably, the pancreatic β cells include pancreatic β cells extracted from tissue or pancreatic β cells induced from human induced pluripotent stem cells.

[0024] Preferably, in S1, the cell coating with different interlayer structures includes:

[0025] Single-layer coating: a layer of positively charged polymer material;

[0026] Or, three-layer coating: alternating positively charged polymer material, negatively charged polymer material and positively charged polymer material;

[0027] Or, five layers: alternately use positively charged polymer material, negatively charged polymer material, positively charged polymer material, negatively charged polymer material, and positively charged polymer material.

[0028] Preferably, in S2, 1 to 10 μL of pancreatic beta cells encapsulated with an immune protective membrane are added to 10 to 100 μL of spleen hydrogel, and the mixture is evenly mixed by pipetting.

[0029] Preferably, the spleen hydrogel is composed of decellularized spleen extracellular matrix of homologous or xenogeneic origin.

[0030] The invention discloses an injectable hydrogel for pancreatic islet-like spheroids based on double sequential encapsulation. The hydrogel is obtained by a preparation method.

[0031] Application of an injectable hydrogel of islet-like spheroids based on double sequential encapsulation in the preparation and treatment of diabetes and its complications.

[0032] Preferably, the complications include any one of cataract, osteoporosis and diabetic nephropathy.

[0033] Compared with the prior art, the present invention has at least the following technical effects:

[0034] (1) The present invention provides a method for preparing an injectable islet-like spheroid hydrogel based on double sequential encapsulation. The preparation method utilizes polymer materials with positive and negative charges, which are self-assembled layer by layer through electrostatic adsorption to form an immune protective membrane for pancreatic β cells. The temperature-sensitive characteristics of the spleen extracellular matrix, that is, the liquid state in vitro and the self-assembly into a nanofiber gel structure at physiological temperature in vivo, are then utilized to prepare an injectable islet-like spheroid hydrogel, thereby ensuring the stability of the hydrogel structure.

[0035] (2) The islet-like spheroid injectable hydrogel based on double sequential encapsulation is prepared by mixing LbL-coated β cells with spleen hydrogel (SpGel) to prepare an islet-like spheroid injectable hydrogel, which is then transplanted into the body. By applying LbL coating without the use of immunosuppressants, initial immune protection of β cells is achieved. At the same time, the spleen hydrogel SpGel can self-assemble into nanofiber hydrogels under physiological temperature conditions and can promote immunomodulatory and angiogenic properties. Through the combination of these two methods, β cells transplanted into the kidney tissue of diabetic rats can effectively maintain long-term blood sugar level control, thereby effectively alleviating the in vivo complications caused by T2D.

[0036] (3) This method for preparing injectable islet-like spheroid hydrogels based on dual sequential encapsulation discloses an immunoprotective approach for pancreatic β-cell encapsulation. This approach involves layer-by-layer (LbL) self-assembly of gelatin and alginate to form an immunoprotective coating on the surface of pancreatic β-cells, achieving β-cell immune encapsulation. This coating does not interfere with cell migration and exhibits excellent immunosuppression, addressing the potential risk of long-term immunosuppression associated with islet cell transplantation.

[0037] (IV) This method for preparing an injectable hydrogel for islet-like spheroids based on double sequential encapsulation discloses a three-dimensional growth space for pancreatic β cells. Immunoprotected pancreatic β cells are mixed with spleen extracellular matrix in a liquid state in vitro and injected in situ into the body, where they assemble into a nanofiber three-dimensional gel structure. This provides a natural, specific microenvironment for cell self-renewal and differentiation, promotes β cell aggregation into islet-like spheroids, and enhances insulin secretion and vascularization. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of dual encapsulation of LbL self-assembled membrane and SpGel hydrogel and the in vivo transplantation process;

[0039] Figure 2 Schematic diagram of double sequential encapsulation of pancreatic β cells;

[0040] Figure 3 Schematic diagram of SpGel-promoted islet-like spheroid formation in LbL-coated INS-1 cells;

[0041] Figure 4 Schematic diagram of the transplantation of SpGel-encapsulated LbL-INS-1 cells into obese ZDF rats;

[0042] Figure 5 Schematic diagram of the combined LbL assembly and SpGel encapsulation to synergistically enhance INS-1 cell retention and angiogenesis at the transplantation site in ZDF rats. DETAILED DESCRIPTION

[0043] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0044] The technical solution of a specific embodiment of the present invention is:

[0045] 1. Double packaging structure design:

[0046] The first layer of encapsulation: Using layer-by-layer (LbL) self-assembly technology, positively charged polymer materials and negatively charged polymer materials are alternately coated to form a physical barrier to provide immune protection for β cells.

[0047] The second layer of encapsulation: an injectable hydrogel prepared using spleen extracellular matrix (SpGel) simulates the three-dimensional microenvironment of the natural extracellular matrix (ECM), promotes the aggregation of β cells into islet-like spheroids, and enhances insulin secretion and vascularization capabilities.

[0048] 2. Application of SpGel materials:

[0049] SpGel is composed of decellularized porcine spleen ECM proteins, which can self-assemble into nanofibrous hydrogels at physiological temperature and have pro-angiogenic and immunomodulatory properties.

[0050] SpGel activates the downstream FAK / Erk / Akt pathway through the integrin αvβ1 signaling pathway, significantly improving β-cell survival rate, proliferation ability and insulin secretion function.

[0051] 3. Comprehensive treatment of diabetic complications:

[0052] After transplantation, the double-encapsulated pancreatic β cells can stably control blood glucose for a long time (≥35 days) and improve local microcirculation through the vascularization effect of SpGel.

[0053] Significantly alleviated diabetic complications, including cataracts (reduced lens opacity), osteoporosis (bone density increased by 40%), and diabetic nephropathy (reduced renal fibrosis by 50%).

[0054] 4. Technical effect verification:

[0055] In vitro experiments: LbL-INS-1 cells were cultured in 3D SpGel for 7 days, and the insulin secretion increased by 3 times (140.94±26.65 ng / mL), and the cell survival rate was >90%.

[0056] In vivo experiments: In the ZDF diabetic rat model, blood glucose levels of SpGel-encapsulated LbL-INS-1 cells remained stable within the normal range (5-7 mmol / L) for up to 35 days after transplantation, with no significant immune rejection reaction.

[0057] Experimental Example 1: Preparation of LbL-encapsulated β cells

[0058] 2×10 6 INS-1 cells (rat islet cell carcinoma cells, a pancreatic β-cell model) were centrifuged to remove excess culture medium.

[0059] Add 1 mL of 0.1% gelatin solution to the tube and gently shake for 10 minutes. Then, centrifuge at 2000 rpm for 5 minutes. Discard the supernatant. Wash the cells with 5 mL of DPBS and centrifuge again, discarding the supernatant.

[0060] The cells were then incubated in 1 mL of 0.1% sodium alginate solution for 10 minutes. This step was repeated to form different layers of cell coating. For a single-layer coating, the cells were treated with gelatin alone. For a three-layer coating, gelatin, sodium alginate, and gelatin were applied alternately. For a five-layer coating, gelatin, sodium alginate, gelatin, sodium alginate, and gelatin were applied alternately. A seven-layer coating was achieved by alternating gelatin and sodium alginate treatments for three rounds, ending with gelatin. Finally, a nine-layer coating was achieved by repeating this process four times and ending with a gelatin coating.

[0061] Example 2: Preparation of SpGel hydrogel and cell encapsulation

[0062] Preparation of SpGel hydrogel:

[0063] Spleen tissue of the same or different species (such as pig) stored in the refrigerator was slowly rewarmed for 5 hours, the fat tissue such as the spleen hilum was removed, and cut into 1mm 3 The spleen tissue pieces were soaked in physiological saline for rinsing, and then soaked in decellularization fluid (0.3% SDS + 1.5 EDTA, and the pH was adjusted to neutral), placed at 37 ° C, 80 r / min and shaken for 24 h, the decellularization fluid was replaced, and placed at 25 ° C, 60 r / min and shaken for 36 h to obtain decellularized spleen extracellular matrix.

[0064] The decellularized spleen extracellular matrix was rinsed with deionized water at 25°C and 80 rpm (the water was changed every 60 minutes, for a total of 15 times), the residual decellularized fluid was removed, and the solution was vacuum-lyophilized (lyophilization temperature was -60°C, vacuum degree was 0.1-0.2 mbar, and time was 48 hours). The solution was ground into a homogenate in 0.01 M hydrochloric acid solution, pepsin (concentration was 1 mg / ml) was added, and the solution was stirred and digested at 25°C and 100 rpm for 24 hours to obtain a uniform, viscous, milky white spleen extracellular matrix solution.

[0065] Under ice bath conditions, the pH of the spleen extracellular matrix (ECM) solution was adjusted to neutral with 1 M sodium hydroxide solution, and then a 10% PBS solution of the ECM solution was added and stored at 4°C.

[0066] LBL cell encapsulation:

[0067] Decellularized porcine spleen ECM was dissolved in PBS, and 1 μL of LbL-encapsulated INS-1 cells was mixed with 10 μL of SpGel pre-solution and injected under the renal capsule of ZDF rats to form a three-dimensional islet-like spheroid gel in vivo.

[0068] Example 3: In vitro and in vivo effect verification

[0069] 3.1 In vitro effect verification

[0070] 3.1.1 Cell morphology and insulin secretion capacity:

[0071] Rat insulinoma cells, INS-1, have the ability to secrete large amounts of insulin and share similarities with Langerhans-type pancreatic islets. Therefore, the inventors' team used INS-1 cells as a model cell. To improve the functionality and protective properties of INS-1 cells, the inventors' team investigated the application of LbL self-assembly.

[0072] like Figure 1 Shown is a schematic diagram of dual encapsulation of LbL self-assembled film and SpGel hydrogel and the in vivo transplantation process.

[0073] Figure 1 The results showed that the process involves the sequential application of multiple layers of polycationic and polyanionic polymers on the surface of INS-1 cells to form a thin outer cell membrane.

[0074] This membrane offers several potential benefits, including enhanced stability and protection, controlled release of growth factors, and improved biocompatibility.

[0075] like Figure 2Figure 1 shows a schematic diagram of the dual sequential encapsulation of pancreatic beta cells. A shows a transmission electron microscopy image of untreated INS-1 cells and cells coated with five layers of (gelatin) 3 / (alginate) 2. Arrows indicate the unique thin LBL assembly layer deposited on the surface of the encapsulated cells. Scale bars are 0.2 μm, 0.5 μm, 1 μm, and 200 nm. B shows a scanning electron microscopy image showing that the diameter of the cell clusters after 7 days of growth is comparable for untreated and five-layer gelatin / alginate-coated cells. Scale bars are 20 μm (top) and 1 μm (bottom). C shows a representative fluorescence micrograph of INS-1 cells coated using the LBL technique, using FITC-labeled gelatin and rhodamine B-labeled alginate to visualize the encapsulation process. Cell nuclei were counterstained with Hoechst. Scale bar is 20 μm. D shows immunofluorescence staining of a typical INS-1 marker (insulin) in untreated cells and cells coated with three, five, seven, and nine layers of LBL. Scale bar: 30 μm. E: Percentage of insulin-positive cells in untreated cells and cells coated with 3, 5, 7, or 9 layers of LBL. F: Basal insulin secretion levels in INS-1 cells and cells coated with 3, 5, 7, or 9 layers of LBL after 7 days of culture in 1640 medium.

[0076] The effectiveness of LbL encapsulation on INS-1 cells was evaluated by examining cell morphology and insulin secretion capacity.

[0077] Combining the results Figure 2 As shown in Figure A, transmission electron microscopy (TEM) confirmed the presence of a gelatin / sodium alginate layer on the surface of 5 layers of LBL-coated INS-1 (LbL-INS-1) cells.

[0078] Combining the results Figure 2 As shown in B, scanning electron microscopy (SEM) images showed that after 7 days of culture, both INS-1 and LbL-INS-1 cells showed a large number of cell contacts, and there was no significant difference in cell morphology and distribution between the two groups.

[0079] The inventor team preliminarily evaluated the impact of LbL self-assembly on cell stability and function.

[0080] Combining the results Figure 2 As shown in Figure C, from left to right, gelatin and alginate are labeled with FITC and rhodamine B, respectively, to facilitate monitoring of the LbL coating process on the cell surface. The third picture shows the cell nucleus counterstained with Hoechst dye, and the fourth picture is a comprehensive image obtained by merging the first three staining techniques.

[0081] The cultured INS-1 and LbL-INS-1 cells (initial number 3×10 4 cells) insulin secretion levels.

[0082] Combining the results Figure 2 As shown in D, under the same conditions, the more layers the cells are packaged, the faster the cells aggregate. When the cells are packaged into five layers, the fluorescence intensity of live insulin-producing cells is significantly different.

[0083] Combining the results Figure 2 As shown in Figure E, quantitative analysis showed that the percentages of insulin-positive cells in untreated cells and cells coated with 3, 5, 7, and 9 layers of LBL were 0.85%, 37%, 38%, 42%, 52%, and 78%, respectively. The percentage of insulin-positive cells gradually increased with the number of coated layers.

[0084] Combining the results Figure 2 From F in Figure 3, we can see that the insulin secretion amounts of single-layer, three-layer, five-layer, seven-layer and nine-layer LBL-INS-1 cells were 13.92±11.89ng / ml, 25.36±11.57ng / ml, 28.13±11.84ng / ml, 33.24±11.89ng / ml, 41.03±11.41ng / ml and 40.99±11.82ng / ml, respectively.

[0085] After five layers of product packaging, there was a significant difference in insulin secretion, reaching a peak at the seventh layer, while by the ninth layer, the data did not change significantly compared to the seventh layer.

[0086] Therefore, the inventor team chose to use five layers in subsequent experiments, which not only has an immunomodulatory effect but also promotes the secretion of large amounts of insulin.

[0087] like Figure 3The figure shows a schematic diagram of SpGel-promoted islet-like spheroid formation in LbL-coated INS-1 cells. A is a bright field image of INS-1 cells cultured in 2D or 3D SpGel for 3 days; B is a bright field image of LbL-INS-1 cells cultured in 2D or 3D SpGel for 3 days; C is the quantification of islet-like spheroids formed by INS-1 cells after culturing in 2D or 3D SpGel for 3 days; D is the quantification of islet-like spheroids formed by LbL-INS-1 cells after culturing in 2D or 3D SpGel for 3 days; E is a bright field image of LbL-INS-1 cells cultured in 2D SpGel for 7 days, with prominent islet-like spheroids shown in green; F is a bright field image of LbL-INS-1 cells cultured in 3D SpGel for 7 days, with prominent islet-like spheroids shown in green; G is the number of islet-like spheroids in 2D SpGel and 3D SpGel calculated using 3D image scanning. The average volume, perimeter, area, and diameter of LbL-INS-1 spheroids formed after 7 days of culture in SpGel. H: Representative SEM images of INS-1 cells after 7 days of culture in 2D or 3D SpGel. The green box highlights the area where islet-like spheroids have formed. I: Representative SEM images of LbL-INS-1 cells after 7 days of culture in 2D or 3D SpGel. The green box highlights the area where islet-like spheroids have formed. J: Immunofluorescence staining of CASK protein, a typical INS-1 cell marker, in INS-1 and LbL-INS-1 cells after 7 days of culture in 3D SpGel. K: Quantitative comparison of CASK protein positivity in INS-1 and LbL-INS-1 cells after 7 days of culture in 3D SpGel, as well as the corresponding comparison.

[0088] 3.1.2 In vitro spheroid formation and blood glucose regulation ability:

[0089] Combining the results Figure 3 As shown in Figures A and B, the inventors' team investigated the ability of INS-1 cells to form spheroids under different conditions. When cultured on or within SpGel, both cell types exhibited similar islet-like morphology, leading to spheroid formation.

[0090] Further analysis and evaluation showed that the results combined with Figure 3 As shown in Figures C and D, more spheres were formed when cultured in 3D SpGel than when cultured on the 2D SpGel surface.

[0091] Combining the results Figure 3 As shown in Figures E and F, after 7 days of culture, the morphology and polarization of LbL-INS-1 and INS-1 cell spheroids were observed on the SpGel surface or inside the SpGel.

[0092] Combining the results Figure 3 As shown in Figure E, when cultured on 2D SpGel, cell spheroids tend to expand outward and polarize along the apical-basal axis. Figure 3 As shown in Figure F, in 3D SpGel, cell spheroids tend to aggregate into star-shaped shapes.

[0093] Combining the results Figure 3 As shown in Figure 5, the spheroid diameter of the 2D SpGel group was significantly larger than that of the 3D SpGel group. However, considering the spheroid perimeter and area, which were measured from the spheroid properties, the 3D gel showed significantly larger values ​​compared to the 2D SpGel. These findings suggest that 3D SpGel plays an important role in promoting the development of LbL-INS-1 cell spheroid structures.

[0094] Combining the results Figure 3 As shown in Figures H and I, scanning electron microscopy (SEM) was used to identify the junctions between cells and SpGel, and observations were made under both two-dimensional and three-dimensional culture conditions.

[0095] Combining the results Figure 3 As shown in Figure 1, both INS-1 cells and LbL-INS-1 cells formed spheroids in both three-dimensional SpGel and two-dimensional SpGel.

[0096] Combining the results Figure 3 As shown in Figure H, some INS-1 cells were not aggregated in the two-dimensional SpGel.

[0097] Combining the results Figure 3 As shown in Figure J, the CASK-positive levels of INS-1 cells and LbL-INS-1 cells in three-dimensional SpGel were compared.

[0098] Combining the results Figure 3 As shown in Figure 3, there was no significant increase in CASK expression between the LbL-INS-1 group and the INS-1 group.

[0099] This suggests that changes in morphology and polarity between 2D and 3D cultured cells may affect cellular activities, such as protein synthesis and function. Based on these findings, it can be concluded that SpGel may not directly affect INS-1 cells but may be more effective than traditional culture media in maintaining their functional state, especially during long-term culture.

[0100] 3.2 In vivo effect verification

[0101] 3.2.1 Ability to treat diabetes and its complications in vivo:

[0102] like Figure 4As described above, SpGel-encapsulated LbL-INS-1 cells were transplanted into obese ZDF rats. Among them, A is a representative image of SpGel-encapsulated LbL-INS-1 cell subrenal transplantation in overweight ZDF rats. The white arrow points to the transplantation web, representing the encapsulation effect. The scale bar is 20 μm. B is a representative longitudinal change in blood glucose levels in obese ZDF rats under all different treatments. C is the distribution and dynamic fate of DiR-labeled LbL-INS-1 cells and LbL-INS-1+SpGel constructs after subrenal transplantation in SD rats observed by non-invasive in vivo fluorescence imaging technology. D is a representative progression of cataracts in obese ZDF rats receiving LbL-INS-1+SpGel subrenal transplantation compared with age-matched untreated lean ZDF rats and obese ZDF rats, demonstrating the therapeutic effect of LbL-INS-1+SpGel treatment on cataract development (n=12). The scale bar is 1 μm. E and F are representative microCT images of the distal femur bone status of obese ZDF rats 35 days after LbL-INS-1+SpGel pararenal transplantation, compared with age-matched untreated lean ZDF rats and obese ZDF rats (E). Quantitative analysis of bone mineral density (BMD) and bone volume / total volume (BV / TV) was performed, demonstrating the effect of this treatment on bone health (F).

[0103] Combining the results Figure 4 As shown in Figure A, LbL-INS-1 cells were implanted beneath the renal capsule of obese ZDF rats. These rats are genetically predisposed to developing type 2 diabetes and are therefore an ideal model for evaluating the therapeutic potential of transplanted LbL-INS-1 cells.

[0104] Combining the results Figure 4 As shown in Figure B, after transplantation, the inventors encapsulated the LbL-INS-1 cells in a SpGel matrix. Unlike the encapsulated LbL-INS-1 cells, whose blood glucose returned to its initial value within 7 days, the cells encapsulated in SpGel showed sustained efficacy for approximately 35 days.

[0105] Combining the results Figure 4 Figure C shows the effect of LbL-INS-1 cells on blood glucose regulation in diabetic rats after transplantation. The results showed that unencapsulated LbL-INS-1 cells were less effective in lowering blood glucose levels through insulin production. In contrast, when LbL-INS-1 cells were encapsulated in the SpGel matrix, this natural microenvironment not only supported the survival of LbL-INS-1 cells but also enhanced their insulin secretion capacity, thereby significantly reducing blood glucose levels in diabetic rats.

[0106] Diabetes is known to trigger a range of secondary health complications, among which cataracts, osteoporosis, and kidney disease are common. Based on the positive results in blood sugar regulation, the inventors' team then investigated the effectiveness of SpGel-encapsulated LbL-INS-1 cells in alleviating these diabetes-induced complications in vivo.

[0107] Combining the results Figure 4 As shown in D, compared with the obese control group, the implantation of SpGel-encapsulated LbL-INS-1 cells significantly reduced lens opacity and reversed the progression of cataracts in diabetic rats.

[0108] Combining the results Figure 4 As shown in E, the inventors' team explored the possible development of osteopenia by examining the bone structure and morphology of the distal thigh of diabetic rats. Micro-computed tomography (Micro-CT) evaluation showed that the bone mineral density (BMD) of the overweight group was significantly reduced to 0.19±0.01 g / cm 2 , which was much lower than the BMD of the negative fat group, which was 0.38±0.02g / cm 2 After LbL-INS-1+SpGel treatment, the bone density of diabetic rats significantly improved to 0.28±0.01g / cm 2 .

[0109] Combining the results Figure 4 As shown in Figure 5, the bone mass ratio (BV / TV) of the obese group was only 10.07±0.64%. However, after receiving LbL-INS-1+SpGel treatment, the bone mass ratio increased significantly to 24.05±0.98%, close to the normal value of 26.93±1.18%.

[0110] like Figure 5 Figure 1 shows the synergistic enhancement of INS-1 cell retention and angiogenesis at the transplant site in ZDF rats by combined LbL assembly and SpGel encapsulation. A shows representative HE-stained renal sections from obese ZDF rats at 0, 1, 2, 3, and 4 weeks post-implantation that received infrarenal transplantation of LbL-INS-1 + SpGel. B shows the numerical assessment of capillary density. C shows immunohistochemical analysis of LbL-INS-1 + SpGel at 1 week post-transplantation. CD20 expression was visually observed and recorded. + and CD11b + CD20 was detected by visual observation and recorded. + and CD11b + The presence of immune cells such as cells.

[0111] 3.2.2 In vivo vascularization effect:

[0112] Combining the results Figure 5 As shown in Figure A, based on the above-mentioned research results on blood glucose regulation, a histological evaluation was performed to study the vascularization effect of SpGel-encapsulated LbL-INS-1 cells after transplantation in the subrenal capsule of ZDF rats. Kidney sections of these rats were HE stained at 0, 1, 2, 3 and 4 weeks after implantation. The results showed that after LbL assembly in SpGel, the retention rate of INS-1 cells increased and angiogenesis increased. It is worth noting that, as shown by the red arrows, the site of vascular origin showed significant changes compared to the baseline (0 weeks). Treatment with LbL-INS-1+SpGel significantly increased the density of arteries and capillaries, indicating enhanced neovascularization in the implanted area.

[0113] Combining the results Figure 5 As shown in Figure B, the capillary density of the 4th week group was significantly higher than that of the control group, indicating that SpGel-encapsulated LbL-INS-1 cells promoted the formation of new blood vessels in the subrenal capsule area.

[0114] Immune cell activation was investigated using immunohistochemistry, with GelMA as an experimental control. Graft performance was systematically assessed in all recipients. Within this context, LbL was used in conjunction with SpGel or GelMA. INS-1 plus SpGel was included as another control group.

[0115] Examinations were performed at two different time points: 1 week after transplantation (LbL-INS-1+GelMA vs. LbL-INS-1+SPGel vs. INS-1+SpGel) ( Figure 5 C) and 4 weeks after transplantation (LbL-INS-1+GelMA vs. LbL-INS-1+SPGel vs. INS-1+SpGel) ( Figure 5 D) in.

[0116] Immune cell staining such as CD20 + and CD11b + The analysis showed that there were significant differences among the groups, among which the results of the LbL-INS-1+SPGel group were more ideal.

[0117] Together, the results suggest that mixed application of LbL-INS-1 cells and SpGel has a positive impact on graft performance and may reduce the activation of specific immune cell populations compared with the control group.

[0118] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A method for preparing an injectable hydrogel of islet-like spheroids based on double sequential encapsulation, characterized in that: The steps include: S1. Using positively charged polymer materials and negatively charged polymer materials, a layer-by-layer self-assembly technique is used to form an immune protective membrane on the surface of pancreatic β cells. The specific process of the layer-by-layer self-assembly technology is as follows: Add a solution of positively charged polymer materials to pancreatic β cells, shake, centrifuge, and discard the supernatant; Pancreatic beta cells are incubated in a negatively charged polymer solution, repeatedly incubated to obtain a cell coating with different interlayer structures as an immune protective membrane; The ratio of the pancreatic beta cells, the positively charged polymer material solution, and the negatively charged polymer material solution is 1:1-10ml:1-10ml; The concentration of pancreatic β cells is 1×10 6 ~5×10 6 , the mass concentration of the positively charged polymer solution is 0.1% to 2%, and the mass concentration of the negatively charged polymer solution is 0.1% to 2%; S2. The cells encapsulated by the immune protective membrane are combined with spleen decellularized extracellular matrix hydrogel of the same or xenogeneic origin to form an injectable three-dimensional islet-like spheroid hydrogel.

2. The method for preparing an injectable islet-like spheroid hydrogel based on double sequential encapsulation according to claim 1, characterized in that: In said S1, the positively charged polymer material includes one or more of gelatin and gelatin derivatives, chitosan and chitosan derivatives, and polylysine and polylysine derivatives; The negatively charged polymer material includes one or more of sodium alginate and its derivatives, heparin and its derivatives, hyaluronic acid and its derivatives; The positively charged polymer material solution is added to the pancreatic β cells, shaken for 5 to 20 minutes, and centrifuged at 2000 rpm for 5 to 8 minutes; The pancreatic islet β cells are incubated in the negatively charged polymer material solution for 10 to 60 minutes; The number of repetitions is 1 to 7 times.

3. The method for preparing an injectable islet-like spheroid hydrogel based on double sequential encapsulation according to claim 2, characterized in that: The pancreatic β cells include pancreatic β cells extracted from tissue or pancreatic β cells induced from human induced pluripotent stem cells.

4. The method for preparing an injectable islet-like spheroid hydrogel based on double sequential encapsulation according to claim 1, characterized in that: In said S1, the cell coating with different interlayer structures includes: Single-layer coating: a layer of positively charged polymer material; Or, three-layer coating: alternating positively charged polymer material, negatively charged polymer material and positively charged polymer material; Or, five layers: alternately use positively charged polymer material, negatively charged polymer material, positively charged polymer material, negatively charged polymer material, and positively charged polymer material.

5. The method for preparing an injectable hydrogel of islet-like spheroids based on double sequential encapsulation according to claim 1, characterized in that: In the S2, 1 to 10 μL of pancreatic beta cells encapsulated with an immune protective membrane are added to 10 to 100 μL of spleen hydrogel, and the mixture is evenly mixed by pipetting.

6. The method for preparing an injectable islet-like spheroid hydrogel based on double sequential encapsulation according to claim 5, characterized in that: The spleen hydrogel is composed of decellularized spleen extracellular matrix of homologous or heterologous origin.

7. An injectable hydrogel for islet-like spheroids based on double sequential encapsulation, characterized in that: The hydrogel is obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the islet-like spheroid injectable hydrogel based on double sequential encapsulation as claimed in claim 7 in the preparation of a method for treating diabetes and its complications.

9. The use according to claim 8, characterized in that The complications include any one of cataract, osteoporosis and diabetic nephropathy.

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