Islet-like spheroid injectable hydrogel based on double sequential encapsulation and preparation and use thereof

Abstract

The application discloses an injectable hydrogel of islet-like spheroids based on double sequential encapsulation and a preparation and application thereof, and belongs to the technical field of bioengineering medical materials. The preparation method utilizes the positive and negative charges of a polymer material, forms an immune protection film of islet beta cells through electrostatic adsorption layer-by-layer self-assembly, and utilizes the extracellular matrix of a spleen, i.e., the characteristics of a liquid state in vitro and self-assembly into a nanofiber gel structure at a physiological temperature in vivo, to prepare the injectable hydrogel of islet-like spheroids, so as to ensure the stability of the hydrogel structure, self-assembly into a nanofiber three-dimensional gel structure in vivo, provide a natural specific microenvironment for cell self-renewal and differentiation, promote the aggregation of beta cells into islet-like spheroids, and enhance the insulin secretion and vascularization capacity. The immune protection film is formed on the surface of the islet beta cells through layer-by-layer self-assembly of gelatin and alginate, so that the beta cell immune protection encapsulation is realized.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering medical materials technology, specifically relating to an injectable hydrogel of islet-like spheres based on dual sequential encapsulation, its preparation and application. Background Technology

[0002] As of 2021, diabetes has affected more than 537 million people worldwide, causing serious health complications and death. People with type 2 diabetes (T2D) experience a variety of glycemic disorders, including: (1) reduced insulin secretion; (2) insulin resistance in muscles, liver and fat cells; and (3) problems with glucose uptake in visceral areas.

[0003] Islet transplantation is typically focused on treating type 1 diabetes. This approach is equally applicable to patients with advanced type 2 diabetes, who experience beta cell dysfunction, which manifests as insulin resistance in the early stages. The later stages of type 2 diabetes are characterized by insulin deficiency. Exogenous islet beta cell transplantation is an attractive strategy for restoring glycemic control and potentially preventing the progression of diabetes and its associated complications. However, the potential risks of long-term immunosuppression and the long-term survival of transplanted cells remain pressing issues that need to be addressed.

[0004] Recent advances in microencapsulation technology for cell delivery have shown promising results. This technology can alleviate rejection reactions in immune cell transplantation without the use of immunosuppressive drugs with side effects. Alginate is the optimal material for microencapsulation, and its performance has been validated in various animal models. To fully utilize the immunoprotective properties of alginate while mitigating immune responses caused by disruption of the encapsulation structure, the applicant's laboratory developed a method to achieve immunoprotective encapsulation of β cells on the cell surface through layer-by-layer (LbL) self-assembly of gelatin and alginate. This coating does not interfere with cell migration and exhibits excellent immunoprotective effects. However, LbL coatings alone cannot provide a favorable three-dimensional environment to promote β cell growth and self-assembly. On the surface, cell movement is two-dimensional, with almost every layer being planar rather than a true three-dimensional shape. 3D environments, such as scaffolds or hydrogels, provide opportunities for cell movement and multidimensional interaction; in this way, it more effectively replicates the tissue environment than a two-dimensional environment. Typically, cells function optimally under the real-world environmental conditions of their life cycle, which resemble the natural tissue structure of "three-dimensional" cells, thus exhibiting different directional interactions. In the LbL system, the layers may lack the structures that allow nutrients, gases, and other factors essential for the survival and proliferation of living cells to pass through. In natural tissues, cells can communicate with each other within a three-dimensional geometry and with the extracellular matrix (ECM). The traditional ECM microenvironment can only provide limited oxygen and nutrients in vivo, leading to restricted vascularization after β-cell 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 available in a truly three-dimensional environment to promote optimal cell growth and function. The extracellular matrix (ECM) within the cell encapsulation provides a natural, specific microenvironment for cell self-renewal and differentiation. Decellularized ECM, composed of collagen, elastin, glycosaminoglycans, and growth factors, exhibits numerous benefits in tissue repair and healing, such as promoting cardiac regeneration (zebrafish heart ECM), reducing oxidative stress (liver ECM), and improving graft survival and integration. Current research indicates that the ECM influences β-cell growth, differentiation, and function through interactions with integrins, cell adhesion receptors. Furthermore, damage to the islet-extracellular matrix interaction during islet separation leads to significant impairment of islet function.

[0006] Related technologies disclose the preparation of hydrogels using silk fibroin; 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 its degradation performance is extremely poor. Related technologies utilize a combination of phenylboronic acid and silk fibroin, with phenylboronic acid promoting the gelation of silk fibroin, thereby ensuring the hydrogel's degradation performance. The above technologies primarily focus on the hydrogel's degradation performance, without addressing its application in diabetes and its complications, particularly in effectively maintaining long-term blood glucose control and alleviating related complications. Summary of the Invention

[0007] The primary objective of this invention is to provide a method for preparing an injectable islet-like spheroid hydrogel based on dual sequential encapsulation. This method utilizes polymer materials carrying positive and negative charges to form an immune protective membrane for pancreatic β-cells through electrostatic adsorption layer by layer. Then, it utilizes the thermosensitive characteristics of the extracellular matrix of spleen cells, namely, the characteristic of self-assembly into a nanofiber gel structure under in vitro liquid state and in vivo physiological temperature, to prepare an injectable islet-like spheroid hydrogel, ensuring the stability of the hydrogel structure.

[0008] A second objective of this invention is to provide an injectable hydrogel for islet-like spheroids based on dual-sequential encapsulation. This hydrogel is prepared by mixing LbL-coated β-cells with spleen hydrogel (SpGel) and then transplanting the mixture into the body. Initial immune protection of β-cells is achieved through LbL coating without the use of immunosuppressants. Simultaneously, the spleen hydrogel (SpGel) can self-assemble into a nanofiber hydrogel under physiological temperature conditions and exhibits 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 glycemic control, thereby effectively alleviating in vivo complications caused by type 2 diabetes (T2D).

[0009] This invention is achieved through the following technical solution:

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

[0011] S1. An immune protective membrane is formed on the surface of pancreatic β cells by using layer-by-layer self-assembly technology with positively charged and negatively charged polymer materials.

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

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

[0014] Pancreatic β cells were incubated in a negatively charged polymer solution, and the process was repeated multiple times to obtain cell coatings with different interlayer structures as immune protective membranes.

[0015] The ratio of pancreatic β cells, positively charged polymer solution, and negatively charged polymer solution is 1:1 to 10 ml: 1 to 10 ml;

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

[0017] S2. Cells encapsulated with an immune protective membrane are combined with spleen decellularized extracellular matrix hydrogels of all or all species to form injectable three-dimensional islet-like spheroid hydrogels.

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

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

[0020] The process involves adding a positively charged polymer solution to pancreatic β cells, shaking for 5–20 min, and centrifuging at 2000 rpm for 5–8 min.

[0021] The pancreatic β cells were incubated in a negatively charged polymer solution for 10–60 min.

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

[0023] Preferably, the pancreatic β cells include tissue-derived pancreatic β cells or pancreatic β cells induced by 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] Alternatively, a three-layer coating: alternating use of positively charged polymer materials, negatively charged polymer materials, and positively charged polymer materials;

[0027] Alternatively, five layers: alternating between positively charged polymer materials, negatively charged polymer materials, positively charged polymer materials, negatively charged polymer materials, and positively charged polymer materials.

[0028] Preferably, in step S2, 1–10 μL of pancreatic β cells encapsulated in an immunoprotective membrane are added to 10–100 μL of spleen hydrogel and mixed thoroughly by pipetting.

[0029] Preferably, the spleen hydrogel is composed of decellularized spleen extracellular matrix from the same or different species.

[0030] An injectable hydrogel for islet-like spheres based on dual sequential encapsulation, the hydrogel being prepared by a method thereof.

[0031] Application of a dual-sequential encapsulation-based injectable hydrogel of islet-like spheres in the preparation of a treatment for diabetes and its complications.

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

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

[0034] (I) This invention provides a method for preparing injectable islet-like spheroid hydrogels based on dual sequential encapsulation. This method utilizes polymer materials with positive and negative charges to form an immune protective membrane for pancreatic β cells through electrostatic adsorption layer by layer. Then, it utilizes the thermosensitive characteristics of the extracellular matrix of spleen cells, namely, the characteristic of self-assembly into a nanofiber gel structure under in vitro liquid state and in vivo physiological temperature, to prepare injectable islet-like spheroid hydrogels, ensuring the stability of the hydrogel structure.

[0035] (II) This is an injectable hydrogel based on dual-sequential encapsulation of islet-like spheroids. The hydrogel is prepared by mixing LbL-coated β-cells with spleen hydrogel (SpGel) and then transplanting the mixture into the body. Initial immune protection of β-cells is achieved through LbL coating without the use of immunosuppressants. Simultaneously, the spleen hydrogel (SpGel) can self-assemble into a nanofiber hydrogel under physiological temperature conditions and promotes immunomodulation and angiogenesis. Through the combination of these two methods, β-cells transplanted into the kidney tissue of diabetic rats can effectively maintain long-term glycemic control, thereby effectively alleviating in vivo complications caused by type 2 diabetes (T2D).

[0036] (III) The method for preparing injectable hydrogels for islet-like spheroids based on dual sequential encapsulation discloses an immunoprotective mechanism for islet β-cell encapsulation. An immunoprotective membrane is formed on the surface of islet β-cells through layer-by-layer (LbL) self-assembly of gelatin and alginate, achieving immunoprotective encapsulation of β-cells. This coating does not interfere with cell migration and exhibits excellent immunomodulatory effects, addressing the potential risk of long-term immunosuppression after islet cell transplantation.

[0037] (iv) The method for preparing injectable hydrogels for islet-like spheroids based on dual sequential encapsulation discloses a three-dimensional growth space for pancreatic β-cells. Immunoprotected pancreatic β-cells are mixed with spleen extracellular matrix in vitro and injected in situ into the body. In vivo, they assemble into a three-dimensional nanofiber gel structure, providing a natural and specific microenvironment for cell self-renewal and differentiation, promoting the aggregation of β-cells into islet-like spheroids, and enhancing insulin secretion and vascularization. Attached Figure Description

[0038] Figure 1 A schematic diagram of dual encapsulation with LbL self-assembly membrane and SpGel hydrogel, and the in vivo transplantation process;

[0039] Figure 2 A schematic diagram of the double-sequence encapsulation of pancreatic β cells;

[0040] Figure 3 A schematic diagram illustrating how SpGel promotes the formation of islet-like spheres in LbL-coated INS-1 cells;

[0041] Figure 4 A schematic diagram of transplanting SpGel-encapsulated LbL-INS-1 cells into obese ZDF rats;

[0042] Figure 5 A schematic diagram illustrating how combined LbL assembly and SpGel encapsulation synergistically enhance INS-1 cell retention and angiogenesis at the ZDF rat transplantation site. Detailed Implementation

[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 for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0044] One specific embodiment of the present invention is as follows:

[0045] 1. Dual packaging structure design:

[0046] The first layer of encapsulation employs layer-by-layer (LbL) self-assembly technology to alternately encapsulate positively charged and negatively charged polymeric materials, forming a physical barrier to provide immune protection for β cells.

[0047] The second layer of encapsulation uses an injectable hydrogel prepared with spleen extracellular matrix (SpGel) to mimic the three-dimensional microenvironment of the natural extracellular matrix (ECM), promoting the aggregation of β cells into islet-like spheres and enhancing insulin secretion and vascularization.

[0048] 2. Applications of SpGel materials:

[0049] SpGel is composed of decellularized porcine spleen ECM proteins and can self-assemble into nanofiber hydrogels at physiological temperatures, possessing pro-angiogenic and immunomodulatory properties.

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

[0051] 3. Comprehensive treatment of diabetic complications:

[0052] Double-encapsulated pancreatic β cells after transplantation can stably control blood glucose for a long period (≥35 days), while improving local microcirculation through the vascularization effect of SpGel.

[0053] Significantly alleviates diabetic complications, including cataracts (reducing lens opacity), osteoporosis (40% increase in bone density), and diabetic nephropathy (50% reduction in renal fibrosis).

[0054] 4. Verification of technical effectiveness:

[0055] In vitro experiments: LbL-INS-1 cells cultured in 3D SpGel for 7 days showed a 3-fold increase in insulin secretion (140.94±26.65 ng / mL) and cell viability >90%.

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

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

[0058] 2×10 6 INS-1 cells (rat islet cell tumor 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 only. 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 formed by three rounds of treatment with alternating gelatin and sodium alginate, ending with gelatin. Finally, a nine-layer coating was formed by repeating the process four times and ending with a gelatin coating.

[0061] Example 2: Preparation and Cell Encapsulation of SpGel Hydrogel

[0062] Preparation of SpGel hydrogel:

[0063] Slowly rewarm the spleen tissue (of the same or different species, such as pig) stored in the refrigerator for 5 hours, remove the fatty tissue such as the splenic hilum, and cut it into 1mm pieces. 3 Small pieces of spleen tissue were rinsed in physiological saline, and then soaked in decellularized solution (0.3% SDS + 1.5% EDTA, with pH adjusted to neutral) and shaken at 37°C and 80 rpm for 24 h. The decellularized solution was then replaced and the spleen tissue was shaken at 25°C and 60 rpm for 36 h to obtain decellularized spleen extracellular matrix.

[0064] The decellularized spleen extracellular matrix was washed with deionized water at 25°C and 80 rpm (water was changed every 60 min for a total of 15 times) to remove residual decellularization solution. The mixture was then freeze-dried under vacuum (60°C, 0.1–0.2 mbar, for 48 h). The mixture was then ground into a homogenate in 0.01 M hydrochloric acid solution, and pepsin (1 mg / ml) was added. The mixture was then stirred and digested at 25°C and 100 rpm for 24 h to obtain a homogeneous, 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 1M sodium hydroxide solution, and then 10% PBS solution of 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 of INS-1 cells encapsulated with 10 μL of SpGel pre-solution was mixed and injected into the subcapsular region of ZDF rat kidneys to form three-dimensional islet-like spherical gels in vivo.

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

[0069] 3.1 In vitro efficacy verification

[0070] 3.1.1 Cell morphology and insulin secretion capacity:

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

[0072] like Figure 1 The diagram shows a double encapsulation of LbL self-assembled membrane and SpGel hydrogel, as well as the in vivo transplantation process.

[0073] Figure 1 The results showed that the process involved the continuous application of multiple layers of polycationic and polyanionic polymers to the surface of INS-1 cells to form a thin extracellular membrane.

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

[0075] like Figure 2The diagram illustrates the double-sequential encapsulation of pancreatic β-cells. A shows transmission electron microscopy images of untreated INS-1 cells and cells coated with five layers of (gelatin) 3 / (algin) 2. Arrows indicate the distinctive 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 scanning electron microscopy images of untreated and gelatin / algin 5-layer coated cells with comparable cell cluster diameters after 7 days of growth. 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, where FITC-labeled gelatin and Rhodamine B-labeled alginate were used to observe the encapsulation process. Cell nuclei were counterstained with Hoechst. Scale bar is 20 μm. D shows immunofluorescence staining of typical INS-1 markers (insulin) in untreated cells and cells coated with 3, 5, 7, and 9 layers of LBL. The scale bar is 30 μm. E represents the percentage of insulin-positive cells in untreated cells and LBL-coated cells in layers 3, 5, 7, and 9. F represents the basal insulin secretion level of INS-1 cells and LBL-coated cells in layers 3, 5, 7, and 9 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] Results combined Figure 2 As shown in A, transmission electron microscopy (TEM) confirmed the presence of a gelatin / sodium alginate layer on the surface of INS-1 (LbL-INS-1) cells coated with 5 layers of LBL.

[0078] Results combined Figure 2 As shown in B, scanning electron microscopy (SEM) images revealed that after 7 days of culture, both INS-1 and LbL-INS-1 cells exhibited extensive cell-to-cell contact, with no significant differences in cell morphology and distribution between the two groups.

[0079] The inventors' team conducted a preliminary assessment of the impact of LbL self-assembly on cell stability and function.

[0080] Results combined Figure 2 As shown in C, from left to right in the image, 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 image shows the cell nucleus counterstained with Hoechst dye. The fourth image is a composite image obtained by combining the first three staining methods using the Merge technique.

[0081] Cultured INS-1 and LbL-INS-1 cells (initially 3 × 10⁻⁶) were observed using confocal laser microscopy. 4 Insulin secretion levels (in cells).

[0082] Results combined Figure 2 As shown in D, under the same conditions, the more packaging layers there are, the faster the cells aggregate. When cells are packaged into five layers, the fluorescence intensity of live insulin-producing cells shows a significant difference.

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

[0084] Results combined Figure 2 As shown in F, the insulin secretion levels of monolayer, trilayer, quinarylayer, septalayer, and quintalayer LBL-INS-1 cells were 13.92±11.89 ng / ml, 25.36±11.57 ng / ml, 28.13±11.84 ng / ml, 33.24±11.89 ng / ml, 41.03±11.41 ng / ml, and 40.99±11.82 ng / ml, respectively.

[0085] Significant differences in insulin secretion were observed after five layers of product packaging, with the seventh layer reaching its peak. However, by the ninth layer, the data showed no significant change compared to the seventh layer.

[0086] Therefore, the inventors chose to use five layers in subsequent experiments, which not only have immunomodulatory effects but also promote the secretion of large amounts of insulin.

[0087] like Figure 3The diagram illustrates how SpGel promotes the formation of islet-like spheres in LbL-coated INS-1 cells. A shows a bright-field image of INS-1 cells cultured in 2D or 3D SpGel for 3 days; B shows a bright-field image of LbL-INS-1 cells cultured in 2D or 3D SpGel for 3 days; C shows the quantitative analysis of islet-like spheres formed by INS-1 cells after 3 days of culture in 2D or 3D SpGel; D shows the quantitative analysis of islet-like spheres formed by LbL-INS-1 cells after 3 days of culture in 2D or 3D SpGel; E shows a bright-field image of LbL-INS-1 cells cultured in 2D SpGel for 7 days, with prominent islet-like spheres highlighted in green; F shows a bright-field image of LbL-INS-1 cells cultured in 3D SpGel for 7 days, with prominent islet-like spheres highlighted in green; G shows the quantitative analysis of islet-like spheres formed by 2D and 3D SpGel using 3D image scanning. The average volume, circumference, area, and diameter of LbL-INS-1 spheres formed after 7 days of culture in SpGel; H shows representative SEM images of INS-1 cells after 7 days of culture in 2D or 3D SpGel. The green box highlights the areas forming islet-like spheres; I shows representative SEM images of LbL-INS-1 cells after 7 days of culture in 2D or 3D SpGel. The green box highlights the areas forming islet-like spheres. J shows CASK protein immunofluorescence staining; CASK protein is a typical marker of INS-1 cells, showing INS-1 and LbL-INS-1 cells after 7 days of culture in 3D SpGel; K shows the quantitative comparison of CASK protein positivity rates between INS-1 and LbL-INS-1 cells after 7 days of culture in 3D SpGel.

[0088] 3.1.2 In vitro formation of spherical structures and ability to regulate blood glucose:

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

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

[0091] Results combined 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 spheres were observed on the surface or inside SpGel.

[0092] Results combined Figure 3 As shown in the middle E, when cultured on 2D SpGel, cell spheroids tend to expand outward and polarize along the apical axis; the results combined with Figure 3 As can be seen from F, in 3D SpGel, cell spheroids tend to aggregate into star-shaped structures.

[0093] Results combined Figure 3 As shown in the data, the spherical diameter of the 2D SpGel group was significantly larger than that of the 3D SpGel group. However, considering the cell spherical perimeter and area measured from the spherical 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 spherical structures in LbL-INS-1 cells.

[0094] Results combined Figure 3 As shown in H and I, scanning electron microscopy (SEM) was used to identify the connection points between cells and SpGel, and observations were performed under both two-dimensional and three-dimensional culture conditions.

[0095] Results combined Figure 3 As can be seen from I, both INS-1 cells and LbL-INS-1 cells form spheres in both three-dimensional and two-dimensional SpGels.

[0096] Results combined Figure 3 As can be seen from H, some INS-1 cells were not aggregated in the two-dimensional SpGel.

[0097] Results combined Figure 3 The results showed that the CASK positivity levels of INS-1 cells and LbL-INS-1 cells in three-dimensional SpGel were compared.

[0098] Results combined Figure 3 As can be seen from the data, there was no significant increase in CASK expression between the LbL-INS-1 group and the INS-1 group.

[0099] This suggests that morphological and polarity changes between two-dimensional and three-dimensional cell cultures 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 conventional culture media in maintaining their functional state, especially during long-term culture.

[0100] 3.2 In vivo efficacy verification

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

[0102] like Figure 4The study described the transplantation of SpGel-encapsulated LbL-INS-1 cells into obese ZDF rats. A shows a representative image of SpGel-encapsulated LbL-INS-1 cells transplanted under the kidneys of overweight ZDF rats. The white arrows pointing to the transplantation webpage indicate the encapsulation effect. The scale bar is 20 μm. B shows representative longitudinal changes in blood glucose levels in obese ZDF rats under all different treatments. C shows the distribution and dynamic fate of DiR-labeled LbL-INS-1 cells and LbL-INS-1+SpGel constructs after kidney transplantation in SD rats, observed using non-invasive in vivo fluorescence imaging. D shows representative cataract progression in obese ZDF rats receiving LbL-INS-1+SpGel kidney transplantation compared to age-matched untreated lean 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 micro-CT images of distal femoral bone condition in obese ZDF rats 35 days after LbL-INS-1+SpGel pararenal transplantation. They are 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 to demonstrate the effect of this treatment on bone health (F).

[0103] Results combined Figure 4 As indicated by A in the diagram, LbL-INS-1 cells were implanted under the renal capsule of obese ZDF rats. These rats have a genetic predisposition to type 2 diabetes, making them an ideal model for evaluating the therapeutic potential of LbL-INS-1 cell transplantation.

[0104] Results combined Figure 4 As shown in section B, after transplantation, the inventors encapsulated LbL-INS-1 cells in a SpGel matrix. Unlike encapsulated LbL-INS-1 cells, whose blood glucose levels returned to their initial values ​​within 7 days, the cells encapsulated in SpGel exhibited effectiveness lasting approximately 35 days.

[0105] Results combined Figure 4 As shown in Figure C, LbL-INS-1 cells have an effect on blood glucose regulation in transplanted diabetic rats. The results indicated that unencapsulated LbL-INS-1 cells were ineffective in lowering blood glucose levels through insulin production. Conversely, when LbL-INS-1 cells were encapsulated in SpGel matrix, this natural microenvironment not only supported their survival but also enhanced their insulin secretion capacity, thereby significantly reducing blood glucose levels in diabetic rats.

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

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

[0108] Results combined Figure 4 As indicated by E in the figure, the inventors' team explored the potential development of osteoporosis by examining the bone structure and morphology of the distal thigh of diabetic rats. Micro-computed tomography (Micro-CT) assessment showed that the bone mineral density (BMD) of the hypertrophic group was significantly reduced, at 0.19 ± 0.01 g / cm³. 2 The BMD was significantly lower than that of the negative fat group, which was 0.38 ± 0.02 g / cm³. 2 After treatment with LbL-INS-1+SpGel, the bone mineral density of diabetic rats was significantly improved, reaching 0.28±0.01 g / cm³. 2 .

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

[0110] like Figure 5 As shown, the combined use of LbL assembly and SpGel encapsulation synergistically enhances INS-1 cell retention and angiogenesis at the transplantation site in ZDF rats. A shows representative HE-stained kidney sections from obese ZDF rats at weeks 0, 1, 2, 3, and 4 post-transplantation; these rats received subrenal transplantation with LbL-INS-1+SpGel. B shows numerical assessment of capillary density. C shows immunohistochemical analysis of LbL-INS-1+SpGel at week 1 post-transplantation (C and D). CD20 was observed and recorded visually. + and CD11b + The presence of immune cells such as cells was observed; D represents the C and D immunohistochemical analysis of LbL-INS-1+SpGel 4 weeks post-transplantation. CD20 levels were observed and recorded visually. + and CD11b + The presence of immune cells such as cells.

[0111] 3.2.2 In vivo vascularization effect:

[0112] Results combined Figure 5 As indicated by A in the text, based on the aforementioned research findings on glycemic regulation, a histological evaluation was conducted to investigate the vascularization effect of SpGel-encapsulated LbL-INS-1 cells after subrenal capsule transplantation in ZDF rats. Kidney sections from these rats were stained with hematoxylin and eosin (HE) at 0, 1, 2, 3, and 4 weeks post-implantation. The results showed that LbL assembly within SpGel increased the retention rate of INS-1 cells and enhanced angiogenesis. Notably, as indicated by the red arrows, a significant change was observed in the site of vascular origin compared to baseline (week 0). Treatment with LbL-INS-1+SpGel significantly increased arterial and capillary density, indicating enhanced neovascularization in the implantation area.

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

[0114] Immunohistochemical staining was used to investigate immune cell activation, with GelMA as an experimental control. Graft performance in all recipients was systematically evaluated. In this context, the LbL setting was used in combination with either SpGel or GelMA. INS-1+SpGel was used as another group.

[0115] The tests were performed at two different time points: 1 week post-transfer (LbL-INS-1+GelMA vs. LbL-INS-1+SPGel vs. INS-1+SpGel) Figure 5 C) and 4 weeks post-graft (LbL-INS-1+GelMA vs. LbL-INS-1+SPGel vs. INS-1+SpGel) Figure 5 (D in the middle).

[0116] Immunocyte staining such as CD20 + and CD11b + Analysis showed significant differences between the groups, with the LbL-INS-1+SPGel group showing more favorable results.

[0117] The results collectively indicate that the combined application of LbL-INS-1 cells and SpGel has a positive impact on graft performance and may reduce the activation of detailed immune cell populations compared to the control group.

[0118] Finally, it should be noted that the above description is merely 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 should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an injectable hydrogel of islet-like spheroids based on dual sequential encapsulation, characterized in that, Includes the following steps: S1. An immune protective membrane is formed on the surface of pancreatic β cells by using layer-by-layer self-assembly technology with positively charged and negatively charged polymer materials. The positively charged polymeric material includes gelatin; The negatively charged polymeric material includes sodium alginate; The specific process of the layer-to-layer self-assembly technology is as follows: Add a positively charged polymer solution to pancreatic β cells, shake for 5-20 min, centrifuge at 2000 rpm for 5-8 min, and discard the supernatant; Then, pancreatic β cells were incubated in a negatively charged polymer solution for 10-60 minutes, and this process was repeated multiple times to obtain cell coatings with different interlayer structures as immune protective membranes. The cell coating with different interlayer structures is: Three-layer coating: Alternating use of positively charged polymer materials, negatively charged polymer materials, and positively charged polymer materials; Alternatively, a five-layer coating: alternating use of positively charged polymer materials, negatively charged polymer materials, positively charged polymer materials, negatively charged polymer materials, and positively charged polymer materials; Wherein, the mass concentration of the positively charged polymer material solution is 0.1%~2%, and the mass concentration of the negatively charged polymer material solution is 0.1%~2%; S2. 1-10 μL of pancreatic β cells encapsulated in an immunoprotective membrane are combined with 10-100 μL of spleen decellularized extracellular matrix hydrogel of all or all species, and mixed evenly by pipetting to form injectable three-dimensional islet-like spherical hydrogel. The pancreatic β cells include tissue-derived pancreatic β cells or pancreatic β cells induced by human induced pluripotent stem cells.

2. An injectable hydrogel based on dual-sequential encapsulation of islet-like spheroids, characterized in that, The hydrogel is obtained by the preparation method described in claim 1.

3. The application of the injectable islet-like spherical hydrogel based on dual sequential encapsulation as described in claim 2 in the preparation of drugs for treating diabetes and diabetic complications, characterized in that, The complications include cataracts and osteoporosis.

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