Hydrogel composition, hydrogel, preparation method of hydrogel and hydrogel kit

Abstract

The invention provides a hydrogel composition, hydrogel, a preparation method of the hydrogel and a hydrogel kit. The hydrogel composition comprises a first component and a second component, wherein the first component is prepared from four-arm polyethylene glycol succinimide glutaric acid ester, and the second component is prepared from four-arm polyethylene glycol amine and polyethyleneimine; and the second component also comprises an exosome. The hydrogel disclosed by the invention is excellent in mechanical properties, including good tensile strength and swelling property, and also shows relatively strong antibacterial activity. The hydrogel can promote angiogenesis and cell migration, effectively regulate the inflammatory response of macrophages, remarkably reduce the inflammatory response and obviously improve the tissue regeneration capacity. In addition, the hydrogel can effectively promote wound closure, reduce the pancreatic leakage risk, and significantly enhance tissue repair without causing immunological rejection.

Description

Technical Field

[0001] The invention relates to a hydrogel composition, a hydrogel and a preparation method thereof, and a hydrogel kit, and belongs to the field of medical materials. Background Art

[0002] Pancreaticojejunostomy is a common surgical procedure for the treatment of pancreatic diseases (such as pancreatic cancer and pancreatitis), and the number of such operations is increasing year by year worldwide. However, postoperative complications, especially pancreatic leakage, remain one of the main factors affecting patient prognosis. Pancreatic leakage is usually caused by incomplete sealing or poor healing of the anastomosis. In severe cases, it can cause peritonitis, infection, abscesses, and even be life-threatening. The speed and quality of anastomotic healing are crucial to the success of the operation, but existing surgical techniques and materials still have limitations and cannot completely solve the problems of pancreatic leakage and poor anastomotic healing.

[0003] In recent years, researchers have proposed a variety of strategies and materials to improve the sealing and healing quality of pancreatic anastomosis. Some materials (such as degradable polymers, collagen membranes and hydrogels) have been used in the market and have shown certain potential in reducing pancreatic leakage and promoting anastomotic healing. For example, polyethylene glycol (PEG) hydrogels and dopamine-modified biomaterials are designed to have high adhesion and good biocompatibility, which can effectively seal the anastomosis and promote neovascularization. However, current materials still have certain defects, such as decreased adhesion after long-term use, which affects the sealing effect. In addition, some degradable materials degrade too quickly and cannot maintain the stability of the anastomosis for a sufficiently long time. At the same time, the regulation of local immune responses is still not ideal, which may lead to excessive inflammatory response or fibrosis. Therefore, there is an urgent need to develop new biomaterials that are more efficient, durable, and have immune regulatory functions to address the problems of pancreatic leakage and tissue healing in pancreaticoduodenal anastomosis.

[0004] Exosomes, due to their remarkable biocompatibility and diverse biological functions, have been widely demonstrated in tissue repair and immune regulation research. However, their inherent properties limit their use. Exosomes from different sources contain varying bioactive components such as proteins and nucleic acids, resulting in varying abilities to promote tissue cell proliferation, differentiation, and angiogenesis. For example, exosomes from certain sources may not effectively stimulate cell regeneration at the anastomosis site, thereby delaying tissue repair.

[0005] Furthermore, exosomes, while promoting tissue repair, may overactivate fibroblasts, leading to excessive collagen deposition and fibrosis at the anastomotic site. Excessive fibrosis can affect the normal structure and function of tissues, reduce tissue elasticity at the anastomotic site, and even lead to anastomotic stenosis. This not only affects the normal passage of food but also increases the risk of postoperative complications such as intestinal obstruction.

[0006] Traditionally, exosomes derived from mesenchymal stem cells (MSCs) have attracted attention for their potential to promote wound healing. MSC-derived exosomes promote tissue repair by regulating cell proliferation, migration, and immune responses. However, recent studies have shown that exosomes derived from M2 macrophages have superior potential in promoting tissue repair. M2 macrophages have significant anti-inflammatory properties and can secrete a variety of growth factors and cytokines that promote angiogenesis, accelerating wound healing. In addition, M2 macrophage exosomes can regulate the local immune microenvironment, reduce inflammatory responses, and promote tissue remodeling and functional recovery. Therefore, compared with MSC-derived exosomes, M2 macrophage exosomes show more obvious advantages in enhancing tissue repair and improving wound healing.

[0007] Therefore, developing an efficient sealing material to enhance the sealing performance of pancreatic anastomosis and promote tissue healing is a key challenge that needs to be addressed in the field of pancreatic surgery. This will not only help to improve the success rate of surgery, but also significantly improve the postoperative prognosis of patients. Summary of the Invention

[0008] Problems to be solved by the invention

[0009] In view of the technical problems existing in the prior art, the present invention first provides a hydrogel composition and hydrogel. The hydrogel of the present invention exhibits excellent mechanical properties, including good tensile strength and swelling properties, while also displaying strong antibacterial activity. The hydrogel can promote angiogenesis and cell migration, and effectively regulate the inflammatory response of macrophages, significantly reducing the inflammatory response and significantly improving tissue regeneration capacity. In addition, the hydrogel can effectively promote wound closure, reduce the risk of pancreatic leakage, and significantly enhance tissue repair without triggering immune rejection.

[0010] Furthermore, the present invention also provides a method for preparing the hydrogel, which is simple and easy to implement, and the raw materials are easily obtained, and is suitable for mass production.

[0011] Solutions for solving problems

[0012] The present invention provides a hydrogel composition comprising: a first component and a second component; wherein,

[0013] The first component includes four-arm polyethylene glycol succinimide glutarate, the second component includes four-arm polyethylene glycol amine and polyethylene imine; and

[0014] The second component also includes exosomes.

[0015] [2] The hydrogel composition according to [1] above, wherein the weight average molecular weight of the four-arm polyethylene glycol succinimidyl glutarate is 5000-20000 Da, the molecular weight of the four-arm polyethylene glycol amine is 5000-20000 Da, and the molecular weight of the polyethyleneimine is 2000-20000 Da.

[0016] [3] The hydrogel composition according to [1] or [2] above, wherein the volume ratio of the first component to the second component is 1:(0.8-1.2), preferably 1:(0.9-1.1).

[0017] [4] The hydrogel composition according to any one of [1] to [3] above, wherein in the second component, the mass ratio of the four-arm polyethylene glycol amine to the polyethylene imine is 1:(0.8-8), preferably 1:(0.9-5).

[0018] [5] The hydrogel composition according to any one of [1] to [4] above, wherein the average diameter of the exosomes is 50-200 nm; and / or the exosomes are derived from macrophages.

[0019] [6] A hydrogel formed by reacting the first component and the second component of the hydrogel composition described in any one of [1] to [5] above; wherein the first component and the second component can bond to form a hydrogel skeleton, and exosomes are loaded in the hydrogel skeleton;

[0020] Preferably, the concentration of the exosomes in the hydrogel is 0.1-10 mg / mL.

[0021] [7] The hydrogel according to [6] above, wherein the hydrogel has at least one of the following characteristics:

[0022] The swelling rate of the hydrogel is 100-200%;

[0023] The hydrogel has a tensile strength of 10-100 kPa and a storage modulus of 5-50 kPa;

[0024] The exosome release time is more than 10 days;

[0025] The hydrogel has a tissue adhesion strength of 10-50 kPa, a peeling force of 0.01-0.5 N / cm, and a bursting strength of 10-50 kPa.

[0026] [8] A method for preparing the hydrogel according to [6] or [7] above, comprising the following steps:

[0027] preparing a solution A comprising a first component;

[0028] preparing a solution B comprising the second component;

[0029] Solution A and solution B were mixed to obtain a hydrogel.

[0030] [9] The preparation method according to [8] above, wherein the mass concentration percentage of the four-arm polyethylene glycol succinimidyl glutarate in the solution A is 1-25% (m / v); and / or,

[0031] In the solution B, the mass concentration percentage of the four-arm polyethylene glycol amine is 1-25% (m / v), and the mass concentration percentage of the polyethyleneimine is 1-80% (m / v).

[0032]

[10] A hydrogel kit comprising the hydrogel composition described in any one of [1] to [5] above; preferably, the first component and the second component of the hydrogel composition are stored separately.

[0033] Effects of the Invention

[0034] The hydrogels of this invention exhibit excellent mechanical properties, including good tensile strength and swelling capacity, while also displaying strong antimicrobial activity. They can promote angiogenesis and cell migration, and effectively regulate the inflammatory response of macrophages, significantly reducing inflammatory responses and significantly improving tissue regeneration. Furthermore, the hydrogels can effectively promote wound closure, reduce the risk of pancreatic leakage, and significantly enhance tissue repair without triggering immune rejection.

[0035] The preparation method of the hydrogel of the present invention is simple and easy, the raw materials are easy to obtain, and it is suitable for mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 A diagram comparing the leakage of conventional pancreaticojejunostomy and pancreaticojejunostomy performed using the hydrogel of the present invention is shown;

[0037] Figure 2 Schematic diagram showing the characterization of different exosomes using TEM and NTA analysis;

[0038] Figure 3Schematic diagram of exosome-induced vascularization; among them, a is the formation of blood vessels observed under an optical microscope, b is the statistics of branching number, c is the statistics of capillary length, d is the 24-hour migration experiment of human umbilical vein endothelial cells (HUVECs), e is the statistics of migration area, and f is VEGF immunofluorescence staining.

[0039] Figure 4 A schematic diagram of the analysis of signaling pathways enriched in exosomes is shown; among them, a is a Venn diagram analyzing the differential expression of genes after BMSCs cells are stimulated by macrophage exosomes from different sources, b is a gene heat map analyzing the genomic differences between M2 macrophage exosomes and M0 and M1 macrophage exosomes, c is a volcano plot analyzing the gene expression differences between M2 macrophage exosomes and M0 macrophage exosomes, d is a KEGG enrichment analysis of the signaling pathway differences between M2 macrophage exosomes and M0 macrophage exosomes, e is a volcano plot analyzing the gene expression differences between M2 macrophage exosomes and M1 macrophage exosomes, and f is a KEGG enrichment analysis of the signaling pathway differences between M2 macrophage exosomes and M1 macrophage exosomes.

[0040] Figure 5 The differences between M2 macrophage exosomes and BMSCs exosomes are shown; among them, a is a gene heat map analysis of the genomic differences between M2 macrophage exosomes and BMSCs exosomes, b is a volcano map analysis of the gene expression differences between M2 macrophage exosomes and BMSCs exosomes, and c is a KEGG enrichment analysis of the signaling pathway differences between M2 macrophage exosomes and BMSCs exosomes.

[0041] Figure 6 The construction process of the hydrogel is shown; among them, a is a schematic diagram of the composition and preparation of the hydrogel, b is the rheological stability of the hydrogel, c is the stress-strain curve of the hydrogel, d is the swelling behavior of the hydrogel, e is the degradation behavior of the hydrogel, and f is the release of exosomes.

[0042] Figure 7 The tissue adhesion test results of the hydrogel are shown; wherein a is PEG / Exo M2 Adhesion of hydrogels to various tissues, b is adhesion strength, c is peeling force, and d is bursting strength.

[0043] Figure 8 The antibacterial study of the hydrogel is shown; among them, a is the antibacterial activity of the hydrogel against Escherichia coli, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus at different concentrations of PEI, b is the anti-biofilm formation activity of the hydrogel against Escherichia coli, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus at different concentrations of PEI, and c is the observation of colony formation of the hydrogel at different concentrations of PEI.

[0044] Figure 9 Shown is the study of hydrogel-induced vascularization; a is microscopic generation observed under an optical microscope, b is branching number statistics, c is intersection number statistics, d is capillary length statistics, e is HUVECs migration experiment, f is cell migration area statistics, g is VEGF / F-actin / DAPI immunofluorescence staining image, and h is semi-quantitative statistics of VEGF fluorescence intensity based on fluorescence images.

[0045] Figure 10 Figure 2 shows the study of the inflammatory regulation ability of hydrogels; a is immunofluorescence staining of M1 pro-inflammatory and M2 anti-inflammatory indicators under LPS stimulation, b is flow cytometry analysis of the inflammatory regulation behavior of hydrogels, and c is quantitative analysis of CD206 + cells, d is a schematic diagram of subcutaneous implantation in vivo, e is H&E staining analysis of implantation 3 and 6 days later, f is analysis of fibrous capsule thickness, g is TNF-α / CD163 / DAPI immunofluorescence staining of tissue, h is a semi-quantitative statistical analysis of the fluorescence intensity of TNF-α (M1 indicator) and CD163 (M2 indicator) based on fluorescence images.

[0046] Figure 11 H&E staining photos showing the heart, liver, spleen, lung and kidney after subcutaneous hydrogel implantation for 6 days;

[0047] Figure 12 Figure 2 shows the research on hydrogel-promoting tissue repair; a is the construction of wound injury model and histological evaluation, b is the real-time observation of wound repair, c is the analysis of wound healing, d is the H&E and Masson's trichrome staining at 6 days, e is the analysis of granulation tissue width, f is the analysis of collagen production, g is the immunofluorescence staining of TNF-α / CD163 / DAPI and VEGF / DAPI, h is the semi-quantitative statistical analysis of the fluorescence intensity of TNF-α (M1 indicator) and CD163 (M2 indicator) based on fluorescence images.

[0048] Figure 13 Figure 2 shows the study on the promotion of wound repair by hydrogel; wherein, a is H&E and Masson's trichrome staining at 12 days, b is analysis of granulation tissue width, c is collagen production analysis, d is VEGF immunofluorescence staining, and e is semi-quantitative analysis of VEGF fluorescence intensity.

[0049] Figure 14 Shown is H&E staining of the heart, liver, spleen, lung, and kidney after subcutaneous hydrogel implantation for 12 days.

[0050] Figure 15Figure 2 shows the application of hydrogel in pancreaticojejunostomy surgery; a is a schematic diagram of spraying hydrogel on the pancreaticojejunostomy wound; b is a flow chart of using hydrogel in pancreaticojejunostomy surgery; c is the statistical volume of drainage fluid 3 days after surgery; d is the detection of α-amylase concentration in the drainage fluid; e is H&E staining analysis of anastomotic repair, and f is a VEGF / α-SMA / DAPI immunofluorescence staining image.

[0051] Figure 16 The figure shows the gross observation of the pancreaticojejunostomy 30 days after surgery.

[0052] Figure 17 The Masson's trichrome staining image of the pancreaticojejunostomy at 30 days is shown.

[0053] Figure 18 Shown is H&E staining of the heart, liver, spleen, lung, and kidney after subcutaneous hydrogel implantation for 30 days.

[0054] Figure 19 Shown is the biocompatibility evaluation of the hydrogel after subcutaneous implantation for 30 days; a is blood routine analysis, b is blood biochemical analysis. Specific implementation plan

[0055] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0056] In addition, numerous specific details are provided in the following specific embodiments to better illustrate the present invention. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0057] Unless otherwise stated, the units used in this specification are international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0058] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0059] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0060] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0061] In this specification, unless otherwise specified, "% (m / v)" refers to the percentage of mass concentration. The percentage of mass concentration refers to the mass of a component in a unit volume of solvent. The percentage of mass concentration in this specification is calculated with mL as the volume unit and g as the mass unit. For example, if 0.003g of a substance component is added to 1mL of solvent, the percentage of mass concentration of the substance component is (0.003g / 1mL)*100%=0.3% (m / v).

[0062] As used herein, "HUVECs" refers to human umbilical vein endothelial cells.

[0063] As used in the present invention, "RAW264.7 cells" means mouse leukemia cells of monocyte macrophage. As used in the present invention, "VEGF" means vascular endothelial growth factor.

[0064] As used in the present invention, "TNF-α" means tumor necrosis factor-α.

[0065] As used in the present invention, “CD206” stands for Cluster of differentiation 206, which belongs to the C-type lectin superfamily and is also known as Mannose Receptor (MR) or Macrophage Mannose Receptor.

[0066] As used in the present invention, "il-4" means interleukin 4 (IL-4).

[0067] As used in the present invention, "IL-6" means interleukin 6 (IL-6).

[0068] As used in the present invention, "CD80" stands for Cluster of differentiation 80, which belongs to the immunoglobulin superfamily.

[0069] As used herein, "α-SMA" means α-smooth muscle actin.

[0070] As used in the present invention, "LPS" means lipopolysaccharide.

[0071] As used in the present invention, "F-actin" means fibrous actin.

[0072] As used herein, "FITC" stands for fluorescein isothiocyanate.

[0073] As used herein, "DAPI" means 4',6-diamidino-2-phenylindole.

[0074] As used in the present invention, "CD163" stands for Cluster of differentiation 163, which belongs to the cysteine-rich B-type scavenger receptor family.

[0075] <First Aspect>

[0076] The first aspect of the present invention provides a hydrogel composition comprising: a first component and a second component; wherein,

[0077] The first component includes four-arm polyethylene glycol succinimide glutarate, the second component includes four-arm polyethylene glycol amine and polyethylene imine; and

[0078] The second component also includes exosomes.

[0079] First component

[0080] The first component of the present invention includes four-arm polyethylene glycol succinimide glutarate. The structural formula of the four-arm polyethylene glycol succinimide glutarate is shown in the following formula (I):

[0081]

[0082] The structural formula of the four-arm polyethylene glycol amine is shown in formula (II):

[0083]

[0084] Where n and m are natural numbers.

[0085] Specifically, the first and second components of the hydrogel composition are separated and mixed several hours before or upon use. Preferably, the four-arm polyethylene glycol amine, polyethylene imine, and exosomes in the second component are stored separately and mixed several hours before or upon use.

[0086] During actual use, the first and second components can be stored separately and then dissolved in a solvent for mixing before or during use. For example, solution A containing the first component can be prepared, followed by solution B containing the second component. Finally, solutions A and B are mixed to form a hydrogel. The present invention does not limit the storage method of the components of the hydrogel composition. Persons skilled in the art can select a specific storage method as needed, and all such methods are within the scope of the present invention.

[0087] In the present invention, four-arm polyethylene glycol succinimidyl glutarate, four-arm polyethylene glycol amine, and polyethyleneimine can be bonded to form a hydrogel skeleton, and exosomes are loaded into the hydrogel skeleton. The inventors of the present invention have discovered that loading exosomes into the hydrogel skeleton can enhance its tissue repair effect.

[0088] In the present invention, four-arm polyethylene glycol succinimidyl glutarate, four-arm polyethylene glycol amine and polyethyleneimine are bonded to form a multi-arm polymer with a three-dimensional spatial structure, which is used as a hydrogel skeleton, and the performance of the obtained hydrogel skeleton is more excellent. Exosomes can be continuously released at the wound site, thereby providing a more lasting biological effect to promote wound healing. And due to the presence of exosomes, the adhesion of the hydrogel can be enhanced, the contact area between the hydrogel and the wound is increased, thereby optimizing the sealing effect and prolonging the release time of the exosomes. In addition, exosomes can continuously regulate the local immune microenvironment, effectively relieve inflammation and further promote wound repair. Therefore, the exosome-loaded hydrogel of the present invention has significant clinical application potential and can further enhance the tissue repair effect.

[0089] In some specific embodiments, the weight average molecular weight of the four-arm polyethylene glycol succinimidyl glutarate is 5000-20000Da, for example: 8000-15000Da; the molecular weight of the four-arm polyethylene glycol amine is 5000-20000Da, for example: 8000-15000Da; the molecular weight of the polyethyleneimine is 2000-20000Da, for example: 4000-15000Da.

[0090] The weight average molecular weight described in the present invention can be measured by methods commonly used in the art, for example, the MALDI-TOF measurement method.

[0091] By adjusting the weight-average molecular weight of four-arm polyethylene glycol succinimidyl glutarate, four-arm polyethylene glycol amine and polyethylene imine, the degradation rate of the hydrogel can be adjusted to meet the degradation and clearance requirements of different tissue sites and avoid inflammatory reactions caused by excessive retention in the body.

[0092] In some specific embodiments, the volume ratio of the first component to the second component is 1:(0.8-1.2), preferably 1:(0.9-1.1). When the volume ratio of the first component to the second component is 1:(0.8-1.2), a hydrogel with good mechanical properties can be obtained.

[0093] In some specific embodiments, in the second component, the mass ratio of the four-arm polyethylene glycol amine to the polyethylene imine is 1:(0.8-8), preferably 1:(0.9-5).

[0094] The ingredients used in this invention, such as four-arm polyethylene glycol succinimidyl glutarate, four-arm polyethylene glycol amine, and polyethyleneimine, all exhibit excellent biocompatibility. Compared to potentially toxic components found in other tissue adhesives, such as glutaraldehyde and cyanoacrylate monomers, these adhesives exhibit superior biocompatibility and are less likely to induce inflammatory responses upon implantation. Generally speaking, although polyethylene glycol macromolecules with a molecular weight below 10,000 are non-degradable and can be excreted through cellular permeation and endocytosis through metabolic circulation, other tissue adhesives, such as BioGlue and α-cyanoacrylate, lack biodegradability due to factors such as high crosslink density and stable intermolecular chemical interactions.

[0095] exosomes

[0096] Exosomes (EVs) are small, double-membrane vesicles produced by cells via paracrine pathways, with diameters ranging from 40 to 1000 nm. Exosomes are widely present in cell culture supernatants, various body fluids (blood, lymph, saliva, urine, semen, and breast milk), and plants (vacuoles). They carry a variety of cell-derived proteins, lipids, and other substances, and participate in processes such as intercellular communication, cell migration, angiogenesis, and immune regulation.

[0097] In some specific embodiments, the average diameter of the exosomes is 50-200 nm, for example, 80-150 nm; and / or the exosomes are derived from macrophages.

[0098] In the present invention, the exosomes are derived from macrophages. The macrophages may be animal macrophages. The present invention is not particularly limited to the animal, as long as the exosomes can be extracted. The macrophages may be non-human animal macrophages, such as pigs, fish, mice, etc.

[0099] Specifically, the exosomes can be derived from M0, M1 and M2 macrophages, preferably M2 macrophages. The present invention does not particularly limit the method for preparing exosomes, and the exosomes can be prepared by a commonly used method in the art.

[0100] The inventors of the present invention have found that the exosomes of M2 macrophages show significant advantages in promoting angiogenesis and regulating the immune microenvironment. The exosomes of M2 macrophages are loaded into a highly adhesive hydrogel. The resulting hydrogel not only effectively promotes angiogenesis, but also significantly regulates local inflammation, providing an ideal biological environment for wound healing. In addition, the hydrogel of the present invention can also effectively enhance the sealing of the anastomosis and promote tissue repair, achieving the expected effect (such as Figure 1 shown).

[0101] <Second Aspect>

[0102] A second aspect of the present invention provides a hydrogel formed by reacting the first and second components of the hydrogel composition described in the first aspect of the present invention; wherein the first and second components bond to form a hydrogel skeleton, and exosomes are loaded into the hydrogel skeleton. The hydrogel of the present invention is a hydrogel material with high adhesion, good biocompatibility, and the ability to promote tissue repair.

[0103] Specifically, in the hydrogel of the present invention, the concentration of the exosomes is 0.1-10 mg / mL, which can be calculated based on the absorbance.

[0104] Figure 6 The hydrogel composition of the present invention is prepared by mixing four-arm polyethylene glycol succinimidyl glutarate, four-arm polyethylene glycol amine and polyethyleneimine to form a cross-linked network structure, thereby gelling.

[0105] Specifically, in the present invention, the volume swelling rate of the hydrogel is 100-200%; the tensile strength of the hydrogel is 10-100 kPa, and the storage modulus is 5-50 kPa; the release time of the exosomes is more than 10 days; the tissue adhesion strength of the hydrogel is 10-50 kPa, the peel force is 0.01-0.5 N / cm, and the bursting strength is 10-50 kPa.

[0106] <Third Aspect>

[0107] The third aspect of the present invention provides a method for preparing the hydrogel according to the second aspect of the present invention, comprising the following steps:

[0108] preparing a solution A comprising a first component;

[0109] preparing a solution B comprising the second component;

[0110] Solution A and solution B were mixed to obtain a hydrogel.

[0111] In some specific embodiments, solution A and solution B are mixed directly on the tissue wound surface, which can directly adhere the wound and promote tissue repair. The hydrogel of the present invention can promote angiogenesis and cell migration, and effectively regulate the inflammatory response of macrophages, significantly reducing fibrosis and inflammatory response, and significantly improving tissue regeneration capacity. In addition, the hydrogel can effectively promote wound closure, reduce the risk of pancreatic leakage, and significantly enhance tissue repair without triggering immune rejection.

[0112] For solution A, four-arm polyethylene glycol succinimidyl glutarate is dissolved in a solvent to obtain it. For the solvent, the present invention is not particularly limited, and it can be any feasible solvent in the art, such as a buffer solution, water, etc. Specifically, a buffer solution can be used to prepare solution A containing the first component. For the buffer solution, the present invention is not particularly limited, and it can be some buffer solutions commonly used in the art. Specifically, the pH value of the buffer solution can be 7.0 to 7.4. When the pH value of the buffer solution is 7.0 to 7.4, the obtained hydrogel is close to neutral, has little irritation to tissues, and is conducive to the growth of cells and tissues. For the buffer solution, the present invention is not particularly limited, and it can be a buffer solution commonly used in the art, such as PBS buffer solution, physiological saline, etc.

[0113] In some specific embodiments, in the solution A, the mass concentration percentage of the four-arm polyethylene glycol succinimidyl glutarate is 1-25% (m / v).

[0114] For solution B, you can choose any of the following preparation methods:

[0115] In some specific embodiments, a solution containing four-arm polyethylene glycol amine, a solution containing polyethyleneimine, and a dispersion containing exosomes are prepared separately, and the three solutions are mixed to obtain the solution B.

[0116] In some specific embodiments, a solution comprising four-arm polyethylene glycol amine and polyethylene imine is prepared, and exosomes are added to the solution comprising four-arm polyethylene glycol amine to obtain the solution B.

[0117] In some specific embodiments, a dispersion containing exosomes is prepared, and four-arm polyethylene glycol amine and polyethyleneimine are added to the dispersion containing exosomes to obtain the solution B.

[0118] In some specific embodiments, four-arm polyethylene glycol amine, polyethyleneimine and exosomes are mixed and added to a solvent to obtain the solution B.

[0119] For a solution containing four-arm polyethylene glycol amine, the four-arm polyethylene glycol amine can be dissolved in a solvent; for a solution containing polyethylene imine, the polyethylene imine can be dissolved in a solvent; for a solution containing four-arm polyethylene glycol amine and polyethylene imine, the four-arm polyethylene glycol amine and polyethylene imine can be dissolved in a solvent; for a dispersion containing exosomes, the exosomes can be placed in a buffer solution.

[0120] As for the solvent, it can be the same as or different from solution A, and can be any feasible solvent in the art, such as a buffer solution, water, etc. Specifically, a buffer solution can be used to prepare a solution B containing the second component. As for the buffer solution, the present invention is not particularly limited, and it can be some buffer solutions commonly used in the art. Specifically, the pH value of the buffer solution can be 7.0 to 7.4. When the pH value of the buffer solution is 7.0 to 7.4, the obtained hydrogel is close to neutral, has little irritation to tissues, and is conducive to the growth of cells and tissues. As for the buffer solution, the present invention is not particularly limited, and it can be a buffer solution commonly used in the art, such as: PBS buffer solution, physiological saline, etc.

[0121] In the solution B, the mass concentration percentage of the four-arm polyethylene glycol amine is 1-25% (m / v), for example: 5%, 10%, 15%, 20%, etc.; the mass concentration percentage of the polyethyleneimine is 1-80% (m / v), for example: 5%, 15%, 25%, 35%, 45%, 55%, 65%, 75%, etc.

[0122] By adjusting the content of four-arm polyethylene glycol succinimidyl glutarate, four-arm polyethylene glycol amine and polyethylene imine, the ratio between the two PEG derivatives in the hydrogel component can be controlled, thereby improving the mechanical properties of the hydrogel component.

[0123] Furthermore, after solution A and solution B in the hydrogel composition of the present invention are mixed, the first component in solution A and the second component in solution B can undergo an amidation reaction, and can quickly form a gel, that is, generate the hydrogel of the present invention.

[0124] <Fourth Aspect>

[0125] The fourth aspect of the present invention provides a hydrogel kit comprising the hydrogel composition according to the first aspect of the present invention; preferably, the first component and the second component of the hydrogel composition are stored separately, and the first component and the second component are mixed when used.

[0126] According to the hydrogel kit of the present invention, preferably, the first component and the second component of the hydrogel components can also be stored separately, and the first component can be mixed a few hours before use or at the time of use, and then mixed with the second component.

[0127] The present invention has no limitation on the storage and use of the components in the hydrogel kit, and suitable storage and use methods can be selected as needed.

[0128] In some specific embodiments, the first component is a solid reagent, for example, the first component is a dry powder of four-arm polyethylene glycol succinimidyl glutarate. In some specific embodiments, the first component is a liquid reagent, for example, the first component is obtained by dissolving four-arm polyethylene glycol succinimidyl glutarate in a solvent. The solvent for dissolving four-arm polyethylene glycol succinimidyl glutarate can be the solvent used to dissolve the second component in the third aspect.

[0129] In some specific embodiments, the second component is a solid reagent, for example, a dry powder of four-arm polyethylene glycol amine, a dry powder of polyethylene imine, and an exosome precipitate. The dry powder of four-arm polyethylene glycol amine, the dry powder of polyethylene imine, and the exosome precipitate can be stored separately or mixed. In some specific embodiments, the second component is a liquid reagent, for example, the second component is obtained by mixing dry powders of four-arm polyethylene glycol amine and polyethylene imine with an exosome dispersion. The solvent for dissolving the dry powders of four-arm polyethylene glycol amine and polyethylene imine can be the solvent used to dissolve the second component in the third aspect.

[0130] <Fifth Aspect>

[0131] The fifth aspect of the present invention provides an application of the hydrogel according to the second aspect of the present invention or the hydrogel prepared by the preparation method of the hydrogel according to the third aspect of the present invention in the preparation of wound repair products, specifically the application in the preparation of wound repair products for pancreaticoduodenostomy.

[0132] Example

[0133] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0134] In the Examples, four-arm polyethylene glycol succinimidyl glutarate (PEG-SG, weight-average molecular weight 10,000 Da) and four-arm polyethylene glycol amine (PEG-NH2, weight-average molecular weight 10,000 Da) were purchased from Nanopeg Co., Ltd., Xiamen, China. Polyethyleneimine (PEI, weight-average molecular weight 10,000 Da) was purchased from Sigma, USA. The pH value of the PBS buffer solution was 7.2-7.4.

[0135] Preparation of M0 exosomes

[0136] RAW264.7 macrophages were cultured at 10 6 Each RAW264.7 cell was seeded in a 10 cm cell culture dish and cultured with 10 mL of cell culture medium for several days. When the number of RAW264.7 cells covered approximately 90% of the dish surface area under a light microscope, the culture medium was replaced with serum-free medium, and the supernatant was collected for exosome extraction. Exosomes were isolated and extracted using differential centrifugation: centrifugation at 300 g for 10 minutes, 2000 g for 20 minutes, and 10,000 g for 30 minutes, followed by ultracentrifugation at 110,000 g for 70 minutes at 4°C. The exosome pellet was collected and resuspended in PBS buffer to obtain an M0 exosome dispersion.

[0137] Preparation of M1 exosomes

[0138] RAW264.7 macrophages were cultured at 10 6 Cells were seeded in 10 cm cell culture dishes and cultured in 10 mL of cell culture medium for several days. When RAW264.7 cells covered approximately 65% ​​of the dish surface area under a light microscope, the culture medium was replaced, and culture medium containing 1 μg / mL LPS was added for another 24 hours. When the RAW264.7 cell population reached approximately 90% of the dish surface area, serum-free culture medium was replaced, and the supernatant was collected for exosome extraction. Exosomes were isolated and extracted using differential centrifugation at 300 g for 10 minutes, 2000 g for 20 minutes, and 10,000 g for 30 minutes, followed by ultracentrifugation at 110,000 g for 70 minutes at 4°C. The exosome pellet was collected and resuspended in PBS buffer to obtain an M1 exosome dispersion.

[0139] Preparation of M2 exosomes

[0140] RAW264.7 macrophages were cultured at 10 6 Cells were seeded in 10 cm cell culture dishes and cultured in 10 mL of cell culture medium for several days. When RAW264.7 cells covered approximately 65% ​​of the dish surface area under a light microscope, the culture medium was changed and cultured for 24 hours with culture medium containing 20 ng / mL IL-4. When the RAW264.7 cells reached approximately 90% of the dish surface area, the culture medium was changed to serum-free medium, and the supernatant was collected for exosome extraction. Exosomes were isolated and extracted using differential centrifugation at 300 g for 10 minutes, 2000 g for 20 minutes, and 10,000 g for 30 minutes, followed by ultracentrifugation at 110,000 g for 70 minutes at 4°C. The exosome pellet was collected and resuspended in PBS buffer to obtain an M2 exosome dispersion.

[0141] Preparation of bone marrow mesenchymal stem cell exosomes

[0142] Bone marrow mesenchymal stem cells were cultured at a rate of 10 6 Cells were seeded in 10 cm cell culture dishes and cultured in 10 mL of cell culture medium for several days. When bone marrow mesenchymal stem cells covered approximately 90% of the dish surface area under a light microscope, the culture medium was replaced with serum-free medium, and the supernatant was collected for exosome extraction. Exosomes were isolated and extracted using differential centrifugation: centrifugation at 300 g for 10 minutes, 2000 g for 20 minutes, and 10,000 g for 30 minutes, followed by ultracentrifugation at 110,000 g for 70 minutes at 4°C. The exosome pellet was collected and resuspended in PBS buffer to obtain an exosome dispersion.

[0143] Comparative Example 1

[0144] 200 mg of PEG-SG was dissolved in 1 mL of PBS buffer, and 200 mg of PEG-NH2 and 200 mg of PEI were dissolved in 1 mL of PBS buffer. The two solutions were then mixed using a syringe at a volume ratio of 1:1 to form a hydrogel, which was designated as PEG.

[0145] Example 1

[0146] The concentration of the extracted M2 exosomes was detected using a BCA kit according to its instructions and diluted to 1 mg / mL with PBS buffer solution. 200 mg of PEG-SG was dissolved in 1 mL of PBS buffer solution, and 200 mg of PEG-NH2 and 100 mg of PEI were dissolved in 1 mL of exosome dilution solution. Subsequently, the two solutions were mixed using a syringe in a volume ratio of 1:1 to form a hydrogel, which was recorded as PEG / Exo M2-10%.

[0147] Example 2

[0148] The concentration of the extracted M2 exosomes was detected using a BCA kit according to its instructions and diluted to 1 mg / mL with PBS buffer solution to obtain an exosome dilution solution. 200 mg of PEG-SG was dissolved in 1 mL of PBS buffer solution, and 200 mg of PEG-NH2 and 200 mg of PEI were dissolved in 1 mL of exosome dilution solution. Subsequently, the two solutions were mixed using a syringe in a volume ratio of 1:1 to form a hydrogel, which was recorded as PEG / Exo M2 -20% or PEG / Exo M2 .

[0149] Example 3

[0150] The concentration of the extracted M2 exosomes was detected using a BCA kit according to its instructions and diluted to 1 mg / mL with PBS buffer solution to obtain an exosome dilution solution. 200 mg of PEG-SG was dissolved in 1 mL of PBS buffer solution, and 200 mg of PEG-NH2 and 300 mg of PEI were dissolved in 1 mL of exosome dilution solution. Subsequently, the two solutions were mixed using a syringe in a volume ratio of 1:1 to form a hydrogel, which was recorded as PEG / Exo M2 -30%.

[0151] Example 4

[0152] The concentration of the extracted M2 exosomes was detected using a BCA kit according to its instructions and diluted to 1 mg / mL with PBS buffer solution to obtain an exosome dilution solution. 200 mg of PEG-SG was dissolved in 1 mL of PBS buffer solution, and 200 mg of PEG-NH2 and 400 mg of PEI were dissolved in 1 mL of exosome dilution solution. Subsequently, the two solutions were mixed using a syringe in a volume ratio of 1:1 to form a hydrogel, which was recorded as PEG / Exo M2 -40%.

[0153] Example 5

[0154] The concentration of the extracted M2 exosomes was detected using a BCA kit according to its instructions and diluted to 1 mg / mL with PBS buffer solution to obtain an exosome dilution solution. 200 mg of PEG-SG was dissolved in 1 mL of PBS buffer solution, and 200 mg of PEG-NH2 and 500 mg of PEI were dissolved in an exosome dilution solution containing 50 ppm of medical-grade brilliant blue. Subsequently, the two solutions were mixed using a syringe in a volume ratio of 1:1 to form a hydrogel, which was recorded as PEG / Exo M2 -50%.

[0155] Performance Testing

[0156] 1. Identification and Characterization of Exosomes

[0157] Transmission electron microscopy (TEM, FEI TECNAI) and nanoparticle analyzer (NanoFCM, N30E) were used to characterize and identify exosomes. Figure 1 shown.

[0158] Exosomes were isolated from three different states of macrophages (M0, M1, and M2). Transmission electron microscopy (TEM) imaging results showed that the three types of exosomes had uniform morphology and particle size, with an average diameter of about 100 nm and a typical cup-shaped structure ( Figure 2 ).

[0159] 2. Exosome-induced vascularization experiment

[0160] 5000 HUVEC cells were seeded in a 48-well plate. When the cells grew to about 80% confluence, they were scratched with a pipette tip (scratch width of about 200 μm), and then cultured for 24 hours in conditioned medium containing different exosomes or without exosomes, and the cell migration distance was measured under an optical microscope (Olympus, Japan). At the same time, HUVEC cells of the same density were seeded on Matrigel (Corning, USA) and cultured in conditioned medium for 24 hours to evaluate the ability to form tubes. The results were observed under an optical microscope. Figure 3 shown.

[0161] For further analysis, HUVEC cells were cultured in conditioned medium for 7 days and stained with primary antibodies against VEGF (ab32152, Abcam, UK), FITC-labeled F-actin, and DAPI. Images were then captured using a confocal laser scanning microscope (CLSM, TCS SP8, Leica, Germany), and semi-quantitative analysis was performed using ImageJ software. The results are shown in Figure 2. Figure 4 shown.

[0162] In addition, RAW264.7 macrophages (10,000 cells / well) were stimulated with 1 μg / mL LPS for 24 hours and then cultured with conditioned medium for another 3 days. At the preset time points, the cells were fixed and immunofluorescence stained with primary antibodies for IL-6 and CD206, respectively, and counterstained with DAPI, and images were taken under CLSM. At the same time, RNA sequencing analysis was performed on BMSC cells treated with conditioned medium to evaluate changes in gene expression profiles. The results are shown in Figure 2. Figure 5 shown.

[0163] In the above-mentioned exosome-induced vascularization experiment, the ability of three exosomes to promote angiogenesis in human umbilical vein endothelial cells (HUVECs) was compared. In the microvessel formation experiment, M2 macrophage exosomes showed the highest number of branch points, approximately 3200μm, while the other groups were less than 2000μm. At the same time, the capillary length was also the longest in the M2 group, reaching approximately 90,000μm per field of view, which was significantly higher than that of the other groups ( Figure 3 ac).

[0164] In addition, in the cell migration experiment, M2 macrophage exosomes significantly enhanced the migration ability of HUVECs, and the area of ​​the migrating cells was approximately 57 mm 2 , while the other groups were all below 40mm 2 ( Figure 3 d,e). Further VEGF immunofluorescence staining results showed that M2 macrophage exosomes can induce stronger angiogenesis effects ( Figure 3 f).

[0165] In order to analyze the differences in angiogenesis ability of exosomes derived from macrophages of different phenotypes, RNA transcriptome analysis was performed on human umbilical vein endothelial cells (HUVECs) treated with exosomes. The results showed that there were significant differences in gene expression in HUVECs of the three exosome-treated groups ( Figure 4 a, b). Specifically, exosomes derived from M2 macrophages are mainly enriched in angiogenesis-related pathways, such as the HIF-1 pathway, and also show a stronger enrichment in inflammatory regulatory pathways (including NF-κB and JAK-STAT pathways) ( Figure 4 cf). These results indicate that M2 macrophages have a stronger regulatory effect on inflammation and provide more favorable conditions for tissue regeneration.

[0166] In addition, the effects of exosomes derived from M2 macrophages and exosomes derived from bone marrow mesenchymal stem cells (BMSCs) in HUVECs were compared. The results showed that M2 macrophage exosomes were more effective in promoting angiogenesis and regulating inflammation ( Figure 5 ac).

[0167] 3. Physical performance test

[0168] Take the PEG / Exo of Example 2 M2 The hydrogel and the PEG hydrogel of comparative example 1 were subjected to relevant experiments, and the results were as follows. Figure 6 The details are as follows:

[0169] 3.1 Swelling experiment

[0170] The initial mass of the hydrogel, M0, was recorded. The hydrogel was then immersed in PBS buffer solution and cultured at 37°C with shaking. The hydrogel was taken out and weighed at different time points, and the mass, M, was recorded. n The swelling rate calculation formula is as follows: Swelling rate (%) = M n / M0×100%.

[0171] 3.2 Degradation experiment

[0172] The hydrogel was freeze-dried and its initial mass M0 was weighed. It was then immersed in PBS and incubated in a constant temperature oscillator at 37°C. Subsequently, the hydrogel was taken out at specific time intervals and its mass M0 was weighed after freeze-drying. n , and the mass degradation rate is calculated using the following formula: Degradation rate (%) = M n / M0×100%.

[0173] 3.3 Tensile test

[0174] The hydrogel solution was poured into a polytetrafluoroethylene mold and demolded after forming to produce a dumbbell-shaped hydrogel. The tensile properties of the two hydrogels were tested using a universal testing machine. The elastic modulus was calculated from the stress-strain curve, based on the slope within the initial 5-15% strain range.

[0175] 3.4 Rheological experiments

[0176] Hydrogels with an 11 mm diameter and a thickness of 4 mm were prepared for testing. A small-amplitude dynamic frequency sweep experiment was performed using a rheometer (Mars40, Thermo Fisher, USA) with a sweep range of 0.1–500 rad / s and logarithmic data collection. The hydrogel sample was placed horizontally on a test plate, and the storage modulus (G') and loss modulus (G") were measured while downward pressure was applied.

[0177] 3.5 Exosome release experiment

[0178] The volume is 1cm 3 PEG / Exo M2 The hydrogel was immersed in 10 mL of deionized water (deionized water has not been treated with enzymes) and incubated in a constant temperature water bath at 37°C for 1, 3, 5, 7, 14, and 21 days. The supernatant was then aspirated and the absorbance value after the supernatant reacted with the BCA reagent was measured according to the product instructions of the BCA kit to obtain the protein concentration. M2 The exosome concentration in the hydrogel was used to calculate the amount of exosomes released.

[0179] Based on the above research results, exosomes derived from M2 macrophages were loaded into the hydrogel skeleton to form an adhesive hydrogel ( Figure 6a). Through tensile and rheological tests, the hydrogel showed good mechanical strength, with a tensile strength of 44kPa and a storage modulus of 25kPa ( Figure 6 b, c). In addition, the results of the swelling and degradation behavior of the hydrogel showed that the hydrogel swelled significantly within 7 days, with the swelling rate reaching 134% on the first day and increasing to 170% on the seventh day, eventually reaching swelling equilibrium ( Figure 6 d). Degradation experiments showed that the hydrogel could be completely degraded within 30 days ( Figure 6 e). Exosome release from the hydrogel lasted up to 21 days ( Figure 6 f), indicating its long-term therapeutic effect in the tissue regeneration process.

[0180] 4. Tissue Adhesion Experiment

[0181] Adhesion strength test includes adhesion strength and peeling strength test, using pig skin as a model, and referring to the American Society for Testing and Materials (ASTM) F standard. M2 The hydrogel and the PEG hydrogel of comparative example 1 were subjected to relevant experiments, and the results were as follows. Figure 7 shown.

[0182] Fresh pig skin was defatted according to ASTM F2258 and cut into 2.5 cm × 2.5 cm pieces. 100 μL PEG / Exo was injected into one of the pieces of pig skin. M2 The two pieces of pigskin were bonded at room temperature. After 1 hour, the force-displacement curve was recorded at a speed of 5 mm / min using an electronic universal testing machine, and the adhesion strength (Pa) was calculated: F max (N) / Area (m 2 ).

[0183] The peeling force test was performed according to ASTM F2256. The pig skin was cut into 4 cm × 2.5 cm pieces and injected with 100 μL PEG / Exo M2 Cover 2.5cm x 1cm and bond on the same side. After 1 hour, measure the force-displacement curve in the shear direction at a speed of 5mm / min.

[0184] The bursting strength test was performed according to ASTM F2392. A 5 mm defect was created in the colon tissue with a diameter of 3 cm, and then 1 mL of PEG / Exo was injected. M2 The defect was covered and allowed to stand at room temperature for 1 hour. The pressure pump was applied at 2 mL / min and the PEG / Exo M2 Bursting strength at rupture.

[0185] In the tissue adhesion experiment, commercial fibrin glue (Fibrin) was used as the control group and the same detection method was used for detection.

[0186] PEG / Exo M2 The tissue adhesion of the hydrogel is attributed to its N-hydroxysuccinimide groups that can chemically bond with primary amino groups in tissues. Therefore, the hydrogel exhibits good adhesion properties on a variety of tissues, including skin, liver, stomach, lungs, and heart. Figure 7 a). PEG / Exo M2 The adhesion strength (26.47±2.95kPa), peeling force (0.23±0.01N / cm) and bursting strength (28.47±3.50kPa) of the hydrogel were significantly better than those of fibrin glue ( Figure 7 bd), indicating that it has high application potential in sealing pancreatic leakage.

[0187] 5. Antibacterial test

[0188] 1 mL of the hydrogel of Example 1-5 was placed in a 50 μL 1×10 6 A 0.1 CFU / mL bacterial suspension was placed in a Petri dish containing 10 mL of TSB medium and incubated at 37°C for 24 hours. After incubation, 100 μL of the solution was transferred to a 96-well plate and the absorbance was measured at 600 nm. Additionally, 1 mL of the solution was transferred to a 1.5 mL EP tube and photographed.

[0189] Take 1 mL of hydrogel and place it in a culture dish, add 50 μL of 1×10 6 CFU / mL bacterial suspension and 10mL TSB medium were added and incubated at 37°C with shaking at 50rpm for 24 hours. After incubation, the samples were removed and placed in a 24-well plate, and 120μL of 0.1% crystal violet solution (dissolved in methanol) was added to each well to stain the biofilm. The dye was removed after standing at room temperature for 10 minutes. Wash three times with PBS and air-dry, then 200μL of 95% ethanol was added to each well to dissolve the crystal violet dye, and the plate was allowed to stand at room temperature for 15 minutes, and the image was taken using a camera. Finally, 100μL of the solution was transferred to a 96-well plate, and the absorbance was measured at 540nm to evaluate the formation of the biofilm. The results are shown in Figure 2. Figure 8 shown.

[0190] Since cationic PEI polymers have broad-spectrum antibacterial activity, the antibacterial properties of the hydrogels increase with increasing PEI concentrations. When the PEI concentration reaches 30%, the antibacterial effect tends to stabilize ( Figure 8 a). In addition, 30% PEI concentration significantly inhibited the biofilm formation of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and methicillin-resistant Staphylococcus aureus (MRSA) ( Figure 8 b). In the colony formation experiment, the 30% PEI hydrogel showed the strongest antibacterial effect ( Figure 8 c).

[0191] 6. Induction of Vascular Differentiation Experiment

[0192] To evaluate the angiogenic potential of the extract, human umbilical vein endothelial cells (HUVECs) were used to culture the PEG / Exo M2 The hydrogel and the PEG hydrogel of comparative example 1 were subjected to relevant experiments, and the results were as follows. Figure 9 The specific experiment is as follows:

[0193] 5,000 HUVECs were seeded in 48-well plates and co-cultured with the extract for 7 days, with the medium replaced every other day. Following incubation, cells were stained with a primary antibody against VEGF (ab32152, Abcam, UK), FITC-conjugated F-actin, and a fluorescent secondary antibody. After incubation in the dark for 30 minutes, VEGF expression was observed using confocal laser scanning microscopy (CLSM), and fluorescence intensity was semi-quantitatively analyzed using Image Pro Plus. Matrigel (Corning, USA) was applied to cell culture plates, and 5,000 HUVECs were seeded and co-cultured with the extract for 12 hours. Subsequently, tubular network formation was observed under a light microscope, and branching and junctional structures were semi-quantitatively measured using ImageJ. 5,000 HUVECs were seeded in 24-well plates. When cells reached approximately 80% confluency, a 200 μm-wide scratch was created using a pipette tip. Cells were then cultured in the extract medium for 24 hours, and cell migration was observed under a light microscope. ImageJ was used to semi-quantitatively analyze the cell migration distance.

[0194] Confocal laser scanning microscopy (CLSM) fluorescence imaging results showed that PEG / Exo M2 The microvessel formation in the group was the most significant, with 243 branch points, 53 intersection points, and a capillary length of 7784 μm, which were significantly higher than those in the blank control group and the PEG hydrogel group without M2 macrophage exosomes ( Figure 9 ad). In addition, in the cell migration experiment, PEG / Exo M2 The migration area of ​​the cells in the group reached 56 mm 2 , while the control group and the PEG hydrogel group without exosomes were 8 mm 2 and 22mm 2 , indicating that PEG / Exo M2 Can significantly enhance cell migration ability ( Figure 9 e,f). PEG / Exo M2 The hydrogel group showed a higher expression of VEGF vascularization markers, and its cyanosis multiple was about 3.5 times and 2.8 times that of the blank control and PEG gel, indicating that it had a higher induction activity of vascularization ( Figure 9 gh).

[0195] 7. Inflammation regulation experiment

[0196] Immunofluorescence staining: RAW264.7 macrophages were cultured at 5×10 4 Cells were seeded in 24-well plates at a density of 100 cells / well and cultured in conditioned medium of different groups for 48 hours. Subsequently, cells were treated with 100 ng / mL LPS for 24 hours, fixed (4% paraformaldehyde, Beyotime, China) for 15 minutes, and permeabilized with 0.3% Triton X-100 (Beyotime, China) for 15 minutes, washed three times with PBS, and blocked with 5% BSA for 1 hour. Subsequently, primary antibodies for IL-6 and CD206 were added and incubated at 4°C overnight. The next day, secondary antibodies were incubated at room temperature for 1 hour, and images were captured using an inverted fluorescence microscope (Nikon, Japan).

[0197] Flow cytometry: Macrophages were collected and washed with PBS, blocked with 5% bovine serum albumin (BSA) (Bioss, China), and incubated with CD80 and CD206 antibodies for 1 hour at 4°C. Finally, the polarization ratio of macrophages was analyzed using a flow cytometer (BD, USA).

[0198] PEG / Exo of Example 2 M2 The hydrogel, the PEG hydrogel of comparative example 1 and the blank control group were compared and analyzed. The results are as follows Figure 10 shown.

[0199] After LPS stimulation of unpolarized macrophages, a significant increase in the pro-inflammatory marker IL-6 was observed, while no significant change in the anti-inflammatory marker CD206 was observed. M2 After hydrogel, the expression of IL-6 was significantly decreased, while the expression of CD206 was significantly increased ( Figure 10 a). To quantify this observation, flow cytometry analysis was performed and the results showed that after LPS stimulation, PEG / Exo M2 CD206 + The cell ratio was the highest, reaching 7.46%, compared with the unstimulated (CD206 + 8.9%), while the control group and PEG group were 1.12% and 0.38% ( Figure 10 b,c).

[0200] This phenomenon was subsequently verified in a subcutaneous implantation model, where LPS was first injected to induce inflammation, followed by implantation of the hydrogel at the injection site. Histological evaluation was performed on days 3 and 6 after implantation ( Figure 10d) H&E staining results showed that the thickness of the fibrous capsule in the control group was 642μm and 347μm on the 3rd and 6th days after surgery, respectively. M2 The fibrous capsule thickness of the 44 and 253 μm groups was significantly lower, respectively. Figure 10 e,f). This indicates that the release of exosomes from M2 macrophages regulates the inflammatory response, thereby reducing the thickness of the fibrous capsule. M2 The expression of CD163 in the PEG / Exo group was higher, and the expression of TNF-α was lower. The ratio of the fluorescence intensity of M2 and M1 showed that the expression of PEG / Exo M2 The M2 / M1 fluorescence intensity of the group was 10.83 (day 6), which was significantly higher than that of the other groups.

[0201] 8. Subcutaneous implantation experiment and wound repair experiment

[0202] The subcutaneous implantation experiment followed the Tianjin Medical Laboratory Animal Care Guidelines and was approved by the Animal Experiment Ethics Committee of Yishengyuan Gene Technology (Tianjin) Co., Ltd. (Ethics Approval Number: YSY-DWLL-2024658). The experimental animals were divided into three groups: 1) injected with 1 mL of sterile saline (control group); 2) subcutaneously implanted with PEG hydrogel (1 cm in diameter and 1 cm in depth); and 3) subcutaneously implanted with PEG / Exo M2 The hydrogel (1 cm in diameter, 1 cm in depth) was implanted and LPS was injected 24 hours later. The experiment was performed on anesthetized SD rats. The rats were sacrificed at different time points (days 3 and 6), and tissues from the implanted sites were removed for H&E staining to assess biocompatibility. TNF-α and CD163 immunofluorescence staining were also performed to assess histological responses. In addition, on day 14 of the experiment, sections of major organs (heart, liver, spleen, lungs, and kidneys) were sectioned to assess their histopathological changes.

[0203] The wound repair experiment followed the Tianjin Medical Laboratory Animal Care Guidelines and was approved by the Animal Experiment Ethics Committee of Yishengyuan Gene Technology (Tianjin) Co., Ltd. (Ethics Approval Number: YSY-DWLL-2024659). SD rats were anesthetized with tribromoethanol (0.2 mL / 10 g) and a 10 mm diameter skin defect was created on the back. The experimental groups were directly treated with PEG or PEG / Exo M2 The hydrogel was applied to the wound surface, while the control group did not receive any treatment. The wound healing process was observed and recorded at specific time points. On the 6th and 12th days, the experimental animals were sacrificed and 1×1 cm 2Skin tissue was fixed in 4% paraformaldehyde. Histological Analysis: Skin samples were dehydrated, embedded in paraffin, and cut into 5 μm tissue sections using a microtome (Leica, Germany). Paraffin sections were dewaxed with xylene, hydrated with graded alcohols, and stained with Hematoxylin and Erythrocyte Embryo (H&E) and Masson's trichrome. Furthermore, antigen retrieval was performed using hyaluronidase, and immunofluorescence staining for TNF-α, CD163, and VEGF was performed. Fluorescence intensity was semiquantitatively analyzed using Image Pro Plus.

[0204] To evaluate PEG / Exo M2 The role of hydrogel in promoting tissue repair was studied using a full-thickness skin wound model ( Figure 12 a). As the implantation time increases, PEG / Exo M2 The wound healing speed of the group was the fastest, and it was almost completely healed on the 12th day, which was significantly better than that of the other groups ( Figure 12 b,c). Histological staining results showed that PEG / Exo M2 The introduction of PEG / Exo did not cause significant inflammatory response in the surrounding tissues. M2 The width of granulation tissue in the PEG group was the smallest, at 3.8 mm, while that in the control group was 6.3 mm and that in the PEG group was 4.5 mm ( Figure 12 d,e). In addition, PEG / Exo M2 The collagen production in the group was more obvious, reaching about 58% on the 6th day ( Figure 12 f). PEG / Exo M2 The hydrogel showed a higher M2 / M1 fluorescence intensity ratio, indicating that PEG / ExoM2 has a significant therapeutic effect in promoting wound repair ( Figure 12 g,h).

[0205] In addition, PEG / Exo M2 No obvious immune rejection reaction was induced on the 6th day after subcutaneous implantation. H&E staining of the heart, liver, spleen, lung and kidney showed no obvious tissue damage in all groups, indicating that the material has excellent biocompatibility ( Figure 11 On day 12, histological staining showed that PEG / Exo M2 The tissue at the wound surface of the group was almost completely repaired, the collagen fibers were arranged tightly and regularly, and the collagen deposition reached 92% ( Figure 13 ). 12 days after implantation, PEG / Exo M2 The H&E staining results of the heart, liver, spleen, lung and kidney of all groups showed normal tissue structure ( Figure 14 ).

[0206] 9. Pancreaticojejunostomy experiment

[0207] The pancreaticojejunostomy experiment followed the Tianjin Medical Laboratory Animal Care Guidelines and was approved by the Animal Experiment Ethics Committee of Yishengyuan Gene Technology (Tianjin) Co., Ltd. (Ethical Approval Number: YSY-DWLL-2024660). Bama pigs were anesthetized by inhalation of isoflurane in a sterile operating room, and the pancreas and jejunum were exposed by midline laparotomy. An electric knife was used to make a 1 cm incision on the pancreatic head and jejunum, respectively, and the pancreatic head wound was anastomosed to the jejunum by suture. The experiment was divided into three groups: 1) suture only, no additional treatment (blank control); 2) PEG hydrogel was covered on the suture; 3) PEG / Exo was covered on the suture. M2 hydrogel. After surgery, the abdominal cavity was closed, and the incisions were sutured layer by layer. At the same time, a drainage tube was placed in the abdominal cavity to collect the peritoneal drainage fluid on the third day after surgery and analyze the α-amylase concentration therein. On the 30th day after surgery, the surgical site was observed with the naked eye, and blood samples were collected for routine hematological and biochemical analysis before the animals were euthanized. The tissues at the surgical site were fixed with formaldehyde and subjected to H&E, Masson's trichrome staining, and VEGF / α-SMA / DAPI immunofluorescence staining. In addition, on the 30th day after surgery, blood was collected for routine blood tests and blood biochemistry analysis, and H&E staining was performed on the heart, liver, spleen, lungs, and kidneys to evaluate potential systemic effects. The results are as follows: Figure 15-19 shown.

[0208] Pancreaticojejunostomy was performed in Bama pigs and compared with clinical practice. M2 Close the wound with sutures ( Figure 15 a, b). On the third day after surgery, drainage fluid was collected from the peritoneal cavity, the drainage volume was recorded, and the α-amylase concentration was measured. When the α-amylase concentration in the drainage fluid exceeded 5000 U / L, pancreatic leakage was considered to have occurred. In the blank control group, α-amylase (α-AMS) was 5040 U / L, indicating that there may be a risk of pancreatic leakage because suturing the wound failed to completely prevent its occurrence. However, after PEG and PEG / Exo M2 After the wound was closed, the α-AMS levels dropped to 891U / L and 847U / L, respectively, successfully preventing pancreatic leakage ( Figure 15 c, d). Thirty days after surgery, tissue sampling was performed. Gross examination showed that PEG / Exo M2 No significant tissue adhesion was observed in the group ( Figure 16 ). Histological analysis of pancreaticojejunostomy tissue showed that PEG / Exo M2 The tissue regeneration and healing of the group were better, especially in terms of collagen deposition, angiogenesis and tissue integrity of the anastomosis site ( Figure 15 e). Masson's trichrome staining shows the difference in collagen fiber deposition at the anastomosis. PEG / Exo M2 group showed more mature collagen ( Figure 15 f, Figure 17 ), suggesting that M2 macrophage-derived exosomes may enhance the healing process by promoting tissue repair. The control group showed a stronger inflammatory response (such as inflammatory cell infiltration), while PEG / Exo M2 The control group showed less inflammatory cell infiltration or better immune regulation, supporting the role of M2 macrophage exosomes in alleviating inflammation.

[0209] PEG / Exo M2 No significant immune rejection reaction was induced after 30 days of implantation. H&E staining of the heart, liver, spleen, lung and kidney of all groups showed normal tissue morphology ( Figure 18 ), and the hematological parameters were not significantly different from those of the control group, indicating that it has excellent biosafety ( Figure 19 ).

[0210] The experimental data of the present invention are expressed as mean ± standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA) using GraphPad Prism 8.0 software. The statistical significance criteria are as follows: ns: no significant difference, * P≤0.05, ** P≤0.01, *** P≤0.001.

[0211] It should be noted that, although the technical solutions of the present invention are described with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0212] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or technological improvements in the marketplace, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A hydrogel composition, characterized in that include: A first component and a second component; wherein, The first component includes four-arm polyethylene glycol succinimide glutarate, the second component includes four-arm polyethylene glycol amine and polyethylene imine; and The second component also includes exosomes.

2. The hydrogel composition according to claim 1, wherein The weight average molecular weight of the four-arm polyethylene glycol succinimide glutarate is 5000-20000 Da, the molecular weight of the four-arm polyethylene glycol amine is 5000-20000 Da, and the molecular weight of the polyethylene imine is 2000-20000 Da.

3. The hydrogel composition according to claim 1 or 2, characterized in that The volume ratio of the first component to the second component is 1:(0.8-1.2), preferably 1:(0.9-1.1).

4. The hydrogel composition according to any one of claims 1 to 3, characterized in that In the second component, the mass ratio of the four-arm polyethylene glycol amine to the polyethylene imine is 1:(0.8-8), preferably 1:(0.9-5).

5. The hydrogel composition according to any one of claims 1 to 4, characterized in that The average diameter of the exosomes is 50-200 nm; and / or the exosomes are derived from macrophages.

6. A hydrogel, characterized in that The hydrogel is formed by reacting the first component and the second component of the hydrogel composition according to any one of claims 1 to 5; wherein the first component and the second component can be bonded to form a hydrogel skeleton, and the exosomes are loaded in the hydrogel skeleton; Preferably, the concentration of the exosomes in the hydrogel is 0.1-10 mg / mL.

7. The hydrogel according to claim 6, characterized in that The hydrogel has at least one of the following characteristics: The swelling rate of the hydrogel is 100-200%; The hydrogel has a tensile strength of 10-100 kPa and a storage modulus of 5-50 kPa; The exosome release time is more than 10 days; The hydrogel has a tissue adhesion strength of 10-50 kPa, a peeling force of 0.01-0.5 N / cm, and a bursting strength of 10-50 kPa.

8. A method for preparing the hydrogel according to claim 6 or 7, characterized in that: The following steps are involved: preparing a solution A comprising a first component; preparing a solution B comprising the second component; Solution A and solution B were mixed to obtain a hydrogel.

9. The preparation method according to claim 8, characterized in that In the solution A, the mass concentration percentage of the four-arm polyethylene glycol succinimidyl glutarate is 1-25% (m / v); and / or, In the solution B, the mass concentration percentage of the four-arm polyethylene glycol amine is 1-25% (m / v), and the mass concentration percentage of the polyethyleneimine is 1-80% (m / v).

10. A hydrogel kit, characterized in that: It comprises the hydrogel composition according to any one of claims 1 to 5; preferably, the first component and the second component of the hydrogel composition are stored separately.

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