Biological patch as well as preparation method and application thereof

Abstract

The invention discloses a biological patch as well as a preparation method and application thereof. The invention provides a biological patch, which comprises: a substrate, the substrate has a cellosilk structure, and the cellosilk has a polar group; wherein the surface of the substrate is grafted with a catechol functional group by using the polar group, and the catechol functional group is derived from a polyphenol compound. The biological patch disclosed by the invention has relatively good elongation at break, modulus and toughness. In addition, the enhanced mechanical properties are similar to the mechanical compatibility of natural intestinal tissues. Meanwhile, the biological patch disclosed by the invention has excellent blood compatibility and cell compatibility. The invention further provides a biological patch with the exosome, the cytocompatibility is further improved, the biological patch has excellent angiogenesis promoting activity and tissue regeneration potential, and meanwhile, the biological patch has the functions of resisting inflammation and promoting tissue repair.

Description

Technical Field

[0001] The present invention relates to a biological patch and its preparation method and use, belonging to the field of biomedical materials. Background Art

[0002] Gastrointestinal anastomosis is a common surgery, mainly used for tumor resection and restoration of the digestive system function. Although surgical techniques have advanced, complications such as anastomotic stenosis, postoperative bleeding, persistent inflammation, poor healing, and infection are still major challenges in clinical practice. Approximately 14 million patients undergo abdominal surgery globally each year, and the incidence of anastomotic leakage ranges from 4% to 21%. Among them, the leakage rate of gastrointestinal anastomosis is 5% to 15%, seriously affecting the recovery and quality of life of patients. Anastomotic leakage can lead to life-threatening conditions such as acute peritonitis, abdominal infection, and sepsis, resulting in a 30% increase in mortality. These persistent complications highlight the urgent need for technological innovation to improve surgical outcomes. The development of novel bioactive patches has particular potential in addressing these issues, promising to promote rapid and effective healing at the anastomotic site, reduce the risk of anastomotic leakage and related complications, and optimize postoperative recovery. An ideal patch should not only provide mechanical support for damaged tissues but also possess bioactivity to promote tissue regeneration and reduce the inflammatory response. Advancing the development of this technology can significantly improve the safety and success rate of gastrointestinal surgery, thereby enhancing the treatment effect and quality of life of patients.

[0003] In gastrointestinal anastomosis, both synthetic patches and biological patches can be selected, and each type of patch has its unique advantages and limitations. Synthetic patches are usually composed of non-biodegradable polymers, capable of providing strong mechanical strength and structural support, but their integration with biological tissues is poor, often causing chronic inflammation and potential immune rejection reactions. On the other hand, biological patches are derived from natural materials such as collagen or extracellular matrix, capable of promoting cell adhesion and proliferation, enhancing tissue regeneration, and thus achieving better tissue integration. However, the mechanical strength of biological patches is usually limited and may not be sufficient to handle high-pressure anastomotic sites or problems with postoperative tissue relaxation. To address these issues, researchers have proposed various strategies. Some methods improve the mechanical properties of biological patches through cross-linking techniques, while others focus on enhancing their healing ability by introducing bioactive components (such as growth factors, cytokines, and active metal ions). These innovations aim to combine the structural advantages of synthetic patches and the biocompatibility of natural materials to optimize the effect of anastomotic repair.

[0004] Among numerous bioactive candidates, exosomes have emerged as important tools in tissue engineering and regenerative medicine. These nanoscale extracellular vesicles can facilitate intercellular communication and carry various biomolecules including proteins, lipids, RNA, and DNA, reflecting the characteristics of their source cells. Exosomes play a key role in regulating physiological and pathological processes and are thus ideal carriers for promoting healing and regeneration. Compared with traditional bioactive additives, exosomes can provide more precise and bioactive therapeutic effects. Although exosomes derived from animals or humans are often used in research, concerns regarding immunogenicity and biosafety still exist. Also, typically, exosomes are introduced into biomaterials by direct mixing, which often results in uneven distribution and rapid release, thereby reducing the therapeutic effect. Summary of the Invention

[0005] Problems to be Solved by the Invention

[0006] The mechanical strength of biological patches is usually limited and may be insufficient to handle high-pressure anastomosis sites or problems with postoperative tissue relaxation. Synthetic patches are usually composed of non-biodegradable polymers, which can provide strong mechanical strength and structural support, but their integration with biological tissues is poor, often causing chronic inflammation and potential immune rejection reactions.

[0007] When introducing bioactive components, exosomes, into biological patches, there are concerns regarding immunogenicity and biosafety in exosomes derived from animals or humans. Also, in the prior art, there are problems such as uneven distribution and rapid release, thereby reducing the therapeutic effect.

[0008] To solve these problems, the present invention provides a biological patch and its preparation method and uses.

[0009] Solutions for Solving the Problems

[0010] In a first aspect of the present invention, a biological patch is provided, the biological patch comprising: a substrate having a fibril structure, and the fibrils having polar groups; wherein, by using the polar groups, catechol functional groups are grafted onto the surface of the substrate, and the catechol functional groups are derived from polyphenolic compounds.

[0011] In some embodiments, the polar groups include carboxyl groups.

[0012] In some preferred embodiments, the carboxyl groups are activated by an activator.

[0013] In some embodiments, the polyphenolic compounds include one or a combination of two or more of dopamine or its salts, tannic acid, catechol, pyrogallol, gallic acid, and catechuic acid.

[0014] In some preferred embodiments, the polyphenolic compound includes dopamine or a salt thereof.

[0015] In some embodiments, exosomes are also attached to the surface of the biological patch.

[0016] In some embodiments, the exosomes are coupled to the surface of the biological patch via hydrogen bonds with the catechol functional groups.

[0017] In some preferred embodiments, the exosomes include plant-derived exosomes.

[0018] In some more preferred embodiments, the exosomes include Allium-derived exosomes.

[0019] In a second aspect of the present invention, there is provided a method for preparing the biological patch according to the first aspect of the present invention, the preparation method comprising: grafting catechol functional groups onto the surface of a substrate; preferably, coupling exosomes to the surface of the substrate using the catechol functional groups.

[0020] In some embodiments, in the step of grafting catechol functional groups onto the surface of the substrate, the substrate is contacted with a solution containing a polyphenolic compound, and the content of the polyphenolic compound in the solution containing the polyphenolic compound is 0.1 - 50 (w / v)%.

[0021] In some embodiments, the reaction time between the substrate and the solution containing the polyphenolic compound is 1 - 18 h.

[0022] In some embodiments, before performing the step of grafting catechol functional groups onto the surface of the substrate, a step of activating the substrate is further included.

[0023] In some optional embodiments, a carbodiimide activator and / or an N-hydroxyimide activator is used to activate the substrate in the step of activating the substrate.

[0024] In some embodiments, in the step of coupling exosomes to the surface of the substrate using the catechol functional groups, the exosomes are coupled to the surface of the substrate by contacting the substrate with a solution containing exosomes.

[0025] In some embodiments, in the solution containing exosomes, the content of exosomes is 1 - 300 μg / mL.

[0026] In some embodiments, after the step of coupling exosomes to the surface of the substrate using the catechol functional groups, a drying step is further included.

[0027] In a third aspect of the present invention, there is provided the use of the biological patch according to the first aspect of the present invention or the biological patch prepared by the preparation method according to the second aspect of the present invention in the preparation of a product for tissue repair.

[0028] In some preferred embodiments, the tissue includes gastrointestinal tissue.

[0029] Effects of the Invention

[0030] In some embodiments of the present invention, the biological patch of the present invention has good elongation at break, modulus and toughness. Moreover, these enhanced mechanical properties are similar to the mechanical compatibility of natural intestinal tissue. Meanwhile, the biological patch of the present invention has excellent blood compatibility and cell compatibility.

[0031] In some embodiments of the present invention, the biological patch with exosomes provided by the present invention further improves cell compatibility, and has excellent angiogenesis-promoting activity, as well as the potential for tissue regeneration, and at the same time has anti-inflammatory and tissue repair-promoting functions. Description of the Drawings

[0032] Figure 1 Schematic diagram for the preparation and characterization of the SIS-dopa patch.

[0033] Figure 1 In a, it is a schematic diagram of the preparation process, starting from the EDC and NHS activation of the SIS patch, followed by dopamine grafting to obtain the SIS-dopa patch (the inset shows the appearance of the SIS-dopa patch); Figure 1 In b, it is the FT-IR spectra of the SIS and SIS-dopa patches; Figure 1 In c, it is the water contact angle study for evaluating the change in hydrophilicity after dopamine modification; Figure 1 In d, it is the DPPH free radical scavenging experiment of the SIS-dopa patch (the inset shows the appearance of the DPPH solution after different treatments); Figure 1 In e, it is the protein adsorption experiment, comparing the SIS-dopa patches with different dopamine grafting times. Statistical significance and P values were determined by ANOVA comparison test, and the data are expressed as mean ± standard deviation (n = 4), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

[0034] Figure 2 Schematic diagram for the sustained release and binding mechanism of exosomes of the SIS-dopa-exo patch.

[0035] Figure 2 In a, it is a schematic diagram of the hypothesized binding mechanism of scallion exosomes to the SIS patch and the role of dopamine as a coupling agent; Figure 2In which, b is a schematic diagram of the real-time release curve of exosomes of SIS-dopa-exo and unmodified SIS-exo patches. Figure 2 In which, c is a schematic diagram of the high-resolution XPS spectrum of the oxygen state on the patch surface; Figure 2 In which, d is a schematic diagram showing possible hydrogen bond interactions in the FT-IR spectrum; Figure 2 In which, e is a schematic diagram of the molecular docking simulation of the interaction between SIS-dopa-exo and aquaporin; Figure 2 In which, f is a schematic diagram of the in-situ FT-IR spectrum describing the dynamic binding behavior. Statistical significance and P values were determined by Student's t-test, and the data are presented as mean ± standard deviation (n = 4), **P ≤ 0.01.

[0036] Figure 3 It is a schematic diagram of angiogenesis of the SIS-dopa-exo patch.

[0037] Figure 3 In which, a is a schematic diagram of the angiogenesis experiment, including preparing the conditioned medium and co-culturing with HUVECs to evaluate the angiogenesis expression; Figure 3 In which, b is a schematic diagram of the scratch experiment showing the promoting effect on cell migration; Figure 3 In which, c is a schematic diagram of the semi-quantitative analysis of the migration distance in the scratch experiment; Figure 3 In which, d is a schematic diagram of the lumen formation experiment to demonstrate angiogenesis; Figure 3 In which, e is a schematic diagram of the semi-quantitative analysis of the microtube length; Figure 3 In which, f is a schematic diagram of immunofluorescence staining of VEGF (red), filamentous actin (F-actin, green), and DAPI (blue) after 7 days of culture; Figure 3 In which, g is an RT-PCR analysis of the VEGF expression level to evaluate angiogenesis differentiation. Statistical significance and P values were determined by ANOVA comparison test, and the data are presented as mean ± standard deviation (n = 4), **P ≤ 0.01, ***P ≤ 0.001.

[0038] Figure 4 It is an evaluation of inflammation regulation and inflammation implantation research.

[0039] Figure 4 In which, a is a schematic diagram of cell inflammation research; Figure 4 In which, b is a quantitative analysis of the expression levels of IL-6 and Arg-1 in RAW264.7 macrophages; Figure 4 In which, c is an immunofluorescence staining of IL-6 (red), DAPI (blue), Arg-1 (green), and DAPI (blue) in RAW264.7 cells after 24 hours of LPS treatment; Figure 4d in it is a schematic diagram of the inflammatory implantation study induced by LPS injection; Figure 4 e in it is the H&E staining of the inflammatory implantation site 3 days after surgery; Figure 4 f in it is the immunofluorescence staining of TNF-α (red), CD163 (green) and DAPI (blue) 3 days after surgery; Figure 4 g in it is the semi-quantitative analysis of TNF-α and CD163 expression in the implantation site. Statistical significance and P values were determined by ANOVA comparison test, and the data are expressed as mean ± standard deviation (n = 4), **P ≤ 0.01, ***P ≤ 0.001.

[0040] Figure 5 is a schematic diagram of the interaction mechanism of SIS-dopa-exo patch in cell signaling.

[0041] Figure 5 a in it is a Venn diagram showing the differentially expressed genes among SIS-dopa-exo, SIS and the control group; Figure 5 b in it is a volcano plot comparison of the up-regulated and down-regulated gene expressions between SIS-dopa-exo and the control group; Figure 5 c in it is a volcano plot comparison of the up-regulated and down-regulated gene expressions between SIS-dopa-exo and SIS; Figure 5 d in it is the KEGG enrichment analysis showing the activated signaling pathways between SIS-dopa-exo and the control group; Figure 5 e in it is the KEGG enrichment analysis showing the activated signaling pathways between SIS-dopa-exo and SIS; Figure 5 f in it is the heatmap clustering analysis of the differentially expressed genes related to pro-inflammation, anti-inflammation and angiogenesis.

[0042] Figure 6 is the gastrointestinal anastomosis study of rat colon injury.

[0043] Figure 6 a in it is a schematic diagram of the rat surgical operation; Figure 6 b in it is to establish a gastrointestinal anastomosis model and implant the SIS-dopa-exo patch at the injured site; Figure 6 c in it is the H&E staining of the injured site in different treatment groups 14 days and 28 days after surgery; Figure 6 d in it is the immunofluorescence staining of CD31 (red) and DAPI (blue) at the injured site 28 days after surgery; Figure 6 e in it is the immunofluorescence staining of TNF-α (red), CD163 (green) and DAPI (blue) at the injured site 28 days after surgery; Figure 6 f in it is the semi-quantitative analysis of the CD31 fluorescence intensity; Figure 6where g is the semi - quantitative analysis of TNF - α and CD163 fluorescence intensity. In Figure 6 in f and e, asterisks indicate the implanted patches. Statistical significance and P - values were determined by ANOVA comparison tests. Data are presented as mean ± standard deviation (n = 3), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

[0044] Figure 7 is a schematic diagram of the gastro - intestinal anastomosis model for porcine colonic injury.

[0045] Figure 7 in a is the establishment of the clinical gastro - intestinal anastomosis model after SIS - dopa - exo patch implantation; Figure 7 in b is the histological evaluation of the injured site in different treatment groups at 28 days after surgery by H&E staining; Figure 7 in c is the Masson's trichrome staining for evaluating the histological changes of the injured site in different treatment groups at 28 days after surgery; Figure 7 in d is the immunofluorescence staining of VEGF (red), α - SMA (green) and DAPI (blue); Figure 7 in e is the immunofluorescence staining of Arg - 1 (red), CD3e (green) and DAPI (blue) for evaluating the injured site at 28 days after surgery; Figure 7 in f is the semi - quantitative analysis of VEGF fluorescence intensity; Figure 7 in g is the semi - quantitative analysis of Arg - 1 and CD3e fluorescence intensity. Statistical significance and P - values were determined by ANOVA comparison tests. Data are presented as mean ± standard deviation (n = 3), **P ≤ 0.01, ***P ≤ 0.001.

[0046] Figure 8 is the characterization of scallion - derived exosomes.

[0047] Figure 8 in a is the particle size distribution of scallion - derived exosomes measured by NTA; Figure 8 in b is the TEM image showing the typical morphology; Figure 8 in c is the HUVECs cell scratch assay for evaluating the wound healing within 24 hours; Figure 8 in d is the vascular tubulogenesis after 24 - hour treatment with scallion exosomes; Figure 8 in e is the immunofluorescence staining of VEGF (red), F - actin (green) and DAPI (blue) in HUVECs after 7 - day treatment with scallion exosomes; Figure 8 in f is the semi - quantitative analysis of the scratch distance and microtubule length. Statistical significance and P - values were determined by Student’s t - test. Data are presented as mean ± standard deviation (n = 4), ***P ≤ 0.001.

[0048] Figure 9 RNA sequencing analysis of scallion-derived exosome-mediated signal transduction in LPS-stimulated RAW264.7 macrophages.

[0049] Figure 9 In a, the Venn diagram shows differentially expressed genes in treated and untreated RAW264.7 macrophages; Figure 9 In b, the heatmap clustering analysis of gene expression levels for scallion exosome treatment; Figure 9 In c, the volcano plot shows upregulated and downregulated genes after scallion exosome treatment compared with the control group, Figure 9 In d, the KEGG signaling pathway enrichment analysis.

[0050] Figure 10 Based on the mechanical properties of SIS patches.

[0051] Figure 10 In a, the stress-strain curve compares the mechanical properties of SIS and SIS-dopa patches; Figure 10 In b, the comparative analysis of the tensile strength and elongation at break of each patch; Figure 10 In c, the comparative analysis of the elastic modulus and toughness of each patch.

[0052] Figure 11 Molecular docking simulation with aquaporin as the membrane protein in scallion-derived exosomes.

[0053] Figure 11 In a, the structural representation of aquaporin; Figure 11 In b, the enlarged structure to show specific regions involved in docking interactions.

[0054] Figure 12 Hemolysis study.

[0055] Figure 12 In a, the appearance of blood after co-culture of various SIS-dopa patches with blood; Figure 12 In b, the quantitative evaluation of the hemolysis rate of various patch types.

[0056] Figure 13 Biocompatibility study.

[0057] Figure 13 In a, live (green) / dead (red) staining of L929 cells cultured on SIS-dopa patches; Figure 13 In b, cytoskeleton staining of L929 cells using rhodamine B-labeled F-actin; Figure 13 In c, the quantitative analysis of cell viability using the CCK-8 reagent; Figure 13The "d" in it is the quantitative analysis of the cell spreading area on different patch surfaces.

[0058] Figure 14 A cell inflammation model was established using RAW264.7 macrophages stimulated with LPS.

[0059] Figure 14 The "a" in it is the immunofluorescence staining of IL-6 (red), DAPI (blue), and Arg-1 (green), DAPI (blue) in the untreated control group; Figure 14 The "b" in it is the staining result after 24 hours of LPS treatment; Figure 14 The "c" in it is the immunofluorescence staining of IL-6 (red), DAPI (blue), and Arg-1 (green), DAPI (blue) in the cells treated with scallion exosomes; Figure 14 The "d" in it is the semi-quantitative analysis of the fluorescence intensities of IL-6 and Arg-1. Statistical significance and P values were determined by ANOVA comparison test, and the data are expressed as mean ± standard deviation (n = 4), ***P ≤ 0.001.

[0060] Figure 15 It is a schematic diagram of the overall appearance after inflammatory implantation.

[0061] In the figure: It is used to evaluate whether there is inflammation or edema after the implantation of the SIS-dopa-exo patch.

[0062] Figure 16 It is a schematic diagram of the evaluation results after inflammatory implantation.

[0063] Figure 16 The "a" in it is the H&E staining of the inflammatory implantation site 3 days and 7 days after the implantation of the SIS-dopa-exo patch; Figure 16 The "b" in it is the Masson's trichrome staining of the inflammatory implantation site 3 days and 7 days after the implantation of the SIS-dopa-exo patch; Figure 16 The "c" in it is the evaluation of the degree of inflammation; Figure 16 The "d" in it is the analysis of the arrangement of collagen fibers 3 days and 7 days after surgery. Statistical significance and P values were determined by ANOVA comparison test, and the data are expressed as mean ± standard deviation (n = 4), *P ≤ 0.05, **P ≤ 0.01.

[0064] Figure 17 It is a schematic diagram of the detailed immunofluorescence staining of the inflammatory implantation site.

[0065] In the figure: It shows the expression of TNF-α (red), CD163 (green), and DAPI (blue). Figure 17 The "a" in it is the schematic diagram of the immunofluorescence staining 3 days after surgery; Figure 17 The "b" in it is the schematic diagram of the immunofluorescence staining 7 days after surgery.

[0066] Figure 18 Schematic diagram of H&E staining 7 days after inflammatory implantation.

[0067] In the figure: including the heart, liver, spleen, lung and kidney to verify the biocompatibility of the SIS-dopa-exo patch.

[0068] Figure 19 Schematic diagram of hematological examination results.

[0069] Figure 19 a in it is the hematological examination 7 days after inflammatory implantation; Figure 19 b in it is the biochemical examination. In the figure: the dotted line range represents the non-pathological range.

[0070] Figure 20 Schematic diagram of the evaluation results of tissue visceral adhesion.

[0071] Figure 20 a in it is the overall appearance of the gastrointestinal anastomosis site at 14 days and 28 days after surgery; Figure 20 b in it is the tissue adhesion score.

[0072] Figure 21 Schematic diagram of the Masson's trichrome staining results of rat colorectal gastrointestinal anastomosis surgery at 14 days and 28 days after surgery.

[0073] Figure 22 Schematic diagram of the histological staining results of the colon of healthy rats.

[0074] Figure 23 Schematic diagram of the histological staining analysis results of the rat colon after 28 days of gastrointestinal anastomosis.

[0075] In the figure: including the heart, liver, spleen, lung and kidney to verify the biocompatibility of the SIS-dopa-exo patch.

[0076] Figure 24 Schematic diagram of hematological examination results.

[0077] Figure 24 a in it is the hematological examination 28 days after rat colorectal gastrointestinal anastomosis; Figure 24 b in it is the biochemical examination. In the figure: the part covered by the dotted line represents the non-pathological range.

[0078] Figure 25 Schematic diagram of the overall appearance of the patch implanted in the pig colon after 28 days.

[0079] Figure 26 H&E staining results of pig colorectal gastrointestinal anastomosis 28 days after surgery.

[0080] In the figure: It includes the heart, liver, spleen, lungs, and kidneys to verify the biocompatibility of the SIS-dopa-exo patch.

[0081] Figure 27 It is a schematic diagram of a novel SIS biological patch loaded with scallion-derived exosomes.

[0082] In the figure: In gastrointestinal anastomosis, the patch continuously releases exosomes, effectively relieving inflammation and promoting angiogenesis, thereby improving tissue repair and enhancing the healing effect. Detailed implementation manners

[0083] The various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The special word "exemplary" used here means "serving as an example, embodiment, or illustration". Any embodiment described as "exemplary" here does not necessarily have to be construed as superior to or better than other embodiments.

[0084] In addition, for a better description of the present invention, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present invention can also be implemented without some 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 gist of the present invention.

[0085] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the systematic errors inevitable in industrial production.

[0086] In this specification, the meaning expressed by using "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0087] In this specification, the so-called "some specific / preferred implementation manners", "other specific / preferred implementation manners", "implementation manners", etc. refer to the specific elements (for example, features, structures, properties, and / or characteristics) related to the implementation manner described herein, which are included in at least one of the implementation manners described herein, and may or may not exist in other implementation manners. In addition, it should be understood that the elements can be combined in various implementation manners in any suitable manner.

[0088] In this specification, the numerical range represented by using "numerical value A to numerical value B" refers to the range including the endpoint numerical values A and B.

[0089] As used in the invention, "HUVECs" represents human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cells).

[0090] As used in the invention, "L929 cells" refers to mouse fibroblast cells (Mouse fibroblast cells, L929 cells).

[0091] As used in the invention, "RAW264.7 cells" refers to mouse leukemic cells of monocyte macrophage (Mouse leukemiacells of monocyte macrophage).

[0092] As used in the invention, "VEGF" refers to vascular endothelial growth factor (Vascular endothelialgrowth factor).

[0093] As used in the invention, "TNF-α" refers to tumor necrosis factor-α (Tumor necrosis factor-α).

[0094] As used in the invention, "CD163" refers to cluster of differentiation 163 (Cluster of differentiation163), which belongs to the cysteine-rich class B scavenger receptor family.

[0095] As used in the invention, "TGF-β" refers to transforming growth factor-β (Transforming growthfactor-β).

[0096] As used in the invention, "TLR4" refers to Toll-like receptor 4 (Toll-like receptor 4).

[0097] As used in the invention, "IL-6" refers to interleukin-6 (Interleukin 6, IL-6).

[0098] As used in the invention, "CD31" refers to cluster of differentiation 31 (Cluster of differentiation 31), also known as platelet-endothelial cell adhesion molecule (Platelet endothelial cell adhesion molecule-1, PECAM-1).

[0099] As used in the invention, "Arg-1" refers to arginase 1 (Arginase 1).

[0100] As used in the invention, "CD3e" refers to the Cluster of Differentiation 3 epsilon subunit, also known as the T-cell surface glycoprotein CD3 epsilon subunit.

[0101] As used in the invention, "α-SMA" refers to α-Smooth muscle actin.

[0102] As used in the invention, "LPS" refers to Lipopolysaccharide.

[0103] As used in the invention, "F-actin" refers to Fibrous actin.

[0104] As used in the invention, "GAPDH" refers to Glyceraldehyde-3-phosphate dehydrogenase.

[0105] <First Aspect>

[0106] The first aspect of the present invention provides a biological patch, which comprises:

[0107] A substrate having a fibril structure, and the fibrils having polar groups; wherein,

[0108] By using the polar groups, the surface of the substrate is grafted with catechol functional groups, and the catechol functional groups are derived from polyphenolic compounds.

[0109] The biological patch of the present invention has good elongation at break, modulus and toughness. Moreover, these enhanced mechanical properties are similar to the mechanical compatibility of natural intestinal tissue. Meanwhile, the biological patch of the present invention has excellent blood compatibility and cell compatibility.

[0110] Substrate

[0111] The substrate of the present invention is a substrate having a fibril structure. The present invention does not particularly limit the substrate, and it can be some substrates commonly used in the art. In the present invention, the substrate is derived from a biodegradable biomaterial, preferably a biomaterial mainly containing collagen, and the collagen can be of natural origin, artificially synthesized, modified or crosslinked, including but not limited to submucosa, dermis, pericardium, collagen, gelatin, etc.

[0112] In some embodiments of the present invention, the substrate is derived from small intestinal submucosa (SIS), preferably from decellularized SIS, that is, the substrate of the present invention can be prepared using SIS. In some embodiments of the present invention, the substrate is derived from the dermis layer, preferably from decellularized dermis layer, that is, the substrate of the present invention can be prepared using the dermis layer. In some embodiments of the present invention, the substrate is derived from bladder submucosa, preferably from decellularized bladder submucosa, that is, the substrate of the present invention can be prepared using bladder submucosa. In some embodiments of the present invention, the substrate is derived from the pericardium layer, preferably from decellularized pericardium layer, that is, the substrate of the present invention can be prepared using the pericardium layer. The submucosa (such as SIS), dermis layer, pericardium layer, etc. are preferably derived from mammals, such as pigs, cows, sheep, dogs, cats, etc.

[0113] In one embodiment of the present invention, the substrate is derived from decellularized porcine SIS. This porcine SIS can be commercially purchased. Porcine SIS has a wide source, is economically available, easy to process, and the decellularized porcine SIS has excellent biocompatibility, is rich in collagen and growth factors, can induce cells to diffuse, adhere, grow and proliferate inward, and promote the repair and regeneration of its own tissue at the tissue defect site. It is suitable for use as the substrate for preparing the biological patch of the present invention. Therefore, the present invention preferably uses the porcine SIS material substrate prepared from porcine SIS as the substrate.

[0114] Regarding the preparation method of the porcine SIS material substrate, the present invention is not particularly limited, and it can be some common preparation methods in the art. The porcine SIS material substrate can also be commercially purchased. As a preference, the porcine SIS material substrate of the present invention can be prepared according to the preparation method in CN107007886A.

[0115] In another embodiment of the present invention, the substrate can also be a fiber membrane prepared using a chemical substance containing collagen. Regarding the fiber membrane, the present invention is not particularly limited, and it can be a common fiber membrane in the art, such as: non-woven fiber membrane or spun fiber membrane, etc.

[0116] The non-woven fiber membrane of the present invention can be obtained by non-woven process using a chemical substance containing collagen and one or more combinations of optionally present other high molecular polymers or their derivatives. The spun fiber membrane of the present invention can be obtained by processes such as electrospinning technology, centrifugal spinning technology, hot melt spinning technology, melt electrospinning technology, etc. using a chemical substance containing collagen and one or more combinations of optionally present other high molecular polymers or their derivatives.

[0117] Other polymer or its derivatives can be various commonly used polymers or their derivatives in the art, such as one or a combination of two or more selected from natural polymers. For example, the commonly used polymer or its derivatives can be a combination of one or two or more of cellulose, chondroitin sulfate, chitosan, modified chitosan, fibrin, silk protein, peptide polymers mimicking elastin, heparin, agar, dextran, alginic acid, cellulose, alginate, starch.

[0118] Preferably, in order to obtain a biological patch with excellent performance in all aspects, the present invention preferably uses a porcine SIS material substrate. Based on the substrate of the present invention, the biological patch of the present invention contains collagen fibers, and due to the presence of collagen fibers, the function of the biological patch of the present invention can be better exerted.

[0119] In some embodiments, the fibrils of the substrate have polar groups.

[0120] As will be described in detail later, using the polar groups, the surface of the substrate is grafted with catechol functional groups, and the catechol functional groups are derived from polyphenolic compounds.

[0121] In some specific embodiments, the polar groups include carboxyl groups; preferably, the carboxyl groups are activated by an activator.

[0122] Catechol Functional Group and Polyphenolic Compounds

[0123] In some embodiments of the present invention, the surface of the substrate is grafted with catechol functional groups, and the catechol functional groups are derived from polyphenolic compounds.

[0124] Grafting catechol functional groups endows the biological patch of the present invention with good elongation at break, modulus and toughness. Moreover, these enhanced mechanical properties are similar to the mechanical compatibility of natural intestinal tissues. At the same time, the biological patch of the present invention has excellent blood compatibility and cell compatibility.

[0125] In some alternative embodiments, the polyphenolic compounds include one or a combination of two or more of dopamine or its salts, tannic acid, catechol, pyrogallol, gallic acid, catechuic acid, etc.

[0126] In some preferred embodiments, the polyphenolic compound includes dopamine or its salt, such as dopamine hydrochloride. In this specification, dopamine, with the chemical name 3,4-dihydroxybenzeneethylamine, is a hydrophilic molecule, and its catechol structure can enhance the binding ability of exosomes by forming strong hydrogen bonds with the primary amino groups in exosomes. This high-affinity binding ensures the continuous interaction between exosomes and the patch. By regulating the dopamine content in the substrate, the release rate of exosomes can be controlled, thereby enhancing its stability and prolonging its therapeutic effect. It not only improves tissue regeneration at the anastomosis site, but also reduces the loss of exosomes, optimizes the treatment cost, and promotes the application progress of the composite material in gastrointestinal surgery.

[0127] To more favorably graft the catechol functional group onto the surface of the substrate, the carboxyl groups of the fibrils of the substrate are activated by an activator.

[0128] In the present invention, there are a large number of polar groups in the substrate, such as the presence of carboxyl groups, and the activator can activate the polar groups. The catechol functional group can couple the polar groups with exosomes to enhance the binding ability of exosomes, and is modified by forming secondary bonds. This secondary bond modification ensures the continuous interaction between exosomes and the substrate, and can also control the release rate of exosomes, thereby enhancing its stability and prolonging its therapeutic effect.

[0129] In the present invention, there is no particular limitation on the type of activator, as long as it is a carboxyl activator that can promote the amidation and / or esterification reaction between the carboxyl group and the polyphenolic compound.

[0130] The activator includes carbodiimide activators, preferably including one or a combination of two or more of dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), carbodiimide (EDAC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and more preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC).

[0131] Furthermore, the purpose of using the activator in the present invention is to be able to activate the polar groups of the substrate and enhance the loading capacity of the substrate. Therefore, the activator also includes N-hydroxyimide activators, preferably including N-hydroxysuccinimide (NHS) or N-hydroxythiobuccinimide, and more preferably N-hydroxysuccinimide (NHS).

[0132] The combined use of the above two activators (carbodiimide activators and N-hydroxyimide activators, such as EDC and NHS) can improve the activation efficiency.

[0133] Exosomes

[0134] In some embodiments, exosomes are also connected to the surface of the biological patch.

[0135] In some embodiments, the exosomes are coupled to the surface of the biological patch via hydrogen bonds with the catechol functional groups.

[0136] "Extracellular vesicles (EVs; or Exosomes, Exos)" refer to vesicular bodies with a bilayer membrane structure produced by cells through the paracrine pathway, with diameters ranging from 40 nm to 1000 nm. Exosomes are widely present in cell culture supernatants, various body fluids (blood, lymph, saliva, urine, semen, milk), and plants (vacuoles), carrying various cell-source-related substances such as proteins and lipids, and participating in processes such as intercellular communication, cell migration, angiogenesis, and immune regulation. In some specific embodiments, the exosomes are derived from the genus Allium, more preferably from scallions. Compared with exosomes derived from animals or humans, plant-derived exosomes, such as the scallion-derived exosomes used in the present invention, have the advantages of low immunogenicity, easy large-scale engineering production, and higher biosafety, opening up new possibilities for their clinical application.

[0137] In some preferred embodiments, the exosomes are exosomes derived from scallions, also known as scallion exosomes. Exosomes derived from scallions have a positive effect on cell migration and the formation of vascular tubular structures, and also have good anti-inflammatory properties that are beneficial to the regenerative repair of gastrointestinal injuries.

[0138] Coupling exosomes derived from scallions to at least one surface of the substrate not only has the advantages of low immunogenicity, easy large-scale engineering production, and higher biosafety, but also can have strong angiogenic activity and excellent anti-inflammatory potential, with a significant regulatory effect on the inflammatory process, and its implantation in the body can promote angiogenesis at the injury site.

[0139] In some embodiments, when preparing the biological patch, the content of exosomes in the exosome-containing solution used is 1-300 μg / mL. Preferably, the content of exosomes in the exosome-containing solution is 5-250 μg / mL, more preferably 10-200 μg / mL, such as 10 μg / mL, 20 μg / mL, 50 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 150 μg / mL, 180 μg / mL, 200 μg / mL, etc. The content (concentration) of exosomes in the exosome-containing solution is characterized by the protein concentration in the solution. For example, the protein concentration of the solution is measured by a method known in the art or a commercially available kit. Exemplarily, a BCA protein detection kit (ThermoFisher, USA) is used for measurement.

[0140] In some embodiments, the exosomes regulate the inflammatory process through the HIF-1 pathway and the TNF, NF-κB, and JAK-STAT pathways.

[0141] In some embodiments, there is no particular limitation on the method for preparing the exosomes. For example, the exosomes are separated and purified by one or both of the ultracentrifugation method and the density gradient centrifugation method. In some preferred embodiments, the exosomes (such as scallion exosomes) are separated and purified by a combination of the ultracentrifugation method and the sucrose gradient centrifugation method.

[0142] In some specific embodiments, the method for preparing scallion exosomes includes extracting scallion juice; first performing ultracentrifugation on the scallion juice; and then performing sucrose gradient centrifugation for separation and purification to obtain scallion exosomes.

[0143] In some specific embodiments, in the present invention, scallion-derived exosomes are combined with the biological patch SIS, and the catechol functional group of dopamine is used as a coupling agent (denoted as SIS-dopa-exo). In the performance test, its effects on angiogenesis and inflammation regulation are explored. As shown by the cell experiments in the subsequent performance test, the patch has excellent biocompatibility, angiogenesis-promoting activity, and inflammation-regulating ability. RNA sequencing analysis further reveals the activation of key signaling pathways (such as HIF-1, TNF, and NF-κB), which are the basis for its function. Animal studies using rat and miniature pig colon models to simulate clinical conditions show that the SIS-dopa-exo patch has significant technical advantages over traditional suturing and conventional SIS patches in promoting anastomotic healing and tissue regeneration. The above results provide strong evidence for the clinical application of this novel biomaterial in improving the efficacy of gastrointestinal surgery ( Figure 27 ).

[0144] <Second Aspect>

[0145] The second aspect of the present invention provides a method for preparing a biological patch according to the first aspect of the present invention, which includes the step of grafting catechol functional groups onto the surface of a substrate, and, preferably, the step of coupling exosomes to the surface of the substrate using the catechol functional groups.

[0146] The method for preparing the biological patch of the present invention is simple and easy to implement, the raw materials are easy to obtain, and it is suitable for mass production.

[0147] Steps of Grafting Catechol Functional Group onto the Surface of the Substrate

[0148] In some embodiments, the substrate is contacted with a solution containing a polyphenolic compound to graft catechol functional groups onto the substrate.

[0149] In some alternative embodiments, in the step of grafting catechol functional groups onto the surface of the substrate, the content of the polyphenolic compound in the solution containing the polyphenolic compound is 0.1 - 50 (w / v)%, where w / v represents solute / solvent. For example, 2.0 g of dopamine hydrochloride (solute) dissolved in 10 mL of deionized water (solvent) is expressed as 20 (w / v)% or abbreviated as 20%, also referred to as a dopamine hydrochloride solution with a concentration of 20%. More preferably, the content of the polyphenolic compound in the solution containing the polyphenolic compound is 1 - 30 (w / v)%, such as 1%, 2%, 4%, 8%, 10%, 20%, 30%. In some exemplary embodiments, the content of the polyphenolic compound in the solution of the polyphenolic compound is 2%, 4%, 8%, 20%. In the step of grafting catechol functional groups onto the surface of the substrate, by adjusting the content of the polyphenolic compound in the solution containing the polyphenolic compound, the amount of the grafted polyphenolic compound in the biological patch can be adjusted, and the release rate of exosomes can be controlled.

[0150] In some preferred embodiments, in the step of grafting catechol functional groups onto the surface of the substrate, the contact time between the substrate and the solution containing the polyphenolic compound is 1 - 18 h, more preferably 1 - 16 h, such as 2 h, 4 h, 8 h, 12 h, etc.

[0151] In some specific embodiments, the polyphenolic compound can be present on at least one surface of the substrate by one or more combinations of spreading, coating, soaking, casting, and spraying, so that catechol functional groups are grafted onto the substrate. In some exemplary embodiments, the substrate can be soaked in a solution containing a polyphenolic compound.

[0152] In some preferred embodiments, the step of grafting catechol functional groups onto the surface of the substrate is carried out in a dark environment. Carrying out the reaction in a dark environment can prevent the oxidative polymerization of polyphenolic compounds such as dopamine.

[0153] In some preferred embodiments, the pH of the solution containing polyphenolic compounds is < 7, and more preferably, the pH of the solution is < 5, such as pH = 1, pH = 2, pH = 3, etc.

[0154] Steps of Activating the Substrate

[0155] In some embodiments, to be more conducive to the grafting of catechol functional groups onto the surface of the substrate, before the step of grafting catechol functional groups onto the surface of the substrate, a step of activating the substrate is further included.

[0156] In some specific embodiments, in the step of activating the substrate, the substrate is treated with an activator so that the carboxyl groups of the substrate of the present invention are activated, which is conducive to grafting catechol functional groups.

[0157] In the present invention, there is no particular limitation on the type of activator, as long as it is a carboxyl activator that can promote the amidation and / or esterification reaction between the carboxyl group and the polyphenolic compound.

[0158] The activator includes carbodiimide activators, preferably including one or a combination of two or more of dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), carbodiimide (EDAC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and more preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC).

[0159] Furthermore, the purpose of using the activator in the present invention is to be able to activate the polar groups of the substrate and improve the loading capacity of the substrate. Therefore, the activator also includes N-hydroxyimide activators, preferably including N-hydroxysuccinimide (NHS) or N-hydroxythiobuccinimide, and more preferably N-hydroxysuccinimide (NHS).

[0160] In some preferred embodiments, the combined use of the above two activators (carbodiimide activators and N-hydroxyimide activators, such as EDC and NHS) can improve the activation efficiency.

[0161] In some specific embodiments, the activator can be present on at least one surface of the substrate by one or a combination of two or more of tiling, coating, soaking, casting, and spraying, thereby activating the substrate. In some exemplary embodiments, the substrate can be soaked in a solution containing the activator.

[0162] In some embodiments, the concentration of the activator in the solution containing the activator is 1-10 mg / mL, where mg / mL represents the ratio of the mass of the solute to the volume of the solvent. For example, 500 mg of EDC (solute) dissolved in 100 mL of deionized water (solvent) is expressed as 5 mg / mL.

[0163] In some specific embodiments, in the solution containing the activator, the concentration of the carbodiimide activator is 3-10 mg / mL, preferably 3-8 mg / mL, more preferably 4-6 mg / mL, such as 4 mg / mL, 5 mg / mL, 6 mg / mL, etc.

[0164] In some specific embodiments, in the solution containing the activator, the concentration of the N-hydroxyimide activator is 0.1-5 mg / mL, preferably 0.5-3 mg / mL, such as 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, etc.

[0165] In some embodiments, the activation time is 30-90 min, preferably, the activation time is 45-75 min, such as 50 min, 55 min, 60 min, 65 min, etc.

[0166] Steps of Coupling Exosomes onto the Surface of the Substrate

[0167] In some embodiments, exosomes are coupled to the surface of the substrate using catechol functional groups.

[0168] In some embodiments, exosomes are coupled to the surface of the substrate by contacting the substrate with a solution containing exosomes.

[0169] In some specific embodiments, the exosomes can be present on at least one surface of the substrate by one or a combination of two or more of tiling, coating, soaking, casting, and spraying, such that the exosomes are coupled to the surface of the substrate.

[0170] In some exemplary embodiments, exosomes are coupled to the surface of the substrate by soaking the substrate in a solution containing exosomes. In some embodiments, the soaking time is 8-24 h, preferably, the soaking time is 10-15 h, such as 11 h, 12 h, 13 h, 14 h, etc.

[0171] In some specific embodiments, in the solution containing exosomes, the content of exosomes is 1-300 μg / mL. Preferably, in the solution containing exosomes, the content of exosomes is 5-250 μg / mL, more preferably 10-200 μg / mL, such as 10 μg / mL, 20 μg / mL, 50 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 150 μg / mL, 180 μg / mL, 200 μg / mL, etc.

[0172] Other Steps

[0173] Before performing the preparation method of the biological patch of the present invention, the activator or the polyphenolic compound can be separately dissolved in a solvent; further, the present invention does not make a special limitation on the solvent, and it can be an inorganic solvent commonly used in the art. In some embodiments, the solvent of the present invention is deionized water.

[0174] In some embodiments, after the step of coupling exosomes to the surface of the substrate, the patch coupled with exosomes can be further dried. For the drying treatment, vacuum drying, freeze-drying, room-temperature drying, etc. can be used. The present invention does not make a special limitation on the method of drying treatment, as long as the biological patch of the present invention can be prepared. In the present invention, freeze-drying is preferably used, and the freeze-dried material maintains its original chemical composition and physical properties.

[0175] <Third Aspect>

[0176] The third aspect of the present invention provides the use of the biological patch described in the first aspect of the present invention or the biological patch prepared by the preparation method described in the second aspect of the present invention for preparing a tissue repair product.

[0177] In some preferred embodiments, the tissue includes gastrointestinal tissue. The biological patch provided by the present invention is particularly suitable for improving the repair of postoperative bleeding, continuous inflammation, poor healing, and infection after gastrointestinal anastomosis surgery.

[0178] In some embodiments, there is provided the use of the biological patch described in the first aspect of the present invention or the biological patch prepared by the preparation method described in the second aspect of the present invention in preparing a product for tissue repair. Preferably, the tissue includes gastrointestinal tissue. Specifically, the product for tissue repair includes a repair material for improving postoperative bleeding after gastrointestinal anastomosis surgery, a repair material for improving postoperative continuous inflammation after gastrointestinal anastomosis surgery, a repair material for improving postoperative poor healing after gastrointestinal anastomosis surgery, and a repair material for improving postoperative infection repair after gastrointestinal anastomosis surgery.

[0179] In some embodiments, there is provided a biological patch as described in the first aspect of the present invention or a biological patch prepared by the preparation method described in the second aspect of the present invention, which is used for tissue repair. Preferably, the tissue includes gastrointestinal tissue. Specifically, the tissue repair includes repair of postoperative defects, repair of postoperative bleeding, repair of postoperative persistent inflammation, repair of poor postoperative healing, and repair of postoperative infection.

[0180] In some embodiments, there is provided a tissue repair method, which includes the step of using the biological patch as described in the first aspect of the present disclosure or a biological patch prepared by the preparation method described in the second aspect of the present invention. Preferably, the tissue includes gastrointestinal tissue. Specifically, the tissue repair includes repair of postoperative defects, repair of postoperative bleeding, repair of postoperative persistent inflammation, repair of poor postoperative healing, and repair of postoperative infection.

[0181] Examples

[0182] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0183] All animal experiments were carried out in accordance with the Tianjin Medical Experimental Animal Care Guidelines, and the animal experiment protocol was approved by the Animal Care and Use Committee of E-Source Gene Technology (Tianjin) Co., Ltd.

[0184] All quantitative data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS software, including Student's t-test and one-way analysis of variance (ANOVA). The difference is considered statistically significant at * P ≤ 0.05, highly significant at ** P ≤ 0.01, and extremely highly significant at *** P ≤ 0.001.

[0185] I. Reagents

[0186] The porcine SIS patch was provided by Beijing Bohui Ruijin Biotechnology Co., Ltd. (Beijing, China). Dopamine hydrochloride was purchased from Sigma (USA). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), and HCl were provided by Aladdin (China). Fresh green onions were purchased from a local store and thoroughly cleaned with deionized water. HUVECs, RAW264.7 macrophages, and L929 fibroblasts were all purchased from Wuhan Pusaisai Life Science and Technology Co., Ltd.

[0187] II. Exosome Extraction and Activity Testing

[0188] Scallions were chopped and soaked in cold phosphate - buffered saline (PBS, 10 mM, pH 7.4), and then scallion juice was extracted by mixing. The scallion juice was centrifuged at 1000 g for 10 minutes, 3000 g for 20 minutes, and 10000 g for 40 minutes (4 °C) in sequence to remove large particles and fibrous debris. The supernatant was collected and centrifuged at 150,000 g for 2 hours using an ultra - centrifuge (Beckman Optima XE - 100, USA). The obtained precipitate was resuspended in PBS and subjected to sucrose gradient centrifugation at 150,000 g for 2 hours to isolate and purify exosomes. The purified exosomes were resuspended in PBS, and their protein concentration was determined using a BCA protein assay kit (ThermoFisher, USA), and the concentration was adjusted to 100 μg mL-1. The exosomes were stored at - 80 °C for later use. For characterization, the morphology of scallion - derived exosomes was observed using a transmission electron microscope (TEM, HT7700, Hitachi, Japan), and the particle size distribution was determined by nanoparticle tracking analysis (NTA, Nanosight NS300, UK).

[0189] Experimental results: The extracted scallion - derived exosomes were characterized by NTA, and the results showed that their average particle size was 121.2 ± 90.8 nm. TEM images further confirmed their intact cup - shaped morphology ( Figure 8 a and b in

[0190] 1. Functional Activity of Scallion Exosomes

[0191] Human umbilical vein endothelial cells (HUVECs) at a density of 5000 cells per well were seeded into 48 - well plates. When the cells reached approximately 80% confluence, a scratch approximately 200 μm wide was created using a pipette tip. Then, the cells were cultured in conditioned medium with or without scallion exosomes. After 24 hours, the migration distance of the cells was measured under an optical microscope (Olympus, Japan). Meanwhile, HUVECs at the same density were seeded onto Matrigel (Corning, USA) and cultured in conditioned medium for 24 hours to evaluate the formation of tubular structures, which were observed under an optical microscope. For further analysis, HUVECs cultured in exosome - conditioned medium for 7 days were stained with VEGF (ab32152, Abcam, UK), FITC - labeled F - actin, and DAPI. Images were taken using a confocal laser scanning microscope (CLSM, TCS SP8, Leica, Germany), and semi - quantitative analysis was performed using ImageJ software.

[0192] Experimental results: In cell experiments, these exosomes significantly promoted cell migration and the formation of vascular tubular structures. The migration distance and the length of the tubular structures were 18.45 ± 8.62 μm and 162.59 ± 39.21 μm, respectively. Compared with the control group (conditioned medium without scallion exosomes), when using the conditioned medium containing scallion exosomes, the healing rate and the length of the microtubules increased by 3.43 times and 2.66 times, respectively. In addition, VEGF immunofluorescence staining in HUVECs showed that the expression level in the scallion-derived exosome group was significantly higher than that in the control group ( Figure 8 c-f in

[0193] ), confirming the strong angiogenic activity of scallion-derived exosomes.

[0194] RAW264.7 macrophages with a density of 10,000 cells per well were stimulated with LPS (1 μg mL -1 ) for 24 hours. After stimulation, the cells were treated in the conditioned medium for 3 days. At predetermined time intervals, the cells were fixed and immunofluorescently stained with antibodies against IL-6 (ab290735, Abcam, UK) and Arg-1 (PA5-29645, Invitrogen, USA), and then stained with DAPI. Images were taken by confocal laser scanning microscopy (CLSM) and quantitatively analyzed using ImageJ software. In addition, the treated cells were also subjected to RNA sequencing to evaluate the gene expression changes induced by the conditioned medium.

[0195] Experimental results: To further explore the anti-inflammatory potential of scallion-derived exosomes, LPS-stimulated RAW264.7 macrophages were induced to enter the M1 polarization state. After treatment with the medium rich in scallion-derived exosomes, compared with the treatment with the medium without scallion-derived exosomes (control group), 525 differentially expressed genes were identified by RNA sequencing. KEGG pathway enrichment analysis showed that these genes were mainly related to the HIF-1 pathway (related to angiogenesis) and the TNF, NF-κB, and JAK-STAT pathways (related to inflammation regulation) ( Figure 9 ). These results not only confirmed the angiogenic effect of scallion-derived exosomes ( Figure 8 ), but also indicated that they had a significant regulatory effect on the inflammatory process.

[0196] Example 1. Preparation of SIS-dopa series patches using a dopamine hydrochloride solution with a dopamine concentration of 2%

[0197] The SIS patch (7 cm × 10 cm) was immersed in 100 mL of deionized water containing 500 mg of EDC and 300 mg of NHS (obtained by dissolving 500 mg of EDC and 300 mg of NHS in 100 mL of deionized water) to activate the carboxyl groups for 60 minutes. Subsequently, the activated SIS patch was placed in 10 mL of deionized water containing 0.2 g of dopamine hydrochloride (obtained by dissolving 0.2 g of dopamine hydrochloride in 10 mL of deionized water, i.e., a dopamine hydrochloride solution with a concentration of 2 (w / v) %), the pH was adjusted to 2, and it was treated (or called reacted, grafted) in the dark for 2 hours, 4 hours, 8 hours, or 12 hours. Then, freeze-drying was carried out to obtain a series of SIS-dopa patches, which were labeled as SIS-dopa-2%.

[0198] Example 2. Preparation of SIS-dopa series patches using a dopamine hydrochloride solution with a dopamine concentration of 4%

[0199] The SIS patch (7 cm × 10 cm) was immersed in 100 mL of deionized water containing 500 mg of EDC and 300 mg of NHS (obtained by dissolving 500 mg of EDC and 300 mg of NHS in 100 mL of deionized water) to activate the carboxyl groups for 60 minutes. Subsequently, the activated SIS patch was placed in 10 mL of deionized water containing 0.4 g of dopamine hydrochloride (obtained by dissolving 0.4 g of dopamine hydrochloride in 10 mL of deionized water, i.e., a dopamine hydrochloride solution with a concentration of 4 (w / v) %), the pH was adjusted to 2, and it was treated in the dark for 2 hours, 4 hours, 8 hours, or 12 hours. Then, freeze-drying was carried out to obtain a series of SIS-dopa patches, which were labeled as SIS-dopa-4%.

[0200] Example 3. Preparation of SIS-dopa series patches using a dopamine hydrochloride solution with a dopamine concentration of 8%

[0201] The SIS patch (7 cm × 10 cm) was immersed in 100 mL of deionized water containing 500 mg of EDC and 300 mg of NHS (obtained by dissolving 500 mg of EDC and 300 mg of NHS in 100 mL of deionized water) to activate the carboxyl groups for 60 minutes. Subsequently, the activated SIS patch was placed in 10 mL of deionized water containing 0.8 g of dopamine hydrochloride (obtained by dissolving 0.8 g of dopamine hydrochloride in 10 mL of deionized water, i.e., a dopamine hydrochloride solution with a concentration of 8 (w / v) %), the pH was adjusted to 2, and it was treated in the dark for 2 hours, 4 hours, 8 hours, or 12 hours. Then, freeze-drying was carried out to obtain a series of SIS-dopa patches, which were labeled as SIS-dopa-8%.

[0202] Example 4. Preparation of SIS-dopa series patches using dopamine hydrochloride solution with a dopamine concentration of 20%

[0203] Immerse the SIS patch (7 cm × 10 cm) in 100 mL of deionized water containing 500 mg of EDC and 300 mg of NHS (obtained by dissolving 500 mg of EDC and 300 mg of NHS in 100 mL of deionized water) to activate the carboxyl groups for 60 minutes. Subsequently, place the activated SIS patch into 10 mL of deionized water containing 2.0 g of dopamine hydrochloride (obtained by dissolving 2 g of dopamine hydrochloride in 10 mL of deionized water, i.e., a dopamine hydrochloride solution with a concentration of 20 (w / v) %), adjust the pH to 2, and treat it in a dark environment for 2 hours, 4 hours, 8 hours, or 12 hours. Then, perform freeze-drying to obtain a series of SIS-dopa patches, which are labeled as SIS-dopa-20%.

[0204] For the SIS-dopa-20% series of patches, according to the treatment time in the dark environment, the patches are specifically labeled as SIS-dopa-2h (treated for 2 hours), SIS-dopa-4h (treated for 4 hours), SIS-dopa-8h (treated for 8 hours), and SIS-dopa-12h (treated for 12 hours). Among them, the patch with a dopamine concentration of 20% and treated for 12 hours in the dark environment, i.e., SIS-dopa-12h, is also denoted as SIS-dopa in the following text.

[0205] Example 5. SIS-dopa-exo 100 Preparation of the patch

[0206] Immerse the SIS patch (7 cm × 10 cm) in 100 mL of deionized water containing 500 mg of EDC and 300 mg of NHS (obtained by dissolving 500 mg of EDC and 300 mg of NHS in 100 mL of deionized water) to activate the carboxyl groups for 60 minutes. Subsequently, place the activated SIS patch into 10 mL of deionized water containing 2.0 g of dopamine hydrochloride (obtained by dissolving 2 g of dopamine hydrochloride in 10 mL of deionized water, i.e., a dopamine hydrochloride solution with a concentration of 20 (w / v) %), adjust the pH to 2, and treat it in a dark environment for 12 hours. After dopamine grafting, immerse the SIS-dopa patch in a solution of scallion exosomes with a concentration of 100 μg mL-1 (prepared by extraction in the "Exosome extraction and activity test" section above) for 12 hours, and then perform freeze-drying. Denote the obtained patch as SIS-dopa-exo 100 , abbreviated as SIS-dopa-exo.

[0207] Example 6. SIS-dopa-exo 10 Preparation of the patch

[0208] Soak the SIS patch (7 cm × 10 cm) in 100 mL of deionized water containing 500 mg of EDC and 300 mg of NHS (obtained by dissolving 500 mg of EDC and 300 mg of NHS in 100 mL of deionized water) to activate the carboxyl groups for 60 minutes. Subsequently, place the activated SIS patch into 10 mL of deionized water containing 2.0 g of dopamine hydrochloride (obtained by dissolving 2 g of dopamine hydrochloride in 10 mL of deionized water, i.e., a dopamine hydrochloride solution with a concentration of 20 (w / v) %), adjust the pH to 2, and treat it in a dark environment for 12 hours. After dopamine grafting, soak the SIS-dopa patch in a solution of scallion exosomes with a concentration of 10 μg mL-1 (prepared according to the extraction in the "Exosome Extraction and Activity Testing" section above, and its protein concentration was determined using a BCA protein assay kit (ThermoFisher, USA) and adjusted to a concentration of 10 μg mL-1) for 12 hours, and then perform freeze-drying. The resulting patch is denoted as SIS-dopa-exo 10 。

[0209] Example 7. SIS-dopa-exo 20 Preparation of the patch

[0210] Soak the SIS patch (7 cm × 10 cm) in 100 mL of deionized water containing 500 mg of EDC and 300 mg of NHS (obtained by dissolving 500 mg of EDC and 300 mg of NHS in 100 mL of deionized water) to activate the carboxyl groups for 60 minutes. Subsequently, place the activated SIS patch into 10 mL of deionized water containing 2.0 g of dopamine hydrochloride (obtained by dissolving 2 g of dopamine hydrochloride in 10 mL of deionized water, i.e., a dopamine hydrochloride solution with a concentration of 20 (w / v) %), adjust the pH to 2, and treat it in a dark environment for 12 hours. After dopamine grafting, soak the SIS-dopa patch in a solution of scallion exosomes with a concentration of 20 μg mL-1 (prepared according to the extraction in the "Exosome Extraction and Activity Testing" section above, and its protein concentration was determined using a BCA protein assay kit (ThermoFisher, USA) and adjusted to a concentration of 20 μg mL-1) for 12 hours, and then perform freeze-drying. The resulting patch is denoted as SIS-dopa-exo 20 。

[0211] Example 8. SIS-dopa-exo 50 Preparation of the patch

[0212] Immerse the SIS patch (7 cm × 10 cm) in 100 mL of deionized water containing 500 mg of EDC and 300 mg of NHS (obtained by dissolving 500 mg of EDC and 300 mg of NHS in 100 mL of deionized water) to activate the carboxyl groups for 60 minutes. Subsequently, place the activated SIS patch into 10 mL of deionized water containing 2.0 g of dopamine hydrochloride (obtained by dissolving 2 g of dopamine hydrochloride in 10 mL of deionized water, i.e., a dopamine hydrochloride solution with a concentration of 20 (w / v) %), adjust the pH to 2, and treat it in a dark environment for 12 hours. After dopamine grafting, immerse the SIS-dopa patch in a solution of scallion exosomes with a concentration of 50 μg mL-1 (prepared according to the extraction and activity test of exosomes in the previous section, and its protein concentration was measured by a BCA protein detection kit (ThermoFisher, USA) and adjusted to a concentration of 50 μg mL-1) for 12 hours, and then perform freeze-drying. Denote the resulting patch as SIS-dopa-exo 50 。

[0213] Example 9. SIS-dopa-exo 200 Preparation of the patch

[0214] Immerse the SIS patch (7 cm × 10 cm) in 100 mL of deionized water containing 500 mg of EDC and 300 mg of NHS (obtained by dissolving 500 mg of EDC and 300 mg of NHS in 100 mL of deionized water) to activate the carboxyl groups for 60 minutes. Subsequently, place the activated SIS patch into 10 mL of deionized water containing 2.0 g of dopamine hydrochloride (obtained by dissolving 2 g of dopamine hydrochloride in 10 mL of deionized water, i.e., a dopamine hydrochloride solution with a concentration of 20 (w / v) %), adjust the pH to 2, and treat it in a dark environment for 12 hours. After dopamine grafting, immerse the SIS-dopa patch in a solution of scallion exosomes with a concentration of 200 μg mL-1 (prepared according to the extraction and activity test of exosomes in the previous section, and its protein concentration was measured by a BCA protein detection kit (ThermoFisher, USA) and adjusted to a concentration of 200 μg mL-1) for 12 hours, and then perform freeze-drying. Denote the resulting patch as SIS-dopa-exo 200 。

[0215] Control 1. SIS

[0216] Porcine SIS patch provided by Beijing Bohui Ruijin Biotechnology Co., Ltd. (Beijing, China).

[0217] Control 2. Preparation of SIS-exo patch

[0218] The SIS patch with a size of 7 cm × 10 cm was immersed in the scallion exosome solution with a concentration of 100 μg mL-1 for 12 hours, and then freeze-dried to obtain the SIS-exo patch.

[0219] Performance test

[0220] I. Performance test of different SIS-dopa patches

[0221] The SIS patch is rich in collagen and contains a large number of polar groups, such as carboxyl groups, making it an ideal platform for surface modification. To utilize this property, as in Examples 1-4, the SIS patch was first immersed in the EDC-NHS solution to activate the carboxyl groups (pre-activation), and then immersed in dopamine solutions with different concentrations under acidic conditions (pH 2), and the reaction was carried out in the dark environment to prevent the oxidative polymerization of dopamine ( Figure 1 a) in). The FT-IR spectrum of the modified SIS-dopa patch (Example 4, SIS-dopa-12h) shows characteristic peaks at 1653 cm-1 and 1532 cm-1, which are attributed to the aromatic rings in dopamine. At the same time, the peak appearing at 812 cm-1 indicates the presence of ortho-substituted aromatic rings, confirming the successful grafting of dopamine ( Figure 1 b) in).

[0222] 1. Water contact angle test

[0223] A water angle measuring instrument (JC2000C1, China) was used to evaluate the hydrophilicity of different patches. 10 μL of water droplets were dropped on the surface of the patch, and the contact angle was recorded using the built-in camera after 10 seconds.

[0224] Experimental results: The water contact angle measurement shows that the hydrophilicity of the dopamine-modified patches (SIS-dopa-2%, SIS-dopa-4%, SIS-dopa-8%, and SIS-dopa-20%, treated in the dark environment for 2 hours, 4 hours, 8 hours, or 12 hours respectively (corresponding to Figure 1 the abscissa 2h, 4h, 8h, 12h in c)) is significantly improved ( Figure 1 c) in), and the reaction time has a greater impact on the hydrophilicity than the dopamine concentration.

[0225] 2. DPPH free radical scavenging experiment

[0226] A DPPH solution with a concentration of 0.05 mg mL-1 was prepared by dissolving DPPH reagent (Sigma, USA) in ethanol. Different 1 cm×1 cm patches were immersed in 10 mL of the DPPH solution and incubated in the dark for 30 minutes. After incubation, the supernatant was collected and the absorbance was measured at 517 nm using a UV-visible spectrophotometer (UV-3600, Shimadzu, Japan). The DPPH radical scavenging efficiency (%) was calculated using the following formula:

[0227]

[0228] where A B represents the absorbance of the DPPH solution after 30 minutes, and A M represents the absorbance of the DPPH solution after co-incubation with the patch. Due to its natural antioxidant properties, ascorbic acid was used as a positive control.

[0229] 3. Protein adsorption experiment

[0230] A bovine serum albumin (BSA) solution with a mass fraction of 0.33% was prepared. Different 1 cm×1 cm patches were immersed in 5 mL of the BSA solution and incubated at room temperature for 30 minutes. After incubation, the supernatant was collected and the residual protein concentration was analyzed using a BCA protein assay kit.

[0231] The results of the above DPPH radical scavenging experiment and protein adsorption experiment showed that, taking SIS-dopa-20% as an example, the DPPH radical scavenging activity and protein adsorption ability of the patches obtained at different grafting times (i.e., SIS-dopa-2h, SIS-dopa-4h, SIS-dopa-8h, and SIS-dopa-12h in Example 4) were evaluated. The DPPH radical scavenging efficiency increased significantly from 4.01±1.39% of the original SIS (Comparative Example 1) to 59.51±2.02% of SIS-dopa-12h, which was attributed to the enhanced antioxidant performance of the catechol group in dopamine ( Figure 1 d in). In addition, the initial protein adsorption amount of SIS (Comparative Example 1) was 1.63±0.23 μg cm-2, and that of SIS-dopa-12h increased significantly to 3.74±0.10 μg cm-2 due to the high affinity of dopamine ( Figure 1 e in).

[0232] 4. Mechanical property test

[0233] The different patches were cut into standard sizes (4 cm × 2 cm) and subjected to mechanical property tests using a universal mechanical testing machine (Instron 5848, USA). At room temperature, the tests were carried out at a tensile speed of 5 mm min-1. The modulus was calculated from the slope of the stress-strain curve, and the toughness was calculated from the area under the curve.

[0234] Mechanical property studies confirmed that dopamine modification had no significant effect on the tensile strength of the patches (SIS-dopa-2h, SIS-dopa-4h, SIS-dopa-8h, and SIS-dopa-12h in Example 4), but significantly increased the elongation at break, modulus, and toughness, which may be due to the strengthening effect of dopamine on collagen fibers ( Figure 10 ). These enhanced mechanical properties were similar to the mechanical compatibility of natural intestinal tissue (about 1 MPa), indicating its suitability for gastrointestinal applications.

[0235] In summary, these research results confirmed the successful preparation of SIS-dopa patches with adjustable dopamine modification.

[0236] 5. Hemolysis experiment of SIS-dopa patches

[0237] Next, the cytocompatibility of different SIS-dopa patches as implantable medical devices was evaluated.

[0238] To evaluate the hemolysis of different SIS-dopa patches, according to the ISO10993 standard, different patches (0.2 g) were first immersed in sterile PBS (1 mL) for 30 minutes. Then the obtained supernatant was mixed with fresh rabbit blood and incubated for 60 minutes. After incubation, the blood samples were centrifuged at 2,000 rpm for 15 minutes, and then the absorbance of the supernatant was measured at 540 nm using a microplate reader. The hemolysis rate (%) was calculated using the following formula:

[0239]

[0240] where A E , A N and A P represent the optical density (OD) values of the experimental patch, negative control, and positive control, respectively. A 0.1% Triton X-100 solution was used as the positive control.

[0241] By detecting the hemolysis of blood cells and the hemolysis rate to evaluate blood compatibility, the results showed that SIS-dopa-2%, SIS-dopa-4%, SIS-dopa-8%, and SIS-dopa-20%, treated in the dark for 2 hours, 4 hours, 8 hours, or 12 hours respectively, the obtained patches were all significantly lower than the 5% threshold, confirming their excellent blood compatibility (Figure 12 a and b) in

[0242] II. Performance Testing of Biological Patches with Exosomes Connected to Different Surfaces

[0243] The SIS-dopa patch (SIS-dopa-12h) in Example 4 was immersed in the exosome solution extracted from scallions. By using the interaction between the catechol groups on SIS-dopa and the polar groups on exosomes, the adsorption and binding efficiency was improved. As in Examples 5 - 9, biological patches with exosomes connected to different surfaces were obtained.

[0244] 1. Mechanical Property Testing

[0245] The different patches were cut into standard sizes (4 cm × 2 cm), and a universal mechanical testing machine (Instron 5848, USA) was used for mechanical property testing. At room temperature, the testing was carried out at a tensile speed of 5 mm min -1 . The tensile strength of the biological patches with exosomes connected to different surfaces obtained with different concentrations of exosomes was obtained.

[0246] The experimental results are shown in Table 1 below:

[0247] Table 1:

[0248]

[0249] From the above results, it can be seen that the introduction of exosomes did not affect the mechanical strength of the SIS substrate.

[0250] 2. DPPH Free Radical Scavenging Experiment

[0251] A DPPH solution with a concentration of 0.05 mg mL-1 was prepared by dissolving DPPH reagent (Sigma, USA) in ethanol. Different 1 cm × 1 cm patches were immersed in 10 mL of the DPPH solution and incubated in the dark for 30 minutes. After incubation, the supernatant was collected, and the absorbance was measured at 517 nm using a UV-visible spectrophotometer (UV-3600, Shimadzu, Japan). The DPPH free radical scavenging efficiency (%) was calculated by the following formula:

[0252]

[0253] where A B represents the absorbance of the DPPH solution after 30 minutes, and A M represents the absorbance of the DPPH solution after co-incubation with the patch. Due to its natural antioxidant properties, ascorbic acid was used as a positive control.

[0254] The experimental results are shown in Table 2 below:

[0255] Table 2:

[0256]

[0257] As can be seen from the above results, the introduction of exosomes did not affect the DPPH scavenging performance of SIS-dopa.

[0258] In subsequent tests, SIS-dopa-exo prepared in Example 5 was used 100 , hereinafter referred to as SIS-dopa-exo for short.

[0259] 3. Release experiment

[0260] The SIS-dopa-exo patch (1 cm × 1 cm; Example 5) was immersed in 5 mL of PBS and incubated at 37 °C. At predetermined time intervals, the supernatant was collected and its protein concentration was analyzed using a BCA kit. The control group was a SIS patch pre-adsorbed with scallion exosomes under the same conditions (denoted as SIS-exo; Comparative Example 2) for comparison. The SIS-dopa-exo and SIS-exo patches were analyzed by X-ray photoelectron spectroscopy (XPS) using a Thermo Scientific K-Alpha USA instrument with aluminum Kα radiation (hν = 1486.6 eV). In addition, in situ FT-IR analysis was also performed on the SIS and SIS-dopa patches at a scanning rate of once every 30 seconds, and the patches were immersed in 3% BSA solution.

[0261] The research results showed that the exosome adsorption ability of the SIS-dopa-exo patch was significantly better than that of the unmodified SIS patch (Comparative Example 2, SIS-exo), and showed extended release characteristics ( Figure 2 a and b in). These results indicate that the SIS-dopa-exo patch can provide a more stable and lasting therapeutic effect, which is crucial for clinical applications that require continuous bioactive support.

[0262] To clarify the differences in exosome release, XPS analysis was performed on the surfaces of SIS-exo and SIS-dopa-exo, and the results showed that an additional peak appeared at 533.9 eV for SIS-dopa-exo ( Figure 2 c in), which may indicate the presence of a weak interaction with the O 1s orbital. Further FT-IR analysis found that a broad peak appeared at 3770 cm -1 for SIS-dopa-exo ( Figure 2 d in), attributed to the formation of hydrogen bonds.

[0263] 4. Molecular docking simulation of exosome membrane protein interactions

[0264] Molecular docking simulations were performed using AutoDock Vina to investigate the binding affinity of hydroxyproline and dopamine-modified hydroxyproline to aquaporin (PDB ID: 6QIM). Protein preparation was carried out according to the procedures of AutoDock Vina, and polar hydrogens were added through AutoDock Tools. The molecular coordinates of the ligand were generated from the SMILES structure using UCSF Chimera software, and non-polar hydrogens were merged during the verification of rotatable bonds using AutoDock Tools. The docking grid was designed to cover the central cavity of the protein, and semi-flexible docking experiments were visualized through AutoDock Tools. The model with the lowest binding energy was manually reviewed and optimized by energy minimization using AMMOS2. The final structure was visualized and analyzed using PyMOL.

[0265] Experimental results: To elucidate the molecular mechanism, molecular docking simulations were performed, focusing on aquaporin as a common membrane protein in plant exosomes (PDB ID: 6QIM, see the structure diagram in Figure 11 ). Hydroxyproline, as the main component of SIS, was used as a ligand after dopamine modification. The docking results showed that the binding energy of the hydroxyproline-dopamine complex to aquaporin was lower than that of unmodified hydroxyproline (-20.20 ± 0.57 kJ mol -1 compared to -16.33 ± 1.67 kJ mol -1 )( Figure 2 e in Figure 2 ), highlighting the enhanced binding achieved through dopamine modification. In addition, in-situ FT-IR spectroscopy showed that SIS-dopa (Example 4, SIS-dopa-12h) had accelerated dynamic protein adsorption compared to SIS (Comparative Example 1)

[0266] 5. Cytocompatibility test of SIS-dopa-exo patch

[0267] 5,000 L929 fibroblasts were seeded onto different patches. After 24 hours of incubation, cell viability was evaluated using a Cell Counting Kit-8 (CCK-8, Dojindo, Japan), calcein-AM / PI (Aladdin, China), and rhodamine B-labeled F-actin / DAPI staining according to the manufacturer's instructions. The OD value was recorded at 450 nm using a microplate reader. The cell viability (%) was calculated using the following formula:

[0268]

[0269] Among them, OD E represents the OD value of cells cultured on the experimental patch, and OD B represents the baseline value from the CCK-8 reagent, and OD C represents the OD value of cells cultured on the tissue culture plate. Fluorescent images of cell spreading were taken by CLSM and semi-quantitatively analyzed using ImageJ software.

[0270] In the cell compatibility test, the results showed that L929 cells were directly inoculated on SIS (Comparative Example 1), SIS-dopa (Example 4, SIS-dopa-12h), and SIS-dopa-exo (Example 5) patches. Live / dead cell staining and cytoskeleton staining showed that cells grew vigorously on all patches, and the cell viability exceeded 90%. Semi-quantitative analysis by ImageJ showed that the cell spreading area on the SIS-dopa-exo patch was larger than that of the unmodified SIS patch, i.e., SIS-dopa (27.55 ± 3.29 μm 2 vs. 20.07 ± 2.03 μm 2 ), indicating that SIS-dopa-exo promoted cell adhesion ( Figure 13 a-d in). These results verified the excellent cell compatibility of the SIS-dopa-exo patch.

[0271] 6. In vitro angiogenesis study of the SIS-dopa-exo patch

[0272] According to the ISO10993 standard, SIS conditioned medium was prepared to culture HUVECs at a cell density of 5,000 cells per well. When the cells reached approximately 80% confluence, a cell scratch of approximately 200 μm was created using a pipette tip. Subsequently, the cells were continuously cultured in the SIS conditioned medium, and the cell migration distance was recorded by optical microscopy after 24 hours. HUVECs with the same density were also seeded on Matrigel and cultured in the conditioned medium for 24 hours to evaluate the formation of tubular networks. Cell morphology and status were observed by optical microscopy. After 7 days of culture, the cells were fixed and immunofluorescently stained with VEGF primary antibody, FITC-labeled F-actin, and DAPI, and images were obtained by CLSM. In addition, total RNA was extracted from HUVECs using Trizol reagent (Invitrogen, USA) for reverse transcription polymerase chain reaction (RT-PCR) analysis. The primer sequences for RT-PCR are listed in Table 3.

[0273] Table 3: Primer sequences for RT-PCR analysis

[0274]

[0275] Experimental results: To evaluate the angiogenic potential of the SIS-dopa-exo patch, as previously described, media extracts from different patches were prepared according to ISO 10993 guidelines and used to culture HUVECs ( Figure 3 as shown in a) of . For the control group, media that was not extracted from the patch was used. The results showed that the SIS-dopa-exo patch (Example 5) significantly promoted the healing rate, with the minimum distance of the remaining scratch being 26.93 ± 3.64 μm, compared to 143.38 ± 12.48 μm for the control group ( Figure 3 as shown in b and c) of . This improvement is consistent with the earlier finding that exosomes derived from scallions have the ability to promote cell migration ( Figure 8 ). In addition, other bioactive components in SIS, such as TGF-β, may also contribute to this effect. In the tube formation assay, SIS-dopa-exo (Example 5) promoted the formation of the longest tubular structures, with the microtubule length reaching 144.37 ± 34.54 μm, significantly exceeding the average length of 35.23 ± 11.59 μm in the control group ( Figure 3 as shown in d and e) of . In addition, immunofluorescence staining showed a significantly increased expression of VEGF in SIS-dopa-exo (Example 5) ( Figure 3 as shown in f) of . RT-PCR analysis also confirmed that the VEGF gene expression increased by 2.62-fold compared to the control group ( Figure 3 as shown in g) of . Collectively, these results confirm the excellent angiogenic activity of the SIS-dopa-exo patch, highlighting its potential in enhancing tissue regeneration.

[0276] 7. Modulating the polarization of RAW264.7 macrophages on the SIS-dopa-exo patch

[0277] RAW264.7 macrophages (10,000 cells per well) were first treated with LPS (1 μg mL -1)Pre-stimulate for 24 hours, and then transfer them to different patches for continued culture for 3 days. To evaluate the expression levels, immunofluorescence staining was performed using IL-6 and Arg-1 antibodies. In addition, after LPS stimulation and subsequent patch culture, the treated cells were subjected to RNA sequencing analysis. Cell lysates were extracted with Triton X-100, and RNA sequencing was performed using the Illumina X Ten system. Quality control ensured the reliability of the data, and then the raw read sequences were filtered and mapped to the reference genome. Subsequently, gene expression levels were quantified, and genes with significantly different expression among groups were identified through differential gene expression analysis. Functional annotation and enrichment analysis were performed using KEGG software to study the biological processes and pathways associated with the differential genes.

[0278] Experimental results: To investigate whether the SIS-dopa-exo patch retained the anti-inflammatory properties of scallion-derived exosomes, a cell inflammation model was established using LPS-stimulated RAW264.7 macrophages. The typical polarization markers IL-6 (M1 pro-inflammatory) and Arg-1 (M2 anti-inflammatory) were detected by immunofluorescence staining. Under LPS stimulation, the expression of IL-6 increased significantly, rising 5.45-fold compared to the unstimulated control group, confirming the effective induction of the M1 pro-inflammatory phenotype ( Figure 14 a and b in). In contrast, the expression of Arg-1 remained unchanged, verifying the successfully constructed M1 polarization state. On this basis, after further treating LPS-stimulated RAW264.7 cells with scallion-derived exosomes, the expression of IL-6 decreased significantly ( Figure 14 c and d in). This result was consistent with the previous RNA sequencing data ( Figure 9 ), indicating that scallion exosomes contributed to reducing the inflammatory response.

[0279] Next, LPS-induced RAW264.7 macrophages were seeded onto different patches and cultured for an extended period of 3 days to evaluate the cell inflammation level ( Figure 4 a in). Through immunofluorescence staining of IL-6 and Arg-1, the results showed that the expression of IL-6 in the SIS (Comparative Example 1) group increased by 1.66-fold and 6.47-fold compared to the SIS-dopa (Example 4, SIS-dopa-12h) and SIS-dopa-exo (Example 5) groups, respectively, while the expression of Arg-1 in the SIS-dopa-exo group increased by 4.98-fold and 4.28-fold compared to the SIS and SIS-dopa groups, respectively ( Figure 4 b and c in).

[0280] 8. Inflammatory implantation model

[0281] To mimic tissue injury by introducing an inflammatory microenvironment, Sprague Dawley (SD) rats received a subcutaneous injection of LPS (100 μL, 1 μg mL -1 ) 24 hours before surgery. After anesthesia with isoflurane, the rats were divided into three groups: 1) injection of sterile saline (1 mL) as the control group; 2) implantation of SIS patch (1 cm × 1 cm; Comparative Example 1); 3) implantation of SIS-dopa-exo patch (1 cm × 1 cm; Example 5). At 3 and 7 days after surgery, the rats were sacrificed, the implantation sites were harvested, fixed in 4% formalin solution, and histological analysis was performed, including hematoxylin-eosin (H&E) staining, Masson's trichrome staining, and immunofluorescence staining of TNF-α (ab220210, Abcam, UK), CD163 (ab182422, Abcam, UK), and DAPI. In addition, hematological analysis was performed, and major organs (such as the heart, liver, spleen, lungs, and kidneys) were examined to evaluate biocompatibility 7 days after implantation.

[0282] Experimental results: To induce inflammation in vivo, as described above, LPS was injected into the implantation site 24 hours before the inflammatory implantation ( Figure 4 d in Figure 15 ). At 3 and 7 days after implantation, no obvious inflammation or edema was observed macroscopically ( Figure 4 e in Figure 16 ), while histological analysis showed fewer inflammatory cells (such as neutrophils) near the SIS-dopa-exo patch and better arrangement of collagen fibers ( Figure 4 f and g in Figure 17 ). Similarly, LPS-induced inflammation significantly increased the TNF-α level in the control group (1.33-fold and 4.78-fold higher than those in the SIS and SIS-dopa-exo groups, respectively), while the CD163 expression in the SIS-dopa-exo group was significantly higher (7.0-fold that of the control group,

[0283] Therefore, both cell and animal inflammation models verified that the SIS-dopa-exo patch could effectively promote the polarization of M2 macrophages, which was beneficial to the regenerative repair of gastrointestinal injuries.

[0284] In addition, 7 days after implantation, histopathological analysis and the results of hematological and biochemical examinations confirmed the excellent biocompatibility of the SIS-dopa-exo patch ( Figure 18 and Figure 19 ).

[0285] To further clarify this phenomenon, macrophages stimulated with LPS were re-planted on different patches, and RNA sequencing was performed after 3 days of culture. The results showed that a total of 3,173 differentially expressed genes were identified among SIS-dopa-exo (Example 5), SIS (Comparative Example 1), and the control group ( Figure 5 a in). Gene expression analysis showed that the SIS-dopa-exo patch up-regulated genes related to angiogenesis, such as VEGF-α, VEGF-β, and TGF-β, while down-regulating pro-inflammatory genes such as TLR4 ( Figure 5 b and c in). KEGG enrichment analysis showed that compared with SIS and the control group, the signaling pathways regulated by SIS-dopa-exo were mainly enriched in the HIF-1, TNF, and NF-κB signaling pathways ( Figure 5 d and e in). Among the differentially expressed genes, genes related to inflammation and angiogenesis were identified. Specifically, compared with SIS and the control group, the SIS-dopa-exo patch significantly reduced the expression of pro-inflammatory M1 genes (such as TNF, TLR4, CCL2, COX2, IFNγ2), while promoting the expression of anti-inflammatory M2 genes (such as TLR7, NFκBie, TGFβ2). In addition, genes related to angiogenesis (such as VEGF) were significantly increased in SIS-dopa-exo ( Figure 5 f in). Therefore, it was concluded that the SIS-dopa-exo patch promoted tissue repair by regulating the angiogenesis signaling pathway and effectively controlling inflammation at the molecular level.

[0286] 9. Rat Gastrointestinal Anastomosis Study

[0287] After isoflurane anesthesia, a 10-mm surgical injury was created on the colon of SD rats. The rats were divided into three groups: 1) The injury site was sutured as the control group; 2) The SIS patch (2 cm × 2 cm; Comparative Example 1) was covered after suturing the injury site; 3) The SIS-dopa-exo patch (2 cm × 2 cm; Example 5) was covered after suturing the injury site. The SIS patch and the SIS-dopa-exo patch were fixed to the injury site by additional suturing at the edges. Visceral adhesions at the surgical site were evaluated at 14 and 28 days after surgery, and then the rats were sacrificed and tissues were collected for histological analysis, including H&E staining, Masson's trichrome staining, and immunofluorescence staining of TNF-α, CD163, CD31, and DAPI. In addition, hematological analysis was performed, and the main organs (such as the heart, liver, spleen, lungs, and kidneys) were examined to evaluate biocompatibility at 28 days after surgery.

[0288] Experimental results: In subsequent animal experiments, as previously described, a 10-mm colonic injury was created in the gastrointestinal tract of rats, disrupting the muscular layer. Anastomosis was then performed, and different SIS-based patches ( Figure 6 a and b in Figure 20 ) were applied at the anastomosis site. Postoperative observations revealed that compared with the control group, visceral tissue adhesions were reduced in the SIS-dopa-exo group (Table 4, Figure 6 ), while the control group showed higher adhesions due to persistent inflammation. Histological staining analysis, including H&E and Masson's trichrome staining, showed neutrophil infiltration and less collagen formation in the control group, while the SIS-dopa-exo group exhibited a more ordered collagen arrangement and fewer neutrophils ( Figure 21 c in Figure 22 ), which may be related to the anti-inflammatory effect of scallion-derived exosomes released by SIS-dopa-exo. It was also found that the SIS patch was able to strengthen the anastomosis site to prevent wound expansion, and the SIS-dopa-exo group was similar to the colon of healthy rats in terms of pathological manifestations and collagen deposition ( Figure 6 ). Immunofluorescence staining results showed that at 28 days postoperatively, the expression of CD31 in the SIS-dopa-exo group increased significantly (3.88 times that of the control group), the expression of TNF-α decreased (0.48 times that of the control group), and the expression of CD163 increased (3.33 times that of the control group) ( Figure 23 d-g in Figure 24 ), indicating that the SIS-dopa-exo group had superior angiogenesis and tissue healing abilities. Further pathological and hematological analyses of the major organs showed no significant differences between the SIS-dopa-exo group and the control group at 28 days postoperatively (

[0289] ), indicating that the SIS-dopa-exo patch had good biocompatibility and the ability to promote anastomotic regeneration in gastrointestinal surgery.

[0290]

[0291] 10. Gastrointestinal Anastomosis Study in Bama Miniature Pigs

[0292] The gastrointestinal anastomosis surgery of Bama minipigs simulated the scenario of human colorectal cancer resection. After isoflurane anesthesia, a 20-mm surgical injury was created on the colon, and the minipigs were divided into three groups: 1) the injury was sutured as the control group; 2) the injury was sutured and covered with an SIS patch (3 cm × 6 cm; Comparative Example 1); 3) the injury was sutured and covered with an SIS-dopa-exo patch (3 cm × 6 cm; Example 5). The patch was fixed to the injury site by additional suturing at the edges. Visceral adhesions were evaluated 28 days after surgery, and then tissues were collected for histological analysis, including H&E staining, Masson's trichrome staining, and immunofluorescence staining of Arg-1, CD3e, VEGF, α-SMA, and DAPI. In addition, major organs were collected 28 days after surgery to evaluate biocompatibility.

[0293] Experimental results: The present invention further consulted clinical gastrointestinal surgeons and, as described above, conducted a 20-mm full-thickness porcine colon injury experiment to simulate the gastrointestinal resection surgery commonly seen in colorectal cancer resection, and applied an SIS patch or an SIS-dopa-exo patch at the injury site ( Figure 7 ) as in a). After obtaining the patch 28 days after surgery ( Figure 25 ), it was found by double-blind evaluation of H&E staining images that after the application of SIS-dopa-exo, the distance of the damaged muscular layer edge decreased ( Figure 7 ) as in b), and the collagen deposition at the wound site increased ( Figure 7 ) as in c). The results of immunofluorescence staining of VEGF / α-SMA / DAPI and Arg-1 / CD3e / DAPI showed that the SIS-dopa-exo patch promoted the regenerative repair of the anastomosis by upregulating the angiogenesis marker VEGF (2.83-fold relative to the control group) and Arg-1 (M2 macrophage phenotype, 4.78-fold relative to the control group) ( Figure 7 ) as in d-g), while the expression level of CD3e had no significant difference, indicating that there was no obvious difference in immune rejection between the SIS-dopa-exo group and the control group. These results further verified the ability of the SIS-dopa-exo patch as a carrier of active scallion exosomes to promote gastrointestinal anastomosis repair in a sustained-release manner. This release process activated the HIF-1 pathway and inhibited the NF-κB pathway, thereby regulating the inflammatory response. In the 28-day pathological analysis, no abnormalities were found in the major organs of the SIS-dopa-exo group ( Figure 26 ), further demonstrating its excellent biocompatibility.

[0294] These results further demonstrated that the SIS-dopa-exo patch, as a carrier of bioactive Welsh onion exosomes, could promote gastrointestinal anastomosis repair by continuously releasing exosomes. This delivery activated the HIF-1 pathway and inhibited the NF-κB pathway, thereby regulating inflammation. Pathological analysis of the major

[0295] organs at 28 days post-operation showed no abnormalities in the SIS-dopa-exo group ( Figure 26 ), indicating its excellent biocompatibility.

[0296] Post-operative complications after gastrointestinal anastomosis, such as bleeding, inflammation, poor tissue healing, and anastomotic leakage, pose a great challenge to the recovery of patients. A novel SIS-dopa-exo patch is provided in the present invention, which is prepared by grafting dopamine onto a biological SIS patch and loading exosomes from plant-derived Welsh onion extract ( Figure 27 ). Specifically, in the present invention, a biological patch based on small intestinal submucosa (SIS) is developed, which is loaded with exosomes from plant-derived Welsh onion extract for promoting wound repair in gastrointestinal anastomosis. By chemically grafting dopamine onto the SIS biological patch (i.e., SIS-dopa), and through strong hydrogen bonding between the exosomes from Welsh onion extract and the catechol groups in SIS-dopa (i.e., SIS-dopa-exo), continuous release of exosomes is achieved. Molecular docking simulations showed that the binding energy between the exosomes and the catechol groups was significantly reduced compared to other chemical groups in SIS. In vitro experiments showed that the patch achieved continuous release of exosomes by reducing the binding energy. Cell experiments showed that the SIS-dopa-exo patch could effectively regulate the inflammatory response and promote angiogenesis and regulate inflammation by activating typical signaling pathways such as HIF-1 and inhibiting NF-κB. RNA sequencing verified this result. In animal studies on gastrointestinal injury repair, the SIS-dopa-exo biological patch showed excellent performance in promoting collagen formation and tissue regeneration, demonstrating significant potential to enhance the healing effect of the gastrointestinal tract. In gastrointestinal anastomosis, the SIS-dopa-exo patch was superior to traditional SIS patches and standard anastomosis in reducing inflammation and promoting collagen deposition. These results highlight the potential of this exosome-modified patch in improving tissue regeneration and providing clinical benefits for gastrointestinal surgery.

[0297] It should be noted that although the technical solutions of the present invention are introduced with specific examples, those skilled in the art can understand that the present invention should not be limited thereto.

[0298] The embodiments of the present invention have been described above. The above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The choice of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to technologies in the market, or to enable other ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A biological patch, characterized in that: The biological patch comprises: A substrate having a fiber structure, and the fiber has polar groups; wherein, The polar groups are used to graft catechol functional groups onto the surface of the substrate, and the catechol functional groups are derived from polyphenol compounds.

2. The biological patch according to claim 1, characterized in that: The polar group includes a carboxyl group; preferably, the carboxyl group is activated by an activating agent.

3. The biological patch according to claim 1 or 2, characterized in that: The polyphenolic compound includes one or a combination of two or more of dopamine or its salt, tannic acid, catechol, pyrogallol, gallic acid and catechin; preferably, the polyphenolic compound includes dopamine or its salt.

4. The biological patch according to any one of claims 1 to 3, characterized in that: Exosomes are also connected to the surface of the biological patch; the exosomes are coupled to the surface of the biological patch via hydrogen bonds with the catechol functional groups; Preferably, the exosomes include plant-derived exosomes, and more preferably, the exosomes include Allium-derived exosomes.

5. A method for preparing a biological patch according to any one of claims 1 to 4, characterized in that: The preparation method comprises: grafting catechol functional groups onto the surface of a substrate; Preferably, the step of coupling the exosomes to the surface of the substrate is performed using catechol functional groups.

6. The preparation method according to claim 5, characterized in that: In the step of grafting the catechol functional group onto the surface of the substrate, the substrate is contacted with a solution containing polyphenolic compounds, wherein the content of the polyphenolic compounds in the solution containing polyphenolic compounds is 0.1-50 (w / v) %, and / or The reaction time of the substrate and the solution containing polyphenol compounds is 1-18 hours.

7. The preparation method according to claim 5 or 6, characterized in that: Before the step of grafting the catechol functional group onto the surface of the substrate, the step of activating the substrate is also included. Optionally, in the step of activating the substrate, a carbodiimide activator and / or an N-hydroxyimine activator is used to activate the substrate.

8. The preparation method according to any one of claims 5 to 7, characterized in that: In the step of coupling the exosomes to the surface of the substrate using the catechol functional group, the exosomes are coupled to the surface of the substrate by contacting the substrate with a solution containing the exosomes.

9. The preparation method according to claim 8, characterized in that: In the solution containing exosomes, the content of exosomes is 1-300 μg / mL.

10. The preparation method according to any one of claims 5 to 9, characterized in that: After the step of coupling the exosomes to the surface of the substrate using the catechol functional group, a drying step is also included.

11. Use of the biological patch according to any one of claims 1 to 4 or the biological patch prepared by the preparation method according to any one of claims 5 to 10 in preparing a product for tissue repair; Preferably, the tissue comprises gastrointestinal tissue.

Images