A biogel carrying nanoplatelet vesicles and its preparation method and application

Abstract

The present invention discloses a biogel carrying nanoplatelet vesicles, and its preparation method and application. The gel is formed by natural activation of nanoplatelets, fibrinogen and thrombin; the gel achieves starvation therapy of proinflammatory cells based on the selective permeation of nutrients, and promotes repair and regeneration through nanoplatelets, and has entered clinical application, providing a new strategy for the preparation, application and clinical transformation of natural source biomaterials.

Description

Technical Field

[0001] The present invention relates to a biogel, and in particular to a biogel carrying nanoplatelet vesicles, and a preparation method and application thereof. Background Art

[0002] Under physiological conditions, the circulatory system provides the body with sufficient oxygen and nutrients, while under pathological conditions, providing sufficient nutrients will have the opposite effect. Under inflammatory conditions of tissue damage, M1 inflammatory macrophages are highly active and the rate of aerobic glycolysis is significantly increased. At the same time, the uptake of glucose by inflammatory cells increases significantly to maintain the high activity of cells, resulting in a chronic, uncontrollable inflammatory response. Excessive inflammation causes tissue damage and inflammatory immune imbalance, thereby hindering tissue repair.

[0003] The extracellular matrix is ​​a very important non-cellular component in the immune microenvironment, providing a structural basis and biochemical support for immune cells. As an effective mass transport medium, the natural extracellular matrix is ​​essential for maintaining sufficient adenosine triphosphate and biosynthetic intermediates to meet the energy and material needs of highly active immune cells. Remodeling of the extracellular matrix microenvironment can cause metabolic changes. For example, cells isolated from the extracellular matrix suffer from a lack of adenosine triphosphate (ATP) due to the loss of glucose transporters. Remodeling and constructing the extracellular matrix may achieve "drug-free" inhibition of the inflammatory environment, thereby promoting tissue regeneration.

[0004] At present, the transformation of extracellular matrix for tissue regeneration is still a major problem, especially the transformation of extracellular matrix with autologous materials. The invention patent with application number CN201810343820.9 discloses an assembled cell-derived extracellular matrix membrane composite bone repair material and its preparation method and application. The composite bone repair material is prepared by completely coating a three-dimensional degradable bioscaffold with a sheet of decellularized matrix membrane under sterile conditions. It realizes the preparation of a bioscaffold that can be degraded and absorbed, but the process is complicated, the cell culture cycle is too long, and exogenous substances are introduced during the decellularization process, which has biosafety risks and does not have inflammation regulation characteristics. The invention patent with application number CN201810956245.X discloses a cosmetic gel for efficiently repairing acne marks and a preparation method. A variety of ingredients are kneaded to obtain a cosmetic gel for efficiently repairing acne marks. It has the advantage of instant preparation, but contains a large amount of exogenous ingredients, has low biosafety, and does not have inflammation regulation characteristics. Therefore, there is a need for a biogel with autologous ingredients and convenient preparation, which is used for inflammation regulation of tissue damage and promotes tissue repair and regeneration.

[0005] During bleeding, platelets and fibrinogen are activated by thrombin to form a gel-like clot. This natural extracellular matrix that forms spontaneously during injury is a key part of tissue repair. Platelets play an important role in tissue healing and angiogenesis, but platelets, as proinflammatory cells, may aggravate inflammation by releasing inflammatory factors after activation, further unbalancing inflammatory immunity, while nano-sized platelets have platelet-like functions and do not significantly release proinflammatory factors after activation. Therefore, a natural-derived biogel carrying nanoplatelet vesicles is needed. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a biogel carrying nanoplatelet vesicles and its preparation method and application. The gel regulates the inflammatory response of proinflammatory cells based on starvation therapy and promotes tissue regeneration through nanoplatelets, thereby promoting the repair of damaged tissues.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The invention provides a biogel carrying nanoplatelet vesicles, wherein the biogel is formed by natural activation of nanoplatelets, fibrinogen and thrombin.

[0009] The nanoplatelets refer to vesicles obtained by nano-processing autologous platelet components, which retain the components and structure of platelets intact; the fibrinogen refers to fibrinogen derived from autologous plasma; and the thrombin refers to productized thrombin.

[0010] The present invention also provides a method for preparing a biogel carrying nanoplatelet vesicles, which comprises the following steps:

[0011] The peripheral blood is obtained, the platelets are separated and enriched, and the platelets are extruded and nanoformed, and fibrinogen and thrombin are added to obtain the biogel after activation.

[0012] In the above technical solution, the methods for separating and enriching platelets include machine apheresis, two-step centrifugation and density gradient centrifugation. Preferably, the two-step centrifugation is performed by the following method:

[0013] Mix the peripheral blood by inverting it upside down, place it in a centrifuge tube, and centrifuge it at 300×g for 10 minutes. Carefully aspirate the upper layer of platelet-containing plasma and transfer it to a new centrifuge tube, and centrifuge it at 800×g for 6 minutes. Aspirate a certain volume of supernatant, and gently pipette and mix the lower layer of plasma and precipitate to obtain platelet-rich plasma.

[0014] Furthermore, the volume ratio of the above-mentioned "certain amount of supernatant" is 1 / 2 to 9 / 10, and preferably, 4 / 5 of the volume of the supernatant is taken to increase the final platelet concentration.

[0015] In the above technical solution, the nano-forming method includes extrusion method, ultrasonic method, freeze-thaw method, preferably, the extrusion method is carried out by the following method:

[0016] The enriched platelets were extruded repeatedly at a constant speed through extrusion membranes with pore sizes of 400 nm, 200 nm and 100 nm.

[0017] Furthermore, the extruded membrane includes PC, PE, PES, and PVDF membranes, and preferably, PES membrane is used.

[0018] In the above technical solution, the method of adding fibrinogen is to add the above "certain volume of supernatant" back to the nanoplatelets, and adjust the concentration of nanoplatelets to 1-4 times the original peripheral blood concentration. Preferably, the ratio is set to 1.5 times.

[0019] In the above technical solution, thrombin is added by adding 50-500U / mL thrombin physiological saline solution at a volume ratio of 10% to 100%. Preferably, the volume ratio is set to 10% and the thrombin concentration is 200U / mL.

[0020] The present invention also provides an application of a biogel carrying nanoplatelet vesicles. The application contents are as follows:

[0021] Biogels loaded with nanoplatelet vesicles or encapsulated with specific drugs will be used to suppress inflammation through starvation therapy and promote regeneration through nanoplatelets, thereby promoting tissue repair.

[0022] Furthermore, the above-mentioned specific drugs include antibiotics and anti-tumor drugs. Preferably, the antibiotic is vancomycin and the anti-tumor drug is doxorubicin.

[0023] Furthermore, the above-mentioned tissue repair includes orthopedic, muscle, skin, organ and other repairs, and is preferably used for difficult-to-heal tissue injuries such as diabetic foot, arthritis, bone infection, and more preferably, for diabetic foot ulcers.

[0024] Beneficial effects of the present invention:

[0025] 1) The biogel carrying nanoplatelet vesicles provided by the present invention has nanosized platelets, which retains the composition and structural integrity of the platelets.

[0026] 2) The biogel carrying nanoplatelet vesicles provided by the present invention effectively realizes the starvation therapy of nutrient blocking of inflammatory cells.

[0027] 3) The biogel carrying nanoplatelet vesicles provided by the present invention effectively promotes tissue repair through nanoplatelets.

[0028] 4) The biogel loaded with nanoplatelet vesicles provided by the present invention has entered clinical application and effectively achieved wound repair of difficult-to-heal tissue damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0030] Figure 1-1 The gelling effect of the biogel carrying nanoplatelet vesicles of Example 1;

[0031] Figure 1-2 The electron microscopic characterization of the biogel loaded with nanoplatelet vesicles of Example 1 shows that nanoplatelet plasma and thrombin can be effectively gelled after mixing, and form a pore size concentration-dependent network structure.

[0032] Figure 2 The determination of permeable substances and mass spectrometry analysis of the biogel loaded with nanoplatelet vesicles of Example 1 show that compared with hyaluronic acid, the biogel loaded with nanoplatelet vesicles has selective permeability to a variety of substances, indicating that the biogel loaded with nanoplatelet vesicles can block nutrients such as glucose.

[0033] Figure 3 This is an electron microscopic characterization of the effect of the biogel loaded with nanoplatelet vesicles of Example 1 on cells, showing that as the concentration increases, the gel network gradually wraps the cells to achieve starvation therapy by blocking nutrients.

[0034] Figure 4 The biogel loaded with nanoplatelet vesicles of Example 1 inhibits the growth of inflammatory cells, showing that as the concentration of the gel increases, the inflammatory cells are inhibited and the consumption of glucose is effectively inhibited.

[0035] Figure 5 The regulation of inflammatory genes of inflammatory cells by the biogel loaded with nanoplatelet vesicles in Example 1 shows that the biogel loaded with nanoplatelet vesicles effectively inhibits the expression and translation of pro-inflammatory proteins such as iNOS in inflammatory cells, and promotes the expression and translation of anti-inflammatory cells such as Arg1, indicating that the biogel loaded with nanoplatelet vesicles can effectively inhibit inflammation.

[0036] Figure 6 The transcriptome and metabolome analysis of the biogel loaded with nanoplatelet vesicles of Example 1 showed that the biogel loaded with nanoplatelet vesicles significantly inhibited the metabolic levels of inflammatory cells, indicating that the biogel loaded with nanoplatelet vesicles can effectively regulate the state of inflammatory cells.

[0037] Figure 7The protein spectrum of the nanoplatelets in the biogel loaded with nanoplatelet vesicles of Example 1 shows that the nanoplatelets retain proteins that promote angiogenesis, cell migration, and cell adhesion, indicating that the nanoplatelets have a strong ability to promote repair.

[0038] Figure 8 The scratch test and ring formation test of the biogel loaded with nanoplatelet vesicles of Example 1 show that the biogel loaded with nanoplatelet vesicles can significantly promote the migration of vascular endothelial cells and vascular ring formation, indicating that the biogel loaded with nanoplatelet vesicles has a strong ability to promote angiogenesis.

[0039] Fig. 9 The histological analysis of the clinical treatment of the biogel loaded with nanoplatelet vesicles of Example 1 showed that the biogel loaded with nanoplatelet vesicles can promote collagen production, endothelial CD31 expression, downregulation of iNOS and upregulation of ARG, indicating that the biogel loaded with nanoplatelet vesicles can promote wound regeneration, angiogenesis and inflammation inhibition in patients. DETAILED DESCRIPTION

[0040] The following is a detailed description of a biogel carrying nanoplatelet vesicles provided by the present invention, a preparation method thereof, and an application thereof in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] The biogel carrying nanoplatelet vesicles provided by the present invention is formed by natural activation of nanoplatelets, fibrinogen and thrombin. "Nanoplatelets" refers to vesicles obtained by nano-processing autologous platelet components, which retain the components and structure of platelets intact. "Fibrinogen" refers to fibrinogen derived from autologous plasma. "Thrombin" refers to productized thrombin.

[0042] The method for preparing the biogel carrying nanoplatelet vesicles provided by the present invention comprises the following steps:

[0043] Step 1: separating and enriching platelets, including machine apheresis, two-step centrifugation and density gradient centrifugation. Preferably, the two-step centrifugation is performed by the following method:

[0044] Mix the peripheral blood by turning it upside down, place it in a centrifuge tube, and centrifuge it at 300×g for 10 minutes. Carefully aspirate the upper layer of platelet-containing plasma, transfer it to a new centrifuge tube, and centrifuge it at 800×g for 6 minutes. Aspirate a certain volume of supernatant, and gently pipette and mix the lower layer of plasma and precipitate to obtain platelet-rich plasma. The volume ratio of the above-mentioned "certain volume of supernatant" is 1 / 2 to 9 / 10. Preferably, 4 / 5 of the volume of supernatant is taken to increase the final platelet concentration.

[0045] Step 2: extrusion nano-crystallization is carried out in the following manner:

[0046] The enriched platelets are extruded through extrusion membranes with pore sizes of 400 nm, 200 nm and 100 nm at a constant speed repeatedly. The extrusion membranes include PC, PE, PES and PVDF membranes, and preferably, PES membranes are used.

[0047] Step 3, adding fibrinogen and thrombin, is performed in the following manner:

[0048] Add the above "certain volume of supernatant" back to the nanoplatelets, and adjust the concentration of the nanoplatelets to 1-4 times the original peripheral blood concentration, add 50-500U / mL of thrombin saline solution at a volume ratio of 10% to 100%, and activate it to form a gel. Preferably, the nanoplatelet concentration ratio is 1.5 times, the volume ratio of thrombin saline solution is 10%, and the thrombin concentration is 200U / mL.

[0049] The application of the biogel carrying nanoplatelet vesicles provided by the present invention is as follows:

[0050] The biogel carrying nanoplatelet vesicles (or encapsulating specific drugs in nanoplatelets) is used to inhibit inflammation through starvation therapy and promote regeneration through nanoplatelets, thereby promoting tissue repair. The above-mentioned specific drugs include antibiotics and anti-tumor drugs. Preferably, the antibiotic is vancomycin and the anti-tumor drug is doxorubicin. The above-mentioned specific drug encapsulation method, preferably ultrasonic encapsulation, is carried out in the following manner: blending nano-enriched platelets with a drug solution, and performing low-temperature bath ultrasonic treatment for 30 minutes. The above-mentioned tissue repair includes orthopedic, muscle, skin, organ repair, etc., preferably, it is used for difficult-to-heal tissue injuries such as diabetic foot, arthritis, bone infection, and more preferably, it is used for diabetic foot ulcers.

[0051] The present invention can also use the above-mentioned other platelet enrichment methods, other nano-forming methods, other gelling methods, and other drug encapsulation methods to achieve the same technical effects.

[0052] Example 1: Biogel with nano-enriched platelets from two-step centrifugation method

[0053] Step 1: Isolation and enrichment of platelets is performed by:

[0054] Mix the peripheral blood by inverting it, place it in a centrifuge tube, and centrifuge it at 300×g for 10 minutes. Carefully aspirate the upper layer of platelet-containing plasma and transfer it to a new centrifuge tube, and centrifuge it at 800×g for 6 minutes. Aspirate 4 / 5 of the supernatant, and gently pipette and mix the lower layer of plasma and precipitate to obtain platelet-rich plasma.

[0055] Step 2: extrusion nano-crystallization is carried out in the following manner:

[0056] The enriched platelets were extruded repeatedly at a constant speed through PES extrusion membranes with pore sizes of 400 nm, 200 nm and 100 nm.

[0057] Step 3, adding fibrinogen and thrombin, is performed in the following manner:

[0058] The above-mentioned "certain volume of supernatant" was added back to the nanoplatelets, and the concentration of the nanoplatelets was adjusted to 1.5 times of the original peripheral blood concentration, and 200U / mL of thrombin saline solution was added at a volume ratio of 10%, and the activated solution was gelled.

[0059] Example 2: Biogel with nano-enriched platelets from two-step centrifugation method

[0060] Step 1: Isolation and enrichment of platelets is performed by:

[0061] Mix the peripheral blood by inverting it upside down, place it in a centrifuge tube, and centrifuge it at 300×g for 10 minutes. Carefully aspirate the upper layer of platelet-containing plasma and transfer it to a new centrifuge tube, and centrifuge it at 800×g for 6 minutes. Aspirate 1 / 2 of the supernatant, and gently pipette and mix the lower layer of plasma and precipitate to obtain platelet-rich plasma.

[0062] Step 2: extrusion nano-crystallization is carried out in the following manner:

[0063] The enriched platelets were extruded repeatedly at a constant speed through PES extrusion membranes with pore sizes of 400 nm, 200 nm and 100 nm.

[0064] Step 3, adding fibrinogen and thrombin, is performed in the following manner:

[0065] The above-mentioned "certain volume of supernatant" was added back to the nanoplatelets, and the concentration of the nanoplatelets was adjusted to 3 times of the original peripheral blood concentration, and 300 U / mL of thrombin saline solution was added at a volume ratio of 15%, and the mixture was activated to form a gel.

[0066] Example 3: Biogel with nano-enriched platelets from two-step centrifugation method

[0067] Step 1: Isolation and enrichment of platelets is performed by:

[0068] Mix the peripheral blood by inverting it, place it in a centrifuge tube, and centrifuge it at 300×g for 10 minutes. Carefully aspirate the upper layer of platelet-containing plasma and transfer it to a new centrifuge tube, and centrifuge it at 800×g for 6 minutes. Aspirate 9 / 10 of the supernatant, and gently pipette and mix the lower layer of plasma and precipitate to obtain platelet-rich plasma.

[0069] Step 2: extrusion nano-crystallization is carried out in the following manner:

[0070] The enriched platelets were extruded repeatedly at a constant speed through PES extrusion membranes with pore sizes of 400 nm, 200 nm and 100 nm.

[0071] Step 3, adding fibrinogen and thrombin, is performed in the following manner:

[0072] The above-mentioned "certain volume of supernatant" was added back to the nanoplatelets, and the concentration of the nanoplatelets was adjusted to 4 times of the original peripheral blood concentration, and 500U / mL of thrombin saline solution was added at a volume ratio of 100%, and the solution was activated to form a gel.

[0073] Example 4: Biogel with nano-enriched platelets from machine apheresis

[0074] Step 1: Isolation and enrichment of platelets is performed by:

[0075] Platelet-rich plasma was obtained using a platelet apheresis instrument, and the platelet enrichment was set to 2-5 times.

[0076] Step 2: extrusion nano-crystallization is carried out in the following manner:

[0077] The enriched platelets were extruded repeatedly at a constant speed through PES extrusion membranes with pore sizes of 400 nm, 200 nm and 100 nm.

[0078] Step 3, adding fibrinogen and thrombin, is performed in the following manner:

[0079] Physiological saline was added to dilute and adjust the nanoplatelet concentration to 1.5 times of the original peripheral blood concentration, and 100 U / mL thrombin physiological saline solution was added at a volume ratio of 20%, and the platelets were activated to form a gel.

[0080] Example 5: Drug-loaded biogel containing nano-enriched platelets derived from density gradient centrifugation

[0081] Step 1: Isolation and enrichment of platelets is performed by:

[0082] Mix the peripheral blood upside down, place it on a plasma separation gel layer with a specific gravity of 1.078-1.082, centrifuge it at 3000×g for 10 minutes, draw a certain volume of supernatant, mix the lower layer of plasma and draw it out, and you will get platelet-rich plasma. The volume ratio of the above "certain volume of supernatant" is 4 / 5 to increase the final platelet concentration.

[0083] Step 2: extrusion nano-crystallization is carried out in the following manner:

[0084] The enriched platelets were extruded repeatedly at a constant speed through PES extrusion membranes with pore sizes of 400 nm, 200 nm and 100 nm.

[0085] Step 3, drug encapsulation, is performed by the following method:

[0086] The nano-enriched platelets were mixed with the vancomycin solution and treated with low-temperature bath ultrasound for 30 minutes.

[0087] Step 4, adding fibrinogen and thrombin, is performed in the following manner:

[0088] The above-mentioned "certain volume of supernatant" was added back to the nanoplatelets, and the concentration of the nanoplatelets was adjusted to 1.5 times of the original peripheral blood concentration, and 500U / mL of thrombin saline solution was added at a volume ratio of 10%, and the activated solution was gelled.

[0089] Gelation effect and electron microscopy characterization of the biogel loaded with nanoplatelet vesicles in Example 1

[0090] 1. Mix different concentrations of nanoplatelet plasma and thrombin and wait for activation to form gel.

[0091] 2. Prepare samples from different groups for scanning electron microscopy and observe them.

[0092] 3. The results show that nanoplatelet plasma and thrombin can be effectively gelled after mixing, and form a network structure that depends on pore size and concentration, as shown in Figure 1.

[0093] Determination of permeation substances and mass spectrometry analysis of the biogel loaded with nanoplatelet vesicles in Example 1

[0094] 1. Place the biogel or hyaluronic acid gel carrying nanoplatelet vesicles on the transwell chamber.

[0095] 2. Add various substances to one side of the chamber, measure the concentration of the substances on the other side after a certain period of time, and perform mass spectrometry analysis.

[0096] 3. Compared with hyaluronic acid, the biogel carrying nanoplatelet vesicles has selective permeability to a variety of substances, indicating that the biogel carrying nanoplatelet vesicles can block nutrients such as glucose. Figure 2 .

[0097] Electron microscopic characterization of the effect of the biogel loaded with nanoplatelet vesicles on cells in Example 1

[0098] 1. Mix different concentrations of nanoplatelet plasma and thrombin, add macrophages, and wait for activation to form gel.

[0099] 2. Prepare samples from different groups for scanning electron microscopy and observe them.

[0100] 3. The results showed that as the concentration increased, the gel network gradually wrapped the cells to achieve starvation therapy by blocking nutrients. Figure 3.

[0101] Inhibition of inflammatory cell growth by the biogel loaded with nanoplatelet vesicles in Example 1

[0102] 1. Mix different concentrations of nanoplatelet plasma and thrombin, wait for activation into gel, and add macrophages.

[0103] 2. After cell culture, cell fluorescence was captured under a fluorescence microscope to observe cell growth and to measure the concentrations of glucose and lactate in the culture medium.

[0104] 3. The results showed that as the gel concentration increased, inflammatory cells were inhibited and glucose consumption was effectively inhibited. Figure 4 .

[0105] Regulation of inflammatory genes in inflammatory cells by the biogel loaded with nanoplatelet vesicles in Example 1

[0106] 1. Mix nanoplatelet plasma and thrombin, wait for activation into gel, and add macrophages.

[0107] 2. After cell culture, the fluorescence signals of iNOS and ARG were captured under a fluorescence microscope, and the cell RNA was extracted and reverse transcribed. RT-PCR was performed on the cDNA to determine the expression of inflammation-related genes.

[0108] 3. The results showed that the biogel loaded with nanoplatelet vesicles effectively inhibited the expression and translation of pro-inflammatory proteins such as iNOS in inflammatory cells, and promoted the expression and translation of anti-inflammatory proteins such as Arg1, indicating that the biogel loaded with nanoplatelet vesicles can effectively inhibit inflammation. Figure 5 .

[0109] Transcriptome and metabolome analysis of the biogel loaded with nanoplatelet vesicles in Example 1

[0110] 1. Mix nanoplatelet plasma and thrombin, wait for activation into gel, and add LPS-stimulated macrophages.

[0111] 2. After cell culture, transcriptome and metabolome analysis was performed on the cell samples.

[0112] 3. The results showed that the biogel carrying nanoplatelet vesicles significantly inhibited the metabolic levels of inflammatory cells, indicating that the biogel carrying nanoplatelet vesicles can effectively regulate the state of inflammatory cells. Figure 6 .

[0113] Protein profile of nanoplatelets in the biogel loaded with nanoplatelet vesicles in Example 1

[0114] 1. Perform protein spectrum analysis on nanoplatelets and compare the composition of platelet-related proteins.

[0115] 2. The results showed that nanoplatelets retained proteins that promote angiogenesis, cell migration, and cell adhesion, indicating that nanoplatelets have strong repair-promoting capabilities. Figure 7 .

[0116] Scratch test and ring formation test of the biogel loaded with nanoplatelet vesicles in Example 1

[0117] 1. Use a small gun tip to scratch the cells, culture umbilical vein endothelial cells, and add physiological saline, nanoplatelets, and biogel loaded with nanoplatelet vesicles.

[0118] 2. Continue culturing and observe cell differentiation. After 72 hours, take photos and measure the cell migration rate.

[0119] 3. The results showed that the biogel loaded with nanoplatelet vesicles can significantly promote the migration of endothelial cells and the formation of blood vessels, indicating that the biogel loaded with nanoplatelet vesicles has a strong ability to promote angiogenesis. Figure 8 .

[0120] Histological analysis of clinical treatment of the biogel loaded with nanoplatelet vesicles in Example 1

[0121] 1. Randomly select clinical patients with diabetic foot ulcers and apply biogel loaded with nanoplatelet vesicles after debridement.

[0122] 2. Obtain patient tissues on days 0, 3, and 7, perform histological analysis, and observe the healing of the patient's wounds.

[0123] 3. The results showed that the biogel loaded with nanoplatelet vesicles can promote collagen production, endothelial CD31 expression, iNOS downregulation and ARG upregulation, indicating that the biogel loaded with nanoplatelet vesicles can promote wound regeneration, angiogenesis and inflammation inhibition in patients. Fig. 9 .

[0124] The biogels loaded with nanoplatelet vesicles obtained in Examples 4 and 5 were respectively subjected to gelation effect and electron microscopic characterization, determination of permeable substances and mass spectrometry analysis, electron microscopic characterization of cell effects, inhibition of inflammatory cell growth, inflammatory gene regulation of inflammatory cells, transcriptome and metabolome analysis, protein spectrum, scratch test and ring formation test, and histological analysis of clinical treatment. The biogel loaded with nanoplatelet vesicles obtained in Example 5 was subjected to drug loading rate determination, drug release curve determination, and in vivo drug concentration determination, and the results were similar to those of the biogel loaded with nano-enriched platelets derived from the two-step centrifugation method in Example 1, which indicates that the preparation and application of biogels loaded with nanoplatelet vesicles can be achieved through the above-mentioned other platelet enrichment methods, other nano-ization methods, other gelation methods, and other drug encapsulation.

[0125] The above is only a preferred embodiment of the present invention. It should be pointed out that although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that several improvements and modifications may be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention and do not deviate from the scope defined by the claims of the present invention.

Claims

1. A biogel carrying nanoplatelet vesicles, characterized in that: The gel is formed by the natural activation of nanoplatelets, fibrinogen, and thrombin; The method for preparing the biogel carrying nanoplatelet vesicles comprises the following steps: Obtain peripheral blood, separate and enrich platelets, extrude and nanoform platelets, add fibrinogen, and add thrombin to obtain biogel after activation; The method of separating and enriching platelets adopts a two-step centrifugation method; the two-step centrifugation method is performed in the following manner: the peripheral blood is mixed upside down, placed in a centrifuge tube, and centrifuged at 300×g for 10 minutes; the upper layer of platelet-containing plasma is aspirated and transferred to a new centrifuge tube, and centrifuged at 800×g for 6 minutes; a portion of the supernatant is aspirated, and the lower layer of plasma and the precipitate are mixed by pipetting to obtain platelet-rich plasma; the aspirated portion of the supernatant volume, wherein the aspirated volume accounts for 1 / 2 to 9 / 10; The nano-sizing method is an extrusion method, which is performed by: repeatedly extruding the enriched platelets through extrusion membranes with pore sizes of 400 nm, 200 nm and 100 nm at a uniform speed; The fibrinogen is added by adding part of the supernatant after peripheral blood separation back to the nanoplatelets, and adjusting the concentration of the nanoplatelets to 1-4 times the original peripheral blood concentration; The method of adding thrombin is to add 200U / mL thrombin physiological saline solution at a volume ratio of 10%.

2. The biogel carrying nanoplatelet vesicles according to claim 1, characterized in that: The partial volume of the supernatant is absorbed, and 4 / 5 of the volume of the supernatant is taken to increase the final platelet concentration.

3. The biogel carrying nanoplatelet vesicles according to claim 1, characterized in that: The extruded films include PC, PE, PES and PVDF films.

4. The biogel carrying nanoplatelet vesicles according to claim 1, characterized in that: The extruded film is a PES film.

5. The biogel carrying nanoplatelet vesicles according to claim 1, characterized in that: The nanoplatelet concentration was adjusted to 1.5 times the original peripheral blood concentration.

6. A tissue repair material, characterized in that: The tissue repair material comprises the biogel carrying nanoplatelet vesicles as claimed in claim 1, or the tissue repair material comprises the biogel carrying nanoplatelet vesicles as claimed in claim 1 and drugs.

7. The tissue repair material according to claim 6, characterized in that: The drugs include antibiotics and anti-tumor drugs.

8. The tissue repair material according to claim 7, characterized in that: The antibiotic is vancomycin and the anti-tumor drug is doxorubicin.

9. The tissue repair material according to claim 6, characterized in that: The tissues include orthopedic tissue, muscle tissue, skin tissue, and organ tissue.

10. Use of the tissue repair material according to claim 6 in preparing products for repairing diabetic foot, arthritis and bone infection.

Images