Hydrogel with glucose and pH dual response and intelligent response to release of platelet-rich plasma, preparation and application

Abstract

The invention relates to glucose and pH dual-response hydrogel capable of intelligently responding to release of platelet-rich plasma, preparation and application, and belongs to the field of cellulose materials.Chitosan is alkalized and then subjected to carboxymethylation treatment, and carboxymethyl chitosan is obtained; the preparation method comprises the following steps: oxidizing nano cellulose to obtain hydroformylation modified nano cellulose; carboxymethyl chitosan and hydroformylation modified nano cellulose are mixed, glucose oxidase, platelet-rich plasma and nano cerium oxide particles are added in the mixing process, and the glucose and pH dual-response hydrogel capable of intelligently responding to release of platelet-rich plasma is obtained. According to the invention, a cross-linking agent is not needed, the operation is simple, the cost is low, the hydrogel can be rapidly formed, and the hydrogel can be automatically and gradually dissolved to release micromolecular substances wrapped in the hydrogel after being stimulated by glucose and pH change, is environment-friendly, has good biocompatibility, and is beneficial to promoting the application of the hydrogel in the fields of diabetes wound dressings and the like.

Description

Technical Field

[0001] The present invention belongs to the field of cellulose materials, and more specifically, relates to a hydrogel with dual glucose and pH-responsive intelligent release of platelet-rich plasma, its preparation and application, and particularly relates to a preparation method of a nano-cellulose and chitosan hydrogel with dual glucose and pH-responsive intelligent release of platelet-rich plasma. Background Art

[0002] In the past few decades, due to the characteristics of natural polymers such as sustainability and degradability, they have great application potential in the fields of sustainable materials, biomedicine, and pharmaceuticals. Among them, hydrogels made of natural polymers (such as chitosan and cellulose, etc.) have attracted extensive attention due to their good biocompatibility and excellent mechanical properties, etc.

[0003] Chitosan is a natural polysaccharide extracted from renewable resources such as crab shells and shrimp shells. Since it can only be dissolved in acids and acidic aqueous solutions, its theoretical research and production applications are restricted. However, chitosan has excellent biocompatibility and biodegradability and is easily made into various derivatives. Among them, carboxymethyl chitosan (CMC) is a chitosan derivative with high solubility under physiological conditions. The hydrogel made of CMC has good biocompatibility and moisturizing ability, but its mechanical properties are usually poor under normal conditions. Nano-cellulose has become an excellent material for improving the mechanical and other properties of hydrogels due to its low density, good mechanical properties, strong hydrophilicity, and low thermal expansion coefficient, etc.

[0004] Compared with other nano- or micro-scale nano-celluloses (such as cellulose nanocrystals (CNC) and cellulose microfibrils (CMF), etc.), cellulose nanofibrils (CNF) exhibit better properties, including high stiffness and strength as well as a larger surface area. Currently, the preparation of CNF-based hydrogels usually adopts cross-linking with metal cations or polymers. However, there are problems such as a longer cross-linking time, the need to use additional cross-linking agents for cross-linking, and some cross-linking agents are toxic, which will limit the application of hydrogels in some fields (such as drug delivery, etc.).

[0005] On the other hand, platelet-rich plasma (PRP) rich in growth factors can effectively accelerate wound healing and promote tissue regeneration by regulating cell proliferation. However, due to the disadvantages such as poor stability of PRP and easy burst release of growth factors, its current applications are limited. Summary of the Invention

[0006] To address the drawbacks and deficiencies of current cellulose hydrogel preparation techniques, the preparation of existing cellulose nanofibril-based hydrogels typically involves crosslinking with metal cations or polymers, which has technical problems such as a relatively long crosslinking time, the need for additional crosslinking agents, and the fact that some crosslinking agents are toxic and inconvenient to use. Additionally, it aims to overcome the shortcomings such as poor stability of platelet-rich plasma and the easy burst release of growth factors. The present invention provides a preparation method for intelligent release of platelet-rich plasma with dual responses to glucose and pH. The method of the present invention effectively combines platelet-rich plasma with chitosan and cellulose nanofibrils to prepare a hydrogel. Compared with traditional methods, the present invention does not require the use of crosslinking agents, has simple operations, a fast hydrogel synthesis rate, and platelet-rich plasma can also be connected to the hydrogel network through Schiff base bonds, thereby enhancing its stability and overcoming drawbacks such as the easy burst release of growth factors. In addition, the hydrogel prepared by the present invention is injectable and can automatically respond and dissolve according to changes in glucose or pH in the environment where the hydrogel is located, which can effectively improve the cellulose hydrogel preparation technology and broaden its application fields.

[0007] According to a first aspect of the present invention, there is provided a preparation method for a hydrogel with dual responses to glucose and pH for intelligent release of platelet-rich plasma, comprising the following steps:

[0008] (1) Alkalize chitosan and then carry out carboxymethylation treatment to obtain carboxymethyl chitosan; oxidize cellulose nanofibrils to obtain aldehyde-modified cellulose nanofibrils;

[0009] (2) Mix the carboxymethyl chitosan obtained in step (1) with the aldehyde-modified cellulose nanofibrils, and add glucose oxidase, platelet-rich plasma, and cerium oxide nanoparticles during the mixing process. The amino groups in the carboxymethyl chitosan and platelet-rich plasma react with the aldehyde groups in the aldehyde-modified cellulose nanofibrils through Schiff base reaction to obtain the hydrogel with dual responses to glucose and pH for intelligent release of platelet-rich plasma.

[0010] Preferably, in step (1), chloroacetic acid is used to carry out carboxymethyl treatment on the alkalized chitosan to obtain carboxymethyl chitosan.

[0011] Preferably, in step (1), sodium periodate is used to oxidize cellulose nanofibrils to obtain aldehyde-modified cellulose nanofibrils.

[0012] Preferably, in step (2), the total amount of the amino groups on the carboxymethyl chitosan and the amino groups on the platelet-rich plasma and the amount of substance of the aldehyde groups on the aldehyde-modified cellulose nanofibrils have a ratio of 4:(1 - 16).

[0013] Preferably, the mass ratio of the glucose oxidase to the cellulose nanofibrils is 5 - 35 μg / g; the mass ratio of the cerium oxide nanoparticles to the cellulose nanofibrils is 60 - 100 μg / g; the mass of the platelet-rich plasma is 5% - 15% of the mass of the carboxymethyl chitosan.

[0014] Preferably, in step (2), the mixing time is 10 - 60 s.

[0015] According to another aspect of the present invention, there is provided a hydrogel with dual glucose and pH responsiveness for intelligent release of platelet-rich plasma.

[0016] According to another aspect of the present invention, there is provided the use of the hydrogel with dual glucose and pH responsiveness for intelligent release of platelet-rich plasma in the preparation of wound dressings, drug delivery carriers or three-dimensional cell culture matrices.

[0017] Preferably, a glycine aqueous solution is sprayed on the surface of the hydrogel to dissolve the hydrogel.

[0018] Preferably, the concentration of glycine in the glycine aqueous solution is 80 - 200 mg / ml.

[0019] Generally speaking, compared with the prior art by the above technical solution conceived by the present invention, the following technical advantages are mainly possessed:

[0020] (1) The existing preparation methods of nanocellulose hydrogels are complex in operation, often require cross-linking agents, have a long cross-linking time, poor biocompatibility, and are opaque, etc., which limit their application and promotion. The hydrogel prepared by combining "carboxymethyl chitosan and aldehyde-modified cellulose nanofibrils" in the present invention has the following main advantages: Carboxymethylation of chitosan can improve its water solubility, enabling it to dissolve without an acidic environment. Selective aldehyde ring-opening of cellulose nanofibrils can further increase its biodegradability. Platelet-rich plasma (PRP) can also be connected to the hydrogel network through Schiff base bonds, thereby enhancing its stability and overcoming the disadvantages such as the easy burst release of growth factors, and further enhancing the biocompatibility of the hydrogel. In addition, the preparation and cross-linking of the hydrogel no longer require additional cross-linking agents, and the hydrogel can be formed generally within 120 s. The prepared hydrogel is neutral, injectable, has good biocompatibility and degradability, has glucose and pH responsiveness, and can be dissolved as needed.

[0021] (2) The preparation mechanism of the hydrogel prepared in this invention is that the Schiff base bonds formed by the aldehyde groups on the aldehyde-modified nanocellulose, the amino groups in carboxymethyl chitosan and PRP achieve the double-network crosslinking of the hydrogel and constitute the framework of the hydrogel. The sugar response of the hydrogel is triggered when the hydrogel contacts glucose. Glucose will be oxidized by glucose oxidase in the hydrogel to generate hydrogen peroxide and glucuronic acid. The generated hydrogen peroxide will be reduced by the cerium oxide nanoparticles loaded in-situ in the hydrogel, and glucuronic acid will make the wound microenvironment acidic, thus triggering the pH response of the hydrogel, and then causing the cleavage of the Schiff base bonds in the hydrogel, thereby realizing the intelligent release of PRP in the hydrogel.

[0022] (3) This invention uses the amino groups in carboxymethyl chitosan and PRP to react with the aldehyde groups on the aldehyde-modified cellulose nanofibrils through Schiff base reaction to rapidly prepare the hydrogel.

[0023] (4) The hydrogel prepared in this invention has good biocompatibility, glucose and pH responsiveness, and degradability. Therefore, it has been widely used in the fields of sustainable materials, chemical production, and green biomedicine.

[0024] (5) The preparation method of this invention is simple in operation, low in cost, and green and environmentally friendly. The prepared hydrogel is neutral, transparent and visible, injectable, has good biocompatibility, and the prepared hydrogel can be dissolved as needed, using glycine aqueous solution for dissolution. In addition, the carboxymethylation of chitosan promotes its water solubility, the selective aldehyde ring-opening of cellulose nanofibrils can enhance its degradability, PRP can further enhance the biocompatibility of the hydrogel, and respondently release various growth factors in PRP, which is beneficial to expanding its applications in different fields. Description of the Drawings

[0025] Figure 1 is the flow chart of this invention.

[0026] Figure 2 is the gelation of the hydrogel prepared in Example 1 provided by this invention.

[0027] Figure 3 is the systematic characterization picture of the hydrogels prepared in Examples 2 and 4 of this invention in terms of biocompatibility (hemolysis and growth factor release kinetics tests); among them Figure 3 A in it is the hemolysis ratio of different hydrogels. The release kinetic curves of PDGF (B), VEGF (C) and EGF (D) in the DAMCNF-CMC-PRP hydrogel (Example 2) in solutions with different glucose concentrations.

[0028] Figure 4System characterization pictures of the prepared hydrogels provided in Examples 2 and 4 of the present invention in terms of biocompatibility (cell viability test and cell live / dead staining detection); among them Figure 4 A in it is the cell viability of NIH3T3 cells treated with the leachate of different hydrogels for 1 day, 3 days, and 5 days. B is the cell viability of HUVECs cells treated with the leachate of different hydrogels for 1 day, 3 days, and 5 days. Calcein-AM / PI staining of NIH3T3 (C) and HUVECs (D) cells after being treated with the leachate of different hydrogels for 1 d. (Data are expressed as SD ± mean value, and the statistical analysis between groups is marked according to no significant difference (ns), *P < 0.5, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0029] Figure 5 Glycine dissolution diagram of the prepared hydrogel provided in Example 2 of the present invention.

[0030] Figure 6 Viscosity test, self-healing and injectability pictures of the prepared hydrogel provided in Example 3 of the present invention.

[0031] Figure 7 Wound healing pictures of the prepared hydrogels provided in Examples 3 and 4 of the present invention for the repair of full-thickness skin wounds in diabetic mice. Detailed implementation manners

[0032] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] As described above, the preparation methods of traditional nanocellulose hydrogels are complex in operation, often require cross-linking agents, have a long cross-linking time, poor biocompatibility, opacity, etc., as well as problems such as poor stability of PRP and easy explosive release of growth factors.

[0034] In view of this, in a specific embodiment of the present invention, a preparation method of a glucose / pH dual-responsive intelligent-responsive platelet-rich plasma-releasing hydrogel is provided. Figure 1 It is a flowchart of the present invention, and the method includes:

[0035] (1) Preparation of carboxymethyl chitosan: After alkalizing chitosan with a sodium hydroxide solution with a certain mass fraction, carboxymethyl chitosan is prepared using chloroacetic acid as an etherifying agent, and the remaining reagents and impurities are removed by dialysis until the pH is neutral and then taken out for drying;

[0036] (2) Preparation of aldehyde - modified cellulose nanofibrils: Using sodium periodate as an oxidant to prepare aldehyde - modified cellulose nanofibrils, dialyzing to remove the remaining reagents and impurities until the pH is neutral, and then taking out for drying.

[0037] (3) Preparation of carboxymethyl chitosan - PRP solution: At room temperature, dissolve the carboxymethyl chitosan obtained in step (1) into a solution at a certain concentration, and then add a certain amount of PRP solution and stir until homogeneous.

[0038] (4) Preparation of aldehyde - modified cellulose nanofibril solution: At a certain temperature, dissolve the aldehyde - modified cellulose nanofibrils obtained in step (2) into a solution at a certain concentration, and then add a certain amount of glucose oxidase and nano - cerium oxide particles, and continue to stir until homogeneous.

[0039] (5) Synthesis of hydrogel: Mix the solutions obtained in step (3) and step (4) in a certain proportion and stir evenly to obtain the hydrogel.

[0040] (6) Reversible dissolution of hydrogel: Use an aqueous glycine solution with a certain concentration to conduct a dissolution test on the hydrogel obtained in step (5).

[0041] In another specific embodiment of the present invention, the preparation of carboxymethyl chitosan: Alkalization treatment: The mass ratio of chitosan to sodium hydroxide is 1:2.5 - 1:3, the mass fraction of the sodium hydroxide solution is 40% - 50%, and the alkalization treatment time is 4 - 8 h. Oxidation treatment: The mass ratio of chitosan to chloroacetic acid is 1:2 - 1:4; The catalyst is potassium iodide, and the dosage is 1% - 3%, the reaction temperature is 50 - 60 °C, and the reaction time is 4 - 10 h. Dialyze for 3 - 5 days and then dry.

[0042] In another specific embodiment of the present invention, the preparation of aldehyde - modified cellulose nanofibrils: The mass ratio of cellulose nanofibrils to sodium periodate, sodium chloride, and deionized water is 1:1:3:60 - 1:1.5:4:70; The oxidation reaction temperature is room temperature, and the reaction time is 36 - 48 h; Dialyze for 3 - 5 days and then dry.

[0043] Before synthesizing the hydrogel, first prepare the aqueous solutions of carboxymethyl chitosan and aldehyde - modified cellulose nanofibrils.

[0044] The conditions for preparing the carboxymethyl chitosan solution are: At room temperature, prepare the solution with a mass fraction of 3% - 5%, the stirring speed is 300 - 600 rpm, and the dispersion time is 0.5 - 1 h; Add platelet - rich plasma (5% - 15% of the mass of chitosan added), the stirring speed is 200 - 600 rpm, and the dispersion time is 10 - 30 min.

[0045] The preparation conditions of the aqueous solution of aldehyde-functionalized cellulose nanofibrils are as follows: at 60-80 °C, the solution is prepared with a mass fraction of 3%-5%, the stirring speed is 300-600 rpm, and the dispersion time is 3-5 h. Glucose oxidase (5-35 μg / g (relative to the mass of nanocellulose)) and cerium oxide nanoparticles (60-100 μg / g (relative to the mass of nanocellulose)) are added, the stirring speed is 200-600 rpm, and the dispersion time is 10-30 min.

[0046] In another specific embodiment of the present invention, the two solutions are mixed at a molar ratio of the amino groups in carboxymethyl chitosan and PRP to the aldehyde groups in aldehyde-functionalized cellulose nanofibrils of 4:1 to 1:4;

[0047] The mixing time is 10-60 s;

[0048] In another specific embodiment of the present invention, the concentration of the glycine solution is 80-200 mg / ml.

[0049] In another specific embodiment of the present invention, a glucose / pH dual-responsive intelligent responsive platelet-rich plasma-releasing hydrogel obtained by the above method is provided.

[0050] In another specific embodiment of the present invention, the application of the above hydrogel in the fields of wound dressings, drug delivery, etc. is provided.

[0051] The following is a further detailed description of the present invention in conjunction with specific examples. It should be noted that the specific examples are explanations of the present invention rather than limitations.

[0052] Example 1

[0053] (1) Alkalization treatment of chitosan: The mass ratio of chitosan to sodium hydroxide solution is 1:2.5, the mass fraction of the sodium hydroxide solution is 40%, and the alkalization treatment time is 6 h;

[0054] (2) Carboxymethylation treatment of chitosan: The mass ratio of alkalized chitosan to chloroacetic acid is 1:3; the amount of potassium iodide used is 3% (relative to the mass of chitosan), the reaction temperature is 60 °C, and the reaction time is 8 h.

[0055] After dialysis for 4 days, freeze-drying is carried out;

[0056] (3) Preparation of aldehyde-functionalized cellulose nanofibrils: The mass ratio of cellulose nanofibrils to sodium periodate, sodium chloride, and deionized water is 1:1.32:3.87:62.5; the oxidation reaction temperature is room temperature, the reaction time is 42 h; after dialysis for 4 days, freeze-drying is carried out.

[0057] (4) Preparation of carboxymethyl chitosan and platelet-rich plasma solution: At room temperature, first prepare a carboxymethyl chitosan solution with a mass fraction of 4%, a stirring speed of 300 rpm, and a dispersion time of 0.5 h; then add 5% platelet-rich plasma, with a stirring speed of 300 rpm and a dispersion time of 10 min.

[0058] (5) Preparation of aldehyde-functionalized cellulose nanofibril aqueous solution: At 80 °C, prepare a solution with a mass fraction of 4%, a stirring speed of 300 rpm, and a dispersion time of 4 h; then add 5 μg / g glucose oxidase and 60 μg / g cerium oxide nanoparticles, with a stirring speed of 300 rpm and a dispersion time of 10 min.

[0059] (6) Synthesis of hydrogel: Mix the two solutions with a molar ratio of amino groups in carboxymethyl chitosan and PRP to aldehyde groups in aldehyde-functionalized cellulose nanofibrils of 2:1, and the mixing time is 30 s.

[0060] (7) Reversible dissolution of hydrogel: Use a 100 mg / ml glycine aqueous solution to conduct a dissolution test on the prepared hydrogel.

[0061] The degree of substitution of the carboxymethyl chitosan prepared in this example is 1.25, and the aldehyde group content of the aldehyde-functionalized cellulose nanofibrils is 7.41 mMol / g (see Table 1).

[0062] Figure 2 This is a picture of the glucose / pH-responsive hydrogel prepared in Example 1 of the present invention. Figure 2 As can be seen, the hydrogel can be formed and prepared quickly.

[0063] Example 2

[0064] (1) Alkalization treatment of chitosan: The mass ratio of chitosan to sodium hydroxide solution is 1:2.5, the mass fraction of the sodium hydroxide solution is 40%, and the alkalization treatment time is 6 h;

[0065] (2) Carboxymethylation treatment of chitosan: The mass ratio of the alkalized chitosan to chloroacetic acid is 1:3; the amount of potassium iodide used is 3% (relative to the mass of chitosan), the reaction temperature is 60 °C, and the reaction time is 4 h.

[0066] After dialysis for 3 days, freeze-dry;

[0067] (3) Preparation of aldehyde-functionalized cellulose nanofibrils: The mass ratio of cellulose nanofibrils to sodium periodate, sodium chloride, and deionized water is 1:1.32:3.87:62.5; the oxidation reaction temperature is room temperature, the reaction time is 48 h; after dialysis for 3 days, freeze-dry.

[0068] (4) Preparation of carboxymethyl chitosan and platelet-rich plasma solution: At room temperature, prepare the solution with a mass fraction of 5%, the stirring speed is 600 rpm, and the dispersion time is 1 h; then add 5% of platelet-rich plasma, the stirring speed is 300 rpm, and the dispersion time is 20 min.

[0069] (5) Preparation of aldehyde-modified cellulose nanofibril aqueous solution: At 60 °C, prepare the solution with a mass fraction of 4%, the stirring speed is 600 rpm, and the dispersion time is 5 h; then add 10 μg / g of glucose oxidase and 100 μg / g of cerium oxide nanoparticles, the stirring speed is 300 rpm, and the dispersion time is 10 min.

[0070] (6) Synthesis of hydrogel: Mix the two solutions with a molar ratio of amino groups in carboxymethyl chitosan and PRP to aldehyde groups in aldehyde-modified cellulose nanofibrils of 2:1, and the mixing time is 180 s.

[0071] (7) Reversible dissolution of hydrogel: Use a 150 mg / ml glycine aqueous solution to conduct a dissolution test on the prepared hydrogel.

[0072] The degree of substitution of the carboxymethyl chitosan prepared in this example is 1.02, and the aldehyde group content of the aldehyde-modified cellulose nanofibrils is 8.01 mMol / g (see Table 1).

[0073] The biocompatibility performance test of the hydrogel prepared in this example is as shown in Figure 3 A and [[ID=I]] Figure 4 shown. The hemolysis test results of A in Figure 3 show that it has good blood compatibility. The cell viability detection in A and B in Figure 4 and the live / dead cell staining results in C and D in Figure 4 show that compared with the control (standard culture medium group) and non-responsive hydrogel (Example 4), this responsive hydrogel not only has good cell compatibility but also can promote cell proliferation, which greatly expands its application in the biomedical field (such as used as a wound repair material). In addition, Figure 3 The B, C, and D in

[0074] Figure 5 are the glycine dissolution diagrams of the hydrogel provided in this example. It can be found that the hydrogel can be dissolved by adding glycine solution, and water is used as a control.

[0075] Example 3

[0076] (1) Alkalization treatment of chitosan: The mass ratio of chitosan to sodium hydroxide solution is 1:3, the mass fraction of the sodium hydroxide solution is 50%, and the alkalization treatment time is 6 h;

[0077] (2) Carboxymethylation treatment of chitosan: The mass ratio of alkalized chitosan to chloroacetic acid is 1:3; the dosage of potassium iodide is 1% (relative to the mass of chitosan), the reaction temperature is 60 °C, and the reaction time is 8 h.

[0078] After dialysis for 5 days, freeze-drying is carried out;

[0079] (3) Preparation of aldehyde-functionalized cellulose nanofibrils: The mass ratio of cellulose nanofibrils to sodium periodate, sodium chloride, and deionized water is 1:1.5:4:70; the oxidation reaction temperature is room temperature, and the reaction time is 48 h; after dialysis for 5 days, freeze-drying is carried out.

[0080] (4) Preparation of carboxymethyl chitosan and platelet-rich plasma solution: At room temperature, a solution is prepared with a mass fraction of 4%, the stirring speed is 300 rpm, and the dispersion time is 0.5 h; then 10% of platelet-rich plasma is added, the stirring speed is 300 rpm, and the dispersion time is 20 min.

[0081] (5) Preparation of aldehyde-functionalized cellulose nanofibril aqueous solution: At 80 °C, a solution is prepared with a mass fraction of 4%, the stirring speed is 300 rpm, and the dispersion time is 4 h; then 20 μg / g of glucose oxidase and 100 μg / g of nano-ceria particles are added.

[0082] (6) Synthesis of hydrogel: Two solutions are mixed at a molar ratio of 2:1 of the amino groups in carboxymethyl chitosan and PRP to the aldehyde groups in aldehyde-functionalized cellulose nanofibrils, and the mixing time is 30 s.

[0083] (7) Reversible dissolution of hydrogel: A 100 mg / ml glycine aqueous solution is used to conduct a dissolution test on the prepared hydrogel.

[0084] The degree of substitution of the carboxymethyl chitosan prepared in this example is 0.87, and the aldehyde group content of the aldehyde-functionalized cellulose nanofibrils is 5.41 mMol / g (see Table 1).

[0085] Figure 6 Pictures of the viscosity test, self-healing, and injectability of the hydrogel prepared in Example 3 of the present invention are shown. It can be found that the prepared hydrogel has a certain viscosity, can quickly adhere to tissues, etc. at the initial stage of use, and has self-healing and injectability.

[0086] The effect of the hydrogel prepared in this example on the repair of full-thickness skin wounds in diabetic mice is as Figure 7As shown, it can be found that this hydrogel can achieve wound closure and repair faster than the control group (PBS + commercially available 3M dressing group), and the wound closure rate reaches over 90% on the 14th day.

[0087] Example 4 (non-responsive hydrogel group)

[0088] (1) Alkalization treatment of chitosan: The mass ratio of chitosan to sodium hydroxide solution is 1:2.5, the mass fraction of sodium hydroxide solution is 40%, and the alkalization treatment time is 4 h;

[0089] (2) Carboxymethylation treatment of chitosan: The mass ratio of alkalized chitosan to chloroacetic acid is 1:3; the dosage of potassium iodide is 3% (relative to the mass of chitosan), the reaction temperature is 60 °C, and the reaction time is 8 h.

[0090] After dialysis for 5 days, freeze-drying is carried out;

[0091] (3) Preparation of aldehyde-functionalized cellulose nanofibrils: The mass ratio of cellulose nanofibrils to sodium periodate, sodium chloride, and deionized water is 1:1.4:3.5:70; the oxidation reaction temperature is room temperature, and the reaction time is 36 h; after dialysis for 5 days, freeze-drying is carried out.

[0092] (4) Preparation of carboxymethyl chitosan solution: At room temperature, a solution is prepared with a mass fraction of 3%, the stirring speed is 300 rpm, and the dispersion time is 0.5 h;

[0093] (5) Preparation of aldehyde-functionalized cellulose nanofibril aqueous solution: At 80 °C, a solution is prepared with a mass fraction of 3%, the stirring speed is 300 rpm, and the dispersion time is 5 h;

[0094] (6) Synthesis of hydrogel: The two solutions are mixed at a molar ratio of amino groups in carboxymethyl chitosan to aldehyde groups in aldehyde-functionalized cellulose nanofibrils of 1:2, and the mixing time is 60 s.

[0095] (7) Reversible dissolution of hydrogel: A 100 mg / ml glycine aqueous solution is used to conduct a dissolution test on the prepared hydrogel.

[0096] The degree of substitution of carboxymethyl chitosan prepared in this example is 0.78, and the aldehyde group content of aldehyde-functionalized cellulose nanofibrils is 7.5 mMol / g (see Table 1).

[0097] The biocompatibility performance test of the hydrogel prepared in this example is as Figure 3 shown in A of Figure 4 and Figure 3 it is found in A of Figure 4Cell viability detection and live / dead cell staining results showed that the cell viability remained in good condition compared with the control (standard medium group).

[0098] The effect of the hydrogel prepared in this example on the repair of full-thickness cortical injury in diabetic mice is as Figure 7 shown. It can be found that this hydrogel can also achieve wound closure repair faster compared with the control group (PBS + commercially available 3M dressing group), and the wound closure rate reached more than 80% on the 14th day.

[0099] Table 1 Characterization results of carboxymethyl chitosan and aldehyde-functionalized nanocellulose in Examples 1-4

[0100]

[0101] It is easy for those skilled in the art to understand that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A preparation method of a glucose and pH dual-responsive intelligent responsive platelet-rich plasma-releasing hydrogel, characterized in that, It includes the following steps: (1) Alkalize chitosan and then carry out carboxymethylation treatment to obtain carboxymethyl chitosan; oxidize cellulose nanofibrils to obtain aldehyde-modified cellulose nanofibrils; (2) Mix the carboxymethyl chitosan obtained in step (1) with the aldehyde-modified cellulose nanofibrils, and add glucose oxidase, platelet-rich plasma and cerium oxide nanoparticles during the mixing process. The amino groups in the carboxymethyl chitosan and platelet-rich plasma react with the aldehyde groups in the aldehyde-modified cellulose nanofibrils to obtain the hydrogel that can release platelet-rich plasma with dual glucose and pH responsiveness.

2. The preparation method of the hydrogel for releasing glucose- and pH-dual responsive intelligent platelet-rich plasma, as claimed in claim 1, wherein In step (1), chloroacetic acid is used to carry out carboxymethyl treatment on the alkalized chitosan to obtain carboxymethyl chitosan.

3. The glucose- and pH-dual-responsive intelligent platelet-rich plasma-releasing hydrogel according to claim 1, wherein In step (1), sodium periodate is used to oxidize cellulose nanofibrils to obtain aldehyde-modified cellulose nanofibrils.

4. The preparation method of the glucose- and pH-dual-responsive intelligent-responsive platelet-rich plasma-releasing hydrogel according to claim 1, wherein, In step (2), the ratio of the total amount of amino groups on the carboxymethyl chitosan and the amino groups on the platelet-rich plasma to the amount of substance of the aldehyde groups on the aldehyde-modified cellulose nanofibrils is 4:(1-16).

5. The preparation method of the glucose- and pH-dual-responsive hydrogel for releasing platelet-rich plasma as claimed in claim 1, wherein, The mass ratio of the glucose oxidase to the cellulose nanofibrils is 5-35 μg / g; the mass ratio of the cerium oxide nanoparticles to the cellulose nanofibrils is 60-100 μg / g; the mass of the platelet-rich plasma is 5%-15% of the mass of the carboxymethyl chitosan.

6. The preparation method of the glucose- and pH-dual-responsive intelligent responsive platelet-rich plasma-releasing hydrogel according to claim 1, wherein In step (2), the mixing time is 10-60 s.

7. A hydrogel that can release platelet-rich plasma with dual glucose and pH responsiveness prepared by the method according to any one of claims 1-6.

8. Use of the hydrogel that can release platelet-rich plasma with dual glucose and pH responsiveness according to claim 7 in the preparation of wound dressings, drug delivery carriers or three-dimensional cell culture matrices.

9. The application according to claim 8, wherein Spray an aqueous glycine solution on the surface of the hydrogel to dissolve the hydrogel.

10. The application according to claim 9, characterized in that, The concentration of glycine in the aqueous glycine solution is 80-200 mg / ml.

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