CN115487350B - Micro-environment hydrogel bracket for regulating immune inflammation and preparation method and application thereof - Google Patents

Abstract

The invention provides a micro-environment hydrogel bracket for regulating immune inflammation, and a preparation method and application thereof. The preparation raw materials of the micro-environment hydrogel scaffold for regulating immune inflammation comprise: cationic high molecular polymer, crosslinked hydrogel and anti-inflammatory cytokine. The invention promotes anti-inflammatory and tissue remodelling actions by removing DAMP which triggers inflammatory reaction and continuously releasing anti-inflammatory cytokines through the difunctional hydrogel, synergistically enhances the regulation and control of immune inflammation microenvironment after spinal cord injury, and better promotes nerve regeneration and function recovery.

Description

A hydrogel scaffold that modulates immune-inflammatory microenvironment and its preparation method and application

Technical field

The invention belongs to the technical field of new medical materials, and specifically relates to a hydrogel scaffold that regulates the immune and inflammatory microenvironment and its preparation method and application. In particular, it relates to a dual-function hydrogel scaffold that regulates the immune and inflammatory microenvironment of spinal cord injury and regulates the immune and inflammatory response. Promote nerve regeneration and functional recovery.

Background technique

Spinal cord injury causes axonal destruction and neuronal death, ultimately leading to functional impairment. Spinal cord injury is divided into two stages: primary injury and secondary injury. The initial trauma results in primary damage, including destruction of neural tissue and necrosis of neurons and glial cells within minutes to hours. The secondary injury process is initiated by the primary injury and lasts for several weeks. Secondary damage mainly includes oxidative stress, secondary apoptosis, neuronal demyelination, immune inflammatory response and scar formation, further exacerbating tissue damage and loss of function; among them, the neuroimmune inflammatory response will continue for several days after the injury. Days or even months hinder the repair of the nervous system.

Damage-associated molecular patterns (DAMPs) are released during spinal cord injury to activate immune cells and non-immune cells to cause inflammation and immune responses. DAMPs activate monocytes/macrophages to release pro-inflammatory cytokines (such as tumor necrosis factor-(TNF-)α, interleukin-(IL-)1β) and chemokines (such as inducible nitric oxide synthase (iNOS) ) and matrix metalloproteinases) and induce inflammatory cell recruitment and further tissue destruction. In addition to macrophages, spinal cord innate immune cells microglia are also activated by DAMPs produced after spinal cord injury and release pro-inflammatory and neurotoxic factors, exacerbating neuronal loss and secondary tissue damage.

In short, DAMP-mediated inflammation and immune microenvironment hinder the regeneration and repair of neural tissue after spinal cord injury. Therefore, regulating spinal cord injury immune inflammation to treat secondary injuries of SCI (spinal cord injury) is important for SCI repair. Researchers have found that inhibiting only one type of DAMPs, such as high mobility group 1 (HMGB1), can significantly attenuate microglia-mediated neuroinflammation, significantly improve motor function, and reduce spinal cord edema.

Although inhibiting HMGB1 expression may be a potential strategy for SCI repair, regulating HMBG1 alone may not be sufficient to establish an optimal microenvironment for SCI treatment. This is mainly due to the fact that DAMPs that trigger the immune-inflammatory microenvironment of spinal cord injury include not only HMGB1, but also other proteins, lipid peroxidation products, nucleic acids, and nucleotide derivatives. Studies have shown that cationic polymers, such as polyamide amide dendrimers, polyethylenimine, etc., can effectively scavenge various DAMPs (such as extracellular DNA and HMBG1), thereby reducing the inflammation caused by them. Additionally, cationic polymers were immobilized on the scaffold to limit systemic exposure, thereby improving biosafety and DAMP neutralizing capacity.

Furthermore, an unbalanced polarization of macrophages occurs in the injured spinal cord: activation levels of pro-inflammatory M1 phenotype macrophages are consistently elevated, while activation of potentially reparative M2 phenotype macrophages is reduced, leading to inflammation. Prolonged duration and failure of tissue remodeling. Therefore, weakening the pro-inflammatory response of M1 macrophages/microglia and promoting the anti-inflammatory properties of M2 macrophages/microglia are both effective ways to effectively regulate the immune microenvironment to treat SCI. IL-10 is a key anti-inflammatory cytokine that inhibits the inflammatory response of monocytes/macrophages, regulates the polarization of microglia and macrophages to the M2 phenotype, and promotes neuronal cell survival and promotes SCI. Role in functional recovery in mice and rats.

Contents of the invention

In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a hydrogel scaffold that regulates immune and inflammatory microenvironment and its preparation method and application. In particular, it provides a bifunctional hydrogel scaffold that regulates immune and inflammatory response/microenvironment and its preparation method. Preparation methods and applications. The hydrogel scaffold that regulates the immune-inflammatory microenvironment can regulate the immune-inflammatory response to spinal cord injury while promoting nerve regeneration and functional recovery.

To achieve this goal, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a hydrogel scaffold that regulates the immune and inflammatory microenvironment. The preparation materials of the hydrogel scaffold that regulates the immune and inflammatory microenvironment include: cationic polymers, cross-linked hydrogels and anti-inflammatory cells. factor.

Existing methods for regulating the microenvironment after spinal cord injury mostly use a single strategy, with limited regeneration and repair effects. In addition, in the process of tissue and organ regeneration and repair under pathological conditions, the immune-inflammatory microenvironment plays an important role in the regeneration and repair process. Therefore, the present invention develops a new intervention strategy to promote tissue and organ regeneration and repair. In order to effectively reshape the immune microenvironment of SCI and promote nerve regeneration and repair, a new hydrogel scaffold with multiple immune regulatory functions to regulate the immune and inflammation microenvironment was developed, which is composed of cationic polymers, cross-linked hydrogels and anti-inflammatory cells. It is composed of factors that can attenuate the acute phase inflammatory response by scavenging DAMP and promote anti-inflammation and tissue remodeling through the sustained release of anti-inflammatory cytokines. Through multiple strategies, we jointly regulate the immune and inflammatory microenvironment after spinal cord injury, inhibit scar formation, support the migration and growth of endogenous stem cells, and promote nerve regeneration and functional recovery. In addition, the functional hydrogel delivers in-situ sustained release of bioactive molecules and improves their stability, extending the effective action time in the damaged area. Compared with systemic administration, the dosage of bioactive molecules during treatment is reduced.

Preferably, the cationic polymer includes PAMAM and/or PEI, preferably PAMAM.

Preferably, the generation number of the PAMAM is 3-5, for example, it can be 3, 4, or 5.

Preferably, the molecular weight of PAMAM is 6909-28826, for example, it can be 6909, 10109, 12927, 14214, 14277, 20112, 20646, 26252, 28826, etc.

Preferably, the molecular formula of PAMAM includes any one or a combination of at least two of C ₃₀₂ H ₆₀₈ N ₁₂₂ O ₆₀ , C ₆₂₂ H ₁₂₄₈ N ₂₅₀ O ₁₂₄ or C ₁₂₆₂ H ₂₅₂₈ N ₅₀₆ O ₂₅₂ .

Preferably, the terminal group of PAMAM includes NH ₂ .

Preferably, the number of terminal groups of the PAMAM is 32-128, for example, it can be 32, 40, 50, 60, 70, 80, 90, 100, 110, 120, 128, etc.

Preferably, the cross-linked hydrogel is gelatin composite collagen and/or hyaluronic acid hydrogel.

Preferably, the gelatin composite collagen is methacrylated gelatin hydrogel.

Preferably, the methacrylated gelatin hydrogel is prepared by the following preparation method: reacting methacrylic anhydride with gelatin and purifying to obtain methacrylated gelatin.

Preferably, the grafting rate of the methacrylated gelatin is 60-95%, for example, it can be 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.

Preferably, the mass ratio of the volume of methacrylic anhydride and gelatin is 1mL:(0.8-2)g, for example, it can be 1mL:0.8g, 1mL:1g, 1mL:1.2g, 1mL:1.4g, 1mL: 1.6g, 1mL:1.8g, 1mL:2g, etc.

Preferably, the reaction temperature is 37-50°C, for example, it can be 37°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, etc., and the reaction time is 4-24h, For example, it can be 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, etc.

Preferably, the reaction is carried out in the presence of a solvent, and the solvent includes any one or a combination of at least two of water, NaCl solution, phosphate buffer or Hank's balanced salt solution.

Preferably, the raw materials for preparing the hydrogel scaffold for regulating immune inflammation microenvironment also include: photoinitiator, solvent and buffer.

Preferably, the photoinitiator includes 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, phenyl-2,4,6-trimethylbenzoyl lithium phosphite Or any one or a combination of at least two of Irgacure.

Preferably, the solvent is water.

Preferably, the buffer includes any one of NaCl solution, phosphate buffer or Hank's balanced salt solution or a combination of at least two.

Preferably, the gel form of the hydrogel scaffold has a three-dimensional porous structure, and the pore diameter of the porous structure is 20-200 μm, for example, it can be 20 μm, 40 μm, 60 μm, 80 μm, 100 μm, 120 μm, 140 μm, 160 μm, 180 μm. , 200μm, etc.

In the present invention, the hydrogel scaffold is a scaffold with a three-dimensional porous structure formed by co-crosslinking of a cationic polymer and a cross-linked hydrogel. Anti-inflammatory cytokines are loaded in the scaffold so that they can pass through Clearing DAMP attenuates the acute phase inflammatory response and promotes anti-inflammation and tissue remodeling through the sustained release of anti-inflammatory cytokines.

In a second aspect, the present invention provides a method for preparing a hydrogel scaffold for regulating an immune-inflammatory microenvironment as described in the first aspect. The method for preparing a hydrogel scaffold for regulating an immune-inflammatory microenvironment is: polymerizing cationic polymers Materials, cross-linked hydrogel and anti-inflammatory cytokines are mixed, and a photocuring reaction is performed to prepare the hydrogel scaffold for regulating immune inflammation microenvironment.

Preferably, the preparation method of the hydrogel scaffold for regulating immune inflammation microenvironment includes the following steps:

(1) Mix cross-linked hydrogel, photoinitiator and solvent to obtain solution A; mix cationic polymer and buffer to obtain solution B; mix anti-inflammatory cytokines and buffer to obtain solution C;

(2) Mix the A solution, B solution and C solution obtained in step (1), and then perform photocuring to obtain the immune-inflammatory microenvironment-regulating hydrogel scaffold.

Preferably, in the solution A, the concentration of the cross-linked hydrogel is 0.08-0.16g/mL, for example, it can be 0.08g/mL, 0.09g/mL, 0.10g/mL, 0.11g/mL, 0.12 g/mL, 0.13g/mL, 0.14g/mL, 0.15g/mL, 0.16g/mL, etc.

Preferably, in the A solution, the concentration of the initiator is 0.002-0.01g/mL, for example, it can be 0.002g/mL, 0.003g/mL, 0.004g/mL, 0.005g/mL, 0.006g/mL , 0.007g/mL, 0.008g/mL, 0.009g/mL, 0.01g/mL, etc.

Preferably, in the solution B, the concentration of the cationic polymer is 2-200 mg/mL, for example, it can be 2 mg/mL, 4 mg/mL, 6 mg/mL, 8 mg/mL, 10 mg/mL, 20 mg/mL. mL, 40mg/mL, 60mg/mL, 80mg/mL, 100mg/mL, 120mg/mL, 140mg/mL, 160mg/mL, 180mg/mL, 200mg/mL, etc.

Preferably, the concentration of the anti-inflammatory cytokine in the C solution is 20-500 μg/mL, for example, it can be 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL, 100 μg/mL, or 120 μg/mL. , 140μg/mL, 160μg/mL, 180μg/mL, 200μg/mL, 250μg/mL, 300μg/mL, 350μg/mL, 400μg/mL, 450μg/mL, 500μg/mL, etc.

Preferably, in step (2), the volume ratio of the A solution, B solution and C solution is (1-4): (0.5-2): (0.5-2);

Among them, "1-4" can be 1, 1.5, 2, 2.5, 3, 3.5, 4, etc.;

Among them, the first "0.5-2" can be, for example, 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2, etc.;

Among them, the first "0.5-2" can be, for example, 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2, etc.

Preferably, in step (2), the light source for photocuring is a visible light source or an ultraviolet light source.

Preferably, in step (2), the photocuring energy is 2-10W, for example, it can be 2W, 3W, 4W, 5W, 6W, 7W, 8W, 9W, 10W, etc., and the photocuring time is 10 -300s, for example, it can be 10s, 20s, 30s, 40s, 60s, 80s, 100s, 120s, 140s, 160s, 180s, 200s, 220s, 240s, 260s, 280s, 300s, etc.

In a third aspect, the present invention provides a hydrogel scaffold that regulates the immune-inflammatory microenvironment as described in the first aspect in the preparation of materials that regulate the immune-inflammatory microenvironment after spinal cord injury and/or materials that promote nerve regeneration and functional recovery. application.

Compared with the existing technology, the present invention has the following beneficial effects:

(1) The present invention has developed a new type of hydrogel scaffold with multiple immunomodulatory functions that regulates the immune-inflammatory microenvironment, which can attenuate the acute phase inflammatory response by clearing DAMP, and promote anti-inflammation and tissue remodeling by sustaining the release of IL-10;

(2) The present invention uses multiple strategies to jointly regulate the immune and inflammatory microenvironment after spinal cord injury, inhibit scar formation, support the migration and growth of endogenous stem cells, and promote nerve regeneration and functional recovery;

(3) The present invention delivers bioactive molecules through in-situ sustained release through functional hydrogels, improves their stability, and prolongs the effective action time in the damaged area. Compared with systemic administration, the dosage of bioactive molecules during treatment is reduced.

Description of the drawings

Figure 1 is a scanning electron microscope image of Scav/Delv-GL provided in Example 1.

Figure 2 is a scanning electron microscope image of the GL stent provided in Comparative Example 1.

Figure 3 is a scanning electron microscope image of the Scav-GL stent provided in Comparative Example 2.

Figure 4 is a scanning electron microscope image of the Delv-GL stent provided in Comparative Example 3.

Figure 5 is a comparison chart of the compression modulus of different stents.

Figure 6 is a comparison chart of the degradation performance of different stents.

Figure 7 is a comparison of the protein clearance efficiencies of different scaffolds in DAMPs.

Figure 8 is a comparison chart of the HMGB1 clearance efficiency of different scaffolds.

Figure 9 is a comparison of the DNA clearing abilities of different scaffolds in DAMPs.

Figure 10 is a comparison chart of the controlled release of IL-10 in vitro by different scaffolds.

Figure 11 is a comparison of IL-1β+ and TNF-α+ fluorescent staining areas in samples implanted with different scaffolds.

Figure 12 is a comparative diagram of semi-quantitative immunostaining analysis of IL-1β+.

Figure 13 is a comparison chart of semi-quantitative immunostaining analysis of TNF-α+.

Figure 14 is a picture of fluorescence staining by immunostaining ED1.

Figure 15 is a graph showing fluorescence staining by immunostaining F4/80.

Figure 16 is a graph showing fluorescence staining by immunostaining Iba-1.

Figure 17 is an iNOS immunofluorescence staining picture.

Figure 18 is an immunofluorescence staining picture of ARG-1.

Figure 19 is a Tuj-1/GFAP immunofluorescence co-staining image after 7 days of treatment.

Figure 20 is a picture of Tuj-1 immunofluorescence staining after 8 weeks of treatment.

Figure 21 is a comparison chart of Tuj-1 gene expression.

Figure 22 shows the waveform of motor evoked potential.

Figure 23 is a graph of the average amplitude of motor evoked potentials.

Figure 24 is a graph of average latency.

Figure 25 is a graph of mouse motor function scores.

Detailed ways

The technical solution of the present invention will be further described below through specific implementations. Those skilled in the art should understand that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

Preparation example

This preparation example provides a kind of methacrylated gelatin, which is prepared by the following preparation method: 15g gelatin and 15mL methacrylic anhydride are placed in 500mL sodium carbonate/sodium bicarbonate buffer solution ( (a mixture of 250 mL of 0.1 mol/L sodium carbonate solution and 250 mL of 0.1 mol/L sodium bicarbonate solution), react at 40°C for 24 hours, and dialyze with a beaker of pure water and an American MFPI dialysis bag (molecular weight cutoff 5000) to remove by-products and unreacted reactants to prepare methacrylated gelatin with a grafting rate of 90%.

Example 1

This embodiment provides a bifunctional hydrogel for clearance and delivery (Scav/Delv-GL). The hydrogel scaffold for regulating immune inflammation microenvironment is prepared by the following preparation method:

(1) Mix 1g of methacrylated gelatin, 0.03g of photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphite) and 10mL of phosphate buffer to obtain A Solution; mix 10 mg of PAMAM-G3 and 1 mL of phosphate buffer to obtain solution B; mix 50 μg of IL-10 and 1 mL of phosphate buffer to obtain solution C;

(2) After mixing the A solution, B solution and C solution obtained in step (1) at a volume ratio of 2:1:1, perform photocuring for 100 seconds under a 5W visible light source to obtain the immune-inflammation regulating microorganism. Environmental hydrogel scaffold.

Among them, Figure 1 is a scanning electron microscope image of the Scav/Delv-GL provided in this embodiment. As shown in Figure 1, the morphology of the Scav/Delv-GL stent is a three-dimensional porous structure, and its pore size ranges from 20 to 200 μm.

Comparative example 1

This comparative example provides a hydrogel scaffold (GL), which is prepared by the following preparation method:

Mix 1 g of methacrylated gelatin, 0.03 g of photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphite) and 10 mL of phosphate buffer to obtain solution A. Solution A and phosphate buffer were mixed at a volume ratio of 1:1, and photocured under a 5W visible light source for 100 s to obtain the hydrogel scaffold (GL) of Comparative Example 1.

Among them, Figure 2 is a scanning electron microscope image of the GL stent. As shown in Figure 2, the morphology of the GL stent is three-dimensional porous, and its pore diameter is 20-200 μm.

Comparative example 2

This comparative example provides a single scavenging function hydrogel scaffold (Scav-GL), which is prepared by the following preparation method:

Mix 1 g of methacrylated gelatin, 0.03 g of photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphite) and 10 mL of phosphate buffer to obtain solution A. Mix 10 mg of PAMAM-G3 and 1 mL of phosphate buffer to obtain solution B; mix solution A, solution B and phosphate buffer at a volume ratio of 2:1:1 and perform light curing for 100 seconds under a 5W visible light source to obtain The Comparative Example 2 single scavenging function hydrogel scaffold (Scav-GL).

Among them, Figure 3 is a scanning electron microscope image of the Scav-GL stent. As shown in Figure 3, the Scav-GL stent has a three-dimensional porous shape with a pore diameter of 20-200 μm.

Comparative example 3

This comparative example provides a single delivery function hydrogel (Delv-GL), which is prepared by the following preparation method:

Mix 1 g of methacrylated gelatin, 0.03 g of photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphite) and 10 mL of phosphate buffer to obtain solution A. Mix 50 μg of IL-10 and 1 mL of phosphate buffer to obtain solution C; mix solution A, solution C and phosphate buffer at a volume ratio of 2:1:1 and perform light curing for 100 seconds with a 5W visible light source to obtain The single delivery functional hydrogel (Delv-GL) of Comparative Example 3.

Among them, Figure 4 is a scanning electron microscope image of the Delv-GL stent. As shown in Figure 4, the Delv-GL stent has a three-dimensional porous shape with a pore diameter of 20-200 μm.

Test example 1

Basic performance testing of hydrogel scaffolds for modulating immune-inflammatory microenvironment

1. Test sample: Example 1, Comparative Examples 1-3 prepared hydrogel scaffold for regulating immune inflammation microenvironment;

2. Test items and test methods:

(1) Compression modulus: Universal material testing machine (USA, Instron 5943);

(2) Degradation performance test: In vitro at 37°C, incubate in buffer for 0, 7, 14, 21 and 28 days respectively, remove the buffer, wash with pure water 3 times, and freeze-dry under vacuum at -20°C. Weighing; degradation rate (initial mass – remaining mass after treatment)/initial mass;

(3) Protein removal efficiency in DAMPs: After incubating with the buffer containing DAMPs (protein content C ₀ ) at 37°C for 4 hours, use the BCA protein quantification kit to measure the protein content of the DAMPs that have not been cleared in the buffer ( C);

The calculation formula for removal efficiency is: removal efficiency (%) = [(C ₀ -C)/C ₀ ]×100%;

(4) HMGB1 clearance: After incubating with the buffer containing DAMPs for 4 hours at 37°C, use an ELISA kit to measure the content of HMGB1 that has not been cleared in the buffer;

(5) DNA removal from DAMPs: After mixing with the buffer containing DAMPs, incubate at 37°C for 4 hours, use the Quant-iT ^TM PicoGreen ^TM dsDNA Assay kit to measure the content of double-stranded DNA that has not been cleared in the buffer;

The test results are shown in Table 1 below (or as shown in Figure 5-9), where "-" means that the test was not performed:

Table 1

It can be seen from the test data in Table 1 that the compression modulus of the Scav/Delv-GL stent prepared by the present invention is approximately 3kPa (Figure 5). In vitro degradation experiments showed (Figure 6) that hydrogel degradation increased in a time-dependent manner, with approximately 25% of the initial scaffold weight decreasing in all groups after 28 days of incubation. After treatment with the Scav-GL scaffold, the amount of extracellular DNA and HMGB1 protein as well as total protein in the DAMP solution were effectively captured and removed, indicating that the scaffold has the ability to effectively remove DAMPs (Figure 7-9).

Test example 2

Testing the controlled release performance of hydrogel scaffolds that regulate immune and inflammatory microenvironments

The functionalized immune-inflammation microenvironment hydrogel scaffold prepared in Example 1 and Comparative Example 3 of the above embodiments was tested for sustained release performance and detected by ELISA;

Among them, Figure 10 is a comparison chart of the controlled release of IL-10 by different scaffolds in vitro; on the first day, approximately 24.4% and 23.7% were released from Delv-GL and Scav/Delv-GL hydrogel respectively. IL-10. Approximately 52.4% of the initially encapsulated IL-10 remained in the Scav/Delv-GL scaffold after 21 days of incubation in Hanks' Balanced Salt Solution (HBSS) at 37°C.

Test example 3

Modulating immune-inflammatory microenvironment hydrogel scaffold modulates inflammatory response after spinal cord injury

(1) The functionalized immune-inflammation microenvironment hydrogel scaffold prepared in the above Example 1 and Comparative Examples 1-3 was tested for controlled release performance. The specific test method is as follows: Detection by immunofluorescence staining:

Among them, Figure 11 shows the comparison of IL-1β+ and TNF-α+ fluorescence staining areas in samples implanted with different scaffolds; after spinal cord injury, macrophages and microglia are rapidly activated by DAMPs, release inflammatory factors and Induces an immune response that lasts for months, further exacerbating the damage. By immunofluorescence staining of IL-1β and TNF-α 7 days after injury, it was found that the IL-1β+ and TNF-α+ stained areas in samples implanted with Scav/Delv-GL and Scav-GL scaffolds were compared with those without implantation. Brackets (Control) or GL and Delv-GL are significantly reduced.

Among them, Figure 12 is a comparison chart of semi-quantitative immunostaining analysis of IL-1β+; Figure 13 is a comparison chart of semi-quantitative immunostaining analysis of TNF-α+. The semi-quantitative immunostaining analysis further confirmed that Scav/Delv-GL and Scav-GL The fluorescence intensity levels of IL-1β and TNF-α in the group were much lower than those in other groups).

The above results indicate that removal of DAMPs by implanting Scav/Delv-GL and Scav-GL scaffolds at the injured site of the spinal cord can reduce the secretion of pro-inflammatory cytokines in the acute phase.

(2) Perform immunostaining investigation on the functionalized immune-inflammation microenvironment hydrogel scaffold prepared in the above Example 1 and Comparative Examples 1-3. The specific test method is as follows: by detecting ED1 (reactive microglia) /macrophage marker), F4/80 (macrophage marker) and Iba-1 (microglia marker) were immunostained to investigate the expression of macrophages and microglia in the lesion area in the acute phase of spinal cord injury. Distribution and immunophenotype;

Among them, Figure 14 is a fluorescence staining chart by immunostaining ED1; Figure 15 is a fluorescence staining chart by immunostaining F4/80; Figure 16 is a fluorescence staining chart by immunostaining Iba-1; as shown 14-15, compared with the other four groups, the number of positively stained macrophages and microglia in the Scav/Delv-GL group was significantly reduced. Implantation of Scav-GL scaffolds significantly reduced the number of ED1, F4/80- and Iba-1-positive cells compared with control and GL-implanted spinal cord injury mice. Furthermore, fewer Iba-1-positive microglia were observed in the Delv-GL group than in the GL group.

(3) Test the inflammatory cell polarization subtype on the functionalized immune-modulating microenvironment hydrogel scaffold prepared in Example 1 and Comparative Examples 1-3. The specific test method is as follows: Detection by immunofluorescence staining :

Among them, Figure 17 is the iNOS immunofluorescence staining picture, and Figure 18 is the ARG-1 immunofluorescence staining picture; as shown in Figure 17-18, the pro-inflammatory M1 phenotype marker iNOS and the anti-inflammatory M2 phenotype marker ARG-1 are stained It was found that compared with the control group and GL group, the number of iNOS-positive cells in the lesion site and surrounding tissue areas of the Scav/Delv-GL, Delv-GL and Scav-GL groups was significantly reduced; the number of ARG-1-positive cells was increased. Scav/Delv-GL regulates macrophage and microglia activation phenotypes in the acute phase of injury.

Test example 4

Regulating the immune-inflammatory microenvironment of spinal cord injury Regulating the immune-inflammatory microenvironment Hydrogel scaffold promotes nerve regeneration and motor function recovery

Tuj-1 immunofluorescence staining was used to observe axonal growth and nerve regeneration at the injured site. The specific test methods are as follows: Detection by immunofluorescence staining and real-time quantitative PCR:

Among them, Figure 19 is a Tuj-1/GFAP immunofluorescence co-staining picture; as shown in Figure 19, 7 days after surgery, the number of Tuj-1-positive newly generated neurons in the Scav/Delv-GL and Delv-GL groups was higher than Scav-GL, GL and control group; Figure 20 shows the immunofluorescence staining of Tuj-1 in the center of the injured area after 8 weeks of treatment; it also shows the long-term promotion of axonal regeneration of the bifunctional scaffold (Scav/Delv-GL).

Among them, Figure 21 shows the expression of Tuj-1 gene 8 weeks after hydrogel transplantation; as shown in Figure 21, in all groups, mice treated with dual immunomodulatory functional hydrogel showed Tuj-1 labeling at the lesion site The largest number of neurons. Compared with the group implanted with GL scaffolds, there was a significant increase in positively stained Tuj-1 neurons at the lesion site in mice implanted with Delv-GL and Scav-GL scaffolds. Almost no Tuj-1 positive cells were seen in the center of the injured area in mice with spinal cord injury (Control). The expression trend of Tuj-1 is consistent with the above immunostaining results. The Tuj-1 expression level of samples from the Scav/Delv-GL group was higher than that of samples from the Delv-GL, Scav-GL, GL and SCI groups.

The formation of neural circuits in bifunctional hydrogel-treated SCI mice was evaluated by measuring motor evoked potential (MEP). Figure 22 is the Tuj-1 motor evoked potential waveform; Figure 23 is the average latency of motor evoked potential; Figure 24 is the average amplitude graph; as shown in Figures 22-24, at 8 weeks after surgery, mice implanted with the Scav/Delv-GL scaffold showed greater improvement in motor-evoked responses, shortened latency, and increased average amplitude. Among them, Figure 25 is a mouse motor function score chart; as shown in Figure 25, the BMS mouse motor function score found that the dual-function modulating immune-inflammatory microenvironment hydrogel scaffold synergistically promotes the recovery of motor function.

The applicant declares that the present invention uses the above examples to illustrate the hydrogel scaffold for regulating immune inflammation microenvironment and its preparation method and application. However, the present invention is not limited to the above examples, which does not mean that the present invention must rely on the above. Embodiments can be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacement of raw materials of the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims (28)

1. The preparation raw materials of the micro-environment hydrogel scaffold for regulating immune inflammation comprise the following steps: cationic high molecular polymer, crosslinked hydrogel and anti-inflammatory cytokine;

the cationic high molecular polymer comprises PAMAM and/or PEI; the crosslinked hydrogel is gelatin composite collagen and/or hyaluronic acid hydrogel; the anti-inflammatory cytokine comprises any one or a combination of at least two of IL-10, IL-4 or IL-33.

2. The immunoinflammatory micro-environment regulating hydrogel scaffold of claim 1, wherein said cationic high molecular polymer is PAMAM.

3. The immunoinflammatory micro-environment regulating hydrogel scaffold of claim 1, wherein the PAMAM is algebraic in the range of 3-5.

4. The immunoinflammatory micro-environment regulating hydrogel scaffold of claim 1, wherein said PAMAM has a molecular weight of 6909-28826.

5. The immunoregulatory inflammatory micro-environment hydrogel scaffold of claim 1, wherein the PAMAM has a molecular formula comprising C ₃₀₂ H ₆₀₈ N ₁₂₂ O ₆₀ 、C ₆₂₂ H ₁₂₄₈ N ₂₅₀ O ₁₂₄ Or C ₁₂₆₂ H ₂₅₂₈ N ₅₀₆ O ₂₅₂ Any one or a combination of at least two of these.

6. The immunoinflammatory micro-environment regulating hydrogel scaffold of claim 1, wherein the terminal groups of PAMAM comprise NH ₂ 。

7. The immunoinflammatory micro-environment regulating hydrogel scaffold of claim 1, wherein the PAMAM has a number of terminal groups ranging from 32 to 128.

8. The immunoregulatory inflammatory micro-environment hydrogel scaffold of claim 1, wherein said gelatin complex collagen is a methacrylate gelatin hydrogel.

9. The immunoregulatory inflammation microenvironment hydrogel scaffold according to claim 8, wherein the methacrylate gelatin hydrogel is prepared by the following preparation method: methacrylic anhydride reacts with gelatin and is purified to obtain the methacrylic acid esterified gelatin.

10. The immunoregulatory inflammation microenvironment hydrogel scaffold according to claim 8, wherein the grafting ratio of the methacrylated gelatin is 60-95%.

11. The immunoinflammatory micro-environment regulating hydrogel scaffold of claim 9, wherein the mass ratio of methacrylic anhydride to gelatin is 1mL (0.8-2) g.

12. The immunoinflammatory micro-environment regulating hydrogel scaffold of claim 9, wherein said reaction temperature is 37-50 ℃ and said reaction time is 4-24 hours.

13. The immunoinflammatory micro-environment modulating hydrogel scaffold of claim 9, wherein said reaction is performed in the presence of a solvent comprising any one or a combination of at least two of water, naCl solution, phosphate buffer, or Hank's balanced salt solution.

14. The immunoregulatory inflammation microenvironment hydrogel scaffold according to claim 1, wherein the preparation raw materials of the immunoregulatory inflammation microenvironment hydrogel scaffold further comprise: photoinitiator, solvent and buffer.

15. The immunoregulatory inflammatory micro-environment hydrogel scaffold of claim 14, wherein said photoinitiator comprises any one or a combination of at least two of 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylbenzophenone, phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite, or Irgacure.

16. The immunoinflammatory micro-environment regulating hydrogel scaffold of claim 14, wherein said solvent is water.

17. The immunoinflammatory micro-environment regulating hydrogel scaffold of claim 14, wherein said buffer comprises any one or a combination of at least two of NaCl solution, phosphate buffer, or Hank's balanced salt solution.

18. The immunoinflammatory micro-environment regulating hydrogel scaffold of claim 1, wherein the gel morphology of said hydrogel scaffold has a three-dimensional porous structure with a pore diameter of 20-200 μm.

19. The method for preparing an immunoregulatory micro-environment hydrogel scaffold according to any one of claims 1-18, wherein the method for preparing the immunoregulatory micro-environment hydrogel scaffold comprises the following steps: mixing the cationic high molecular polymer, the crosslinked hydrogel and the anti-inflammatory cytokine, and performing a photocuring reaction to prepare the immune inflammation regulating micro-environment hydrogel scaffold.

20. The method for preparing an immunoregulatory micro-environment hydrogel scaffold according to claim 19, wherein the method for preparing the immunoregulatory micro-environment hydrogel scaffold comprises the following steps:

(1) Mixing the crosslinked hydrogel, a photoinitiator and a solvent to obtain a solution A; mixing a cationic high molecular polymer and a buffer solution to obtain a solution B; mixing anti-inflammatory cytokines with a buffer solution to obtain a solution C;

(2) And (3) mixing the solution A, the solution B and the solution C obtained in the step (1), and performing photocuring to obtain the immune inflammation regulating micro-environment hydrogel scaffold.

21. The method for preparing a micro-environmental hydrogel scaffold for modulating immunity according to claim 20, wherein the concentration of the cross-linked hydrogel in the solution a is 0.08-0.16g/mL.

22. The method for preparing a micro-environmental hydrogel scaffold for modulating immune inflammation according to claim 20, wherein the concentration of the initiator in the solution a is 0.002-0.01g/mL.

23. The method for preparing a micro-environmental hydrogel scaffold for modulating immune inflammation according to claim 20, wherein the concentration of the cationic high molecular polymer in the solution B is 2-200mg/mL.

24. The method for preparing an immunoinflammatory micro-environment regulating hydrogel scaffold according to claim 20, wherein the concentration of said anti-inflammatory cytokine in said solution C is 20-500 μg/mL.

25. The method for preparing a micro-environmental hydrogel scaffold for modulating immune inflammation according to claim 20, wherein in the step (2), the volume ratio of the solution A to the solution B to the solution C is (1-4): 0.5-2.

26. The method of claim 20, wherein in step (2), the light source for photo-curing is a visible light source or an ultraviolet light source.

27. The method for preparing a micro-environmental hydrogel scaffold for modulating immune inflammation according to claim 20, wherein in the step (2), the energy of the photo-curing is 2-10W, and the time of the photo-curing is 10-300s.

28. Use of a hydrogel scaffold for modulating an immunoinflammatory microenvironment according to any one of claims 1-18 for the preparation of a material for modulating the immunoinflammatory microenvironment following spinal cord injury and/or a material for promoting nerve regeneration and functional recovery.