Injectable hydrogel capable of releasing exosome in microenvironment responsiveness and preparation method and application of injectable hydrogel

Abstract

The invention belongs to the technical field of hydrogel, and particularly relates to injectable hydrogel capable of releasing exosome in a microenvironment responsive mode and a preparation method and application thereof.The injectable hydrogel takes sodium alginate and gelatin as main chains and is combined with exosome, and the system comprises a double-network structure formed by boric acid ester bonds and Schiff base bonds. A boric acid ester bond and a Schiff base bond react to a main chain of biomacromolecule gelatin and sodium alginate, a bioactive molecule exosome is combined, a microenvironment responsive injectable hydrogel combination combined with the exosome is constructed, and at the early stage of spinal cord injury, through the interaction of the boric acid ester bond and active oxygen in a microenvironment, the injectable hydrogel combination can be used for treating spinal cord injury. Meanwhile, active oxygen removal and exosome release are achieved, the microenvironment is purified in time, a beneficial support space is provided for nerve growth by utilizing retention of a Schiff base bond network in the SC I chronic stage, the combination is beneficial to movement and recovery of bladder functions, and clinical application is more facilitated through injectability of the combination.

Description

Technical Field

[0001] The invention relates to an injectable hydrogel capable of releasing exosomes in response to a microenvironment, and a preparation method and application thereof, and belongs to the technical field of hydrogels. Background Art

[0002] Spinal cord injury (SCI) is a destructive disease of the central nervous system, and there is currently no effective treatment. The pathological process of SCI is mainly characterized by vascular rupture, axonal severity and cell membrane damage immediately after injury, followed by a large amount of reactive oxygen species (ROS) that diffuse into the injured area, forming an early severe oxidative stress microenvironment. If ROS cannot be effectively removed, oxidative stress can activate macrophages to synthesize proinflammatory cytokines, causing more serious secondary cascade injuries such as neuronal apoptosis and glial cell proliferation. At the same time, the proliferation of glial scars and the loss of extracellular matrix (ECM) form a cavity around the injury that hinders nerve growth. If spatial support is not provided in the chronic stage of SCI, it will lead to a lack of attachment space for nerve repair and growth, hindering the recovery of motor function in SCI patients.

[0003] In addition, neurogenic bladder (NGB) caused by SCI is one of the main factors that reduce the quality of life of patients. The recovery of NGB also represents the repair of spinal cord tissue and is an important post-healing indicator for judging the degree of spinal cord tissue repair. Therefore, in addition to improving motor function, the repair of SCI also needs to pay attention to NGB. Multi-faceted reconstruction will be more conducive to improving the quality of life of patients and reducing mortality, which has important clinical and social significance.

[0004] Hydrogel is a promising tissue repair material that can better simulate the ECM in the spinal cord microenvironment. However, due to the lack of bioactive molecules, hydrogel alone cannot specifically promote neurogenesis and is often ineffective. Since the size, shape and degree of SCI injuries vary in clinical practice, implanting pre-formed stents or drug delivery devices may further damage the spinal cord tissue due to inappropriate size. Urinary system complications caused by SCI seriously affect the quality of life of patients, and the recovery of bladder function also reflects the repair of spinal cord tissue. However, there is often a lack of attention to the treatment of urinary system complications caused by SCI in clinical practice. Summary of the invention

[0005] The present invention aims at the early spread of ROS in spinal cord injury, the lack of attachment space for nerve growth in the chronic stage, and the shortcomings of urinary system complications often ignored by current treatment methods. It provides an injectable hydrogel that releases exosomes responsively to a microenvironment and a preparation method and application thereof, which are used to treat spinal cord injury and improve movement and bladder function.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] One of the purposes of the present invention is to provide an injectable hydrogel that releases exosomes in response to a microenvironment, which has sodium alginate and gelatin as the main chain and is combined with exosomes, and the system contains a double network structure formed by borate bonds and Schiff base bonds.

[0008] Furthermore, in the hydrogel system, the concentration of gelatin was 10±1 wt %, the concentration of sodium alginate was 10±1 wt %, and the concentration of exosomes was 100 μg / 60 μL.

[0009] Furthermore, the exosomes are derived from adipose-derived mesenchymal stem cells (ADSCs).

[0010] The second object of the present invention is to provide a method for preparing the above-mentioned injectable hydrogel for microenvironment-responsive release of exosomes, which mainly comprises the following steps:

[0011] (1) Preparation of oxidized sodium alginate:

[0012] The hydroxyl group in sodium alginate is oxidized by sodium periodate to form an aldehyde group, thus obtaining the product OSA;

[0013] (2) Gelatin grafted with phenylboric acid:

[0014] The amino group on phenylboronic acid reacts with the carboxyl group on gelatin to form an amide bond, and phenylboronic acid is grafted onto the main chain of gelatin to obtain the product Gel-PBA;

[0015] (3) Aminated gelatin grafted with phenylboronic acid:

[0016] The Gel-PBA obtained in (2) is mixed with ethylenediamine and reacted under acidic conditions to graft the amino group into the gelatin backbone to obtain the product Gel-PBA-NH2;

[0017] (4) Construction of injectable hydrogels for microenvironment-responsive release of exosomes:

[0018] The two precursor materials OSA and Gel-PBA-NH2 prepared in (1) and (3) were diluted with a PBS solution with an exosome concentration of 100 μg / 30 μL, and then the two were mixed. After being mixed evenly, the gel will be in a viscous and stringy state, and then it will be placed in a constant temperature incubator for cross-linking to obtain a light yellow hydrogel.

[0019] Furthermore, in (1), the mass ratio of sodium alginate to sodium periodate is 5:2 to 2.2.

[0020] Furthermore, in (2), EDC·HCl and NHS are used as catalysts, and the mass ratio of phenylboric acid to gelatin is 2.4-3.4:5.

[0021] Furthermore, in (3), after Gel-PBA and ethylenediamine are mixed, the pH is adjusted to 5±0.5 with hydrochloric acid, EDC·HCl is used as a catalyst, and the mass ratio of Gel-PBA to ethylenediamine is 4-5:14-15.

[0022] Furthermore, in (4), the two precursor materials OSA and Gel-PBA-NH2 were diluted with a PBS solution containing exosomes at a concentration of 100 μg / 30 μL to a concentration of 10±1 wt% for both precursor materials, and the two were mixed in a volume ratio of 2:1.

[0023] Furthermore, in (4), the temperature of the constant temperature incubator is 37±5° C., and the cross-linking time is 1 hour.

[0024] The third object of the present invention is to provide an application of the above-mentioned injectable hydrogel for releasing exosomes in a microenvironment-responsive manner in the preparation of drugs for treating spinal cord injury and improving movement and bladder function.

[0025] The beneficial effects of the present invention are as follows: the present invention reacts borate bonds and Schiff base bonds on the main chains of biomacromolecule gelatin and sodium alginate, combines bioactive molecules exosomes, and constructs an injectable hydrogel assembly (GEL-EXO assembly) that is microenvironment responsive and combines exosomes. In the early stage of SCI, the borate bonds interact with ROS in the microenvironment, and the removal of ROS and the release of exosomes are simultaneously achieved, thereby purifying the microenvironment in a timely manner. In the chronic stage of SCI, the retention of the Schiff base bond network is utilized to provide a favorable support space for nerve growth. The assembly helps to restore movement and bladder function, and its injectability is more conducive to clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The synthesis process of the hydrogel of the embodiment;

[0027] Figure 2 is an identification diagram of the hydrogel synthesized in the embodiment;

[0028] Figure 3 This is an identification diagram of exosomes in the embodiment;

[0029] Figure 4 Electron microscopic images of hydrogels prepared in Examples and Comparative Examples;

[0030] Figure 5 The BBB score of SCI rats was performed at six weeks;

[0031] Figure 6 This is the bladder morphology image of SCI rats at six weeks;

[0032] Figure 7 Schematic diagram of the structure of the hydrogel of the present invention. DETAILED DESCRIPTION

[0033] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0034] Example

[0035] A microenvironment-responsive injectable hydrogel (GEL-EXO assembly) for releasing exosomes, the preparation steps of which include (1) synthesis of oxidized sodium alginate; (2) synthesis of gelatin grafted with phenylboronic acid; (3) synthesis of aminated gelatin grafted with phenylboronic acid; (4) synthesis of the GEL-EXO assembly; natural polymer sodium alginate and gelatin are used as the main chain of the hydrogel, borate ester bonds and Schiff base bonds are used to construct the hydrogel, and the precursor material is diluted with an exosome solution, and the gel is formed into a GEL-EXO assembly. Specifically, the following steps are included:

[0036] (1) Preparation of oxidized sodium alginate

[0037] 2.5 g of sodium alginate (SA) was dispersed in 12.5 mL of ethanol to prepare solution 1, and 1.0875 g of sodium periodate was dissolved in 12.5 mL of deionized water to prepare solution 2. After the preparation, solution 1 and solution 2 were mixed, stirred at room temperature in the dark for 6 hours, and then 1.25 mL of ethylene glycol was added, stirred for 30 minutes, dialyzed with ultrapure water for 5 days, the water was changed 3 times a day, and then freeze-dried with a freeze dryer for 48 hours to obtain the precursor material OSA;

[0038] (2) Gelatin grafted with phenylboronic acid

[0039] 2.5 g of gelatin was dissolved in 250 mL of deionized water at 37 ± 5 °C, 3.46 g of EDC·HCl, 0.83 g of NHS and 1.5 g of phenylboronic acid (PBA) were added to the gelatin solution in sequence, and then stirred at 37 °C for 48 hours, dialyzed with ultrapure water for 5 days, with the water changed 3 times a day, and then freeze-dried with a freeze dryer for 48 hours to obtain Gel-PBA;

[0040] (3) Aminated gelatin grafted with phenylboronic acid

[0041] 2.4 g Gel-PBA was dissolved in 100 mL water, heated to 35 ° C and dispersed evenly, then 8 mL ethylenediamine (EDA) was added, the pH was adjusted to 5 with hydrochloric acid, and then 2.3 g EDC·HCl was added, and the reaction was started at 35 ° C for 24 hours. After the reaction was completed, it was dialyzed with ultrapure water for 5 days, and the water was changed 3 times a day, and then freeze-dried with a freeze dryer for 48 hours to obtain the precursor material Gel-PBA-NH2;

[0042] (4) Construction of GEL-EXO assembly

[0043] The two precursor materials OSA and Gel-PBA-NH2 prepared in (1) and (3) were respectively prepared into solutions with a concentration of 20±1wt%, and then diluted with a PBS solution with an exosome concentration of 100μg / 30μL to a precursor material concentration of 10±1wt%, and then mixed in a volume ratio of 2:1. After uniform mixing, the gel will be in a viscous and stringy state. It is then placed in a 37°C constant temperature incubator for cross-linking for 1 hour to obtain a GEL-EXO assembly, and the color turns to light yellow.

[0044] The synthesis process of the hydrogel in this embodiment is as follows Figure 1 As shown, the precursor materials Gel-PBA-NH2 and OSA are diluted separately in liquid state. After the two are evenly mixed, they are stirred at room temperature for one minute to form gel immediately, and borate bonds are formed. The hydrogel is placed at 37°C and the gel is light yellow after 1 hour, and a double network of borate bonds and Schiff base bonds is formed.

[0045] Figure 2 The identification diagram of the synthetic hydrogel in this example is shown in Figure 1. Freeze-dried OSA and Gel-PBA-NNH2 were mixed with potassium bromide and ground into powder for Fourier transform infrared spectroscopy (FTIR) detection. The results are shown in Figure 1. Figure 2 A and Figure 2 B, where Figure 2 A is the Fourier transform infrared spectroscopy (FTIR) result of OSA molecules, Figure 2 B is the Fourier transform infrared spectroscopy (FTIR) result of Gel-PBA-NH2 molecule. The freeze-dried products OSA and Gel-PBA-NNH2 were dissolved in deuterium oxide (D2O) respectively. 1 H NMR was used for detection, and the results are shown in Figure 2 C and Figure 2 D, where Figure 2 C is the OSA molecule 1 H NMR spectrum, 5.3 ppm and 5.6 ppm are aldehyde peaks; Figure 2 D is the Gel-PBA-NH2 molecule 1 H NMR spectrum, 7.3-8.5 ppm is the phenyl peak.

[0046] The extraction process of exosomes in this embodiment:

[0047] When the P3 generation SD rat adipose mesenchymal stem cells (ADSCs) were cultured to 70%-80% confluence, the cell culture medium containing exosome-free serum was replaced and cultured for 48 hours, the supernatant was collected, and the cell supernatant was concentrated by tangential flow (Vivaflow 200). Every 800 ml of supernatant was concentrated to 25 ml, and then ultracentrifuged twice at 100000g for 90 minutes each using an XPN-100 ultracentrifuge (Beckman Coulter, USA) SW32 rotor to extract exosomes. Finally, the exosome precipitate was dissolved in an appropriate PBS solution to prepare a PBS solution with an exosome concentration of 100 μg / 30 μL.

[0048] Identification of exosomes:

[0049] ①Western blotting detection (WB):

[0050] BCA protein quantification reagent was used to quantify the protein of exosomes, and finally Western blotting was used to detect the expression of exosome surface specific marker proteins CD63, TSG101 and Alix. The band results are shown in Figure 3 A, Immunoblotting to detect the expression of membrane surface proteins Ailx, CD63 and TSG101. The strip results showed that the expression of exosome surface specific marker proteins CD63, TSG101 and Alix could be detected, proving that the extracted substances were exosomes.

[0051] ② Transmission electron microscopy (TEM)

[0052] The morphology of exosomes was detected by transmission electron microscope (TEM). 5 μL of exosome particle solution was dropped onto a Formvar / carbon-coated 200-mesh copper electron microscope grid, incubated at room temperature for 5 minutes, and then stained with standard uranyl acetate and observed under the microscope. The results are shown in Figure 3 C, Transmission electron microscopy shows the morphology of exosomes, which are cup-shaped or saucer-shaped with clear edges.

[0053] ③Particle size distribution (NTA)

[0054] The particle size distribution of exosomes was detected by Malvern zata potential and nanoparticle size analyzer. Figure 3 B, is the particle size distribution measured by NTA, which shows that the exosome particle size is between 30 and 150 nm.

[0055] Hydrogels are a promising class of tissue repair materials that can better simulate the ECM in the spinal cord microenvironment. In addition to being non-invasive and controllable, stimuli-responsive hydrogels that interact with the injury microenvironment can also give the gel itself a certain therapeutic effect. Injectable hydrogels can fill irregularly shaped wounds and achieve personalized treatment. Therefore, the preparation of multifunctional hydrogels that are microenvironmentally responsive and injectable is of great significance for SCI repair and clinical treatment.

[0056] Sodium alginate (SA) and gelatin (Gel) are both natural polymer materials with no immunogenicity, reproducibility and plasticity. The borate bond has good ROS responsiveness and the Schiff base bond is a strong covalent bond with good biocompatibility.

[0057] Comparative Example

[0058] Different from the embodiment, step (4) of this comparative example is hydrogel synthesis: two precursor materials OSA and Gel-PBA-NH2 with a concentration of 20±1wt% are diluted with PBS to a concentration of 10±1wt%, stirred and dispersed evenly, and then mixed in a ratio of 2:1. After uniform mixing, the gel will be in a viscous and stringy state. Thereafter, it is placed at 37±5°C for cross-linking for 1 hour to obtain a hydrogel, and the color becomes light yellow.

[0059] Figure 4 The electron microscope images of the hydrogels prepared in the examples and comparative examples are shown in FIG. Figure 4 It is shown that in the GEL-EXO assembly prepared in the embodiment, the exosomes are attached to the inner surface of the hydrogel pores ( Figure 4 B), the measured diameter is 111.6nm ( Figure 4 D), in contrast, the hydrogel prepared in the comparative example has a smooth pore surface ( Figure 4 A, C), confirming the successful construction of the GEL-EXO assembly of this example.

[0060] Animal experiments investigated the effects of hydrogels on the recovery of motor function and bladder function after SCI:

[0061] ① Modeling

[0062] Thirty female SD rats weighing 220-250 g were selected and anesthetized by intraperitoneal injection of 2% sodium pentobarbital. The back hair was shaved and disinfected with iodine. The skin was cut along the midline of the back with the T10 spinous process as the center, the fascia tissue was bluntly separated, the muscles were cut and the lamina was fully exposed, the T9-T11 lamina was removed, and the spinal cord tissue was fully exposed. The spinal cord was completely transected with microscissors, and the broken ends were checked with a tooth hook to ensure that they were completely transected, and the blood and tissue in the fracture were aspirated with a sterile cotton ball.

[0063] After complete hemostasis, the hydrogels prepared in the example and comparative example were injected into the fracture (GEL-EXO group and GEL group, respectively). The SCI group without hydrogel injection and the Sham group with only the vertebral lamina opened without damaging the spinal cord tissue served as controls.

[0064] The muscle tissue and skin were sutured, and penicillin (80 units / animal / day) was injected intraperitoneally within one week to prevent infection. Bladder massage was given twice a day to assist the animals in urination until the bladder's autonomous urination function was restored. There were 10 animals in each experimental group and control group, and they were fasted overnight before and after the operation.

[0065] ②BBB rating

[0066] The behavioral outcomes of the rats were assessed weekly after surgery. The rats moved freely in an open room, and BBB motility was scored by an observer blinded to the treatment groups, and the results were analyzed using GraphPad, see Figure 5 According to the BBB scoring table, the motor function of rats in the Sham group was completely unaffected, the function of rats in the SCI group could not be restored without treatment and they were almost completely paralyzed, and the motor function of rats treated with GEL alone was partially restored. After 6 weeks, they could move the three large joints of the hind limbs in a large range of motion. After 6 weeks of treatment with the GEL-EXO system, the recovery of the motor function of the hind limbs was effectively promoted, and weight-bearing movements were visible. The different points on the vertical axis in the figure represent different motor performances, and 10 points represent visible weight-bearing movements.

[0067] Figure 5 The data are expressed as mean ± SD. The GEL-EXO group was compared with the SCI group. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; GEL-EXO compared with GEL group, # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001.

[0068] ③Bladder morphology

[0069] After six weeks, the bladder morphology of the rats Figure 6In the sham group, the bladder wall pressure decreased when the bladder was empty, and the bladder contracted into a ball shape. When it was full, the bladder wall pressure increased, making the bladder pear-shaped or round. However, the shape of the bladder in the SCI group was close to pear-shaped even when it was empty, and the volume was larger. This should be because after the injury, the bladder expanded to the surroundings due to the storage of urine, but could not shrink. The bladder in the GEL group was smaller than that in the SCI group, but the bladder after treatment with the GEL-EXO system was closer to normal in shape and relatively smaller in volume.

[0070] The present invention reacts borate ester bonds and Schiff base bonds to the main chains of biomacromolecule gelatin and sodium alginate to synthesize microenvironment-responsive injectable hydrogels, which are more conducive to clinical applications; combined with mesenchymal stem cell-derived exosomes, a GEL-EXO assembly is constructed (see Figure 7 ) to eliminate ROS at the damaged site in the early stage, purify the microenvironment, and provide a favorable space for nerve growth in the chronic stage; at the same time, it promotes the therapeutic effect of movement and bladder function recovery.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An injectable hydrogel for releasing exosomes in a microenvironment-responsive manner, characterized in that: It uses sodium alginate and gelatin as the main chain and is combined with exosomes. The system contains a double network structure formed by borate bonds and Schiff base bonds.

2. The injectable hydrogel for releasing exosomes in response to microenvironment according to claim 1, characterized in that: In the hydrogel system, the concentration of gelatin was 10±1wt%, the concentration of sodium alginate was 10±1wt%, and the concentration of exosomes was 100μg / 60μL.

3. The injectable hydrogel for releasing exosomes in response to microenvironment according to claim 2, characterized in that: The exosomes are derived from adipose-derived mesenchymal stem cells.

4. A method for preparing an injectable hydrogel for releasing exosomes in a microenvironment-responsive manner as claimed in any one of claims 1 to 3, characterized in that: The main steps include: (1) Preparation of oxidized sodium alginate: The hydroxyl group in sodium alginate is oxidized by sodium periodate to form an aldehyde group, thus obtaining the product OSA; (2) Gelatin grafted with phenylboric acid: The product Gel-PBA was obtained by reacting phenylboronic acid with gelatin to form an amide bond and grafting phenylboronic acid into the gelatin backbone; (3) Aminated gelatin grafted with phenylboronic acid: The Gel-PBA obtained in (2) is mixed with ethylenediamine and reacted under acidic conditions to graft the amino group into the gelatin backbone to obtain the product Gel-PBA-NH2; (4) Construction of injectable hydrogels for microenvironment-responsive release of exosomes: The two precursor materials OSA and Gel-PBA-NH2 prepared in (1) and (3) were diluted with a PBS solution with an exosome concentration of 100 μg / 30 μL, respectively, and then the two were mixed. After mixing, they were placed in a constant temperature incubator for cross-linking to obtain a light yellow hydrogel.

5. The preparation method according to claim 4, characterized in that: In the above (1), the mass ratio of sodium alginate to sodium periodate is 5:2 to 2.

2.

6. The preparation method according to claim 4, characterized in that: In the method (2), EDCHCl and NHS are used as catalysts, and the mass ratio of phenylboric acid to gelatin is 2.4-3.4:

5.

7. The preparation method according to claim 4, characterized in that: In the method (3), after mixing Gel-PBA and ethylenediamine, the pH value is adjusted to 5±0.5 with hydrochloric acid, EDC·HCl is used as a catalyst, and the mass ratio of Gel-PBA to ethylenediamine is 4-5:14-15.

8. The preparation method according to claim 4, characterized in that: In (4), the two precursor materials OSA and Gel-PBA-NH2 were diluted with a PBS solution with an exosome concentration of 100 μg / 30 μL to a concentration of 10±1 wt% for both precursor materials, and the two were mixed in a volume ratio of 2:

1.

9. The preparation method according to claim 8, characterized in that: In the above (4), the temperature of the constant temperature incubator is 37±5°C, and the cross-linking time is 1 hour.

10. Use of the injectable hydrogel capable of releasing exosomes in response to microenvironment as claimed in any one of claims 1 to 3 in the preparation of drugs for treating spinal cord injury and improving movement and bladder function.

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