Multifunctional hydrogel for treating traumatic brain injury and preparation method and application thereof

Abstract

The present application relates to a multifunctional hydrogel for treating traumatic brain injury and a preparation method and application thereof. The preparation method of the hydrogel comprises the following steps: step 1, dissolving a tetracycline drug in a buffer to prepare a tetracycline drug solution; step 2, adding a multi-arm polyethylene glycol with a double bond end group into the solution prepared in step 1 to form a solution containing the multi-arm polyethylene glycol with a double bond end group and the tetracycline drug; step 3, adding a multi-sulfhydryl compound into the solution prepared in step 1 to form a solution containing the multi-sulfhydryl compound and the tetracycline drug; and step 4, mixing the solutions prepared in steps 2 and 3 to prepare the hydrogel. In the acute phase, the active oxygen rapidly increases to make the hydrogel release more drugs to regulate the inflammatory environment and play a neuroprotective role, and in the chronic phase, the hydrogel continuously releases drugs to continuously regulate the anti-inflammatory polarization of microglia cells, enhance neural plasticity and restore nerve function.

Description

Technical Field

[0001] This invention belongs to the field of drug carrier technology, specifically relating to a multifunctional hydrogel for treating traumatic brain injury, its preparation method, and its application. Background Technology

[0002] Traumatic brain injury (TBI) is a structural or physiological injury to the brain caused by external force, and it is one of the most common brain diseases treated in neurosurgery and emergency departments. Globally, there are approximately 5-6 million TBI patients annually. In developed countries, the leading cause of TBI is falls (53%), followed by road traffic accidents (26%), while in developing countries, the leading cause is road traffic accidents (57%), followed by falls (20%). In addition, various forms of violence, including war, regional conflicts, and armed clashes, are also significant contributing factors to TBI.

[0003] In addition to primary brain injury caused by external forces, patients with total brain injury (TBI) often suffer secondary damage. Following primary injury, various pathological changes occur in the damaged tissue, such as oxidative stress and inflammatory responses. If these early pathological changes are not addressed promptly, they can lead to secondary brain injury, which plays a more significant role in the progression of TBI than primary brain injury. Recruitment of reactive astrocytes and activated microglia to damaged tissue further enhances neuroinflammation, releases reactive oxygen species (ROS), inhibits the integration of newly formed neurons, and prevents axonal development. Furthermore, the accumulation of ROS in the brain trauma microenvironment creates a toxic environment for neighboring neurons and releases large amounts of inflammatory factors. These factors can induce apoptosis and necrosis of neurons through activated microglia, further leading to neuronal loss. These secondary injuries can further impair neurological function, resulting in motor disorders, cognitive decline, emotional instability, speech disorders, and behavioral changes. However, due to the complex pathology, insufficient clinical drugs, and difficulties in drug delivery and targeting to the brain, there is currently no universally accepted successful treatment for TBI in clinical practice.

[0004] While various drugs have been reported in the literature for the rehabilitation of TBI, effective drug delivery to the brain remains a challenge. As the blood-brain barrier gradually recovers after TBI, traditional intravenous injections become increasingly ineffective in delivering drugs to the brain. Therefore, multiple doses are often required to achieve therapeutic effects, which frequently leads to various side effects such as hepatotoxicity, gut dysbiosis, and gastrointestinal discomfort. Some patients with mild TBI and over 90% with moderate to severe TBI require surgery to remove blood clots, bone fragments, and foreign bodies. Post-surgery, more space is created in the brain, forming a fillable cavity. Currently, clinical practice only provides limited post-operative care such as hemostatic sponges. Therefore, utilizing the cavity created after TBI surgery and employing hydrogel carriers to load drugs can not only directly deliver drugs to the disease site but also achieve long-term drug release through the sustained-release properties of hydrogels, continuously promoting recovery after TBI. Injectable hydrogels, administered in liquid form, can form hydrogels in situ and continuously release drugs in situ, making them ideal for treating brain diseases. Their injectability ensures that drugs directly reach the target lesion. Furthermore, it can be endowed with intelligent release behavior, such as ROS-responsive release, to significantly improve drug bioavailability and efficacy.

[0005] Previously, the inventors prepared an injectable hydrogel for the treatment of stroke via Michael addition of 4-arm polyethylene glycol acrylate (4-PEGA) and DL-dithiothreitol (DTT). Furthermore, this sulfur-containing hydrogel exhibits ROS-sensitive release behavior, enabling it to release more drug in microenvironments with high ROS accumulation to modulate microglia polarization (e.g., see Shulei Zhang, et al. Curcumin loaded hydrogel with double ROS-scavenging effect regulates microglia polarization to promote poststroke rehabilitation, Materials Today Bio, Vol. 28, 2024, 101177). However, the inventors' previous studies showed that the injectable hydrogel drug delivery system was not ideal for the treatment of TBI.

[0006] Subsequently, through extensive research and screening of the injectable hydrogel delivery system and the drugs it delivers, the inventors proposed a multifunctional hydrogel delivery system for treating traumatic brain injury (TBI). In this invention, by introducing polyethylene glycol acrylate with a greater number of arms of the same molecular weight to form a hydrogel with DTT, each unit can contain a higher sulfur content, thus exhibiting higher ROS sensitivity to adapt to the TBI microenvironment with high ROS accumulation. During the acute phase of TBI, the rapid increase in ROS can trigger the hydrogel to release more tetracycline drugs to modulate the local inflammatory environment and exert a neuroprotective effect. As ROS levels gradually decrease in the chronic phase, the hydrogel can achieve long-term, continuous drug release, continuously modulating the anti-inflammatory polarization of microglia, enhancing neuroplasticity, and restoring neurological function. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a multifunctional hydrogel for treating traumatic brain injury, its preparation method, and its applications.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a method for preparing a multifunctional hydrogel for treating traumatic brain injury, the method comprising the following steps:

[0010] Step 1: Dissolve an appropriate amount of tetracycline drug in buffer solution to prepare a tetracycline drug solution;

[0011] Step 2: Add an appropriate amount of multi-arm polyethylene glycol with double-chain end groups to the tetracycline drug solution prepared in Step 1 to form a solution containing multi-arm polyethylene glycol with double-chain end groups and tetracycline drug.

[0012] Step 3: Add an appropriate amount of polythiol compound to the tetracycline drug solution prepared in Step 1 to form a solution containing polythiol compound and tetracycline drug;

[0013] Step 4: Mix the solutions obtained in Step 2 and Step 3 in a ratio of 5:1, stir evenly, and let stand for a period of time until there is no liquid flow to obtain the hydrogel.

[0014] Alternatively, in the preparation method, the tetracycline drug is selected from one or more of the following: minocycline, doxycycline, tetracycline, methacycline, desmethyltetracycline, octocycline, ricocycline, or tigecycline.

[0015] Preferably, the tetracycline drug is selected from minocycline.

[0016] Alternatively, in the preparation method, the double bond groups of the multi-arm polyethylene glycol are selected from one or more of the following: acryloyl, methacryl, norbornene, or vinyl, and the number of arms of the multi-arm polyethylene glycol is greater than 4, and the number of thiol groups of the multi-thiol compound is not less than 2.

[0017] Preferably, the double bond group of the multi-arm polyethylene glycol is selected from acryloyl, and the number of arms of the multi-arm polyethylene glycol is 6-10, and the number of thiol groups in the multi-thiol compound is not less than 2.

[0018] More preferably, the multi-arm polyethylene glycol is 8-arm polyethylene glycol acrylate (8-PEGA), and the thiol compound is dithiothreitol (DDT).

[0019] Preferably, the molecular weight of the multi-arm polyethylene glycol is in the range of 1000-20000 Da.

[0020] More preferably, the molecular weight range of the multi-arm polyethylene glycol is 5000-15000 Da.

[0021] Alternatively, in the preparation method, in step 1, the concentration of tetracycline in the tetracycline solution is 0.1-4 mg / mL.

[0022] Preferably, in step 1, the concentration of tetracycline in the tetracycline solution is 0.5-2 mg / mL.

[0023] More preferably, in step 1, the concentration of tetracycline in the tetracycline solution is 1 mg / mL.

[0024] Alternatively, in the preparation method, in step 2, the concentration of the multi-arm polyethylene glycol with double-terminated groups is 100-200 mg / mL in the solution containing the multi-arm polyethylene glycol with double-terminated groups.

[0025] Preferably, in step 2, in the solution containing multi-arm polyethylene glycol with double-terminated groups and tetracycline drugs, the concentration of multi-arm polyethylene glycol with double-terminated groups is 120-180 mg / mL.

[0026] More preferably, in step 2, in the solution containing multi-arm polyethylene glycol with double-terminated groups and tetracycline drugs, the concentration of multi-arm polyethylene glycol with double-terminated groups is 165 mg / mL.

[0027] Alternatively, in the preparation method, in step 3, the concentration of the polythiol compound in the solution containing the polythiol compound and the tetracycline drug is 10-100 mg / mL.

[0028] Preferably, in step 3, the concentration of the polythiol compound in the solution containing the polythiol compound and the tetracycline drug is 30-60 mg / mL.

[0029] More preferably, in step 3, the concentration of the polythiol compound in the solution containing the polythiol compound and the tetracycline drug is 50 mg / mL.

[0030] Alternatively, in the preparation method, in step 4, the standing conditions are set at 37°C for 5-15 minutes.

[0031] More preferably, in the preparation method, in step 4, the standing condition is to stand at 37°C for 10 minutes.

[0032] In a second aspect, the present invention provides a multifunctional hydrogel prepared by the preparation method described in the first aspect above.

[0033] Preferably, during the acute phase of the traumatic brain injury, the rapid increase in reactive oxygen species triggers the hydrogel to release more tetracycline drugs to regulate the local inflammatory environment and exert a neuroprotective effect. As the level of reactive oxygen species gradually decreases during the chronic phase, the hydrogel achieves long-term and continuous drug release behavior, continuously regulating the anti-inflammatory polarization of microglia, enhancing neuroplasticity, and restoring neurological function.

[0034] In a third aspect, the present invention provides the use of the multifunctional hydrogel described in the second aspect above in the preparation of products for treating traumatic brain injury.

[0035] Alternatively, in the above-described applications, the product is an injectable hydrogel filler.

[0036] Compared with the prior art, the present invention has the following advantages and positive effects:

[0037] (1) In the preparation method of the present invention, multi-arm polyethylene glycol acrylate with more than 4 arms and dithiothreitol are used as cross-linking backbones to encapsulate tetracycline drugs in hydrogel. The preparation method is mild, simple and efficient.

[0038] (2) The multifunctional hydrogel prepared by the method of this invention has a significant therapeutic effect on traumatic brain injury. It can not only repair the wound, but more importantly, it can restore the damaged nerve function. In the acute phase of traumatic brain injury, the rapid increase of reactive oxygen species triggers the hydrogel to release more tetracycline drugs to regulate the local inflammatory environment and exert a neuroprotective effect. As the level of reactive oxygen species gradually decreases in the chronic phase, the hydrogel achieves long-term and continuous drug release behavior, continuously regulating the anti-inflammatory polarization of microglia, enhancing neuroplasticity, and restoring nerve function. Attached Figure Description

[0039] Figure 1 Preparation and performance study of MHH. This includes: a. Chemical reaction equation of MHH; b. Inverted vial experiment; c. Scanning electron microscopy observation of hydrogel morphology; d. Infrared spectra of blank hydrogel, MH, and MHH; e. In vitro degradation curves of MHH under ROS simulated by PBS and H2O2; f. Drug release curves of MHH under ROS simulated by PBS and H2O2; g. Live / dead cell viability determination after 48 hours using the live / dead assay; h. Quantitative analysis of N2a cell viability; and i. CCK-8 assay for BV2 cell viability.

[0040] Figure 2 Results of in vitro cell culture experiments of MHH. Schematic diagram of in vitro cell culture. a. PCR expression of IL-1β and b. TGF-β, c. Schematic diagram of co-culture of N2a and BV2 cells, d. Viability of N2a cells co-cultured with BV2 cells before and after LPS treatment, e. Representative WB stripes of synaptophysin and PSD95 expression in co-cultured with BV2 cells before and after LPS treatment, f. Quantitative WB results of synaptophysin and g. PSD95.

[0041] Figure 3 Results of MHH experiments in TBI model mice. a. Schematic diagram of the animal experimental protocol; b. Injury image after TBI; c. In vivo fluorescence images of the brain at different time points after implantation; d. Brain injury images; e. Results of the new object recognition experiment; f. Behavioral results of the balance beam test and g. Rotary bar test.

[0042] Figure 4 Systemic toxicity analysis. H&E staining of the heart, liver, spleen, lungs, and kidneys. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0045] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0046] Preparation Example:

[0047] 1. Materials

[0048] 8-arm polyethylene glycol acrylate (8-PEGA, Mw 10000, Beijing Jenkem Technology Co., Ltd.), dithiothreitol (DTT) (J&K Chemical Technology Co., Ltd.), minocycline hydrochloride (MH, Shanghai Maclean Biochemical Technology Co., Ltd.).

[0049] 2. Preparation method of MHH

[0050] Minocycline hydrochloride (MH) was dissolved in PBS to prepare a 1 mg / mL MH solution (MHS). Then, 165 mg of 8-PEG was added to 1 mL of MH solution to form a 165 mg / mL 8-PEG solution, and 50 mg of DTT was added to 1 mL of MH solution to form a 50 mg / mL DTT solution. The 8-PEG and DTT solutions were mixed at a 5:1 ratio and allowed to stand at 37°C for 10 min to prepare minocycline hydrochloride hydrogel (MHH).

[0051] The MHH used in the following effect examples are all MHH prepared in the "Preparation Examples" section.

[0052] Example of effect:

[0053] Example 1: Characterization and performance study results of the multifunctional hydrogel of this invention 1. Experimental Methods

[0054] 1.1 Gelation

[0055] The gelation time of MHH was determined using the inverted vial method. A timer was started when the hydrogel precursor solution was thoroughly mixed. After 10 minutes, the vial was inverted, the solution stopped flowing, and the sol-gel transition occurred. The time taken for this process is the gelation time.

[0056] 1.2 Morphological observation

[0057] The freeze-dried MHH sample was attached to the sample tray of a scanning electron microscope with conductive adhesive, sputtered with gold using an ion sputtering instrument, and then the microstructure of the sample was observed using a scanning electron microscope (SEM, Regulus S8230).

[0058] 1.3 Fourier Transform Infrared Spectroscopy (FT-IR)

[0059] The freeze-dried MHH was pressed into transparent thin sheets and measured using a Fourier transform infrared spectrometer.

[0060] 1.4 Degradation status

[0061] Weigh the lyophilized MHH and record the mass as m0. Place the MHH into a 10mL centrifuge tube containing 8mL of PBS solution. Place the centrifuge tube in a water bath and shake gently at 37°C. Remove the sample at the required experimental time intervals, lyophilize, and weigh the sample, recording the mass as mx. The degradation rate (DR) of MHH is calculated using the following formula: DR = (m0 - mx) / m0 × 100%.

[0062] 1.5MH of in vitro release

[0063] The in vitro release of MH was determined using a UV-Vis spectrophotometer (UV-1800). First, a standard curve was established by measuring the absorbance of a series of MH solutions along a concentration gradient. Then, 1 mL of MH was completely immersed in a 50 mL centrifuge tube containing 10 mL of PBS solution and gently mixed in a shaker at 37°C. The release solution was then removed at predetermined time points and replaced with 0.5 mL of fresh PBS. The solution was then measured using a UV-Vis spectrophotometer (UV-1800) to obtain an in vitro MH release chromatogram.

[0064] 1.6 MHH cytotoxicity

[0065] BV2 cells (mouse microglia, Wuhan Pronosei Biotechnology Co., Ltd.) were cultured with the hydrogel extract to evaluate the cytotoxicity of the hydrogel. A dried hydrogel (0.2 mg) was placed in 20 mL of culture medium and extracted at 37 °C for 24 h. The extract was then filtered through a membrane. The extraction was performed at a concentration of 1 x 10⁻⁶. 4 BV2 cells were seeded into 96-well plates at 100% per well and cultured adherently for 24 hours. Then, the culture medium was replaced with serially diluted extraction buffer (100%, 50%, 25%, 0%). Cells were cultured at 37°C in a 5% CO2 incubator (SANYO (XD101), Japan). After 2 days, cell viability was assessed using a CCK-8 assay according to the manufacturer's instructions.

[0066] Biocompatibility of 1.7 MHH

[0067] N2a cells (mouse neuroblastoma cells, Wuhan Pronosei Biotechnology Co., Ltd.) were cultured using MHH-extracted medium to evaluate the biocompatibility of MHH. MHH (0.1 mL) was prepared in a glass vial, and then 5 mL of culture medium was added to the vial. MHH was extracted at 37℃ for 24 h to obtain the MHH extract. N2a cells were cultured in 96-well plates (1×10⁻⁶). 4Cells were cultured in 100 cells / well for 24 h, and then the medium was replaced with different MHH extracts (0%, 50%, 100%) diluted with normal medium. Cells were cultured in a humid environment at 37°C and 5% CO2 for 24 h. Cell viability was then determined by measuring live and dead cells according to the manufacturer's instructions, and fluorescence images were obtained by fluorescence imaging microscopy (Olympus BX51, Japan).

[0068] 2. Experimental Results

[0069] In vitro characterization of MHH, such as Figure 1 As shown. Figure 1 a shows the chemical reaction equation for a blank hydrogel, in which 8-PEG and DTT are mixed in a certain proportion to form a blank hydrogel with a cross-linked network structure. Figure 1 b. The formation of MHH was characterized using the inverted vial method. A gel formed from the solution in 10 minutes at 37°C. Figure 1 The SEM image of c depicts the porous structure of MHH, where the lighter-colored part in the left image with a scale bar of 20 μM is likely MH crystals loaded in the gel. Figure 1 d represents the FT-IR spectra of the 8-arm PEG, MH, and MHH. The IR spectrum of the 8-arm PEG is shown at 2931 cm⁻¹. -1 This is the stretching vibration peak of the methylene (-CH2) group in PEG, at 1730 cm⁻¹. -1 The peak at 1688 cm⁻¹ represents the stretching vibration of the carbonyl group on the 8-arm PEG. On the MH spectrum, this peak is at 1688 cm⁻¹. -1 The absorption peak at 3357 cm⁻¹ is the amide I band of MH. -1 This corresponds to the stretching vibration peak of the hydroxyl group on MH. These absorption peaks can all be found in the IR spectrum of MHH, which preliminarily demonstrates the successful preparation of the MH-loaded hydrogel.

[0070] Considering the applications of hydrogels in the biomedical field, Figure 1The degradation performance of the hydrogel was tested in vitro using PBS (pH 7.4) to simulate the normal physiological microenvironment and H2O2 solution to simulate the ROS environment. As shown in the figure, the hydrogel curves in both environments exhibited a similar pattern of slow initial degradation followed by rapid degradation. This is consistent with the degradation pattern of PEG hydrogels reported in the literature. The slower initial weight loss curve mainly corresponds to the gradual destruction of the intact cross-linked network in the hydrogel; before the cross-linked network is completely destroyed, the gel loses little weight. The rapid weight loss curve in the later stage corresponds to the process of rapid weight loss as a large amount of PEG components in the gel dissolves in the solution after the cross-linked network is completely destroyed. However, there are significant differences in the timing of the rapid initial degradation and the time to complete gel degradation between the two environments. Under PBS conditions, the change point appears on day 14, and the hydrogel is not completely degraded by day 21. In the ROS environment simulated by H2O2 solution, the change point in the hydrogel degradation rate appears on day 5, and complete degradation occurs around day 10. These results not only demonstrate that MHH has good degradation characteristics when applied in vivo, but also further prove that the hydrogel is responsive to the ROS environment, i.e., ROS can significantly promote the degradation of the hydrogel.

[0071] Figure 1 Further investigation confirmed that ROS-responsive hydrogels (MHH) can release more drugs under ROS conditions. As shown in the figure, the MH release curves from the gel in the two environments correspond to their respective degradation curves under the same conditions. Under PBS conditions, MH release was slow before day 15; after day 15, with the rapid disintegration of the hydrogel, the MH release rate significantly accelerated, reaching approximately 80% drug release by day 23. In the ROS environment simulated by H2O2 solution, the MH release rate significantly increased, and was completed by approximately day 10. These results indicate that the ROS environment can regulate the more rapid release of MH loaded in the hydrogel, a characteristic highly suitable for drug carriers in TBI treatment. Oxidative stress in the brain after TBI can promote faster MH release from the gel, regulate the local inflammatory environment, and promote TBI recovery.

[0072] Figure 1 g and Figure 1 The viability of N2a cells was determined using the h-cell live / dead assay. Compared to the control group, the three groups cultured in MHH-extracted medium showed a similar number of live cells after 48 hours of incubation. Furthermore, they showed very few dead cells, comparable to the control group. These results indicate that MHH has low cytotoxicity and good biocompatibility. In addition, in… Figure 1 In h, the group using 100% MHH extraction medium had more dead cells than the groups using 25% and 50% MHH extraction medium.

[0073] Figure 1The cytotoxicity of MHH on BV2 cells was further demonstrated by CCK-8 assay. The three groups of cells cultured in MHH-extracted medium showed similar cell viability to the control samples, again indicating the good biocompatibility of MHH. The cell viability of the group using 50% MHH-extracted medium was slightly higher than that using 100% MHH-extracted medium. Therefore, considering cytotoxicity and efficacy, 50% MHH-extracted medium was selected for subsequent cell experiments.

[0074] Example 2: In vitro cell culture experiment results of the multifunctional hydrogel of the present invention 1. Experimental Methods

[0075] 1.1 Regulatory effect of MHH on microglial cell phenotype in vitro

[0076] BV2 cells were cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin / streptomycin (1 million U / L each). Cells were cultured in a humid environment at 37°C and 5% CO2. Cells were cultured under a microscope until confluence density exceeded 80%, with the medium changed every 2 days and passaged every 3 days. BV2 cells were stimulated with 1 μg / mL LPS for 24 h, and cells were collected and whole-cell lysates were prepared. The expression levels of IL-1β and TGF-β in BV2 cells were detected by RT-qPCR.

[0077] 1.2 In vitro regulation of synaptic plasticity by BV2 cells

[0078] Four groups of BV2 cells (①BV2, ②BV2+MHH, ③LPS BV2, and ④LPS BV2+MHH) were seeded in the upper layer of nested chambers in a Transwell system. Groups ③ and ④ were cultured with 1 μg / mL LPS for 24 h, and then replaced with fresh culture medium. Two groups of N2a cells (N2a and LPS N2a) were seeded in the lower layer of the Transwell system, and N2a cells were stimulated with 1 μg / mL LPS for 24 h, and then replaced with normal culture medium (BV2:N2a = 1:1).

[0079] Four BV2 cells and two N2a cells were paired and assembled to form eight complete Transwell systems. BV2 and N2a cells were co-cultured in the Transwell systems for 24 hours, then the BV2 cells were removed, and the N2a cells were collected. Cell viability was assessed using the CCK-8 assay, and the expression of Synaptophysin and PSD95 proteins was detected by Western blotting.

[0080] 1.3 PCR detection of mRNA

[0081] RT-PCR was performed using the RNAqueous-Micro Total RNA Isolation Kit (Thermo Fisher Scientific). Primer sequences are as follows:

[0082] Positive IL-1β: 5'-GCTTTCAGGAATGGAGGGCTA-3',

[0083] Reverse IL-1β: 5'-GACAGCCCAGGTCAAAGGTT-3';

[0084] Positive TGF-β: 5'-GAGCCGGGACGGTTCTG-3',

[0085] Reverse TGF-β: 5'-GGCAGTCGTAGGAGGGGTTA-3';

[0086] Forward IRF4: 5'-TCCGACAGTGGTTGATCGAC-3',

[0087] Reverse IRF4: 5'-CCTCACGATTGTAGTCCTGCTT-3';

[0088] Forward IRF5: 5'-GGTCAACGGGGAAAAGAAACT-3',

[0089] Reverse IRF5: 5'-CATCCACCCCTTCAGTGTACT-3';

[0090] Forward GAPDH: 5'-TGGATTTGGACGCATTGGTC-3';

[0091] Reverse GAPDH: 5'-TTTGCACTGGTACGTGTTGAT-3'.

[0092] 2. Experimental Results:

[0093] Experimental results are as follows Figure 2 As shown. Since IL-1β and TGF-β are typical pro-inflammatory and anti-inflammatory factors secreted by BV2 cells, respectively, in order to demonstrate that MHH can regulate the polarization of BV2 cells and cause them to develop an anti-inflammatory phenotype, therefore... Figure 2 a and Figure 2b. PCR was used to determine the expression of inflammatory factors IL-1β and TGF-β in LPS-induced BV2 cells with and without MHH treatment. After LPS treatment, IL-1β expression increased and TGF-β expression decreased in BV2 cells, indicating that LPS treatment successfully induced pro-inflammatory polarization of microglia in vitro. The low IL-1β expression and high TGF-β expression in the MHH group after different treatments demonstrate that MHH can effectively regulate the anti-inflammatory polarization of BV2 cells.

[0094] To further verify whether MHH-regulated anti-inflammatory microglia can enhance neural plasticity in vitro, such as... Figure 2 As shown in Figure c, co-culture of N2a cells and BV2 cells was performed in the Transwell system. Figure 2 Figure d shows the cell viability of N2a cells after co-culture. MHH treatment had no significant effect on the proliferation rate when N2a was co-cultured with normal BV2 cells. However, cell viability was significantly reduced when N2a was co-cultured with pro-inflammatory BV2 cells. This suggests that pro-inflammatory microglia may secrete neurotoxic inflammatory factors, leading to reduced neuronal proliferation. Furthermore, LPS treatment of N2a significantly reduced cell viability. In particular, cell viability was reduced to the lowest level when LPS-treated N2a and LPS-treated BV2 were co-cultured, indicating that pro-inflammatory microglia are detrimental to neuronal plasticity. However, after treating LPS-induced pro-inflammatory microglia with MHH to induce anti-inflammatory polarization, and then co-culturing them with N2a cells, the cell viability of LPS-treated N2a cells significantly increased. These results suggest that anti-inflammatory microglia may be able to enhance neuronal plasticity in vitro.

[0095] To further confirm this result, Western blotting was used to detect the expression of synaptophysin and PSD95, which are markers of synaptic plasticity, such as... Figure 2 e Figure 2 f and Figure 2 As shown in g, there was no significant difference in the expression of synaptophysin and PSD95 in the four groups of normal N2a cells. After LPS treatment, the expression of these two proteins was significantly reduced in the four groups of LPS-treated N2a cells compared with the four groups of normal N2a cells, indicating that LPS simulation successfully mimicked the inflammation model of TBI, resulting in a significant reduction in in vitro neural plasticity. Among the four groups of LPS-treated N2a cells, the expression of the two proteins was lowest in the group co-cultured with LPS-treated BV2 cells. However, if LPS-treated BV2 cells were first treated with MHH and then co-cultured with LPS-treated N2a cells, the expression of synaptophysin and PSD95 rebounded significantly. This result indicates that MHH-regulated anti-inflammatory microglia can upregulate the expression of synaptophysin and PSD95 in LPS-treated N2a cells. This suggests that MHH-regulated anti-inflammatory microglia can enhance in vitro neural plasticity. Example of effect 3: This invention Pharmacodynamic study results of multifunctional hydrogels in TBI animal model 1. Experimental Methods

[0096] 1.1 Animals

[0097] All in vivo experiments were approved by the Animal Experiment Ethics Committee of Beijing Rehabilitation Hospital, Capital Medical University. All animal care, feeding, surgery, and anesthesia procedures were conducted in accordance with the "Regulations on the Management of Laboratory Animals in China". Male C57BL / 6j mice (8 weeks old, 24.0±1.5g) were used in the experiments. They were fed and watered in the laboratory for 7 days to acclimatize to the environment.

[0098] 1.2 Establishment of a mouse model of moderate brain injury (TBI)

[0099] Mice were anesthetized with isoflurane (2% induction, 1.5% maintenance), their heads were shaved, and their scalps were disinfected. The mice were then fixed in a stereotaxic apparatus (Shenzhen Ruiward Technology Co., Ltd.), and a midline incision was made along the head-tail axis to separate the skull from the skin, exposing the skull. The TBI animal model employed the typical Feeney free-fall method. A 1.5cm incision was made along the midline of the scalp, and a craniotomy was performed 2mm to the right of the midline and 3mm posterior to the anterior fontanelle. A 4mm diameter skull fragment was carefully removed, and the impactor was placed perpendicular to the dura mater. When the impactor contacted the dura mater, a 40g weight was dropped from a height of 7.5cm, causing moderate brain injury. TBI mice were randomly divided into 5 groups of 6 mice each: Sham, TBI, TBI+Blank, TBI+MHS, and TBI+MHH groups. The Sham group underwent sham surgery, with the scalp sutured directly without impact; the TBI group received no treatment after impact. After debridement and hemostasis in other groups, 7 μL of blank gel, MHS, and MHH were immediately injected into the brain injury model cavity using a 10 μL microsyringe over a period of 1 minute. Finally, the mouse head skin was sutured and placed on a heating pad (37°C) for 10 minutes. After the mice recovered from anesthesia, they were placed in a new cage and given food and water as needed.

[0100] In vivo imaging at 1.3 MHz

[0101] Immediately after brain injury, 7 μL of FITC-labeled MHH was injected into the brain cavity of mice, and small animal fluorescence imaging was performed on days 1, 3, 7, 14, and 21 post-brain injury. Lumina III (PerkinElmer, USA) observed the in vivo degradation of the hydrogel.

[0102] 1.4 Animal behavioral assessment

[0103] Rotating bar test: During the test, the speed of the rotating bar was uniformly accelerated from 4 rpm to 25 rpm over 3 minutes, and the time required for the mouse to fall off the rotating shaft was recorded.

[0104] Balance beam test: Motor coordination is quantified by counting the number of times the right hind paw slips off the balance beam (approximately 80cm long, 1.5mm wide, and 100cm off the ground) during the experiment.

[0105] The above experiments were conducted with a 3-day training period before the formal testing, and the experiments were repeated three times for each mouse.

[0106] New Object Recognition Test: For the first 30 minutes of evaluation, mice were placed in a behavior chamber to acclimatize to the environment. On Day 1, mice spent 10 minutes in a box without any objects. On Day 2, mice were exposed to two identical objects and allowed to explore the box for 5 minutes. One hour later, one of the objects was replaced with a new object, and the mice explored the box again for 5 minutes. The exploration time for each object was recorded using a camera and software (exploration was defined as the mouse's mouth or nose being within approximately 2-3 cm of the object). The number of times, duration, and distance the animal moved while exploring each object were measured within 5 minutes. Mouse behavior was recorded and analyzed using animal tracking software (Noldus) through video clips. The time spent on the new object was compared to the time spent on the old object. The New Object Recognition Index was calculated using the following formula: (Time spent on the new object) / (Time spent on the new object + Time spent on the old object). A result greater than 50% indicates recognition of the new object, less than 50% indicates no recognition of the new object, and 50% indicates equal recognition.

[0107] 1.5 Overall safety

[0108] The systemic biocompatibility of the hydrogel was evaluated in vivo. On day 3 after gel injection, H&E staining was performed on the vital organs (heart, liver, spleen, lung, and kidney) of mice in each group, with normal C57 mice serving as the control group.

[0109] 1.6 Statistical Analysis Methods

[0110] Immunofluorescence staining results were analyzed using ImageJ software to count positive areas. Statistical analysis was performed using GraphPad Prism software (version 5.01). Results are expressed as mean ± standard deviation (SD). The t-test was used to test differences between two groups. One-way ANOVA was used to test differences among multiple groups. All analyses were considered statistically significant with p < 0.05, where *p < 0.05, **p < 0.01, and ***p < 0.001.

[0111] 2. Experimental Results

[0112] Experimental results are as follows Figure 3 As shown. Figure 3 a describes the protocol for animal testing. Figure 3 b shows the surgical wound from TBI.

[0113] Figure 3 Image c shows an in vivo immunofluorescence image of MHH in vivo using an animal fluorescence imaging system. The fluorescent molecule FITC was used to replace MH in the hydrogel. After implantation of the fluorescent solution and fluorescent hydrogel into the intracranial cavity of TBI mice, strong fluorescence signals were observed in both groups, indicating successful intracranial implantation. With prolonged implantation time, the signal intensity gradually decreased in both groups, indicating gradual release of FITC. Notably, only a very weak fluorescence signal was detected in the fluorescent solution group on day 14, and no signal was detected by day 21, while a significant fluorescence signal was still detected in the hydrogel group on day 21. This demonstrates the sustained release behavior of the hydrogel, allowing MHH to act on the brain for a longer period. Furthermore, it also shows that the fluorescent solution is easily removed by cerebrospinal fluid.

[0114] Figure 3 Figure d shows the brain damage in different groups on days 3 and 21. On day 3, significant brain damage was present in all groups, with the TBI group showing the most severe damage. However, by day 21, the injured area in the TBI group had only slightly decreased, while the TBI+MHS and TBI+MHH groups showed a much greater reduction in injured area compared to the TBI group. Furthermore, the area of ​​damage appeared to have decreased more in the TBI+MHH group than in the TBI+MHS group.

[0115] Normal mice tend to prefer new objects to old ones, and their cognitive abilities are reflected in the ratio of time spent exploring new objects to old ones. Figure 3 The results of the novel object exploration experiment performed on day 14 after TBI are shown. The proportion of time mice spent exploring novel objects decreased after TBI, but all treatment groups showed varying degrees of recovery. The TBI+MHH group showed the most significant recovery in novel object exploration time, indicating that MHH has a significant effect on improving cognitive abilities after TBI. Figure 3 f and Figure 3g shows the results of the balance beam test and experiments used to assess the recovery of motor function in TBI mice. In the balance beam test, TBI significantly increased the number of falls in mice, but the number of falls decreased in all TBI groups as treatment progressed. By day 7, significant differences were observed between the treatment groups (TBI+MHH and TBI+MHS) and the non-treatment groups (TBI+Blank and TBI). The number of falls in the MHS group was initially lower than that in the MHH group; however, after day 14, the number of falls in the MHH group was lower than that in the MHS group. By day 21, the MHH group had the fewest falls among all TBI mice, with a number of falls close to that of the sham-operated group, but without statistical significance. In the rotarod test, the fall time decreased in all mice after TBI. Over time, the fall time gradually recovered in all TBI groups, with the two treatment groups showing the best recovery. Between these two groups, the fall time was longer in the TBI+MHS group compared to the MHH group, until day 14 after TBI. After day 14, the MHH group had a longer fall duration than the MHS group, while there was no statistically significant difference between the TBI+MHH group and the sham-operated group. These results indicate that MH treatment can effectively promote the recovery of motor function in TBI mice. Both behavioral tests showed similar trends: the TBI+MHS group performed better in the acute phase (within a few days after TBI), while the TBI+MHH group showed better performance in the chronic phase. This trend may be related to the sustained drug release from the hydrogel carrier in the TBI+MHH group, which continuously modulates microglia polarization and enhances neural plasticity in the chronic phase.

[0116] Finally, H&E staining was used to further examine the systemic biosafety of MHH in vital organs such as the heart, liver, spleen, lungs, and kidneys. Figure 4 As shown, H&E staining of the Sham, TBI, TBI+blank, TBI+MHS, and TBI+MHH groups showed no significant changes in these organs, indicating that neither the hydrogel nor the drug had significant biotoxicity.

[0117] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a multifunctional hydrogel for treating traumatic brain injury, characterized in that: The preparation method includes the following steps: Step 1: Dissolve an appropriate amount of tetracycline drug in buffer solution to prepare a tetracycline drug solution; Step 2: Add an appropriate amount of multi-arm polyethylene glycol with double-chain end groups to the tetracycline drug solution prepared in Step 1 to form a solution containing multi-arm polyethylene glycol with double-chain end groups and tetracycline drug. Step 3: Add an appropriate amount of polythiol compound to the tetracycline drug solution prepared in Step 1 to form a solution containing polythiol compound and tetracycline drug; Step 4: Mix the solutions obtained in Step 2 and Step 3 in a ratio of 5:1, stir evenly, and let stand for a period of time until there is no liquid flow to obtain the hydrogel.

2. The preparation method according to claim 1, characterized in that: In the preparation method, the tetracycline drug is selected from one or more of the following: minocycline, doxycycline, tetracycline, methacycline, desmethyltetracycline, octocycline, ricocycline, or tigecycline.

3. The preparation method according to claim 1, characterized in that: In the preparation method, the double bond groups of the multi-arm polyethylene glycol are selected from one or more of the following: acryloyl, methacryl, norbornene, or vinyl, and the number of arms of the multi-arm polyethylene glycol is greater than 4, and the number of thiol groups of the multi-thiol compound is not less than 2.

4. The preparation method according to claim 1, characterized in that: In step 1, the concentration of tetracycline in the tetracycline solution is 0.1-4 mg / mL.

5. The preparation method according to claim 1, characterized in that: In step 2, the concentration of multi-arm polyethylene glycol with double-terminated groups and tetracycline drugs in the solution is 100-200 mg / mL.

6. The preparation method according to claim 1, characterized in that: In step 3, the concentration of the polythiol compound in the solution containing the polythiol compound and the tetracycline drug is 10-100 mg / mL.

7. The preparation method according to claim 1, characterized in that: In step 4, the settling conditions are: settling at 37°C for 5-15 minutes.

8. The multifunctional hydrogel prepared by the preparation method according to any one of claims 1 to 7, characterized in that: During the acute phase of the traumatic brain injury, the rapid increase of reactive oxygen species triggers the hydrogel to release more tetracycline drugs to regulate the local inflammatory environment and exert a neuroprotective effect. As the level of reactive oxygen species gradually decreases in the chronic phase, the hydrogel achieves long-term and continuous drug release behavior, continuously regulating the anti-inflammatory polarization of microglia, enhancing neuroplasticity, and restoring neurological function.

9. Use of the multifunctional hydrogel of claim 8 in the preparation of products for treating traumatic brain injury.

10. The use according to claim 9, characterized in that: The product is an injectable hydrogel filler.

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