Hydrogen and magnesium ion pump catheter used for peripheral nerve defect repair and provided with Jingxiang structure and preparation method of hydrogen and magnesium ion pump catheter

Abstract

The invention relates to the technical field of nerve repair, and discloses a hydrogen and magnesium ion pump catheter with a Jingxiang structure for peripheral nerve defect repair and a preparation method of the hydrogen and magnesium ion pump catheter. The pump guide pipe is composed of a hydrophilic inner layer, a hydrophobic outer layer and a storage cavern middle layer, the hydrophilic inner layer and the hydrophobic outer layer are made of degradable high polymer materials, and the storage cavern middle layer is made of a mixture of one or more of polylactic acid, polyglycolide or polylactic acid-glycolic acid and a magnesium-based material. The pump catheter is of a two-sided structure, the hydrophilic inner layer provides biophysical clues, the cell compatibility of the catheter is improved, adhesion and migration of nerve cells are promoted, the hydrophobic outer layer prevents adverse cells such as fibroblasts from adhering and invading damaged parts, and the magnesium ion and hydrogen storage middle layer can continuously release hydrogen and magnesium ions to play a biological role. The nerve regeneration microenvironment is improved, and the nerve injury repairing effect of the stent is improved.

Description

Technical Field

[0001] The present invention relates to the field of nerve repair, and in particular to a hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects and a preparation method thereof. Background Art

[0002] Peripheral nerve injuries are often caused by trauma and tumor resection, severely impairing motor and sensory functions. Although peripheral nerves have some regenerative capacity, regeneration becomes challenging when long segment defects occur. Autologous nerve transplantation has been considered the standard approach for treating peripheral nerve defects, but this presents significant limitations, such as limited donor nerve tissue, donor site morbidity, and neuropathic pain caused by neuroma formation. Advances in tissue engineering and regenerative medicine have led to the development of tissue-engineered nerve grafts as a promising alternative. However, conventional nerve-supporting scaffolds are unable to restore nerve structure and function because they lack the essential biochemical or biophysical cues required to create a favorable regenerative microenvironment. The microenvironment that controls peripheral nerve regeneration is the result of a dynamic interaction of biochemical and biophysical signals that influence tissue regeneration and cellular behavior. Therefore, nerve conduits that incorporate appropriate biophysical and biochemical cues may significantly enhance nerve regeneration.

[0003] Oxidative stress at the injury site is one of the typical characteristics of the neural regeneration microenvironment, mainly caused by excessive reactive oxygen species (ROS). It can damage cell structures including lipids, proteins, and DNA. Excessive ROS can aggravate neural damage and delay repair. Hydrogen (H 2 ) was discovered to be a therapeutic medical gas with anti-inflammatory properties. 2 Effectively reduces highly cytotoxic oxidative free radicals, including hydroxyl radicals (•OH) and peroxynitrite (ONOO − ), without interfering with metabolic redox reactions in healthy cells or disrupting physiological ROS involved in cell signaling. In addition, even at high concentrations, H 2 is also nontoxic, emphasizing its potential for safe therapeutic applications. 2 The high diffusivity, low water solubility and dose-dependent therapeutic effect of H limit its application. 2 Local delivery of drugs remains a major challenge.

[0004] Magnesium-based materials, such as magnesium (Mg), magnesium hydride (MgH 2 ), magnesium-containing alloys, react with water to release H 2 , is an ideal local release H 2 In addition, magnesium-based materials can also promote nerve regeneration as a source of magnesium ions. 2+Essential for neuronal survival and function; it is involved in the formation of membrane phospholipids, signal transduction, myelin and synapse formation, and regulates neurotransmitters such as dopamine and serotonin. Oral Mg 2+ Supplementation has been shown to improve peripheral nerve regeneration after crush injuries. Magnesium-based materials react with water to form magnesium hydroxide (Mg(OH) 2 ), which will hinder the magnesium-based materials from reacting with water to release hydrogen; and Mg(OH) 2 Ionization to generate hydroxide ions will make the local area alkaline and have poor cell compatibility. In addition, Mg(OH) 2 It is difficult to dissolve in water and cannot provide enough magnesium ions to play a biological role. 2 ), magnesium-based materials such as magnesium alloys can provide potential biochemical clues for nerve regeneration: H 2 and Mg 2+ , promoting nerve regeneration, but its application still faces great difficulties.

[0005] Traditional catheter materials are usually hydrophobic, resulting in poor cell compatibility, so improving their hydrophilicity is beneficial to improving their biocompatibility. On the other hand, catheter materials must maintain certain hydrophobic characteristics to prevent fibroblasts from adhering to the catheter and invading the injured area. Therefore, it is necessary to rationally design catheter materials so that their biophysical cues are appropriate and promote nerve regeneration.

[0006] The patent application with publication number CN114699560A discloses a double-layer tubular product for promoting regeneration of defective nerves, which is composed of a tubular outer layer and a tubular inner layer closely attached to the inner wall thereof, wherein the tubular outer layer is formed of a human body compatible biodegradable material, and the tubular inner layer is a hydrogel layer formed of polyethylene glycol. The human body compatible biodegradable material is selected from: polylactic acid or its derivatives, gelatin and its derivatives, chitosan, polyurethane, polycaprolactone, water-insoluble cellulose and its derivatives, etc. The polylactic acid derivative is selected from: polylactic acid-polyethylene glycol copolymer, lactic acid-glycolic acid copolymer (PLGA), PLGA-polyethylene glycol copolymer, lactic acid-caprolactone copolymer (PCLA), PCLA-polyethylene glycol copolymer. The weight ratio of the tubular outer layer to the tubular inner layer is 100:20-100, and the thickness of the tube wall of the double-layer tubular product is 50-500 μm. The double-layer tubular product of the present invention presents the technical effects described in the specification.

[0007] The catheter in the prior art does not carry biochemical cues to regulate the regenerative microenvironment of regenerated nerves or directly exert biological effects on related cells involved in nerve regeneration. Therefore, this type of nerve repair material still needs to be improved to further enhance the nerve repair effect of the catheter stent.

[0008] Therefore, a new technical solution is needed to solve the above technical problems. Summary of the invention

[0009] The purpose of the present invention is to provide a hydrogen and magnesium ion pump catheter with a Janus structure for peripheral nerve defect repair and a preparation method thereof. The rationally designed nerve stent can use magnesium-based materials as H 2 and Mg 2+ The release source provides biochemical clues and also carries appropriate biophysical clues to improve the effect of the stent in repairing nerve damage.

[0010] The technical solution adopted by the present invention is: A hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects. The pump catheter consists of a hydrophilic inner layer, a hydrophobic outer layer and a reservoir middle layer. The hydrophilic inner layer and the hydrophobic outer layer are made of degradable polymer materials, and the reservoir middle layer is made of a mixture of one or more of polylactic acid, polyglycolide or polylactic acid-glycolic acid and a magnesium-based material.

[0011] Preferably, the degradable polymer material is a degradable polymer material of artificial synthetic origin, such as polycaprolactone, poly-L-lactide-caprolactone, polylactic acid, polyglycolide or polylactic acid-glycolic acid, or a degradable polymer material of biological origin, such as chitosan, silk fibroin or collagen, and the materials of the hydrophilic inner layer and the hydrophobic outer layer are one or a mixture of several of the above substances.

[0012] Preferably, the magnesium-based material is one or more of magnesium, magnesium hydride, and magnesium-containing alloys.

[0013] Preferably, the pump catheter can be used for the repair and regeneration of organs and tissues in vivo such as Achilles tendon tissue, spinal cord tissue, blood vessels, esophagus, trachea and bronchi.

[0014] By adopting the above structure, the hydrophilic inner layer improves the cell compatibility of the catheter and promotes the adhesion and migration of nerve cells. The hydrophobic outer layer blocks the invasion of adverse cells such as fibroblasts into the damaged site. The middle layer of the magnesium ion and hydrogen storage can continuously release hydrogen and magnesium ions to exert biological effects.

[0015] Preferably, a method for preparing a hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects comprises the following steps: Step 1: Preparation of the hydrophilic inner layer: dissolving the degradable polymer material in a solvent, transferring the solution into a syringe, and performing electrospinning to make the material hydrophilic by its own properties, or coating the polymer catheter with a hydrophilic polymer coating, plasma treatment, chemical treatment, or nanomaterial modification to obtain a hydrophilic inner layer; Step 2: preparing the intermediate layer, dissolving one or more of polylactic acid, polyglycolide or polylactic-co-glycolic acid in a solution, adding a magnesium-based material and mixing evenly, transferring the mixture into a syringe, and performing electrospinning; Step 3: Preparation of a hydrophobic outer layer, dissolving the biodegradable polymer material in a solvent, transferring the solution into a syringe, and performing electrospinning, using the hydrophobic properties of the material itself, or performing chemical treatment and surface coating with a hydrophobic layer to make the outer layer hydrophobic, to obtain a three-layer structure catheter, i.e., a hydrogen and magnesium ion pump catheter with a Janus structure.

[0016] By adopting the above process, when the pump catheter is used, the magnesium-based material reacts with water to generate H 2 and Mg(OH) 2 , the acidic degradation products of the middle polymer material neutralize Mg(OH) 2 Ionization produces OH − ions and decompose Mg(OH) 2 , which enables the reaction between magnesium-based materials and water to proceed, and also promotes Mg 2+ This process enables the middle layer to continuously release H like a pump. 2 and Mg 2+ .

[0017] Preferably, in step 1 and step 3, the molecular weight of the degradable polymer in the degradable polymer material is 10,000 to 500,000, preferably 20,000 to 200,000, such as 30,000, 50,000, or 120,000; the solution concentration is 5 to 30% w / w, preferably 5% w / w, 10% w / w, or 15% w / w.

[0018] Preferably, in step 2, the molecular weight of the degradable polymer is 10,000 to 500,000, preferably 30,000 to 300,000, such as 70,000, 150,000, or 200,000; the solution concentration is 5 to 30% w / w, preferably 5% w / w, 10% w / w, or 15% w / w; the mass ratio of the magnesium-based material to the polymer material is 0.5:100 to 30:100, preferably 2:100 to 20:100, such as 5:100 or 10:100.

[0019] Preferably, in step 1, step 2 and step 3, the conditions for electrospinning are: using a 10~30G needle, preferably 15-25G, such as 18G, 20G, 21G; the distance from the needle to the receiver is 10~30 cm, preferably 10~20 cm, such as 15cm, 18cm; the flow rate is 0.05~1 mm / min, preferably 0.1~0.5 mm / min, such as 0.15 mm / min, 0.2 mm / min, 0.3 mm / min; the voltage is +10~30 kV and -1~10 kV, preferably, +15~25 kV and -2~8 kV, such as +18kV and -4 kV, +20 kV and -4 kV; the diameter of the receiving rod is 0.5~30 mm, preferably 1~20 mm, such as 1.5 mm, 2 mm, 5 mm, 10 mm. The spinning time of step 1, step 2 and step 3 is 10 to 300 minutes, preferably 20 to 200 minutes, such as 30 minutes, 60 minutes, 120 minutes. The operation is carried out at room temperature, and the humidity is 10 to 80%, preferably 15 to 50%, such as 30% and 40%.

[0020] The H released by the pump catheter produced by the above process 2 It can effectively remove harmful free radicals, reduce inflammation, and help create a good microenvironment for nerve regeneration. The Mg released by the pump catheter 2+ It can reduce cellular oxidative stress and promote Schwann cells to express neurotrophic factors, playing a biological role that is beneficial to nerve regeneration.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. The pump catheter of the present invention has a Janus structure. The hydrophilic inner layer improves the cell compatibility of the catheter and promotes the adhesion and migration of nerve cells. The hydrophobic outer layer blocks the adhesion and invasion of unfavorable cells such as fibroblasts to the damaged site. The middle layer of the magnesium ion and hydrogen reservoir can continuously release hydrogen and magnesium ions to play a biological role and promote nerve regeneration.

[0022] 2. The rationally designed neural stent of the present invention can use magnesium-based materials as H 2 and Mg 2+ The release source provides biochemical clues and also carries appropriate biophysical clues to improve the effect of the stent in repairing nerve damage.

[0023] 3. H released from the pump catheter of the present invention 2 It can effectively remove harmful free radicals, reduce inflammation, and help create a good microenvironment for nerve regeneration. The Mg released by the pump catheter 2+ It can reduce cellular oxidative stress and promote Schwann cells to express neurotrophic factors, playing a biological role that is beneficial to nerve regeneration.

[0024] 4. The pump catheter of the present invention showed enhanced functional, electrophysiological and histological recovery in the rat sciatic nerve defect model, highlighting the potential of Janus Mg 2+ / H 2 Pump catheters are a promising approach for peripheral nerve defect repair by providing a favorable microenvironment to promote nerve regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a SEM image of the pump catheter of the present invention; Figure 2 The water contact angles of the inner PCL layer and the outer PCL@PDA layer of the pump catheter of the present invention; Figure 3 The morphology of RSC96 cells on the PCL and PCL@PDA membranes of the pump catheter of the present invention; Figure 4 The pump catheter of the present invention has MgH in 28 days. 2 @PLGA membrane and MgH 2 Accumulated Mg of particles 2+ Release amount; Figure 5 The pump catheter of the present invention is MgH in 7 days. 2 @PLGA membrane and MgH 2 Particle H 2 Release curve; Figure 6 is the ABTS free radical scavenging rate of the pump catheter of the present invention; Figure 7 is the hydroxyl radical scavenging rate of the pump catheter of the present invention; Figure 8 Statistical analysis of the mean fluorescence intensity ratio of JC-1 aggregates to JC-1 monomers; Fig. 9 Relative mRNA expressions of iNOS, TNF-α, IL-6, and IL-1β in RAW 264.7 cells cultured with different media; Fig.10 Relative mRNA expressions of NGF, c-Jun, BDNF, and P75 in RSC96 cells cultured with different media; Fig.11 Immunostaining of NF200 and S100β in longitudinal sections of nerve samples. b) Length of axons in the duct; Fig.12 Electrophysiological assessment of CMAP amplitude for the three groups; Fig.13Macroscopic image of the gastrocnemius muscle. The left side is the affected side. Wet weight ratio of the gastrocnemius muscle (affected side to unaffected side); Fig.14 a) Masson staining of gastrocnemius muscle, b) mean minimum Feret diameter; Fig.15 Immunofluorescence staining of neuronal NF200 and S00β after 8 weeks of repair; Fig.16 a) TEM image of remyelinated axons, b) number of myelinated axons in the region of interest (ROI), c) thickness of myelin sheath.

[0026] Data are presented as mean ± SEM. Statistical analysis was performed using GraphPad Prism 8. Unpaired Student's t-test, one-way analysis of variance (ANOVA) followed by Tukey's test, p values ​​less than 0.05 were considered significant. The significance levels were set as follows: p < 0.05, p < 0.01, **p < 0.001, ****p < 0.0001. DETAILED DESCRIPTION Example

[0027] A hydrogen and magnesium ion pump catheter with a Janus structure for peripheral nerve defect repair, the pump catheter is composed of a hydrophilic inner layer, a hydrophobic outer layer and a reservoir middle layer, the hydrophilic inner layer is polydopamine-coated polycaprolactone (PCL), the hydrophobic outer layer is a PCL outer layer, and the reservoir middle layer is MgH 2 @PLGA middle layer.

[0028] A method for preparing a hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects, comprising the following steps: Step 1: Preparation of the hydrophilic inner layer: PCL with a molecular weight of 30,000 was dissolved in dichloromethane / dimethylformamide with a volume ratio of 7:3 to a concentration of 10% w / w. The solution was transferred to a 10 mL syringe for electrospinning. The electrospinning conditions were: a 21G needle, a needle-to-receiver distance of 20 cm, a flow rate of 0.1 mm / min, a voltage of +18 kV and -4 kV, a receiving rod diameter of 2 mm, a spinning time of 30 minutes, and operation at room temperature with a humidity of 40-50%. Then, the PCL tube was coated with polydopamine (PDA) to obtain PCL@PDA. The operation of PCL tube coating with polydopamine (PDA) was as follows: the PCL tube was immersed in Tris-HCl buffer containing 2 mg / mL dopamine hydrochloride for 16 hours, Tris-HCl buffer 10 mM, pH=8.5, and the PCL@PDA was washed with deionized water and freeze-dried; Step 2: Preparation of the intermediate layer: insert a 1.5 mm receiving rod into the center of the PCL@PDA tube, dissolve PLGA with a molecular weight of 70,000 in hexafluoroisopropanol at a concentration of 15% w / w, and add MgH 2 After the particles were mixed, they were transferred to a 10 mL syringe for electrospinning. The electrospinning conditions were as follows: an 18G needle was used, the distance from the needle to the receiver was 15 cm, the flow rate was 0.2 mm / min, the voltage was +20 kV and -4 kV, the spinning time was 40 min, and the operation was performed at room temperature with a humidity of 40-50%; Step 3: PCL with a molecular weight of 30,000 was dissolved in dichloromethane / dimethylformamide with a volume ratio of 7:3 to a concentration of 10% w / w. The solution was transferred to a 10 mL syringe for electrospinning. The electrospinning conditions were: a 21G needle, a needle-to-receiver distance of 20 cm, a flow rate of 0.1 mm / min, a voltage of +18 kV and -4 kV, a spinning time of 1 hour, and operation at room temperature with a humidity of 40%. A three-layer structured catheter was obtained, i.e., a hydrogen and magnesium ion pump catheter with a Janus structure. Example

[0029] A hydrogen and magnesium ion pump catheter with a Janus structure for peripheral nerve defect repair, the pump catheter is composed of a hydrophilic inner layer, a hydrophobic outer layer and a reservoir middle layer, the hydrophilic inner layer is polydopamine-coated polycaprolactone (PCL), the hydrophobic outer layer is a PCL outer layer, and the reservoir middle layer is MgH 2 @PLGA middle layer.

[0030] A method for preparing a hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects, comprising the following steps: Step 1: Preparation of the hydrophilic inner layer: PCL with a molecular weight of 40,000 was dissolved in dichloromethane / dimethylformamide with a volume ratio of 7:3 at a concentration of 15% w / w. The solution was transferred to a 10 mL syringe for electrospinning. The electrospinning conditions were: a 21G needle, a needle-to-receiver distance of 20 cm, a flow rate of 0.1 mm / min, a voltage of +18 kV and -4 kV, a receiving rod diameter of 2 mm, a spinning time of 30 minutes, and operation at room temperature with a humidity of 40%. The PCL tube was then coated with polydopamine (PDA) to obtain PCL@PDA. The operation of PCL tube coating with polydopamine (PDA) was as follows: the PCL tube was immersed in Tris-HCl buffer containing 2 mg / mL dopamine hydrochloride for 16 hours. The Tris-HCl buffer was 10 mM, pH = 8.5. The PCL@PDA was washed with deionized water and freeze-dried. Step 2: Preparation of the intermediate layer: PLGA with a molecular weight of 100,000 was dissolved in hexafluoroisopropanol at a concentration of 20% w / w, and Mg and MgH2O with a mass ratio of 0.5:100 were added. 2 After mixing, the mixture was transferred to a syringe for electrospinning. The electrospinning conditions were as follows: a 20G needle was used, the distance from the needle to the receiver was 20 cm, the flow rate was 0.3 mm / min, the voltage was +25 kV and -6 kV, the spinning time was 80 min, and the operation was performed at room temperature with a humidity of 40%; Step 3: Preparation of the hydrophobic outer layer. Dissolve PCL with a molecular weight of 40,000 in the solution at a concentration of 15% w / w. Transfer the solution to a syringe for electrospinning. The electrospinning conditions are: use a 21G needle, a needle-to-receiver distance of 20 cm, a flow rate of 0.1 mm / min, a voltage of +18 kV and -4 kV, a spinning time of 1 hour, operate at room temperature, and a humidity of 40%. A three-layer structured catheter, i.e., a hydrogen and magnesium ion pump catheter with a Janus structure, was obtained. A three-layer structured catheter, i.e., a hydrogen and magnesium ion pump catheter with a Janus structure was obtained. Example

[0031] A hydrogen and magnesium ion pump catheter with a Janus structure for peripheral nerve defect repair, the pump catheter is composed of a hydrophilic inner layer, a hydrophobic outer layer and a reservoir middle layer, the hydrophilic inner layer is polydopamine-coated polycaprolactone (PCL), the hydrophobic outer layer is a PCL outer layer, and the reservoir middle layer is MgH 2 @PLGA middle layer.

[0032] A method for preparing a hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects, comprising the following steps: Step 1: Preparation of the hydrophilic inner layer: PCL with a molecular weight of 50,000 was dissolved in dichloromethane / dimethylformamide with a volume ratio of 7:3 to a concentration of 10% w / w. The solution was transferred to a 10 mL syringe for electrospinning. The electrospinning conditions were: a 21G needle, a needle-to-receiver distance of 20 cm, a flow rate of 0.1 mm / min, a voltage of +18 kV and -4 kV, a receiving rod diameter of 2 mm, a spinning time of 30 minutes, and operation at room temperature with a humidity of 40-50%. Then, the PCL tube was coated with polydopamine (PDA) to obtain PCL@PDA. The operation of PCL tube coating with polydopamine (PDA) was as follows: the PCL tube was immersed in Tris-HCl buffer containing 2 mg / mL dopamine hydrochloride for 16 hours. The Tris-HCl buffer was 10 mM, pH=8.5. The PCL@PDA was washed with deionized water and freeze-dried. Step 2: Preparation of the intermediate layer: insert a 1.5 mm receiving rod into the center of the PCL@PDA tube, dissolve PLGA with a molecular weight of 100,000 in hexafluoroisopropanol at a concentration of 15% w / w, and add MgH 2 After the particles were mixed, they were transferred to a 10 mL syringe for electrospinning. The electrospinning conditions were as follows: an 18G needle was used, the distance from the needle to the receiver was 15 cm, the flow rate was 0.2 mm / min, the voltage was +20 kV and -4 kV, the spinning time was 40 min, and the operation was performed at room temperature with a humidity of 40-50%; Step 3: PCL with a molecular weight of 50,000 was dissolved in dichloromethane / dimethylformamide with a volume ratio of 7:3 to a concentration of 10% w / w. The solution was transferred to a 10 mL syringe for electrospinning. The electrospinning conditions were: a 21G needle, a needle-to-receiver distance of 20 cm, a flow rate of 0.1 mm / min, a voltage of +18 kV and -4 kV, a spinning time of 1 hour, and operation at room temperature with a humidity of 40%. A three-layer structured catheter was obtained, i.e., a hydrogen and magnesium ion pump catheter with a Janus structure.

[0033] The pump catheter made by the process of the above-mentioned embodiment 1 of the present invention was observed by SEM at 20 kV. The SEM showed that the catheter had a three-layer structure and an inner diameter of 2 mm. As shown in FIG1 , the stress-strain curve of the catheter was tested by a dynamic mechanical analyzer at room temperature at a strain rate of 20% / min and a strain range of 0-150%. The PLGA catheter and 10% MgH 2 The ultimate stress of PLGA conduits was 4.62 ± 0.05 MPa and 4.99 ± 0.21 MPa, respectively. 2 The increase in the ultimate stress of PLGA catheters is due to the incorporation of MgH 2 The outer PCL layer, the middle MgH 2 Water contact angles of the outer PCL membrane and the middle PLGA membrane are hydrophobic with water contact angles exceeding 100 degrees. On the contrary, the inner layer becomes hydrophilic due to the polydopamine coating with a contact angle less than 90 degrees, as shown in Figure 2. On the PCL membrane, RSC96 cells (a type of Schwann cell) appear more rounded, while on the PCL@PDA membrane, the cells tend to spread out, as shown in Figure 3.

[0034] Janus 2+ / H 2 Mg for pump catheter 2+ and H2 Release behavior investigation: Mg 2+ Release behavior: The membrane was immersed in 1X PBS at a 20 mg / mL membrane-solution ratio and an equal amount of MgH 2 The pellet was also immersed in an equal volume of 1X PBS, and 10% of the release solution was removed at each time point and replaced with fresh 1X PBS. 2+ Detection kit quantifies accumulated Mg 2+ Release amount, release curve shows that MgH 2 The microparticle group generates Mg(OH) 2 Poor solubility in water, unable to release Mg continuously 2+ In contrast, MgH 2 @PLGA membranes showed sustained Mg over 28 days 2+ Release, and the released Mg 2+ The amount of MgH 2 The particle content increases, as shown in Figure 4; H 2 Release behavior: MgH 2 The PLGA membrane was immersed in 1X PBS in a sealed bottle at a membrane-solution ratio of 2 mg / mL, with an equal amount of MgH 2 The particles were also immersed in 1X PBS, and as shown in Figure 5, the membrane exhibited similar 2 Particle H 2 The release behavior was rapid in the first two days, then the release rate slowed down and reached almost 100% on the 7th day.

[0035] In summary, this example demonstrates that MgH 2 @PLGA membrane can act as a pump to continuously release Mg 2+ and H 2 .

[0036] Janus 2+ / H 2 The free radical scavenging properties of hydrogen released from the pump catheter: Free Radical Scavenging Test: For the •OH scavenging test, the assay solution consisted of equal parts of 6 mM salicylic acid solution (dissolved in ethanol) and 6 mM FeSO 4 and 6 mM HO 2 O 2 The aqueous solution was composed of PLGA membrane and MgH 2@PLGA membrane, the final mass-volume ratio was 2 mg / mL, and the absorbance at 510 nm was measured every day using a microplate reader. For the ABTS free radical scavenging test, 7 mM ABTS solution was reacted with 2.45 mM potassium persulfate in the dark for 16 h, and then diluted three times with 1X PBS to obtain the assay solution, to which PLGA membrane and MgH 2 @PLGA membrane to a final mass-volume ratio of 2 mg / mL, and the absorbance at 734 nm was measured every day using a microplate reader.

[0037] The results showed that H released from the membrane 2 The proportion of scavenging •OH and ABTS radicals increased with time, e.g. Figure 6 , Figure 7 shown.

[0038] Janus 2+ / H 2 Protective effect of pump catheter on RSC96 cells under oxidative stress conditions: Preparation of extracts: Complete medium consisting of 10% FBS, 0.5% penicillin-streptomycin, 25 mM HEPES and basal medium was used to coat the PLGA membrane and MgH 2 PLGA membranes were immersed in this medium at a mass-volume ratio of 2 mg / mL for 48 h, and the extracts were collected and filtered through a 0.22 μm membrane. 2+ Assay kit to quantify MgH 2 @PLGA releases Mg in solution and complete medium 2+ concentration, MgCl 2 Add to complete medium to match MgH 2 @Mg in PLGA extract 2+ For cell experiments.

[0039] JC-1 staining: RSC96 cells were seeded in the wells and cultured overnight. The medium was replaced with complete medium, membrane extract solution, and Mg 2+ Complete medium was supplemented with or without the addition of 100 μM H 2 O 2After 48 hours, the cells were stained with a JC-1 staining kit and observed using a laser confocal microscope. When the mitochondrial membrane potential was high, JC-1 formed red fluorescent J aggregates (Ex / Em=585 / 590 nm). On the contrary, when the membrane potential was low, JC-1 remained as a green fluorescent monomer (Ex / Em=510 / 527 nm). The decrease in mitochondrial membrane potential, as an early sign of apoptosis, was reflected in the red-green fluorescence ratio. Statistical analysis showed that compared with the untreated group, the PLGA extract (p = 0.0016) and the control group (p = 0.0008) had a significantly higher red-green fluorescence ratio in the H 2 O 2 The red-green fluorescence ratio is significantly reduced during treatment, such as Figure 8 As shown, four H 2 O 2 In the treatment group, MgH 2 @PLGA membrane extract and Mg 2+ The red-green fluorescence ratio of the supplemented medium group was significantly higher than that of the PLGA and control groups (p<0.01), indicating that Mg 2+ It indeed alleviates oxidative damage by stabilizing mitochondrial membrane potential.

[0040] Overall, this example demonstrates that Mg 2+ It protects RSC96 cells under oxidative stress by stabilizing mitochondrial membrane potential.

[0041] Janus 2+ / H 2 The pump catheter regulates inflammation and promotes the secretion of neurotrophic factors by Schwann cells: RAW 264.7 cells were seeded in 12-well plates and cultured for 12 h. The medium was then replaced with fresh medium containing 100 ng / mL LPS for 12 h. Subsequently, the medium was replaced with complete medium, membrane extract solution, and Mg supplemented. 2+ The cells were cultured for another 24 h, and total RNA was extracted for qRT-PCR analysis. -ΔΔCt Methods Relative mRNA expression was calculated, with GAPDH as the control. The primer sequences are shown in Table 1. After LPS stimulation, the relative mRNA expression levels of all four factors increased significantly, especially IL-6 (>1000-fold) and IL-1β (>300-fold). Fig. 9 Compared with the control group, Mg 2+ The supplemented groups showed higher relative IL-6 and TNF-α mRNA expression levels, while MgH 2@PLGA membrane extracts significantly reduced the expression of these two markers, MgH 2 @PLGA membrane extract and Mg 2+ The relative IL-1β mRNA expression levels were significantly reduced in the supplemented medium groups. 2 @PLGA membrane extract-treated cells showed lower relative iNOS mRNA expression, but no significant difference from other LPS-treated groups.

[0042] In general, Mg 2+ It seems to affect the expression of various pro-inflammatory factors through different mechanisms, which may involve multiple signaling pathways. 2 It exerts anti-inflammatory effects by inhibiting the expression of pro-inflammatory factors.

[0043] Schwann cells are components of the peripheral nervous system. They play a vital role in the formation of myelin and are essential for maintaining the environment for axonal growth. After peripheral nerve injury, Schwann cells rapidly proliferate, differentiate, and secrete various protein molecules, thereby promoting nerve self-repair and regeneration. 2+ and H 2 Effects on Schwann cells were assessed by qRT-PCR using extracts and Mg 2+ Expression of repair-related genes in RSC96 cells cultured with supplemented medium RSC96 cells were seeded in 12-well plates and cultured for 12 h, and then the medium was replaced with complete medium, membrane extract solution, and Mg supplemented. 2+ The cells were cultured for another 48 hours, and total RNA was extracted for qRT-PCR analysis. -ΔΔCt Methods Relative mRNA expression was calculated, with GAPDH as a control. Table 1: qRT-PCR primer sequences

[0044] The experimental results show that Fig.10 As shown, compared with PLGA membrane extracts, MgH 2 @PLGA membrane extract and Mg 2+ Supplementation of the medium significantly enhanced the relative mRNA expression of P75, which limits distal atrophy after nerve injury, and in addition, in the presence of MgH 2In cells cultured with PLGA membrane extracts, we observed an increase in the mRNA expression of the transcription factor c-Jun, which plays a key role in activating the Schwann cell repair program to support regeneration, although this increase was not statistically significant. The relative mRNA expression of neurotrophic factors, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), was also increased in the presence of MgH 2 @PLGA membrane extract and Mg 2+ Supplementation of the culture medium increases the number of cells cultured.

[0045] In summary, this example shows that MgH 2 H released from PLGA membrane 2 Has anti-inflammatory effects, while MgH 2 Mg released from PLGA membrane 2+ Promote RSC96 cells to secrete more neurotrophic factors.

[0046] Janus 2+ / H 2 Evaluation of nerve repair effect after pump catheter bridging nerve stumps 4 weeks later: Animals and surgical procedures: Twenty-seven male Sprague-Dawley rats (6-8 weeks old) were randomly divided into three groups: autologous transplantation group, PLGA catheter group, and MgH 2 In the PLGA catheter group, rats were anesthetized with isoflurane, the lower back was shaved and disinfected with povidone-iodine, and a subcutaneous incision of approximately 4-5 cm was made along the right femur to find the gluteus maximus and biceps femoris. The sciatic nerve was exposed through the gluteal cleft, and 10 mm of the sciatic nerve was removed with surgical scissors. For the PLGA catheter group and MgH 2 In the PLGA catheter group, a 14 mm catheter was used to bridge the gap by suturing a 2 mm nerve segment with 8-0 nylon suture. In the autologous transplantation group, the resected nerve was turned over and sutured back with 8-0 suture, the muscle was sutured with 6-0 nylon suture, and the skin was sutured with 4-0 nylon suture and disinfected with povidone-iodine.

[0047] After 4 weeks, the nerve grafts were harvested, the outer and middle layers of the conduits were removed, the samples were embedded in Tissue-TekO.CT compound and frozen with liquid nitrogen, and the longitudinal sections were subjected to immunofluorescence staining to determine the expression of NF200 and S100β, and DAPI was used to stain the nuclei. The images showed that the autologous graft group showed a well-aligned structure, with NF200 and S100β expression extending from the proximal end to the distal end, and the conduits were filled with matrix, e.g. Fig.11 a. In MgH 2In the @PLGA catheter group, axon extension was observed in the middle of both the proximal and distal ends, whereas in the PLGA catheter group, extension was only seen in the proximal end. 2 The length of NF200-positive axons in the @PLGA conduit group was longer than that in the PLGA conduit group (9.9 ± 1.3 mm vs 6.3 ± 0.6 mm). Fig.11 As shown in b.

[0048] The above results show that the MgH 2 @PLGA conduits create a microenvironment that is conducive to axonal extension.

[0049] Janus 2+ / H 2 Evaluation of the repair effect of pump catheter bridging the nerve stump 8 weeks later: Gastrocnemius electrophysiology: At 8 weeks postoperatively, the rats were anesthetized with isoflurane, the surgical site was reopened, the gastrocnemius muscle was exposed, two electrodes were inserted 2 mm and 4 mm proximal to the catheter, two electrodes were implanted at the origin of the Achilles tendon, and a ground electrode was inserted at the midpoint between the stimulating electrode and the recording electrode. Subsequently, 5 mA, 0.1 ms square wave pulses were applied to the sciatic nerve, and CMAPs were recorded using a biosignal acquisition and analysis system. The PLGA catheter group showed the lowest compound muscle action potential (CMAP) amplitude (0.22 ± 0.11 mV), indicating poor nerve conduction, as shown in Figure 3 . Fig.12 In contrast, the autologous transplantation group and MgH 2 The @PLGA catheter group showed higher CMAP amplitudes, 0.53±0.16 mV and 0.74±0.19 mV, respectively, reflecting better nerve conduction.

[0050] The rats were euthanized and the gastrocnemius muscles on both sides were collected. Macroscopically, the gastrocnemius muscles on the injured side in the three groups experienced significant atrophy due to loss of innervation after nerve injury, as shown in Figure 13a. 2 The muscle weight ratio of the @PLGA catheter group was higher than that of the PLGA catheter group, as shown in Figure 13b, which corresponds to its better electrophysiological performance.

[0051] Masson staining was performed on the gastrocnemius muscles on both sides. We analyzed the diameter and distribution of muscle fibers. The diameter of muscle fibers in the autologous transplantation group ranged from 15 to 25 μm. In contrast, the distribution of the catheter group shifted to the left. The diameter of the PLGA catheter group was mainly between 5 and 10 μm, while the diameter of the MgH 2 The diameter of the @PLGA catheter group ranged from 5 to 15 μm, as shown in Figure 14a.2 The average muscle fiber diameter of the PLGA catheter group was significantly larger than that of the PLGA catheter group (p = 0.0405). Fig.14 As shown in b.

[0052] The regenerated nerve grafts were harvested and the nerve tissues were cross-sectioned into 15 μm thick slices for immunofluorescence staining of NF-200 (1:200) and S100β (1:400) to mark axons and myelin sheaths, respectively. The autologous transplanted nerves were denser, while the regenerated nerves in the conduits appeared looser, as shown in Figure 2. Fig.15 shown.

[0053] The regenerated nerves were fixed with 2.5% glutaraldehyde, embedded in resin, and cross-sectioned into ultrathin sections with a thickness of 70 nm. The sections were observed using TEM, and the diameter of myelinated axons and the thickness of myelin sheaths were quantified using ImageJ software. Irregular black circles represent regenerated myelin sheaths, e.g. Fig.16 The autologous transplant group showed the largest number of myelinated axons, surrounded by the thickest myelin sheath, with an average thickness of 0.59±0.02 μm. Fig.16 b. MgH 2 The myelination of regenerated axons in the PLGA catheter group was superior to that in the PLGA catheter group, both in terms of myelin thickness (0.43±0.04 μm vs 0.32±0.02 μm) and the number of myelinated axons (9±1 / ROI vs 5±1 / ROI). Fig.16 b and 16c.

[0054] Overall, this example shows that from MgH 2 Mg released from PLGA catheters 2+ and H 2 Effectively promotes nerve regeneration and myelination.

[0055] Conclusion: In conclusion, the Janus Mg 2+ / H 2 The innovative design of the pump catheter and its favorable microenvironment have the potential to achieve effective peripheral nerve repair. The hydrophilic inner layer promotes cell adhesion, while the hydrophobic outer layer prevents cell penetration. MgH 2 @PLGA catheters function effectively as pumps, continuously releasing Mg 2+ and H 2 , thereby regulating the regenerative microenvironment, and importantly, H 2 Selectively scavenges ROS and reduces inflammation, and Mg 2+MgH plays a key role in protecting cells under oxidative stress and promoting the transformation of Schwann cells into a pro-regenerative state, as evidenced by animal functional and histological recovery results. 2 @PLGA conduits significantly enhanced nerve repair.

[0056] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by those skilled in the art should be included in the protection scope determined by the claims of the present invention.

Claims

1. A hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects, characterized in that: The pump catheter is composed of a hydrophilic inner layer, a hydrophobic outer layer and a reservoir middle layer. The hydrophilic inner layer and the hydrophobic outer layer are made of degradable polymer materials, and the reservoir middle layer is made of a mixture of one or more of polylactic acid, polyglycolide or polylactic acid-glycolic acid and a magnesium-based material.

2. The hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects according to claim 1, characterized in that: The degradable polymer material is a degradable polymer material of artificial synthesis origin, such as polycaprolactone, poly-L-lactide-caprolactone, polylactic acid, polyglycolide or polylactic acid-glycolic acid, or a degradable polymer material of biological origin, such as chitosan, silk fibroin or collagen, and the materials of the hydrophilic inner layer and the hydrophobic outer layer are one or a mixture of several of the above substances.

3. The hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects according to claim 1, characterized in that: The magnesium-based material is one or more of magnesium, magnesium hydride, and magnesium-containing alloy.

4. The hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects according to claim 1, characterized in that: The pump catheter can be used for the repair and regeneration of organs and tissues in vivo such as Achilles tendon tissue, spinal cord tissue, blood vessels, esophagus, trachea and bronchus.

5. A method for preparing a hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects, characterized in that: The following steps are involved: Step 1: Preparation of the hydrophilic inner layer: dissolving the degradable polymer material in a solvent, transferring the solution into a syringe, and performing electrospinning to make the material hydrophilic by its own properties, or coating the polymer catheter with a hydrophilic polymer coating, plasma treatment, chemical treatment, or nanomaterial modification to obtain a hydrophilic inner layer; Step 2: preparing the intermediate layer, dissolving one or more of polylactic acid, polyglycolide or polylactic-co-glycolic acid in a solution, adding a magnesium-based material and mixing evenly, transferring the mixture into a syringe, and performing electrospinning; Step 3: Preparation of a hydrophobic outer layer, dissolving the biodegradable polymer material in a solvent, transferring the solution into a syringe, and performing electrospinning, using the hydrophobic properties of the material itself, or performing chemical treatment and surface coating with a hydrophobic layer to make the outer layer hydrophobic, to obtain a three-layer structure catheter, i.e., a hydrogen and magnesium ion pump catheter with a Janus structure.

6. The method for preparing a hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects according to claim 5, characterized in that: In the step 1, the molecular weight of the degradable polymer in the degradable polymer material is 10,000 to 500,000, and the solution concentration is 5 to 30% w / w.

7. The method for preparing a hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects according to claim 5, characterized in that: In the step 1, the conditions for electrospinning are: using a 10~30G needle, the distance from the needle to the receiver is 10~30 cm, the flow rate is 0.05~1 mm / min, the voltage is +10~30 kV and -1~10 kV, the diameter of the receiving rod is 0.5~30 mm, the spinning time is 10~300 minutes, the operation is carried out at room temperature, and the humidity is 10~80%.

8. The method for preparing a hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects according to claim 5, characterized in that: In the step 2, the molecular weight of the degradable polymer is 10,000 to 500,000, the concentration of the polymer solution is 5 to 30% w / w, and the mass ratio of the magnesium-based material to the polymer material is 0.5:100 to 30:

100.

9. The method for preparing a hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects according to claim 5, characterized in that: In the step 2, the conditions for electrospinning are: using a 10-30G needle, the distance from the needle to the receiver is 10-30 cm, the flow rate is 0.05-1 mm / min, the voltage is +10-30 kV and -1-10 kV, the spinning time is 10-300 minutes, the operation is carried out at room temperature, and the humidity is 10-80%.

10. The method for preparing a hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects according to claim 5, characterized in that: In the step 3, the molecular weight of the degradable polymer is 10,000 to 500,000, the solution concentration is 5 to 30% w / w, and the conditions for electrospinning are: using a 10 to 30G needle, the distance from the needle to the receiver is 10 to 30 cm, the flow rate is 0.05 to 1 mm / min, the voltage is +10 to 30 kV and -1 to 10 kV, the diameter of the receiving rod is 0.5 to 30 mm, the spinning time is 10 to 300 minutes, the operation is carried out at room temperature, and the humidity is 10 to 80%.

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