A hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects and a preparation method thereof

Abstract

The present invention relates to the field of nerve repair technology, and discloses a hydrogen and magnesium ion pump catheter with a Janus structure for peripheral nerve defect repair and a preparation method thereof. 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-glycolic acid and a magnesium-based material. The pump catheter has a Janus structure, the hydrophilic inner layer provides biophysical clues, 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 area. The magnesium ion and hydrogen reservoir middle layer can continuously release hydrogen and magnesium ions, exerting biological effects, improving the nerve regeneration microenvironment, and improving the effect of the stent in repairing nerve damage.

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 injury is commonly caused by trauma and tumor resection, severely impairing motor and sensory function. 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 has 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 basic biochemical or biophysical cues required to create a favorable regenerative microenvironment. The microenvironment that controls peripheral nerve regeneration is the result of a dynamic interplay of biochemical and biophysical signals, which influence tissue regeneration and cellular behavior. Therefore, nerve conduits that incorporate appropriate biophysical and biochemical cues have the potential to significantly enhance nerve regeneration.

[0003] Oxidative stress at the injury site is a hallmark of the neural regeneration microenvironment, primarily caused by excessive reactive oxygen species (ROS). It damages cellular structures, including lipids, proteins, and DNA. Excessive ROS exacerbate neural damage and delay repair. Hydrogen (H2) has been found to be a therapeutic medical gas with anti-inflammatory properties. H2 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. Furthermore, H₂ is nontoxic even at high concentrations, highlighting its potential for safe therapeutic applications. However, H₂'s high diffusivity, low water solubility, and dose-dependent therapeutic effects limit its application. Achieving localized H₂ delivery remains a significant challenge.

[0004] Magnesium-based materials, such as magnesium (Mg), magnesium hydride (MgH2), and magnesium-containing alloys, react with water to release H2 and are ideal sources of local H2 release. In addition, magnesium-based materials can also promote nerve regeneration as a source of magnesium ions. This is because Mg 2+ Essential for neuronal survival and function; it is involved in the formation of membrane phospholipids, signal transduction, myelin sheath and synapse formation, and regulates neurotransmitters such as dopamine and serotonin. Oral Mg 2+Supplements have 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 release of hydrogen by the reaction between magnesium-based materials and water; and the ionization of Mg(OH)2 to form hydroxide ions will cause local alkalinity and poor cell compatibility. In addition, Mg(OH)2 is poorly soluble in water and cannot provide enough magnesium ions to exert biological effects. Therefore, although magnesium (Mg), magnesium hydride (MgH2), magnesium-containing alloys and other magnesium-based materials can provide potential biochemical clues for nerve regeneration: H2 and Mg 2+ , promoting nerve regeneration, but its application still faces great difficulties.

[0005] Traditional catheter materials are often hydrophobic, resulting in poor cytocompatibility. Therefore, improving their hydrophilicity can improve their biocompatibility. On the other hand, catheter materials must maintain a certain degree of hydrophobicity to prevent fibroblasts and other cells from adhering to the catheter and invading the injury site. Therefore, it is necessary to rationally design catheter materials to optimize their biophysical cues and promote nerve regeneration.

[0006] Patent application publication number CN114699560A discloses a double-layer tubular product for promoting regeneration of damaged nerves. The product comprises a tubular outer layer and a tubular inner layer adhered to the inner wall. The tubular outer layer is formed from a human-compatible biodegradable material, while the tubular inner layer is a hydrogel layer formed from polyethylene glycol. The human-compatible biodegradable material is selected from the group consisting of polylactic acid or its derivatives, gelatin and its derivatives, chitosan, polyurethane, polycaprolactone, water-insoluble celluloses and their derivatives, etc. The polylactic acid derivative is selected from the group consisting of polylactic acid-polyethylene glycol copolymers, lactic acid-co-glycolic acid copolymers (PLGA), PLGA-polyethylene glycol copolymers, lactic acid-co-caprolactone copolymers (PCLA), and PCLA-polyethylene glycol copolymers. The weight ratio of the tubular outer layer to the tubular inner layer is 100:20-100, and the thickness of the tubular wall of the double-layer tubular product is 50-500 μm. The double-layer tubular product of the present invention exhibits the technical effects described in the specification.

[0007] The catheter in this 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, which can use magnesium-based materials as H2 and Mg2+ pumps and a rationally designed nerve stent. 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 in the present invention is:

[0011] 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-glycolic acid and a magnesium-based material.

[0012] 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.

[0013] Preferably, the magnesium-based material is one or more of magnesium, magnesium hydride, and magnesium-containing alloys.

[0014] Preferably, the pump catheter can be used for the repair and regeneration of organs and tissues in the body such as Achilles tendon tissue, spinal cord tissue, blood vessels, esophagus, trachea and bronchi.

[0015] 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.

[0016] 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:

[0017] 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 utilize the hydrophilic nature of the material, or coating the polymer catheter with a hydrophilic polymer coating, plasma treatment, chemical treatment, or nanomaterial modification to obtain a hydrophilic inner layer;

[0018] Step 2: Preparation of 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;

[0019] Step 3: Preparation of the hydrophobic outer layer: dissolving the degradable polymer material in a solvent, transferring the solution into a syringe, and performing electrospinning. The hydrophobic nature of the material itself is utilized, or chemical treatment or surface coating with a hydrophobic layer is performed to make the outer layer hydrophobic, thereby obtaining a three-layer structure catheter, i.e., a hydrogen and magnesium ion pump catheter with a Janus structure.

[0020] When the pump catheter produced by the above process is used, the magnesium-based material reacts with water to generate H2 and Mg(OH)2, and the acidic degradation products of the middle polymer material neutralize the OH generated by the ionization of Mg(OH)2. − 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 H2 and Mg as a pump. 2+ .

[0021] 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.

[0022] 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.

[0023] 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, or 120 minutes. The operation is carried out at room temperature and the humidity is 10 to 80%, preferably 15 to 50%, such as 30% or 40%.

[0024] The H2 released by the pump catheter produced by the above process 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 the expression of neurotrophic factors in Schwann cells, thereby playing a biological role that is beneficial to nerve regeneration.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 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 adverse 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, exert biological effects, and promote nerve regeneration.

[0027] 2. The rationally designed neural stent of the present invention can use magnesium-based materials as H2 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.

[0028] 3. The H2 released by the pump catheter of the present invention can effectively remove harmful free radicals, reduce inflammation, and help create a good microenvironment for nerve regeneration. The Mg released by the pump catheter can effectively remove harmful free radicals, reduce inflammation, and help create a good microenvironment for nerve regeneration. 2+ It can reduce cellular oxidative stress and promote the expression of neurotrophic factors in Schwann cells, thereby playing a biological role that is beneficial to nerve regeneration.

[0029] 4. The pump catheter of the present invention showed enhanced functional, electrophysiological and histological recovery in the rat sciatic nerve defect model, highlighting the Janus Mg 2+ / H2 pump catheters serve as a promising approach for peripheral nerve defect repair by providing a favorable microenvironment to promote nerve regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a SEM image of the pump catheter of the present invention;

[0031] 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;

[0032] Figure 3 The morphology of RSC96 cells on the PCL and PCL@PDA membranes of the pump catheter of the present invention;

[0033] Figure 4 The cumulative Mg of MgH2@PLGA membrane and MgH2 particles in the pump catheter of the present invention within 28 days is 2+ Release amount;

[0034] Figure 5 H2 release curves of the MgH2@PLGA membrane and MgH2 microparticles of the pump catheter of the present invention within 7 days;

[0035] Figure 6 is the ABTS free radical scavenging rate of the pump catheter of the present invention;

[0036] Figure 7 is the hydroxyl radical scavenging rate of the pump catheter of the present invention;

[0037] Figure 8 Statistical analysis of the mean fluorescence intensity ratio of JC-1 aggregates to JC-1 monomers;

[0038] Figure 9 Relative mRNA expressions of iNOS, TNF-α, IL-6, and IL-1β in RAW 264.7 cells cultured with different media;

[0039] Figure 10 Relative mRNA expressions of NGF, c-Jun, BDNF, and P75 in RSC96 cells cultured with different media;

[0040] Figure 11 Immunostaining of NF200 and S100β in longitudinal sections of nerve samples. b) Length of axons in the conduit;

[0041] Figure 12Electrophysiological assessment of CMAP amplitude for the three groups;

[0042] Figure 13 Macroscopic images of the gastrocnemius muscle. The affected side is on the left. Gastrocnemius muscle wet weight ratio (affected to unaffected side);

[0043] Figure 14 a) Masson staining of gastrocnemius muscle, b) mean minimum Feret diameter;

[0044] Figure 15 Immunofluorescence staining of neural NF200 and S00β after 8 weeks of repair;

[0045] Figure 16 a) TEM image of remyelinated axons, b) number of myelinated axons in the region of interest (ROI), and c) thickness of the myelin sheath.

[0046] Data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism 8. Unpaired Student's t-test and one-way analysis of variance (ANOVA) followed by Tukey's test were used. P values ​​less than 0.05 were considered significant. The following significance levels were set: p < 0.05, p < 0.01, **p < 0.001, ****p < 0.0001. DETAILED DESCRIPTION Example

[0047] A hydrogen and magnesium ion pump catheter with a Janus structure for peripheral nerve defect repair. The pump catheter consists 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 a MgH2@PLGA intermediate layer.

[0048] A method for preparing a hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects comprises the following steps:

[0049] Step 1: Preparation of the hydrophilic inner layer: PCL with a molecular weight of 30,000 was dissolved in dichloromethane / dimethylformamide (7:3 by volume) at a concentration of 10% w / w. The solution was transferred to a 10 mL syringe for electrospinning. The electrospinning conditions were as follows: a 21G needle, a needle-to-receiver distance of 20 cm, a flow rate of 0.1 mm / min, voltages 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%. The PCL tube was then coated with polydopamine (PDA) to obtain PCL@PDA. 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.

[0050] Step 2: Preparation of the intermediate layer: A 1.5 mm receiving rod was inserted into the center of the PCL@PDA tube. PLGA (molecular weight 70,000) was dissolved in hexafluoroisopropanol at a concentration of 15% w / w. MgH2 particles were added at a mass ratio of 10:100 and mixed. The mixture was then transferred into a 10 mL syringe for electrospinning. The electrospinning conditions were: an 18G needle, a needle-to-receiver distance of 15 cm, a flow rate of 0.2 mm / min, voltages of +20 kV and -4 kV, a spinning time of 40 minutes, and operation at room temperature with a humidity of 40-50%.

[0051] 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

[0052] A hydrogen and magnesium ion pump catheter with a Janus structure for peripheral nerve defect repair. The pump catheter consists 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 a MgH2@PLGA intermediate layer.

[0053] A method for preparing a hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects comprises the following steps:

[0054] Step 1: Preparation of the hydrophilic inner layer: PCL with a molecular weight of 40,000 was dissolved in dichloromethane / dimethylformamide (7:3 by volume) at a concentration of 15% w / w. The solution was transferred to a 10 mL syringe for electrospinning. The electrospinning conditions were as follows: a 21G needle, a needle-to-receiver distance of 20 cm, a flow rate of 0.1 mm / min, voltages 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 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.

[0055] 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. Mg and MgH2 at a mass ratio of 0.5:100 were added and mixed. The mixture was then transferred to a syringe for electrospinning. The electrospinning conditions were: a 20G needle, a needle-to-receiver distance of 20 cm, a flow rate of 0.3 mm / min, voltages of +25 kV and -6 kV, a spinning time of 80 minutes, and operation at room temperature and 40% humidity.

[0056] Step 3: Preparation of the hydrophobic outer layer. PCL with a molecular weight of 40,000 was dissolved in a solution with a concentration of 15% w / w. The solution was transferred to a 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, 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

[0057] A hydrogen and magnesium ion pump catheter with a Janus structure for peripheral nerve defect repair. The pump catheter consists 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 a MgH2@PLGA intermediate layer.

[0058] A method for preparing a hydrogen and magnesium ion pump catheter with a Janus structure for repairing peripheral nerve defects comprises the following steps:

[0059] Step 1: Preparation of the hydrophilic inner layer: PCL with a molecular weight of 50,000 was dissolved in dichloromethane / dimethylformamide (7:3 by volume) at a concentration of 10% w / w. The solution was transferred to a 10 mL syringe for electrospinning. The electrospinning conditions were as follows: a 21G needle, a needle-to-receiver distance of 20 cm, a flow rate of 0.1 mm / min, voltages 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%. The PCL tube was then coated with polydopamine (PDA) to obtain PCL@PDA. 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.

[0060] Step 2: Preparation of the intermediate layer: A 1.5 mm receiving rod was inserted into the center of the PCL@PDA tube. PLGA (molecular weight: 100,000) was dissolved in hexafluoroisopropanol at a concentration of 15% w / w. MgH2 particles (mass ratio: 10:100) were added and mixed. The mixture was then transferred into a 10 mL syringe for electrospinning. The electrospinning conditions were: an 18G needle, a needle-to-receiver distance of 15 cm, a flow rate of 0.2 mm / min, voltages of +20 kV and -4 kV, a spinning time of 40 minutes, and operation at room temperature with a humidity of 40-50%.

[0061] 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.

[0062] The pump catheter produced according to the process of Example 1 of the present invention was observed using a scanning electron microscope (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 measured using a dynamic mechanical analyzer at room temperature at a strain rate of 20% / min and a strain range of 0-150%. The ultimate stresses of the PLGA catheter and the 10%MgH2@PLGA catheter were 4.62 ± 0.05 MPa and 4.99 ± 0.21 MPa, respectively. The increase in the ultimate stress of the MgH2@PLGA catheter was due to the incorporation of MgH2 particles, which increased the diameter of the PLGA fibers and enhanced the mechanical strength of the catheter. The water contact angles of the outer PCL layer, the middle MgH2@PLGA layer, and the inner PCL@PDA layer were measured using a video optical contact angle meter. The outer PCL and middle PLGA membranes are hydrophobic, with water contact angles exceeding 100 degrees. In contrast, 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.

[0063] Janus Mg 2+ Mg / H2 pump conduit 2+ And H2 release behavior investigation:

[0064] Mg 2+ Release behavior: The membrane was immersed in 1X PBS at a ratio of 20 mg / mL to solution. An equal amount of MgH2 particles was also immersed in an equal volume of 1X PBS. At each time point, 10% of the release solution was removed and replaced with fresh 1X PBS. 2+ Detection kit quantifies accumulated Mg 2+ Release amount, release curve shows that the MgH2 microparticle group cannot release Mg continuously due to the poor solubility of the generated Mg(OH)2 in water. 2+ In contrast, the MgH2@PLGA membrane showed sustained Mg 2+ Released, and the released Mg 2+ The amount increases with the increase of MgH2 particle content in the fiber, as shown in Figure 4;

[0065] H2 release behavior: The MgH2@PLGA membrane was immersed in 1X PBS in a sealed bottle at a membrane-solution ratio of 2 mg / mL. An equal amount of MgH2 particles was also immersed in 1X PBS. As shown in Figure 5, the membrane exhibited H2 release behavior similar to that of MgH2 particles, with rapid release in the first two days, followed by a slowing release rate that reached almost 100% on the 7th day.

[0066] In summary, this example demonstrates that the MgH2@PLGA membrane can act as a pump to continuously release Mg 2+ and H2.

[0067] Janus Mg 2+ / H2 The free radical scavenging performance of hydrogen released from the pump catheter:

[0068] Free radical scavenging test: For the •OH scavenging test, the assay solution consisted of equal amounts of 6 mM salicylic acid solution (dissolved in ethanol) and 6 mM FeSO4 and 6 mM H2O2 aqueous solutions. PLGA membranes and MgH2@PLGA membranes were added to a final mass-to-volume ratio of 2 mg / mL, and the absorbance at 510 nm was measured daily 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 hours and then diluted threefold with 1X PBS to obtain the assay solution. PLGA membranes and MgH2@PLGA membranes were added to a final mass-to-volume ratio of 2 mg / mL, and the absorbance at 734 nm was measured daily using a microplate reader.

[0069] The results showed that the ratio of •OH and ABTS radicals scavenged by H2 released from the membrane increased with time, e.g. Figure 6 、 Figure 7 shown.

[0070] Janus Mg 2+ Protective effect of H2 pump catheter on RSC96 cells under oxidative stress conditions:

[0071] Preparation of extracts: PLGA membranes and MgH2@PLGA membranes were immersed in complete medium consisting of 10% FBS, 0.5% penicillin-streptomycin, 25 mM HEPES, and basal medium for 48 h at a mass-volume ratio of 2 mg / mL. The extracts were collected and filtered through a 0.22 μm membrane. 2+ Detection kit to quantify Mg in MgH2@PLGA release solution and complete culture medium 2+ MgCl2 was added to the complete culture medium to match the Mg concentration in the MgH2@PLGA extract. 2+ For cell experiments.

[0072] JC-1 staining: RSC96 cells were seeded in the wells and cultured overnight. The culture medium was replaced with complete culture medium, membrane extract solution, and Mg2+. 2+Complete culture medium was supplemented with or without the addition of 100 μM H2O2. After 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). In contrast, 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 the red-green fluorescence ratio of the PLGA extract (p = 0.0016) and the control group (p = 0.0008) was significantly reduced when treated with H2O2 compared with the untreated group. Figure 8 As shown, in the four H2O2 treated groups, MgH2@PLGA membrane extracts and Mg 2+ The red-green fluorescence ratio of the supplemented culture 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.

[0073] Overall, this example demonstrates that Mg 2+ It protects RSC96 cells under oxidative stress by stabilizing mitochondrial membrane potential.

[0074] Janus Mg 2+ / H2 pump catheter regulates inflammation and promotes Schwann cells to secrete neurotrophic factors:

[0075] 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 and analyzed by qRT-PCR. -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). Figure 9 Compared with the control group, Mg 2+ The supplemented groups showed higher relative IL-6 and TNF-α mRNA expression levels, while MgH2@PLGA membrane extract significantly reduced the expression of these two markers. 2+The supplemented culture medium groups all showed significantly reduced relative IL-1β mRNA expression levels, and cells treated with MgH2@PLGA membrane extract showed lower relative iNOS mRNA expression, but no significant difference was found with other LPS-treated groups.

[0076] In general, Mg 2+ It seems that H2 affects the expression of various proinflammatory factors through different mechanisms, which may involve multiple signaling pathways. H2 exerts anti-inflammatory effects by inhibiting the expression of proinflammatory factors.

[0077] 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+ The effects of H2 on Schwann cells were evaluated 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, after which 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

[0078]

[0079] The experimental results show that Figure 10 As shown, compared with PLGA membrane extracts, MgH2@PLGA membrane extracts and Mg 2+ Supplementation of the culture medium significantly enhanced the relative mRNA expression of P75, which limits distal atrophy after nerve injury. In addition, 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, in cells cultured with MgH2@PLGA membrane extracts, 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 cells cultured with MgH2@PLGA membrane extracts and Mg 2+ Supplementation of culture medium increases cells in culture.

[0080] In conclusion, this example shows that H2 released from MgH2@PLGA membrane has anti-inflammatory effect, while Mg released from MgH2@PLGA membrane has 2+ Promote RSC96 cells to secrete more neurotrophic factors.

[0081] Janus Mg 2+ Evaluation of nerve repair effect after bridging nerve stumps with H2 pump catheter 4 weeks later:

[0082] Animals and surgical procedures: Twenty-seven male Sprague-Dawley rats (6-8 weeks old) were randomly divided into three groups: autologous transplantation group, PLGA conduit group, and MgH2@PLGA conduit group. The rats were anesthetized with isoflurane, and the lower back was shaved and disinfected with povidone-iodine. A subcutaneous incision of approximately 4-5 cm was made along the right femur. The gluteus maximus and biceps femoris muscles were found, and the sciatic nerve was exposed through the gluteal fissure. A 10-mm segment of the sciatic nerve was removed with surgical scissors. For the PLGA conduit group and the MgH2@PLGA conduit group, a 14-mm conduit was used to bridge the gap by suturing a 2-mm nerve segment with 8-0 nylon suture. In the autologous transplantation group, the removed nerve was flipped 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.

[0083] 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. Longitudinal sections were subjected to immunofluorescence staining to determine the expression of NF200 and S100β. 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, as shown in Figure 5. Figure 11 As shown in a. In the MgH2@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. The length of NF200-positive axons in the MgH2@PLGA catheter group was longer than that in the PLGA catheter group (9.9 ± 1.3 mm vs 6.3 ± 0.6 mm). Figure 11 As shown in b.

[0084] The above results indicate that the MgH2@PLGA conduit constructed in the present invention creates a microenvironment that is conducive to axonal extension.

[0085] Janus Mg 2+ Evaluation of the repair effect of H2 pump catheter bridging nerve stumps 8 weeks later:

[0086] Gastrocnemius electrophysiology: At 8 weeks after surgery, 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 biological signal 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. Figure 12 In contrast, the autologous transplantation group and the MgH2@PLGA catheter group showed higher CMAP amplitudes of 0.53±0.16 mV and 0.74±0.19 mV, respectively, reflecting better nerve conduction.

[0087] Rats were euthanized and the gastrocnemius muscles were harvested from both sides. Macroscopically, the gastrocnemius muscles on the injured side in all three groups experienced significant atrophy due to loss of innervation following nerve injury, as shown in Figure 13a. Weighing revealed that the muscle to weight ratio was higher in the MgH2@PLGA conduit group than in the PLGA conduit group, as shown in Figure 13b, corresponding to their improved electrophysiological properties.

[0088] Masson staining was performed on the gastrocnemius muscles on both sides, and we analyzed the diameter and distribution of muscle fibers. The diameter of the muscle fibers in the autologous transplant group ranged from 15 to 25 μm. In contrast, the distribution in the conduit group shifted to the left. The diameters of the PLGA conduit group were mainly between 5 and 10 μm, while those in the MgH2@PLGA conduit group were between 5 and 15 μm, as shown in Figure 14a. The average muscle fiber diameter in the MgH2@PLGA conduit group was significantly larger than that in the PLGA conduit group (p = 0.0405). Figure 14 As shown in b.

[0089] The regenerated nerve grafts were harvested and the nerve tissue was 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 more compact, while the regenerated nerves in the conduit appeared looser, as shown in Figure 2. Figure 15 shown.

[0090] 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, as shown in Figure 16As shown in a. 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. Figure 16 As shown in b, the myelin sheath formation of regenerated nerve axons in the MgH2@PLGA conduit group was superior to that in the PLGA conduit group, both in terms of myelin sheath 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). Figure 16 b and 16c.

[0091] Overall, this example demonstrates that Mg released from MgH2@PLGA catheters 2+ and H2 effectively promoted nerve regeneration and myelination.

[0092] Conclusion: In conclusion, the Janus Mg 2+ The innovative design of the H2 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. The MgH2@PLGA catheter effectively functions as a pump, continuously releasing Mg. 2+ and H2, thereby regulating the regenerative microenvironment. Importantly, H2 selectively scavenges ROS and reduces inflammation, and Mg 2+ It plays a key role in protecting cells under oxidative stress and promoting the transformation of Schwann cells into a pro-regenerative state. Animal functional and histological recovery results demonstrated that MgH2@PLGA conduits significantly enhanced nerve repair.

[0093] 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 scope of protection determined by the claims of the present invention.

Claims

1. A hydrogen and magnesium ion pump catheter with a Janus structure for peripheral nerve defect repair, characterized by: 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 peripheral nerve defect repair 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 peripheral nerve defect repair according to claim 1, characterized in that: The magnesium-based material is one or more of magnesium, magnesium hydride, and magnesium-containing alloys.

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 repairing and regenerating organs and tissues in the body such as Achilles tendon tissue, spinal cord tissue, blood vessels, esophagus, trachea and bronchi.

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 utilize the hydrophilic nature of the material, 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: Preparation of 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 the hydrophobic outer layer: dissolving the degradable polymer material in a solvent, transferring the solution into a syringe, and performing electrospinning. The hydrophobic nature of the material itself is utilized, or chemical treatment or surface coating with a hydrophobic layer is performed to make the outer layer hydrophobic, thereby obtaining 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 step 1, the electrospinning conditions are as follows: using a 10-30G needle, a distance from the needle to the receiver of 10-30 cm, a flow rate of 0.05-1 mm / min, a voltage of +10-30 kV and -1-10 kV, a receiving rod diameter of 0.5-30 mm, the spinning time of 10-300 minutes, operating at room temperature, and a humidity of 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 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 step 2, the electrospinning conditions are as follows: using a 10-30G needle, a distance from the needle to the receiver of 10-30 cm, a flow rate of 0.05-1 mm / min, a voltage of +10-30 kV and -1-10 kV, the spinning time of 10-300 minutes, operating at room temperature, and a humidity of 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 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 electrospinning conditions are: using a 10 to 30G needle, a distance from the needle to the receiver of 10 to 30 cm, a flow rate of 0.05 to 1 mm / min, a voltage of +10 to 30 kV and -1 to 10 kV, a receiving rod diameter of 0.5 to 30 mm, the spinning time is 10 to 300 minutes, the operation is performed at room temperature, and the humidity is 10 to 80%.

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