A donor of nitric oxide that releases slowly in response to reactive oxygen species cascade and a nerve repair catheter based thereon

Abstract

The present invention relates to a nitric oxide donor that slowly releases nitric oxide in response to an active oxygen cascade and a nerve repair conduit based thereon. The nitric oxide donor that slowly releases nitric oxide in response to an active oxygen cascade has the following structural formula: wherein m=5-30, n=14-238; the nitric oxide donor that slowly releases nitric oxide in response to an active oxygen cascade provided by the present invention can release nitric oxide under an active oxygen cascade response, eliminate local excess active oxygen, and reduce the release of nitric oxide as the concentration of active oxygen decreases, thereby realizing the on-demand release of nitric oxide, greatly improving the immune microenvironment of nerve regeneration, and promoting the structural and functional recovery of damaged nerves.

Description

Technical Field

[0001] The present invention belongs to the technical field of prosthetic materials or prosthetic coating materials, and particularly relates to a donor of nitric oxide that slowly releases in response to an active oxygen cascade and a nerve repair catheter based thereon. Background Art

[0002] Peripheral nerve injuries are commonly caused by congenital defects, accidents, surgical complications, and other traumatic events, accounting for 2-5% of traumatic cases. Severe cases can lead to motor and sensory impairments, resulting in long-term disability and significant economic and psychosocial burdens. Due to the complex structure and function of peripheral nerve tissue and the difficulty in treatment, peripheral nerve repair remains a major clinical challenge. Currently, the standard clinical intervention for injured nerve gaps smaller than a few millimeters is direct tension-free end-to-end suturing. However, for larger gaps (>5 mm in adults), autologous transplantation remains the gold standard. However, limited donor resources, secondary injury, size mismatch, and neuroma formation contribute to poor treatment outcomes, necessitating the urgent need for alternative strategies to alleviate this dilemma. Artificial nerve grafts (nerve conduits) are an alternative approach to repairing peripheral nerve gaps and have made considerable research progress in recent years. However, this field still faces challenges in rapidly regulating the neural regenerative microenvironment, directing the migration of regenerative cells, and ensuring precise axonal docking with the target organ, resulting in poor neurological recovery and unsatisfactory clinical outcomes. Therefore, how to construct a nerve conduit that can improve the regenerative microenvironment and promote the rapid and directional migration of nerve regeneration-related cells is of great significance for the structural and functional repair of peripheral nerves.

[0003] After peripheral nerve injury, it is inevitable to face the problem of oxidative stress, which is one of the important factors of the regenerative microenvironment. Peripheral nerve injury causes bleeding at the lesion site, retraction of nerve endings and inflammatory response, which will cause a large amount of reactive oxygen species to be produced and aggregated. Endogenous antioxidants such as glutathione can actively regulate the production of reactive oxygen species and maintain the balance of the microenvironment to a certain extent. However, the expression of related proteins caused by damage may be disturbed and cannot be fully restored, resulting in the oxidative stress level in the regenerative microenvironment still being too high. Studies have shown that consuming excessive reactive oxygen species to inhibit oxidative stress and inflammatory response can regulate the nerve regeneration microenvironment and accelerate the structural and functional recovery of peripheral nerves [Xianzhen Dong et al. An injectable and adaptable hydrogen sulfide delivery system formodulating neuroregenerative microenvironment. Sci. Adv. 9, eadi1078 (2023)].

[0004] Research has found that nitric oxide (NO) participates in multiple physiological functions, including wound healing, neurotransmission, and immune response regulation, and exhibits bactericidal activity against various bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). It was the first gasotransmitter discovered by Furchgott, Ignarro, and Murad. Its production in vivo primarily depends on NO synthase (NOS), which converts L-arginine into NO and L-citrate. There are three NOS isoforms: neuronal NOS (NOS1, nNOS), inducible NOS (NOS2, iNOS), and endothelial NOS (NOS3, eNOS), each of which plays a role in distinct physiological and pathological processes. NO produced by eNOS plays a crucial role in regulating vasodilation. Through a cGMP-mediated signaling pathway, it exerts antiproliferative effects on smooth muscle cells and maintains cardiovascular health by regulating vasodilation and blood pressure. However, the biological effects of NO are closely dose-dependent. An appropriate NO concentration has a positive regulatory effect. Excessively low concentrations will not achieve therapeutic effects, while excessively high concentrations can easily produce toxic side effects. In addition, a too fast release rate can also cause rapid accumulation of NO, leading to adverse consequences such as inflammatory reactions. Therefore, how to achieve a controlled release of NO with "on-demand administration" of NO donors is the key to adaptively regulating the peripheral nerve regeneration microenvironment with NO, thereby accelerating peripheral nerve regeneration and functional recovery.

[0005] CN116942896A discloses a conductive biomimetic double-porous electrospun fiber scaffold for promoting the regeneration of long-segment peripheral nerve defects. This scaffold is constructed by loading a self-assembled nanocomposite material (BP-BNN6) of black phosphorus (BP) and the photosensitive nitric oxide (NO) donor N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine (BNN6) onto an oriented double-porous scaffold. However, the nitric oxide donor used in this scheme is a nitroso-type nitric oxide donor, which lacks long-term stability and is prone to decomposition or conversion. Furthermore, its release mechanism in response to near-infrared light irradiation does not allow for "on-demand" release based on the actual microenvironment of the injury site, nor does it provide feedback regulation. This can easily lead to nitric oxide accumulation, resulting in neurotoxicity and inflammatory reactions. Therefore, developing a donor capable of precise and controllable NO release is a bottleneck in peripheral nerve repair. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to address the above-mentioned deficiencies in the prior art and provide a donor of nitric oxide that slowly releases in response to reactive oxygen species cascade and a nerve repair catheter based thereon. The donor has stable chemical properties at room temperature, and the nerve repair catheter prepared based thereon can release nitric oxide in response to reactive oxygen species, eliminate local excess reactive oxygen species, inhibit macrophage inflammatory response, accelerate the vascularization process of human umbilical vein endothelial cells, reduce oxidative damage to Schwann cells and enhance the production of the energy supply substance adenosine triphosphate (ATP), greatly improving the immune microenvironment of nerve regeneration, promoting the structural and functional recovery of damaged nerves, and having good biocompatibility and degradability. The preparation method and synthesis process are simple.

[0007] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0008] Provided is a donor of nitric oxide that responds to an active oxygen cascade and releases slowly, the structural formula of which is as follows:

[0009] ;

[0010] Among them, m=5~30, n=14~238.

[0011] The present invention also includes a method for preparing the above-mentioned donor of nitric oxide that releases in response to active oxygen cascade, and the specific steps are as follows:

[0012] 1) Synthesis of 4-(chloromethyl) benzyl methacrylate (4-CBM): A mixture of methacryloyl chloride and anhydrous dichloromethane (DCM) was slowly added dropwise to a mixture of 4-(chloromethyl)benzyl alcohol, dichloromethane, and triethylamine (TEA). The solution was stirred in the dark at room temperature for 12-48 hours. 4-CBM was post-treated to obtain an oily product.

[0013] 2) Synthesis of 4-((nitrooxy)methyl)benzylmethacrylate (4-NMBM): Dissolve 4-(chloromethyl)benzylmethacrylate obtained in step 1) and silver nitrate in tetrahydrofuran (THF). Stir the solution in the dark for 8-48 hours. Filter the solution using a fritted funnel to remove the residue. Purify the filtrate to obtain liquid 4-((nitrooxy)methyl)benzylmethacrylate.

[0014]

[0015] 3) Synthesis of 3,3'-(propane-2,2-diylbis(sulfanediyl)) dipropanoic acid (TK-COOH): 3-Mercaptopropionic acid, anhydrous acetone, and trifluoroacetic acid (TFA, to provide an acidic environment) were mixed and stirred at room temperature (15-35°C) for 3-10 hours. The mixture was then cooled in an ice-water bath to allow crystallization. The resulting crystalline product was filtered and purified to obtain 3,3'-(propane-2,2-diylbis(sulfanediyl)) dipropanoic acid as a white powder.

[0016]

[0017] 4) Synthesis of 5,5,15-trimethyl-9,14-dioxo-10,13-dioxa-4,6-dithiohexa-15-enoic acid (TK-HEMA): 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid was dissolved in dichloromethane, and dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) were added. After activation, hydroxyethyl methacrylate (HEMA) was added and stirred for 12-48 hours. The insoluble matter was filtered out using a G4 fritted funnel, and the filtrate was concentrated under reduced pressure. 5,5,15-trimethyl-9,14-dioxo-10,13-dioxa-4,6-dithiohexa-15-enoic acid was obtained by column chromatography.

[0018]

[0019]

[0020] 5) NO Donor (PTKNO) Synthesis: 5,5,15-trimethyl-9,14-dioxo-10,13-dioxa-4,6-dithiohexadecene-15-enoic acid (from step 4) was added to a reaction flask along with 4-((nitrooxy)methyl)benzyl methacrylate (from step 2), mPEG-BCSPA, azobisisobutyronitrile (AIBN), and N,N-dimethylformamide (DMF). After deoxygenation, RAFT polymerization was performed. After completion of the reaction, the solution was added to diethyl ether for precipitation to yield the product, mPEG-P[(TK-HEMA)-(4-NMBM)], abbreviated as PTKNO.

[0021] According to the above scheme, the mass volume ratio of methacryloyl chloride to anhydrous dichloromethane in the mixed solution of methacryloyl chloride and anhydrous dichloromethane in step 1) is 1g:5~200mL.

[0022] According to the above scheme, the molar ratio of methacryloyl chloride to 4-(chloromethyl)benzyl alcohol in step 1) is 0.2-5:1.

[0023] According to the above scheme, in the mixed solution of 4-(chloromethyl)benzyl alcohol, dichloromethane and triethylamine in step 1), the molar ratio of 4-(chloromethyl)benzyl alcohol to triethylamine is 1:0.2~1, and the molar volume ratio of 4-(chloromethyl)benzyl alcohol to dichloromethane is 1mmol:5~20mL.

[0024] According to the above scheme, in step 2), the molar ratio of 4-(chloromethyl)benzyl methacrylate to silver nitrate is 1:1-3, and the mass volume ratio of 4-(chloromethyl)benzyl methacrylate to tetrahydrofuran is 1 g:10-50 mL.

[0025] According to the above scheme, the molar ratio of 3-mercaptopropionic acid to anhydrous acetone in step 3) is 1:0.5~4, and the molar volume ratio of 3-mercaptopropionic acid to trifluoroacetic acid is 1 mol:20~60 mL.

[0026] According to the above scheme, the mass volume ratio of 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid and dichloromethane in step 4) is 1 g: 10-40 mL.

[0027] According to the above scheme, the molar ratio of 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid to dicyclohexylcarbodiimide and 4-dimethylaminopyridine in step 4) is 1:1-3:0.1-0.5.

[0028] According to the above scheme, the molar ratio of 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid to hydroxyethyl methacrylate in step 4) is 1:1-5.

[0029] According to the above scheme, the molar ratio of 5,5,15-trimethyl-9,14-dioxo-10,13-dioxa-4,6-dithiohexadecenoic acid to 4-((nitrooxy)methyl)benzyl methacrylate, mPEG-BCSPA, and azobisisobutyronitrile in step 5) is 5-20:5-20:1:0.1-0.5.

[0030] According to the above scheme, the RAFT polymerization reaction conditions in step 5) are: reaction at 60-80°C for 8-24 h.

[0031] The present invention also includes a nerve repair catheter based on the above-mentioned donor of nitric oxide that responds to the reactive oxygen species cascade and releases slowly, which includes a hollow catheter skeleton and a hydrogel filled inside the catheter skeleton, wherein the donor of nitric oxide that responds to the reactive oxygen species cascade and releases slowly is evenly distributed inside the hydrogel.

[0032] According to the above solution, the hollow catheter skeleton is obtained by spinning biodegradable polymer fibers. The catheter skeleton provides the main mechanical support during the nerve repair process, helps transport nutrients and discharge metabolic waste.

[0033] According to the above scheme, the biodegradable polymer includes any one or more combinations of L-alanine-caprolactone copolymer, gelatin-caprolactone copolymer, lactide-caprolactone copolymer, L-alanine-glycolic acid-lactic acid copolymer, lactic acid-glycolic acid copolymer, and lactic acid-glycolic acid-silk fibroin copolymer, and the biodegradable polymer can be spun using an electrospinning process. Ideally, the degradation process of the biodegradable polymer can be aligned with the nerve repair process. Different degrees of damage result in different repair times, so the degradation rate of the copolymer can be selected to suit different application scenarios.

[0034] According to the above scheme, the inner diameter of the catheter skeleton lumen is 1-5 mm, the tube wall thickness is 0.5-1.5 mm, and the catheter length is 10-50 mm.

[0035] According to the above scheme, the hydrogel is polymerized from spherical microgel modules with uniform particle size. These modules fill 40-80% of the hollow catheter framework by volume. The hydrogel of the present invention, composed of spherical microgel modules, has a significantly increased specific surface area compared to conventional hydrogels, significantly improving nutrient exchange, oxygen transport, and adhesion of regenerative cells during nerve regeneration.

[0036] According to the above scheme, the spherical microgel modules have a particle size of 100-200 μm and a light response wavelength range of 365-410 nm. The spaces between the spherical microgel modules and the pores within the spherical microgel modules create a multi-level interconnected porous structure within the nerve repair conduit, providing a large specific surface area for the adhesion of cells related to nerve regeneration and providing sufficient space for nerve regeneration.

[0037] According to the above scheme, the particle size of the donor for sustained release of nitric oxide in response to reactive oxygen species cascade is 10-450 nm. These donor nanoparticles can release nitric oxide in a cascade response to reactive oxygen species (ROS) for a long time, reducing local ROS concentrations, inhibiting oxidative damage to nerves, significantly downregulating the expression of inflammatory factors in regenerated nerves, and upregulating the expression of antioxidant enzymes, vascular endothelial growth factor, and genes related to cellular energy metabolism. This actively regulates the immune microenvironment of nerve regeneration and promotes the structural and functional recovery of damaged nerves.

[0038] Another object of the present invention is to provide a method for preparing the above-mentioned nerve repair catheter, comprising the following steps:

[0039] 1) Dissolving a biodegradable polymer in an organic solvent to obtain an electrospinning solution, and then electrospinning to obtain a hollow catheter skeleton;

[0040] 2) dissolving a nitric oxide donor in tetrahydrofuran to obtain a donor solution, then slowly adding the resulting donor solution dropwise to a PBS buffer solution under ultrasonic vibration, removing the tetrahydrofuran by vacuum distillation, and then filtering through a 0.45 μm pore size aqueous filter membrane to remove aggregates, thereby obtaining a nitric oxide donor dispersion for sustained release in response to an active oxygen species cascade;

[0041] 3) preparing a PBS solution of a hydrogel precursor, adding the dispersion of the reactive oxygen species cascade response sustained-release nitric oxide donor obtained in step 2) and mixing the mixture to obtain a mixed solution, loading the solution into a syringe, and injecting the solution into a silicone tube flowing with paraffin wax under near-infrared light irradiation (the purpose of the near-infrared light irradiation is to prevent the microgel droplets from solidifying before UV curing, thereby avoiding needle clogging) to produce spherical microdroplets. Irradiating the outlet of the silicone tube with UV light to solidify the microdroplets to form microgels, and removing the paraffin wax to obtain spherical microgel modules.

[0042] 4) injecting the spherical microgel module obtained in step 3) into the hollow catheter skeleton obtained in step 1), and irradiating the microgel module with ultraviolet light to further cross-link the microgel module to obtain a nerve repair catheter that responds to the reactive oxygen species cascade and slowly releases nitric oxide.

[0043] According to the above scheme, the organic solvent in step 1) is one or more of hexafluoroisopropanol, chloroform, acetone, N,N-dimethylformamide, dimethyl sulfoxide, and dichloromethane; and the concentration of the biodegradable polymer in the obtained electrospinning solution is 0.1-0.3 g / mL.

[0044] According to the above scheme, the electrospinning parameters in step 1) are voltage -2.5~12 kV, liquid propulsion rate 0.5~1.5 mL / h, roller receiving distance 8~15 cm, and roller speed 30~250 r / min.

[0045] According to the above scheme, the concentration of the donor solution in step 2) is 0.05-0.2 g / mL, and the volume ratio of tetrahydrofuran to PBS buffer is 1:50-100.

[0046] According to the above scheme, in step 2), the ultrasonic frequency is 30~50kHz and the ultrasonic power is 150~200W.

[0047] According to the above scheme, the hydrogel precursor in step 3) is a mixture of any one or more of gelatin-lipoic acid polymer, gelatin-selenooctanoic acid polymer, gelatin-lipoic acid-selenooctanoic acid polymer, chitosan-lipoic acid polymer, chitosan-selenooctanoic acid polymer, chitosan-lipoic acid-selenooctanoic acid polymer, hyaluronic acid-lipoic acid polymer, hyaluronic acid-selenooctanoic acid polymer, hyaluronic acid-lipoic acid-selenooctanoic acid polymer, polyethylene glycol-lipoic acid polymer, polyethylene glycol-selenooctanoic acid polymer, and polyethylene glycol-lipoic acid-selenooctanoic acid polymer, and its number average molecular weight is 10,000 to 100,000.

[0048] According to the above scheme, the concentration of the PBS solution of the hydrogel precursor in step 3) is 75-150 mg / mL.

[0049] According to the above scheme, the concentration of the nitric oxide donor in the mixed solution in step 3) is 0.05~650 μM, the concentration of the hydrogel precursor is 50~100 mg / mL, the injection speed of the mixed solution into the silicone tube is 0.5~2 mL / h, the wavelength range of the ultraviolet light is 365~410 nm, and the droplet curing time is 5~15 s.

[0050] According to the above scheme, the method for removing the paraffin in step 3) is: washing the microgel with n-hexane and water respectively, and then centrifuging.

[0051] According to the above scheme, in step 4), the wavelength of ultraviolet light irradiation is 365-410 nm, and the ultraviolet light irradiation time is 10-60 s.

[0052] Another object of the present invention is to provide the use of the above-mentioned nerve repair conduit in the preparation of nerve repair materials or tissue engineering scaffold materials.

[0053] The nitric oxide (NO) donor (PTKNO) designed in this invention, which releases nitric oxide in a cascade response to reactive oxygen species (ROS), carries both nitrate and thioketal functional groups. It can recognize and respond to excess ROS molecules in the pathological microenvironment of nerve damage. The specific mechanism of NO release is as follows: In the pathological microenvironment of nerve damage, when ROS levels rise, the thioketal functional group on PTKNO reacts with the excess ROS, releasing mercaptopropionic acid. Mercaptopropionic acid then undergoes a reduction reaction with the nitrate on PTKNO under the action of a specific enzyme to produce NO gas. Therefore, PTKNO only releases NO in a cascade response in the presence of ROS, regulating the regenerative microenvironment. Once NO takes effect, the pathological microenvironment is significantly improved, thereby reducing ROS production through feedback regulation. This in turn reduces the rate of NO production by PTKNO, achieving on-demand release and feedback regulation.

[0054] Compared with the existing technology, the present invention triggers the nitric oxide donor loaded in the nerve conduit through the excessive production of reactive oxygen species in the local microenvironment of peripheral nerve injury, thereby achieving adaptive release of nitric oxide, promoting angiogenesis, inhibiting macrophage inflammatory response and Schwann cell oxidative damage, and reducing reactive oxygen species to avoid excessive release of nitric oxide, achieving on-demand drug administration, and greatly improving the regenerative microenvironment of peripheral nerve injury.

[0055] The nerve repair conduit of the present invention is used to repair peripheral nerve injuries. After implantation, tissue fluid gradually penetrates the injured area. The microgel hydrogel in the conduit comes into contact with the surrounding tissue fluid. The loaded nitric oxide donor (PTKNO) reacts with the ROS, releasing mercaptopropionic acid. Nitrate, triggered by specific intracellular enzymatic processes and thiols, releases NO. NO exerts anti-inflammatory and antioxidant effects, promoting rapid cell migration and reducing excessive ROS production. PTKNO is also chemically stable between -20°C and 50°C. The specific ROS cascade prevents further nitric oxide release, ultimately improving the microenvironment for peripheral nerve regeneration.

[0056] The beneficial effects of the present invention are as follows: 1. The nitric oxide donor provided by the present invention, which releases nitric oxide in a cascade response to reactive oxygen species, can release nitric oxide in response to the reactive oxygen species cascade, scavenging local excess reactive oxygen species. As the reactive oxygen species concentration decreases, the release of nitric oxide decreases, enabling on-demand release of nitric oxide, significantly improving the immune microenvironment for nerve regeneration, and promoting structural and functional recovery of damaged nerves. 2. The preparation method of the present invention has a simple synthesis process and is amenable to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The mPEG-BCSPA prepared in this example 1 H NMR spectrum;

[0058] Figure 2 The 4-CBM synthesized in step 1) and the 4-NMBM synthesized in step 2) of Example 1 are 1 H NMR spectrum;

[0059] Figure 3 The TK-COOH synthesized in step 3) and the TK-HEMA synthesized in step 4) of Example 1 are 1 H NMR spectrum;

[0060] Figure 4 The PTKNO prepared in Example 1 1 H NMR spectrum;

[0061] Figure 5 Gel permeation chromatography (GPC) of PTKNO prepared in Example 1;

[0062] Figure 6 The molecular structure of PTKNO prepared in Example 1 changes with time in the presence of hydrogen peroxide 1 H NMR spectrum;

[0063] Figure 7 This is a labeling diagram of NO released by PTKNO prepared in Example 1 in macrophages;

[0064] Figure 8 The PTKNO prepared in Example 1 was stored at room temperature for 0, 7, and 14 days. 1 H NMR spectrum;

[0065] Figure 9 This is a graph showing the change in the amount of NO released spontaneously by nitrate donors at room temperature over time;

[0066] Figure 10 Transmission electron microscope image and dynamic light scattering (DLS) particle size diagram of PTKNO nanoparticles prepared in Example 2;

[0067] Figure 11 This is a fluorescence image of the microgel module loaded with donor nanoparticles that release nitric oxide in response to active oxygen species prepared in Example 2 after being stained with rhodamine B;

[0068] Figure 12 The microgel module loaded with donor nanoparticles that release nitric oxide in response to active oxygen species prepared in Example 2 forms a complete hydrogel image after ultraviolet light irradiation;

[0069] Figure 13 This is a scanning electron micrograph of a microgel module loaded with donor nanoparticles that release nitric oxide in response to active oxygen species, prepared in Example 2;

[0070] Figure 14 is a graph showing the relationship between the gel transition of the hydrogel precursor solution and the photocrosslinking time in step 3) of Example 2;

[0071] Figure 15 Comparison of stress-strain curves, Young's modulus, and ultimate compressive strain of Gel-LA-SA microgel and microgel hydrogel of Gel-LA-SA hydrogel;

[0072] Figure 16 Schematic diagram of the nerve regeneration process of the nerve repair catheter prepared in Example 2;

[0073] Figure 17 This is a picture of the nerve repair conduit prepared in Example 2 implanted in a 10 mm sciatic nerve defect in a SD rat;

[0074] Figure 18This is a comparative chart of the neurological function recovery evaluation of the three groups of mice with sciatic nerve defects after 12 weeks of treatment in Example 2. DETAILED DESCRIPTION

[0075] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below with reference to the accompanying drawings.

[0076] The gel strength of the gelatin used in the embodiment of the present invention is 300g Bloom.

[0077] The synthesis method of the gelatin-lipoic acid-selenooctanoic acid polymer used in the embodiment of the present invention is as follows:

[0078] 1. Add 2 g (50 mmol) of NaOH to 25 mL of water, then add 3.95 g (50 mmol) of selenium powder and 100 mg (0.274 mmol) of hexadecyltrimethylammonium bromide to obtain solution 1. Slowly add 0.25 g (6.6 mmol) of NaBH4 and 0.2 g (5 mmol) of NaOH to 5 mL of water in an ice-water bath to dissolve and obtain solution 2. Under nitrogen protection, slowly add solution 2 dropwise to solution 1, then react at room temperature for 1 h, then heat to 90 °C and react for 0.5 h to obtain a brown-red Na2Se2 aqueous solution.

[0079]

[0080] 2. Synthesis of selenooctanoic acid: 6.01 g (25 mmol) of ethyl 6,8-dichlorooctanoate was added to a 250 mL three-necked flask, followed by 10 mL of ethanol, 2 g of NaOH (50 mmol) and 38 mL of water. The temperature was raised to 50 °C and the reaction was continued for 2 h. The temperature was further raised to 70 °C and the above-mentioned Na2Se2 aqueous solution was added dropwise. The solution was added after about 2 h. The reaction was continued with stirring for 3 h. After the reaction was completed, the temperature was lowered to 40 °C. 4 g of activated carbon was added to the reaction solution and stirred for 30 min to adsorb unreacted raw materials and impurities. The solution was then filtered while hot and 350 mL of water was added to the filtrate. The solution was cooled to 0 °C while stirring. ℃, then dilute hydrochloric acid solution (concentration 5wt%) was added dropwise to adjust the pH value of the system to 1-2. After a period of time, a large amount of brown solid adhered to the magnetic bar. The supernatant was poured off, and the remaining liquid was vacuum dried to initially remove water. The product was then dissolved in dichloromethane and dried over anhydrous magnesium sulfate. After filtration and rotary evaporation, 4.06 g of the final product, selenooctanoic acid, was obtained with a yield of 55.57%. All processes were carried out under strict light-shielding conditions.

[0081] 3. Synthesis of gelatin-lipoic acid-selenoic acid copolymer (Gel-LA-SA): 0.402 g (1.954 mmol, 1 Equiv.) of lipoic acid, 0.349 g (2.150 mmol, 1.1 Equiv.) of N,N'-carbonyldiimidazole (CDI), and 20 mL of dimethyl sulfoxide (DMSO) were added to a 50 mL single-necked bottle and reacted for 2 h to obtain CDI-activated lipoic acid; 0.156 g (0.487 mmol, 0.25 Equiv.) of selenoic acid, 0.087 g (0.537 mmol, 0.275 Equiv.) of CDI, and 5 mL of DMSO were added to a 50 mL single-necked bottle and reacted for 2 h to obtain CDI-activated selenoic acid; 5 g (6.1 mmol amino group, 3.125 Equiv.) of gelatin were added to 50 mL of water and stirred at 40 °C for 2 h. The gelatin aqueous solution was dissolved by heating at 35°C. CDI-activated lipoic acid and CDI-activated selenoic acid were added to the gelatin aqueous solution to adjust the mass ratio of selenoic acid: lipoic acid: gelatin to 1:1-5:100-500. The reaction was carried out at 35°C for 24 h. The resulting reaction solution was placed in a dialysis bag (molecular weight cutoff, 10,000 Da) and dialyzed for 3 days. After freeze-drying, it was stored at -20°C until use (the product had a number average molecular weight of 80,000-120,000).

[0082] The synthetic route is as follows:

[0083]

[0084] Gel-LA-SA

[0085] The preparation method of the gelatin-lipoic acid polymer used in the examples of the present invention is as follows: 1 g of (R)-α-lipoic acid and 0.85 g of CDI are dissolved in 50 mL of DMSO and stirred at room temperature for 2 hours to obtain solution a; 10 g of gelatin is then dissolved in 100 mL of deionized water to obtain solution b; solution a and solution b are mixed, stirred in the dark, and reacted for 24 hours. After the reaction is completed, the resulting solution is placed in a dialysis bag (molecular weight cutoff 10,000 Da) and dialyzed for 72 hours, and then freeze-dried to obtain the gelatin-lipoic acid polymer.

[0086] The chain transfer agent mPEG-BCSPA used in the examples of the present invention was prepared as follows: 0.890 g (1.5 equiv.) of BCSPA (prepared in reference literature, see Macromolecules 2005, 38, 6, 2191–2204) was added to 50 mL of DCM for dissolution, and then 0.600 g of CDI (1.5 equiv.) was added and reacted for 1 hour. Then, 10 g of mPEG4000 (purchased from TCI, Shanghai) was added and the reaction was continued for two days. After the reaction, 100 mL of ethyl acetate was added, the mixture was washed three times with saturated brine, dried over anhydrous magnesium sulfate overnight, filtered through a G4 sand core funnel, and rotary evaporated to retain a small amount of liquid. The concentrated liquid was added to anhydrous ether for precipitation, filtered, and the resulting solid was dissolved in a small amount of DCM, precipitated again, and dried to obtain mPEG-BCSPA. 1 H NMR spectrum Figure 1 shown.

[0087] The reaction route is as follows:

[0088]

[0089] Example 1

[0090] A donor of PTKNO that releases nitric oxide in response to active oxygen, the specific preparation method of which is as follows:

[0091] 1) Synthesis of 4-(chloromethyl)benzyl methacrylate (4-CBM): A mixture of 5 g of methacryloyl chloride (47.83 mmol, 1.5 eq.) and 250 mL of anhydrous DCM was slowly added dropwise to a mixture of 7.491 g of 4-(chloromethyl)benzyl alcohol (31.93 mmol, 1.5 eq.), 250 mL of DCM, and 9.68 g of triethylamine. The solution was then stirred in the dark at room temperature for 24 h, then spin-dried and dissolved in ethyl acetate. The product was then washed once with saturated sodium bicarbonate solution and twice with deionized water to remove impurities. The product was then dried over anhydrous sodium sulfate, filtered, and the solvent removed under reduced pressure. Chromatographic purification using a 30:1 ratio of hexane to ethyl acetate afforded 4-CBM as an oil.

[0092] 2) Synthesis of 4-((nitrooxy)methyl)benzylmethacrylate (4-NMBM): 4-(Chloromethyl)benzylmethacrylate (1 g, 4.45 mmol, 1 equiv.) and silver nitrate (1.059 g, 6.27 mmol, 1.4 equiv.) were dissolved in tetrahydrofuran (10 mL). The solution was stirred in the dark at room temperature for 12 h. The solid was removed by filtration using a fritted funnel. Acetonitrile was removed by vacuum distillation. The resulting product was diluted with 50 mL of ethyl acetate and washed three times with water and saturated brine, respectively. The product was dried over MgSO4, concentrated, and dried to obtain liquid 4-NMBM, which was then stored at -20°C in an oxygen-free environment.

[0093] 3) Propanoic acid (3,3'-(propane-2,2-diylbis(sulfanediyl)) dipropanoic acid, TK-COOH): 3-Mercaptopropionic acid (10.46 g, 98.2 mmol), anhydrous acetone (2.90 g, 41.9 mmol), and 4 mL of trifluoroacetic acid were mixed and stirred at room temperature (25°C) for 6 h. The mixture was then cooled in an ice-water bath to allow crystallization. The resulting crystalline product was filtered, washed three times with deionized water and three times with n-hexane, and dried under vacuum to obtain a white powder (yield 90.18%). The product was then stored at 4°C in the absence of oxygen.

[0094] 4) Synthesis of 5,5,15-trimethyl-9,14-dioxo-10,13-dioxa-4,6-dithiohexadecenoic acid (TK-HEMA): 5 g TK-COOH (1 equiv.) was dissolved in 100 mL DCM. 6.14 g dicyclohexylcarbodiimide (1.5 equiv.) and 0.36 g 4-dimethylaminopyridine (0.15 equiv.) were added, respectively. After activation for 2 h, 2.6 g hydroxyethyl methacrylate (HEMA, 1 equiv.) was added and stirred for 24 h. The insoluble matter was filtered out using a G4 fritted funnel, and the solution was concentrated under reduced pressure. The product was separated by column chromatography using a mixed solvent of DCM:ethyl acetate:triethylamine = 30:1:0.15 (volume ratio) and 300-400 mesh silica gel to obtain the target product, TK-HEMA, in a yield of 42.85%.

[0095] 5) Synthesis of NO Donor (PTKNO): 0.386 g (1 equiv.) mPEG-BCSPA, 3.128 mg (0.2 equiv.) azobisisobutyronitrile (AIBN), 0.5 g (15 equiv.) TK-HEMA, 0.346 g (15 equiv.) 4-NMBM, and 5 mL DMF were added to a reaction flask. After freezing with liquid nitrogen, the mixture was evacuated for 15 min, filled with argon, and dissolved at 30°C. This process was repeated three times. The reaction flask was then immersed in a 65°C oil bath for 16 h. The solution was then added to diethyl ether to allow precipitation to obtain the product, mPEG-P[(TK-HEMA)-(4-NMBM)], abbreviated as PTKNO, in a yield of 75.37%.

[0096] In this example, the 4-CBM synthesized in step 1) and the 4-NMBM synthesized in step 2) 1 H NMR test picture Figure 2 As shown, step 3) synthesis of TK-COOH and step 4) synthesis of TK-HEMA 1 H NMR spectrum Figure 3 shown.

[0097] Figure 4 The PTKNO prepared in this embodiment 1 H NMR spectrum, according to 1 The H NMR spectrum confirmed its structural formula:

[0098] ,

[0099] Where m=14, n=180.

[0100] Figure 5 is the GPC curve of PTKNO prepared in this example, where Mn is the number average molecular weight, M P is the peak molecular weight, Mw is the weight average molecular weight, and Mz is the Z average molecular weight. The Mn of the obtained PTKNO was measured to be 14216, and the polydispersity index (PD) was 1.049.

[0101] In order to evaluate the responsiveness of PTKNO donor molecules to ROS, PTKNO was added to a mixed solvent of CD3CN and D2O containing H2O2 (volume ratio CD3CN: D2O = 3:1). The concentration of PTKNO was 1.67 mg / mL and the concentration of H2O2 was 1 mM. The test solution was placed at different times. 1 H NMR spectrum, observe the changes in the molecular structure of PTKNO donor in the presence of H2O2, the test diagram is as follows Figure 6As shown in the figure, it can be seen that under the action of H2O2, the thioketal in PTKNO will react to generate mercaptopropionic acid and acetone, and its active oxygen response rate can be measured by 1 The peak area change at 1.54 ppm in the H NMR spectrum was calculated based on the decrease in the proton peak of the methyl group on the thioketal.

[0102] To test the labeling of NO released by PTKNO in macrophages, the DAF-FM DA fluorescent probe can react with NO inside the cells to produce a triazole fluorescent compound with a significant fluorescent signal. The specific test method is as follows: macrophage RAW264.7 cells were seeded into a 6-well plate containing 2 mL of DMEM medium, cultured to 80% confluence, the medium was discarded, and then washed with DMEM medium without FBS. Then, the cells were incubated with DMEM medium without FBS (the culture medium contained PTKNO and NO fluorescent probe DAF-FM DA prepared in this example, and the concentration of PTKNO was 1 mg / mL) for 30 minutes. After washing with PBS to remove extracellular materials and DAF-FM DA, the cells were incubated with lipopolysaccharide (LPS) (500 ng / mL) in DMEM medium without FBS for 3 hours. A control sample without LPS was used. After rinsing with PBS, the cells were imaged using an IX71 inverted fluorescence microscope (Olympus, Japan) at an excitation wavelength of 495 nm and an emission wavelength of 515 nm. Cell images under white light, NO probe, and Merge were taken. The test figures are shown in the figure below. Figure 7 As shown (scale bar: 100 μm), Figure 7 The upper left center image shows a white light image of macrophages incubated with a PTKNO donor, and the upper right shows a white light image of macrophages incubated with LPS and a PTKNO donor; the lower left shows a fluorescence image of macrophages incubated with a PTKNO donor and reacted with a DAF-FM DA probe; the lower right shows a fluorescence image of macrophages incubated with LPS and a PTKNO donor and reacted with a DAF-FM DA probe. The results show that weak fluorescence can be observed in the control sample (denoted as PTKNO), indicating that PTKNO can be converted into NO in a small amount by enzymes in RAW264.7 cells. After induction with lipopolysaccharide (denoted as PTKNO+LPS), strong triazole fluorescence can be clearly observed in RAW264.7 cells, indicating that LPS can stimulate RAW264.7 cells to release ROS, thereby causing PTKNO to release NO. This shows that the thioketal in the PTKNO molecular structure has good ROS responsiveness, can sensitively respond to release mercaptopropionic acid, and then activate nitrates to release NO, which can be used for precise treatment of inflammatory sites in the body.

[0103] The PTKNO prepared in this example was stored at room temperature for 0, 7, and 14 days, and its 1 H NMR spectrum changes, the test graph is as follows Figure 8 As shown in the figure, it can be seen that the donor has almost no spontaneous decomposition at room temperature and is chemically stable. The common nitrate donor (preparation method see: https: / / authors.elsevier.com / a / 1jAZo6CFjZU1Ie) spontaneously releases NO at room temperature over time. Figure 9 The NO concentration in a 0.5 mg / mL nitrate donor solution was tested at room temperature using the Griess reagent (S0021S, Beyotime Biotechnology) at different times (0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, and 12 hours). It can be seen that the first six hours are the burst release period, during which approximately 20% of NO gas can be released, indicating that the donor is not chemically stable at room temperature.

[0104] Example 2

[0105] A nerve repair conduit for sustained release of nitric oxide in response to an active oxygen cascade comprises a hollow conduit framework and a hydrogel filled within the conduit framework. The conduit framework is an electrospun fiber conduit made of an L-alanine-caprolactone copolymer. The conduit is filled with a gelatin-lipoic acid-selenooctanoic acid microgel hydrogel. The hydrogel is loaded with PTKNO, a donor for releasing nitric oxide in response to active oxygen, prepared in Example 1. The specific preparation method is as follows:

[0106] 1) 1 g of lactide-caprolactone copolymer (prepared in-house, reference: Biomaterials 280 (2022) 121288) with a number-average molecular weight of 100,000 was weighed and dissolved in 5 mL of hexafluoroisopropanol to obtain an electrospinning solution with a concentration of 0.2 g / mL. The electrospinning process parameters were set as follows: positive pressure of 8.5 kV, negative pressure of 2.5 kV, liquid propulsion rate of 1 mL / h, roller receiving distance of 10 cm, roller speed of 30 r / min, and needle type of 22G. The electrospinning solution was electrospun to collect nanofibers (average diameter of 643.70 ± 127.76 nm). The nanofibers were then wrapped around a polytetrafluoroethylene mold to prepare a catheter skeleton with an inner diameter of 1.2 mm, a thickness of 1 mm, and a length of 10 mm.

[0107] 2) Weigh 20 mg of PTKNO, a nitric oxide donor for reactive oxygen species (ROS) release, and dissolve it in 0.2 mL of tetrahydrofuran. The mixture was slowly added dropwise to 10 mL of PBS buffer (pH 7.4) under ultrasound (ultrasonic frequency 40 kHz, power 150 W). The tetrahydrofuran was then removed by vacuum distillation. Aggregates were then removed by filtration using a 0.45 μm aqueous filter membrane to obtain a dispersion of 2 mg / mL nitric oxide donor nanoparticles for sustained ROS cascade release.

[0108] 3) 1 g of gelatin-lipoic acid-selenooctanoic acid copolymer was weighed and dissolved in 10 mL of PBS buffer to obtain a hydrogel precursor solution with a concentration of 0.1 g / mL. 10 mL of a dispersion of reactive oxygen species-responsive nitric oxide-releasing donor nanoparticles was then added and stirred to obtain a mixture. The mixture was then injected into a syringe and injected at a rate of 1 mL / h into a silicone tubing (200 μm inner diameter) filled with ice paraffin under near-infrared light (wavelength 700-2500 nm, power 0.5-10 W) to produce spherical microdroplets. The microdroplets were solidified at the outlet of the silicone tubing with ultraviolet light (365 nm) (irradiation time approximately 10 s) to form microgels. The spherical microgels were then washed with n-hexane and water, and centrifuged to remove the paraffin on the surface of the spherical microgels, thereby obtaining microgel modules loaded with reactive oxygen species-responsive nitric oxide-releasing donor nanoparticles.

[0109] 4) Use a standard syringe without a needle to draw up the aforementioned microgel modules and inject them into the catheter framework at a volume of 50% of the catheter framework's internal volume. Then, irradiate the catheter framework with ultraviolet light (365 nm) for 15 seconds until the microgel modules further cross-link with each other to form a nerve repair conduit with the function of releasing nitric oxide gas in response to reactive oxygen species.

[0110] According to theoretical calculations and SEM testing, the hydrogel composed of spherical microgel modules within the nerve repair conduit of this embodiment has pores of three sizes: small (50-100 microns), medium (400-500 microns), and large (700-800 microns), forming a multi-level pore structure. Because this hydrogel is composed of spherical microgel modules, its total specific surface area can be increased by 5-10 times compared to conventional hydrogels of the same filling volume. This significantly improves nutrient exchange and oxygen transport during nerve regeneration, as well as the adhesion of regenerative cells.

[0111] like Figure 10 Shown are transmission electron microscopy images (scale: 100 nm) and dynamic light scattering (DLS) particle size distribution diagrams of PTKNO nanoparticles obtained after freeze-drying the dispersion prepared in step 2) of this example. The diagrams show that PTKNO can self-assemble into spherical structures. The average particle size measured by DLS is 13.97 nm, with a particle size range of 10 to 450 nm.

[0112] like Figure 11 Shown is a fluorescence image of the microgel module loaded with donor nanoparticles that release nitric oxide in response to active oxygen species prepared in step 3) of this example after being stained with rhodamine B. The image shows that the microgel module is uniformly spherical and has a size of 300-400 μm (in a hydrated state).

[0113] The microgel modules loaded with the donor nanoparticles that release nitric oxide in response to active oxygen species prepared in step 3) of this example were dyed with rhodamine B and methylene blue, respectively. The rhodamine B-dyed microgel modules and the methylene blue-dyed microgel modules were then mixed in a mold and irradiated with ultraviolet light (365 nm) for 10 seconds to obtain a complete hydrogel, as shown in the photo. Figure 12 As shown, it shows that the microgel module loaded with donor nanoparticles that release nitric oxide in response to active oxygen species prepared in step 3) still has a certain cross-linking ability.

[0114] like Figure 13 Shown is a scanning electron micrograph of the microgel module loaded with donor nanoparticles that release nitric oxide in response to reactive oxygen species, prepared in step 3) of this example (scale bars: 500 μm and 100 μm). The image shows that the microgel module has pores within it, with an internal pore size ranging from 50 to 100 μm, and the pore size between modules ranging from 200 to 400 μm.

[0115] The relationship between the gel transition and photocrosslinking time of the hydrogel precursor solution in step 3) of this example was tested. The specific method was as follows: a parallel plate rotor with a diameter of 25 mm and a platform with a UV lamp were selected, and the distance between the two was set to 1 mm. The hydrogel precursor solution in step 3) of this example was injected into the gap between the parallel plate rotor and the platform. After the UV lamp (365 nm) was turned on, the real-time changes in the storage modulus (G') and loss modulus (G'') of the resulting hydrogel were measured by performing a time scan (10 rad / s; 1% strain). The test diagram is shown in FIG. Figure 14 As shown in the figure, it can be seen that after 10 s of photocrosslinking, the hydrogel exhibits solid-like elastic behavior (storage modulus G'>loss modulus G").

[0116] like Figure 15Shown are the Gel-LA-SA hydrogel (denoted as Gel-LA-SA Hydrogel) prepared by crosslinking the hydrogel precursor solution in step 3) of this example in a polytetrafluoroethylene mold (10 mm in diameter, 5 mm in height) by ultraviolet light (365 nm) for 10 s, and the microgel hydrogel (denoted as Gel-LA-SA Microgel Hydrogel) prepared by further crosslinking the microgel module prepared in step 3) by ultraviolet light (365 nm) for 10 s. The results are shown in FIG. 1 . The stress-strain curves, Young's modulus and ultimate compressive strain comparison diagrams measured at a speed of 100 mm / min and a deformation rate of 50% show that Gel-LA-SA hydrogel has higher compressive strength and compression deformation rate than microgel hydrogel, while its elastic modulus is lower than that of microgel hydrogel. This is because Gel-LA-SA hydrogel is a uniformly cross-linked whole, while microgel hydrogel is composed of smaller microgel modules, so its compressive strength and compression deformation capacity are lower. However, due to the longer total UV irradiation time, the cross-linking degree of microgel hydrogel is higher than that of Gel-LA-SA hydrogel, so its elastic modulus is greater than that of Gel-LA-SA hydrogel.

[0117] Figure 16Figure 3 is a schematic diagram of the nerve regeneration process of the nerve repair conduit prepared in this example. PTKNO NPs prepared by ultrasonic self-assembly were loaded into the Gel-LA-SA solution. The PTKNO NPs / Gel-LA-SA microgel module was prepared by microfluidics technology. The microgel module was filled into the conduit skeleton (lactide-caprolactone copolymer, P(LLA-CL)). Microgel hydrogel was further formed under ultraviolet light (365 nm). The nerve repair conduit was then implanted into the 10 mm sciatic nerve defect site (Nerve conduit implanation) of SD rats for nerve repair. The mechanism by which this nerve repair conduit promotes nerve regeneration is that in terms of microenvironment regulation, ischemia and excessive inflammation will cause a large accumulation of reactive oxygen species (ROS), leading to microenvironment imbalance, which will trigger the PTKNO NPs cascade reaction to release NO. Under the anti-inflammatory, antioxidant, angiogenesis and mitochondrial fusion and fission effects of NO, the microenvironment is rebalanced; in terms of nerve growth, the interconnected multi-level pore structure of the microgel hydrogel provides sites for cell adhesion and provides sufficient space for cell migration and axon growth (Microgel interconnected pore structure provides channels for nerve and cells growth).

[0118] The effect of the nerve repair conduit prepared in this example on rat nerve repair was tested. The specific experimental method is as follows: Before the experiment, 72 SD rats were housed in an animal room for 1 week to acclimate to the environment, with 3 rats in each cage. The rats were randomly and evenly divided into three groups: a PTK (no NO release) group, a PTKNO (slow-release NO) group, and an autograft group. The rats were provided with a suitable temperature, sufficient water, and feed. After anesthesia with an intraperitoneal injection of 5% sodium pentobarbital (400 mg / kg), each rat was fixed on an operating table in a prone position. An incision was made at the hind leg muscle of the rat, and the skin and subcutaneous tissue were separated in sequence to expose the sciatic nerve. A 7 mm long nerve segment was resected in the middle of the nerve trunk. Due to nerve retraction, a 10 mm long sciatic nerve defect was formed. The PTKNO group was anesthetized with 8-0 The nerve repair conduit prepared in this example was sutured to both ends of the damaged nerve using PLGA sutures. The nerve repair conduit without NO release function was sutured to both ends of the damaged nerve using PTK group. The preparation method of the nerve repair conduit without NO release function was as follows: 4-NMBM in step 5) of Example 1 was replaced with 4-CBM in an equal molar amount to obtain a PTK molecule without NO release function. The nerve repair conduit without NO release function was then prepared using the same method as in this example. The autologous transplantation group used 8-0 PLGA sutures to reverse the above-mentioned resected nerve segment of the rat 180° and then sutured it to both ends of the damaged nerve by docking the two ends of the broken end. The muscle and epidermis were sutured using 4-0 PLGA sutures. After the operation, iodine was applied to the incision and adequate diet was ensured. Figure 17 The picture shows the implantation of the nerve repair conduit prepared in this example into the 10 mm sciatic nerve defect of SD rats. Figure 18 This is a comparative diagram of the neurological function recovery evaluation of the three groups of mice with sciatic nerve defects in this example after 12 weeks of treatment. A is a representative photograph of footprints, B is the statistical results of the sciatic nerve function index (SFI), C is the statistical results of CMAP peak-to-peak potential, D is the statistical results of regenerated nerve conduction velocity, and E is the electrophysiological evaluation of regenerated nerves in the PTK (no NO release) group, PTKNO (slow-release NO) group, and autograft group. The sciatic nerve function index (SFI) value is calculated based on the following formula:

[0119] SFI=

[0120] Toe length (PL), toe width (TW), and medial toe width (MTW) ​​are assessments of hindlimb function in the non-operated (non) and experimental (exp), respectively. The SFI value ranges from -100 to 0, with -100 indicating complete loss of neurological function and 0 indicating normal neurological function.

[0121] Electrophysiology: After anesthesia, a ground electrode was fixed to the tail of SD rats, and then the rat sciatic nerve at the surgical site was exposed. A 20 mV electrical stimulus was applied to the proximal trunk of the regenerated nerve, and a monopole was placed in the gastrocnemius muscle to record the compound motor action potential (CMAP).

[0122] from Figure 18 It can be seen that the SFI value of the PTKNO group was higher than that of the PTK group (p<0.05), but lower than that of the autologous transplantation group (p<0.05). The CMAP amplitude of the PTKNO group (20.29 mV) was closer to that of the autologous transplantation group (28.93 mV), but there was a significant difference (p<0.05). The amplitude of the PTK group was lower (12.17 mV). In addition, the nerve conduction velocity of the PTKNO group (27.18 ms) was higher than that of the autologous transplantation group (28.93 mV). -1 ) was higher than that of the PTK group (20.18 ms -1 ), which was lower than that of the autologous transplantation group (34.15 ms -1 ), these findings indicate that the nerve repair catheter prepared in this example improves the recovery of nerve function after peripheral nerve injury.

[0123] The nerve repair conduit obtained in this embodiment is applied to the repair of peripheral nerve injury. As the time after implantation increases, the tissue fluid gradually penetrates into the damaged area. The microgel hydrogel in the conduit comes into contact with the surrounding tissue fluid, and the reactive oxygen species loaded therein responds to release the donor nanoparticles of nitric oxide. After contact with the excessive reactive oxygen molecules in the tissue fluid, the thioketone unit reacts with ROS and releases mercaptopropionic acid. Nitrate releases NO under specific enzymatic processes and thiol triggering in the cell. Further, NO exerts anti-inflammatory, antioxidant and rapid cell migration effects, reducing the excessive production of reactive oxygen species. At the same time, the donor chemical properties are stable. Since there is no large amount of thiol substances in the peripheral nerve injury microenvironment, the specific reactive oxygen cascade response avoids the further release of nitric oxide, thereby ultimately achieving the purpose of improving the peripheral nerve regeneration microenvironment.

[0124] Example 3

[0125] A nerve repair catheter, the specific preparation method is as follows:

[0126] 1) 1 g of lactide-caprolactone copolymer (molecular weight 80,000) was weighed and dissolved in 6.67 mL of hexafluoroisopropanol to obtain an electrospinning solution. The electrospinning process parameters were set as follows: positive pressure of 10 kV, negative pressure of 2 kV, liquid propulsion rate of 1.2 mL / h, roller receiving distance of 8 cm, roller speed of 50 r / min, and needle size of 22G. The electrospinning solution was electrospun to collect nanofibers, which were then wrapped around a polytetrafluoroethylene mold to prepare a catheter skeleton with an inner diameter of 1.5 mm, a thickness of 0.5 mm, and a length of 15 mm.

[0127] 2) Weigh 60 mg of PTKNO, a nitric oxide donor for ROS cascade release, and dissolve it in 1.2 mL of tetrahydrofuran. Slowly add the resulting mixture dropwise to 75 mL of PBS buffer under ultrasonic vibration. Then, vacuum distillation is performed to remove the tetrahydrofuran. Filter the mixture through a 0.45 μm aqueous filter to remove aggregates, yielding a 2 mg / mL dispersion of nitric oxide donor nanoparticles for ROS cascade release.

[0128] 3) 1 g of gelatin-lipoic acid-selenooctanoic acid copolymer (MW 90,000) was weighed and dissolved in 12.5 mL of PBS buffer to obtain a hydrogel precursor solution. 75 mL of a dispersion of donor nanoparticles that release nitric oxide in response to reactive oxygen species was then added and stirred to mix thoroughly. The solution was then loaded into a syringe and injected at a rate of 1.5 mL / h into a silicone tube (200 μm inner diameter) filled with ice paraffin under near-infrared light to produce spherical microdroplets. The microdroplets were then solidified (irradiation time: approximately 15 s) at the outlet of the silicone tube by irradiation with a UV lamp (410 nm) to form a microgel. The spherical microgel was then washed with n-hexane and water, and centrifuged to remove the paraffin on the surface of the spherical microgel, thereby obtaining a microgel module loaded with donor nanoparticles that release nitric oxide in response to reactive oxygen species.

[0129] 4) Use a standard syringe without a needle to draw up the aforementioned microgel modules and inject them into the catheter framework at a volume of 60% of the catheter framework's internal volume. Then, irradiate the catheter framework with ultraviolet light (365 nm) for 10 s until the microgel modules further cross-link with each other to obtain an intraluminal microgel-filled peripheral nerve repair catheter with the function of releasing nitric oxide gas in response to active oxygen.

[0130] Example 4

[0131] A nerve repair catheter, the specific preparation method is as follows:

[0132] 1) 1 g of lactide-caprolactone copolymer (molecular weight: 90,000) was weighed and dissolved in 10 mL of hexafluoroisopropanol to obtain an electrospinning solution. The electrospinning process parameters were set as follows: positive pressure of 12 kV, negative pressure of 1.5 kV, liquid propulsion rate of 1.5 mL / h, roller receiving distance of 12 cm, roller speed of 100 r / min, and needle size of 22G. The electrospinning solution was electrospun to collect nanofibers, which were then wrapped around a polytetrafluoroethylene mold to prepare a catheter skeleton with an inner diameter of 1 mm, a thickness of 1.5 mm, and a length of 20 mm.

[0133] 2) 30 mg of PTKNO, a nitric oxide donor for reactive oxygen species (ROS) release, was weighed and dissolved in 0.45 mL of tetrahydrofuran (THF). The resulting mixture was slowly added dropwise to 25 mL of PBS buffer under ultrasonic vibration. The THF was then removed by vacuum distillation. Aggregates were then removed by filtration using a 0.45 μm aqueous filter to obtain a 2 mg / mL dispersion of nitric oxide donor nanoparticles for ROS cascade response.

[0134] 3) 0.2 g of gelatin-lipoic acid-selenooctanoic acid copolymer (MW 100,000) was weighed and dissolved in 1 mL of PBS buffer to obtain a hydrogel precursor solution. 25 mL of a dispersion of reactive oxygen species-responsive nitric oxide-releasing donor nanoparticles was then added and stirred to mix thoroughly. The solution was then loaded into a syringe and injected at a rate of 2 mL / h into a silicone tube (200 μm inner diameter) filled with ice paraffin under near-infrared light to produce spherical microdroplets. The microdroplets were then solidified (irradiation time approximately 8 s) at the outlet of the silicone tube using a UV lamp (365 nm) to form a microgel. The spherical microgel was then washed with n-hexane and water, and centrifuged to remove the paraffin on the surface of the spherical microgel, thereby obtaining a microgel module loaded with reactive oxygen species-responsive nitric oxide-releasing donor nanoparticles.

[0135] 4) Use a standard syringe without a needle to draw up the aforementioned microgel modules and inject them into the catheter framework at a volume of 80% of the catheter framework's internal volume. Then, irradiate the catheter framework with ultraviolet light (365 nm) for 20 seconds until the microgel modules further cross-link with each other to obtain an intraluminal microgel-filled peripheral nerve repair catheter with the function of releasing nitric oxide gas in response to active oxygen.

[0136] The above description is a preferred embodiment of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principles of the present invention. These improvements and changes are also considered to be within the scope of protection of the present invention.

Claims

1. A donor for sustained release of nitric oxide in response to an active oxygen cascade, characterized in that: Its structural formula is as follows: ； Among them, m=5~30, n=14~238.

2. A method for preparing the donor of nitric oxide that releases in response to active oxygen cascade according to claim 1, characterized in that: The specific steps are as follows: 1) Synthesis of 4-(chloromethyl)benzyl methacrylate: A mixture of methacryloyl chloride and anhydrous dichloromethane was slowly added dropwise to a mixture of 4-(chloromethyl)benzyl alcohol, dichloromethane, and triethylamine. The solution was then stirred at room temperature in the dark for 12-48 hours. 4-(chloromethyl)benzyl methacrylate was then post-treated to obtain an oily product. 2) Synthesis of 4-((Nitrooxy)methyl)benzyl methacrylate: Dissolve 4-(chloromethyl)benzyl methacrylate obtained in step 1) and silver nitrate in tetrahydrofuran. Stir the solution in the dark for 8-48 hours. Filter using a fritted funnel to remove the residue. Purify the filtrate to obtain liquid 4-((Nitrooxy)methyl)benzyl methacrylate. 3) Synthesis of 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid: 3-Mercaptopropionic acid, anhydrous acetone, and trifluoroacetic acid were mixed and stirred at room temperature for 3-10 hours. The mixture was then cooled in an ice-water bath to allow crystallization. The resulting crystalline product was filtered and purified to obtain 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid as a white powder. 4) Synthesis of 5,5,15-trimethyl-9,14-dioxo-10,13-dioxa-4,6-dithiohexa-15-enoic acid: Dissolve 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid in dichloromethane, then add dicyclohexylcarbodiimide and 4-dimethylaminopyridine, respectively. After activation, add hydroxyethyl methacrylate and stir for 12-48 hours. Filter the insoluble matter using a G4 fritted funnel, concentrate the filtrate under reduced pressure, and separate by column chromatography to obtain 5,5,15-trimethyl-9,14-dioxo-10,13-dioxa-4,6-dithiohexa-15-enoic acid. 5) NO Donor Synthesis: 5,5,15-trimethyl-9,14-dioxo-10,13-dioxa-4,6-dithiohexadecene-15-enoic acid obtained in step 4) was added to a reaction flask along with 4-((nitrooxy)methyl)benzyl methacrylate obtained in step 2), mPEG-BCSPA, azobisisobutyronitrile, and N,N-dimethylformamide. After deoxygenation, RAFT polymerization was performed. After completion of the reaction, the solution was added to an ether solution for precipitation to obtain the product. The structural formula of mPEG-BCSPA is as follows: 。 3. The method for preparing a donor for sustained release of nitric oxide in response to active oxygen cascade according to claim 2, characterized in that: In step 2), the molar ratio of 4-(chloromethyl)benzyl methacrylate to silver nitrate is 1:1-3, and the mass volume ratio of 4-(chloromethyl)benzyl methacrylate to tetrahydrofuran is 1 g:10-50 mL.

4. The method for preparing a donor for sustained release of nitric oxide in response to active oxygen cascade according to claim 2, characterized in that: In step 3), the molar ratio of 3-mercaptopropionic acid to anhydrous acetone is 1:0.5-4, and the molar volume ratio of 3-mercaptopropionic acid to trifluoroacetic acid is 1 mol:20-60 mL.

5. The method for preparing a donor for sustained release of nitric oxide in response to active oxygen cascade according to claim 2, characterized in that: In step 4), the molar ratio of 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid to dicyclohexylcarbodiimide and 4-dimethylaminopyridine is 1:1-3:0.1-0.

5.

6. The method for preparing a donor for sustained release of nitric oxide in response to active oxygen cascade according to claim 2, characterized in that: In step 5), the molar ratio of 5,5,15-trimethyl-9,14-dioxo-10,13-dioxa-4,6-dithiohexadecene-15-enoic acid to 4-((nitrooxy)methyl)benzyl methacrylate, mPEG-BCSPA, and azobisisobutyronitrile is 5-20:5-20:1:0.1-0.5; and the RAFT polymerization reaction conditions are: reaction at 60-80°C for 8-24 hours.

7. A nerve repair catheter based on the donor of reactive oxygen species cascade response and sustained release of nitric oxide according to claim 1, characterized in that: The invention comprises a hollow catheter skeleton and a hydrogel filled in the catheter skeleton, wherein the donor of the reactive oxygen species cascade response and slow-release nitric oxide according to claim 1 is uniformly distributed in the hydrogel.

8. The nerve repair catheter according to claim 7, characterized in that: The hollow catheter skeleton is obtained by fiber spinning from a biodegradable polymer, wherein the biodegradable polymer includes any one or more combinations of L-alanine-caprolactone copolymer, gelatin-caprolactone copolymer, lactide-caprolactone copolymer, L-alanine-hydroxyacetic acid-lactic acid copolymer, lactic acid-hydroxyacetic acid copolymer, and lactic acid-hydroxyacetic acid-silk fibroin copolymer, and the biodegradable polymer is spun using an electrospinning process; the hydrogel is obtained by polymerizing spherical microgel modules with uniform particle size, wherein the spherical microgel modules fill a volume percentage of 40-80% in the hollow catheter skeleton, the particle size of the spherical microgel modules is 100-200 μm, and the light response wavelength range is 365-410 nm.

9. The method for preparing the nerve repair catheter according to claim 7, characterized in that: The following steps are involved: 1) Dissolving a biodegradable polymer in an organic solvent to obtain an electrospinning solution, and then electrospinning to obtain a hollow catheter skeleton; 2) dissolving a donor for sustained nitric oxide release in response to an active oxygen species cascade in tetrahydrofuran to obtain a donor solution, then slowly adding the obtained donor solution dropwise to a PBS buffer solution under ultrasonic vibration, then removing the tetrahydrofuran by vacuum distillation, and then filtering through a 0.45 μm pore size aqueous filter membrane to remove aggregates, thereby obtaining a dispersion of the donor for sustained nitric oxide release in response to an active oxygen species cascade; 3) preparing a PBS solution of a hydrogel precursor, adding the reactive oxygen species cascade response sustained-release nitric oxide donor dispersion obtained in step 2) and mixing to obtain a mixed solution, loading the solution into a syringe, and injecting the solution into a silicone tube flowing with paraffin wax under near-infrared light to produce spherical microdroplets. Irradiating the outlet of the silicone tube with an ultraviolet lamp solidifies the microdroplets to form microgels, and removing the paraffin wax to obtain spherical microgel modules. 4) injecting the spherical microgel module obtained in step 3) into the hollow catheter skeleton obtained in step 1), and irradiating the microgel module with ultraviolet light to further cross-link the microgel module to obtain a nerve repair catheter based on the donor of nitric oxide that slowly releases in response to the reactive oxygen species cascade.

10. Use of the nerve repair catheter according to claim 7 in the preparation of nerve repair materials or tissue engineering scaffold materials.

Images