Multifunctional drug delivery nerve conduits and methods of making the same

Abstract

This application provides a multifunctional drug delivery nerve conduit and its preparation method, belonging to the field of biomaterials technology. The multifunctional drug delivery nerve conduit is internally filled with a biodegradable microgel-based hydrogel and multi-effect drug-releasing nanoparticles (methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothiol ethyl)-methacrylamide-co-methyl methacrylate] nanoparticles loaded within the biodegradable microgel-based hydrogel. This multifunctional drug delivery nerve conduit combines multi-effect drug-releasing nanoparticles with a microgel-based hydrogel, not only providing more cell adhesion sites for nerve regeneration but also addressing the challenges of timely drug release and on-demand drug release through the sustained release of nitric oxide and reactive oxygen species in response to p-hydroxybenzyl alcohol. This achieves dynamic regulation of the regenerative microenvironment at the nerve injury site, promoting nerve regeneration and functional recovery.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a multifunctional drug delivery nerve conduit and its preparation method. Background Technology

[0002] Currently, nerve injury repair mainly relies on nerve conduits and nerve transplantation techniques. Among these, nerve conduits have received widespread attention as an alternative due to their non-invasiveness, ease of manipulation, and good adaptability. However, existing nerve conduits still face some challenges in terms of repair efficacy, such as a lack of effective cell adhesion sites, insufficient nutrient supply, unstable microenvironment, and inconsistent drug release. These issues severely affect the effectiveness and speed of nerve repair.

[0003] To address these challenges, researchers have explored the use of various biomaterials to design neural repair conduits. Biodegradable polymers such as polylactic acid, polycaprolactone, and polyglycolic acid have been used in the fabrication of neural conduits; however, these materials often only provide basic mechanical support and cannot offer sufficient cellular support and drug regulation at the injury site. Although studies have explored various drug delivery systems and hydrogel materials, the challenge remains in endowing conduits with topological structures and precisely controlling the timing, range, and rate of drug release. Therefore, optimizing the repair effect of neural conduits by introducing topological structures and multifunctional drug delivery systems has become a research hotspot in the field of neural repair in recent years.

[0004] Patent application CN118772352A discloses a nitric oxide donor that releases nitric oxide in response to a reactive oxygen species (ROS) cascade. This donor can release nitric oxide in response to an ROS cascade, clearing excess ROS locally, and reducing nitric oxide release as the ROS concentration decreases, thus achieving on-demand nitric oxide release. However, this method can only release nitric oxide in the presence of ROS, and cannot achieve sustained nitric oxide release or ROS-responsive release of p-hydroxybenzyl alcohol. It also cannot achieve timely release or synergistic effects of dual-drug therapy, and its effect on regulating the microenvironment for nerve regeneration around damaged tissues is limited. Summary of the Invention

[0005] In view of the technical problems existing in the background art, this application provides a multifunctional drug delivery nerve conduit and its preparation method. This method combines multi-effect drug sustained-release nanoparticles with microgel-based hydrogels, which not only provides more cell adhesion sites for nerve regeneration, but also releases p-hydroxybenzyl alcohol in response to the sustained release of nitric oxide and reactive oxygen species. This sequential release design simultaneously solves the problems of responsive drug release not being able to treat in a timely manner and sustained-release drugs not being able to be released on demand, realizing the dynamic regulation of the regenerative microenvironment at the nerve injury site, and promoting nerve regeneration and functional recovery.

[0006] This application provides a multifunctional drug delivery neural conduit, comprising a biodegradable polymer conduit, wherein the interior of the biodegradable polymer conduit is filled with a biodegradable microgel-based hydrogel and multi-effect drug sustained-release nanoparticles loaded within the biodegradable microgel-based hydrogel; the multi-effect drug sustained-release nanoparticles are methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothiol ethyl)-methacrylamide-co-methyl methacrylate] nanoparticles.

[0007] Furthermore, the loading concentration of the methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothioethyl)-methacrylamide-co-methyl methacrylate] nanoparticles is 0.1-100 mg / mL, and the drug release period is 7-28 days.

[0008] Furthermore, the biodegradable microgel-based hydrogel is one of methacrylamide gelatin-lipoic acid or methacrylamide gelatin-selenoic acid microgel-based hydrogel.

[0009] Furthermore, the biodegradable polymer catheter is an electrospun catheter with a lumen diameter of 1–5 mm, a wall thickness of 1–3 mm, and a length of 3–100 mm.

[0010] This application also provides a method for preparing a multifunctional drug delivery nerve conduit, comprising the following steps:

[0011] Microgels loaded with multi-effect drug sustained-release nanoparticles were added to a 0.1–4% concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate photoinitiator solution for 5–10 min, and then injected into a biodegradable polymer conduit. Under ultraviolet light irradiation, the microgels were further cross-linked to obtain a multifunctional drug delivery nerve conduit.

[0012] Furthermore, the preparation method of the multi-effect drug sustained-release nanoparticles is as follows:

[0013] 1) Weigh out the predetermined amounts of methoxy polyethylene glycol-2-[[(butylthio)thiomethyl]thio]propionic acid, diethylaminoethyl methacrylate, methacrylic acid, methyl methacrylate, and azobisisobutyronitrile. Mix them and add anhydrous N,N-dimethylformamide. Perform freeze-thaw deoxygenation 2-3 times. Then immerse the reaction flask in an oil bath at 65-75℃ for 12-18 hours. After the reaction is complete, add the solution to diethyl ether to precipitate and obtain methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-methacrylic acid-co-methyl methacrylate].

[0014] 2) Dissolve methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-methacrylic acid-co-methyl methacrylate] in 10 times (w / v) of dichloromethane, then add 1-5 times the molar amount of N,N-carbonyldiimidazole, 1-1.5 times the molar amount of cysteine ​​hydrochloride, and 2-6 times the molar amount of triethylamine. React at room temperature in the dark for 2-3 days. After vacuum distillation, dialyzing, and freeze-drying, the product methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] is obtained.

[0015] 3) Take methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] and one molar amount of 4-(bromomethyl)phenylboronic acid (a tertiary amine) and add them to methanol and N,N-dimethylformamide solution. Stir and react at room temperature for 24-36 h under argon protection. After dialyzing and freeze drying, the product methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] is obtained.

[0016] 4) Take methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] and add it to a 1-10 mM dithiothreitol methanol solution. React at room temperature for 2-4 h, then add it to diethyl ether for precipitation. After drying, you will get methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-mercaptoethyl)-methacrylamide-co-methyl methacrylate].

[0017] 5) Add methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-mercaptoethyl)-methacrylamide-co-methyl methacrylate] to a methanol solution of 100-400 mM tert-butyl nitrite, react at room temperature for 10-15 min, then place in a -20℃ refrigerator to react overnight. After precipitation and drying, methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothiol ethyl)-methacrylamide-co-methyl methacrylate] is obtained.

[0018] 6) Dissolve methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothioethyl)-methacrylamide-co-methyl methacrylate] in ice-cold methanol, then add it dropwise to PBS under ultrasonic conditions and disperse it by ultrasonication. Remove methanol by vacuum distillation, and filter the resulting solution with an aqueous filter membrane to remove macromolecular aggregates, thus obtaining a multi-effect drug sustained-release nanoparticle dispersion.

[0019] Further, in step S1, the molar ratio of methoxy polyethylene glycol-2-[[(butylthio)thiomethyl]thio]propionic acid, diethylaminoethyl methacrylate, methacrylic acid, methyl methacrylate, and azobisisobutyronitrile is 1:(30-50):(20-40):(10-30):0.2.

[0020] Furthermore, the preparation method of the microgel loaded with multi-effect drug sustained-release nanoparticles is as follows:

[0021] Prepare an aqueous solution of 5–20% (w / v) methacrylamide gelatin-lipoic acid or methacrylamide gelatin-selenoic acid, then add the dispersion of multi-effect drug sustained-release nanoparticles, mix well, and load into a syringe. Inject the solution into a silicone tube containing paraffin at a rate of 0.1–5 mL / h. Irradiate the outlet of the silicone tube with a UV lamp to solidify the microgel. Centrifuge and wash the filtered microgel to obtain the microgel loaded with multi-effect drug sustained-release nanoparticles.

[0022] Furthermore, the curing time of the microgel is 8-12 seconds.

[0023] The beneficial effects of this application are as follows:

[0024] 1. This application proposes a method for preparing a multifunctional drug delivery nerve conduit. By combining multi-effect drug sustained-release nanoparticles with microgel-based hydrogels, not only are more cell adhesion sites provided for nerve regeneration, but also p-hydroxybenzyl alcohol is released in response to the sustained release of nitric oxide and reactive oxygen species. This sequential release design simultaneously solves the problems of timely treatment by responsive drugs and the inability of sustained-release drugs to be released on demand, realizing the dynamic regulation of the regenerative microenvironment at the nerve injury site and promoting nerve regeneration and functional recovery.

[0025] 2. The multifunctional drug delivery nerve conduit prepared in this application has good biocompatibility and biodegradability, and can provide more cell adhesion sites for nerve regeneration. The conduit has the function of sequentially releasing NO and p-hydroxybenzyl alcohol, providing a good regenerative microenvironment for damaged nerves, and promoting the docking and functional recovery of damaged nerves.

[0026] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of this application, the accompanying drawings used in this application will be briefly described below. Obviously, the drawings described below are merely some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without any creative effort.

[0028] Figure 1 A schematic diagram of the synthesis process of multi-effect drug sustained-release nanoparticles;

[0029] Among them, step (1) synthesizes methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-methacrylate-co-methyl methacrylate].

[0030] Step (2) Synthesize methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate].

[0031] Step (3) Synthesize methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate].

[0032] Step (4) Synthesize methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-mercaptoethyl)-methacrylamide-co-methyl methacrylate].

[0033] Step (5) Synthesize methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothioethyl)-methacrylamide-co-methyl methacrylate].

[0034] Figure 2 This is a schematic diagram of the polymer self-assembling into nanoparticles in step (6).

[0035] Figure 3 This is a particle size distribution diagram of nanoparticles for sustained-release of multi-effect drugs.

[0036] Figure 4 This is a transmission electron microscope image of multi-effect drug sustained-release nanoparticles.

[0037] Figure 5 The results of the 1H NMR spectrum of the methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-methacrylate-co-methyl methacrylate] synthesized in step 1) of Example 1 are shown.

[0038] Figure 6The results of the 1H NMR spectrum of the methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] synthesized in step 2) of Example 1 are shown.

[0039] Figure 7 The results of the 1H NMR spectrum of the methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-mercaptoethyl)-methacrylamide-co-methyl methacrylate] synthesized in step 4) of Example 1 are shown.

[0040] Figure 8 The results of the 1H NMR spectrum of the methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothioethyl)-methacrylamide-co-methyl methacrylate] synthesized in step 5 of Example 1 are shown.

[0041] Figure 9 The release curve of nitric oxide sustained-release from the multi-effect drug sustained-release nanoparticles prepared in Example 1.

[0042] Figure 10 The NMR spectrum of p-hydroxybenzyl alcohol released by the multi-effect drug sustained-release nanoparticles prepared in Example 1 under the action of hydrogen peroxide.

[0043] Figure 11 This is an assessment of the recovery of nerve function in rats; (A) represents the recovery of sciatic nerve function, (B) represents the conduction velocity of regenerated nerves, and (C) represents the peak potential of nerve electrophysiology. In the figure, represent P <0.05, represent P <0.01.

[0044] Figure 12 This is a schematic diagram illustrating the mechanism of action of the multi-effect drug sustained-release nanoparticles prepared in Example 1 in releasing nitric oxide and reactive oxygen species in response to p-hydroxybenzyl alcohol. Detailed Implementation

[0045] The embodiments of the technical solution of this application will now be described in detail with reference to the accompanying drawings. These embodiments are only used to more clearly illustrate the technical solution of this application and are therefore merely examples, and should not be used to limit the scope of protection of this application.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0047] Microgel-based hydrogels, as novel biomaterials, possess excellent biodegradability and good cell adhesion properties. Combining multi-effect sustained-release drugs with microgel-based hydrogels can not only improve drug stability and duration but also regulate drug release within the microenvironment, thereby providing effective therapeutic support. Simultaneously, multi-effect sustained-release nanoparticles can carry multiple drugs, meeting the needs of sequential release and targeted delivery, further enhancing the effect of nerve repair.

[0048] p-Hydroxybenzyl alcohol has been extensively studied for its use in the field of nerve repair because it can play important roles in areas of nerve injury, such as anti-oxidation, anti-inflammation, and promoting axonal regeneration. In particular, nitric oxide and p-hydroxybenzyl alcohol can produce sustained therapeutic effects on damaged nerves through a sequential release mechanism, improving the microenvironment of the nerve injury area and promoting nerve regeneration.

[0049] This application provides a multifunctional drug delivery neural conduit, comprising a biodegradable polymer conduit filled with a biodegradable microgel-based hydrogel and multi-effect drug-releasing nanoparticles loaded within the biodegradable microgel-based hydrogel. The multi-effect drug-releasing nanoparticles are methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothioethyl)-methacrylamide-co-methyl methacrylate] nanoparticles.

[0050] Among them, the biodegradable microgel-based hydrogel is one of the methacrylated gelatin-lipoic acid and methacrylated gelatin-selenoic acid microgel-based hydrogels.

[0051] The loading concentration of methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothioethyl)-methacrylamide-co-methyl methacrylate] nanoparticles is 0.1-100 mg / mL, and the drug release period is 7-28 days.

[0052] The biodegradable polymer catheter is an electrospun catheter with a lumen diameter of 1–5 mm, a wall thickness of 1–3 mm, and a length of 3–100 mm.

[0053] This application also provides a method for preparing a multifunctional drug delivery nerve conduit, which includes the following steps:

[0054] Microgels loaded with multi-effect drug sustained-release nanoparticles were added to a 0.1–4% concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate photoinitiator solution for 5–10 min, and then injected into a biodegradable polymer conduit. Under ultraviolet light irradiation, the microgels were further cross-linked to obtain a multifunctional drug delivery nerve conduit.

[0055] The preparation method of multi-effect drug sustained-release nanoparticles is as follows:

[0056] 1) Weigh out the predetermined amounts of methoxy polyethylene glycol-2-[[(butylthio)thiomethyl]thio]propionic acid, diethylaminoethyl methacrylate, methacrylic acid, methyl methacrylate, and azobisisobutyronitrile. Mix them and add anhydrous N,N-dimethylformamide. Perform freeze-thaw deoxygenation 2-3 times. Then immerse the reaction flask in an oil bath at 65-75℃ for 12-18 hours. After the reaction is complete, add the solution to diethyl ether to precipitate and obtain methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-methacrylic acid-co-methyl methacrylate].

[0057] The molar ratio of methoxy polyethylene glycol-2-[[(butylthio)thiomethyl]thio]propionic acid, diethylaminoethyl methacrylate, methacrylic acid, methyl methacrylate, and azobisisobutyronitrile is 1:(30-50):(20-40):(10-30):0.2.

[0058] 2) Dissolve methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-methacrylic acid-co-methyl methacrylate] in 10 times (w / v) of dichloromethane, then add 1-5 times the molar amount of N,N-carbonyldiimidazole, 1-1.5 times the molar amount of cysteine ​​hydrochloride, and 2-6 times the molar amount of triethylamine. React at room temperature in the dark for 2-3 days. After vacuum distillation, dialyzing, and freeze-drying, the product methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] is obtained.

[0059] 3) Take methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] and one molar amount of 4-(bromomethyl)phenylboronic acid (a tertiary amine) and add them to methanol and N,N-dimethylformamide solution. Stir and react at room temperature for 24-36 h under argon protection. After dialyzing and freeze drying, the product methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] is obtained.

[0060] 4) Take methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] and add it to a 1-10 mM dithiothreitol methanol solution. React at room temperature for 2-4 h, then add it to diethyl ether for precipitation. After drying, you will get methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-mercaptoethyl)-methacrylamide-co-methyl methacrylate].

[0061] 5) Add methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-mercaptoethyl)-methacrylamide-co-methyl methacrylate] to a methanol solution of 100-400 mM tert-butyl nitrite, react at room temperature for 10-15 min, then place in a -20℃ refrigerator to react overnight. After precipitation and drying, methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothiol ethyl)-methacrylamide-co-methyl methacrylate] is obtained.

[0062] 6) Dissolve methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothioethyl)-methacrylamide-co-methyl methacrylate] in ice-cold methanol, then add it dropwise to PBS under ultrasonic conditions and disperse it by ultrasonication. Remove methanol by vacuum distillation, and filter the resulting solution with an aqueous filter membrane to remove macromolecular aggregates, thus obtaining a multi-effect drug sustained-release nanoparticle dispersion.

[0063] In this application, the preparation method of the microgel loaded with multi-effect drug sustained-release nanoparticles is as follows:

[0064] Prepare an aqueous solution of 5–20% (w / v) methacrylamide gelatin-lipoic acid or methacrylamide gelatin-selenoic acid, then add the dispersion of multi-effect drug sustained-release nanoparticles, mix well, and load into a syringe. Inject the solution into a silicone tube containing paraffin at a rate of 0.1–5 mL / h. Irradiate the outlet of the silicone tube with a UV lamp to solidify the microgel. Centrifuge and wash the filtered microgel to obtain the microgel loaded with multi-effect drug sustained-release nanoparticles.

[0065] The curing time of the microgel is 8-12 seconds.

[0066] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0067] Example 1

[0068] Please see Figure 1-4 As shown, this embodiment provides a method for preparing a multifunctional drug delivery nerve conduit, including the following steps:

[0069] 1) Take 1.2 g (1 Equiv.) methoxy polyethylene glycol-2-[[(butylthio)thiomethyl]thio]propionic acid, 17.751 mg (0.2 Equiv.) azobisisobutyronitrile, 3.870 g (50 Equiv.) diethylaminoethyl methacrylate, 1.396 g (30 Equiv.) methacrylic acid, 0.541 g (10 Equiv.) methyl methacrylate, and 8 mL N,N-dimethylformamide and add them to the reaction flask. Perform freeze-thaw deoxygenation three times. Then immerse the reaction flask in a 65℃ oil bath for 16 h. After the reaction is complete, add the solution to diethyl ether for precipitation to obtain methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-methacrylic acid-co-methyl methacrylate]; its 1H NMR spectrum results are as follows: Figure 5 As shown.

[0070] 1H NMR results: δ (chemical shift) 4.03 (single peak, 100H), 3.68-3.60 (double peak, 180H), 2.74-2.60 (double peak, 200H), 1.96 (multiple peak, 180H), 1.27-0.85 (multiple peak, 480H).

[0071] 2) Dissolve 2 g of methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-methacrylate-co-methyl methacrylate] (carboxyl molar amount 4.629 mmol, 1 Equiv.) in 20 mL of DCM. Then add 1.126 g (1.5 Equiv.) N,N-carbonyldiimidazole, 1.251 g (1.2 Equiv.) cysteine ​​dihydrochloride, and 2.249 g triethylamine (4.8 Equiv.). React at room temperature in the dark for 48 h. Remove DCM by vacuum distillation, and dialyze the remaining solution (MwCO3500 Da) for 24 hours. After freeze-drying, store the product methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] at 4 °C under argon atmosphere; its 1H NMR spectrum results are as follows. Figure 6 As shown.

[0072] 1H NMR results: δ (chemical shift) 4.11 (single peak, 100H), 3.69-3.60 (multiple peak, 180H), 3.20-2.68 (multiple peak, 440H), 1.90-1.73 (multiple peak, 180H), 1.31-0.94 (multiple peak, 480H).

[0073] 3) Take 1 g of methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] (tertiary amine molar amount 2.933 mmol, 1 Equiv.) and 0.630 g (1 Equiv.) 4-(bromomethyl)phenylboronic acid and add them to 10 mL of methanol and 10 mL of N,N-dimethylformamide, respectively. After dissolution, mix the two solutions and stir at room temperature for 24 h under argon protection. Dialyze the reaction solution for 3 days, freeze-dry, and store the product (methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate)) at 4℃ under argon atmosphere;

[0074] 4) Dissolve 1 g of methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] in 10 mL of methanol, add 0.154 g of dithiothreitol, react at room temperature for 2 h, then add to diethyl ether for precipitation, and dry to obtain methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-mercaptoethyl)-methacrylamide-co-methyl methacrylate]; its 1H NMR spectrum results are as follows: Figure 7 As shown.

[0075] 1H NMR results: δ (chemical shift) 7.82-7.40 (multiple peak, 200H), 4.68-4.25 (multiple peak, 300H), 3.65-3.60 (multiple peak, 180H), 3.47-2.98 (multiple peak, 440H), 1.96-1.52 (multiple peak, 180H), 1.30-0.92 (multiple peak, 480H).

[0076] 5) The obtained methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-mercaptoethyl)-methacrylamide-co-methyl methacrylate] was added to 10 mL of methanol, dissolved, and then 500 μL of tert-butyl nitrite was added. The mixture was reacted at room temperature for 10 min, and then placed in a -20°C refrigerator to react overnight. The resulting solution was added to ice-cold diethyl ether for precipitation, and then dried under vacuum to obtain methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothiol ethyl)-methacrylamide-co-methyl methacrylate]; its 1H NMR spectrum results are shown below. Figure 8 As shown.

[0077] 1H NMR results: δ (chemical shift) 7.82-7.40 (multiple peak, 200H), 4.56-4.25 (multiple peak, 300H), 3.67-3.60 (multiple peak, 180H), 3.42-2.66 (multiple peak, 440H), 1.93-1.53 ​​(multiple peak, 180H), 1.32-0.92 (multiple peak, 480H).

[0078] 6) Take 10 mg of methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothioethyl)-methacrylamide-co-methyl methacrylate] and dissolve it in 200 μL of ice-cold methanol. Then add it dropwise to 4 mL of PBS under sonication (ice-water bath) and sonicate for 10 min. Remove methanol by vacuum distillation. Filter the resulting solution through a 0.45 μm aqueous filter membrane to remove macromolecular aggregates and obtain a dispersion of multi-effect drug sustained-release nanoparticles.

[0079] 7) Prepare a 10% (w / v) aqueous solution of methacrylamide gelatin-lipoic acid. Then, add the dispersion of multi-effect drug sustained-release nanoparticles to the solution and mix well. Load the solution into a syringe and inject it into a silicone tube containing paraffin at a rate of 1 mL / h. Irradiate the outlet of the silicone tube with a UV lamp (365 nm) to solidify the microgel (approximately 10 s). After filtering, add water to the obtained microgel, centrifuge to remove residual paraffin, and further wash with 70% ethanol to obtain a microgel-based hydrogel loaded with multi-effect drug sustained-release nanoparticles.

[0080] 8) The microgel loaded with multi-effect drug sustained-release nanoparticles was added to a 1% concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate photoinitiator solution for 5 min, and then injected into a biodegradable polymer conduit. Under 365 nm ultraviolet light irradiation, the microgel was further cross-linked to obtain a multifunctional drug delivery nerve conduit.

[0081] Figure 9 The release curve of nitric oxide from the multi-effect drug sustained-release nanoparticles prepared in Example 1 is shown. It can be seen that NO can be released relatively quickly to reach its maximum concentration and can maintain this concentration for a long time.

[0082] Figure 10 The NMR spectrum shows the release of p-hydroxybenzyl alcohol by the multi-effect drug sustained-release nanoparticles prepared in Example 1 under the action of hydrogen peroxide. It can be seen that the phenylboronic acid group exhibits good reactive oxygen species responsiveness, and can continuously release p-hydroxybenzyl alcohol under the action of hydrogen peroxide.

[0083] The ability of nerve conduits to induce nerve regeneration was studied using an SD rat peripheral nerve defect model. Results were as follows: Figure 11 As shown.

[0084] Please see Figure 12 As shown, the mechanism of action of the multifunctional drug delivery nerve conduit of this application is as follows:

[0085] When methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothiol ethyl)-methacrylamide-co-methyl methacrylate] nanoparticles are introduced into the solution, the nitrosothiol bonds break, releasing NO; upon further addition of hydrogen peroxide, the phenylboronic acid on the nanoparticles reacts with the hydrogen peroxide to release p-hydroxybenzyl alcohol.

[0086] Comparative Example 1

[0087] The main difference between Comparative Example 1 and Example 1 is that step 3 was not performed, resulting in drug-release nanoparticles that could only release nitric oxide. The specific process is as follows:

[0088] 1) Take 1.2 g (1 Equiv.) methoxy polyethylene glycol-2-[[(butylthio)thiomethyl]thio]propionic acid, 17.751 mg (0.2 Equiv.) azobisisobutyronitrile, 3.870 g (50 Equiv.) diethylaminoethyl methacrylate, 1.396 g (30 Equiv.) methacrylic acid, 0.541 g (10 Equiv.) methyl methacrylate, and 8 mL N,N-dimethylformamide and add them to the reaction flask. Perform freeze-thaw cycles to remove oxygen three times. Then immerse the reaction flask in a 65°C oil bath for 16 h. After the reaction is complete, add the solution to diethyl ether to allow precipitation, yielding methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-methacrylic acid-co-methyl methacrylate].

[0089] 2) Dissolve 2 g of methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-methacrylate-co-methyl methacrylate] (carboxyl molar amount 4.629 mmol, 1 Equiv.) in 20 mL of DCM, then add 1.126 g (1.5 Equiv.) N,N-carbonyldiimidazole, 1.251 g (1.2 Equiv.) cysteine ​​dihydrochloride and 2.249 g triethylamine (4.8 Equiv.), and react at room temperature in the dark for 48 h. Remove DCM by vacuum distillation, and dialyze the remaining solution (MwCO3500 Da) for 24 hours. After freeze-drying, store the product methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] at 4 °C under argon atmosphere.

[0090] 3) Dissolve 1 g of methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] in 10 mL of methanol, add 0.154 g of dithiothreitol, react at room temperature for 2 h, then add to diethyl ether to precipitate, and dry to obtain methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(2-mercaptoethyl)-methacrylamide-co-methyl methacrylate].

[0091] 4) The obtained methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(2-mercaptoethyl)-methacrylamide-co-methyl methacrylate] was added to 10 mL of methanol, dissolved, and then 500 μL of tert-butyl nitrite was added. The mixture was reacted at room temperature for 10 min, and then placed in a -20°C refrigerator to react overnight. The resulting solution was added to ice-cold diethyl ether to precipitate, and then dried under vacuum to obtain methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(2-nitrosothiolethyl)-methacrylamide-co-methyl methacrylate].

[0092] 5) Take 10 mg of methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(2-nitrosothiol ethyl)-methacrylamide-co-methyl methacrylate] and add it to 200 μL of ice-cold methanol to dissolve it. Then add it dropwise to 4 mL of PBS under sonication (ice-water bath) and sonicate for 10 min. Remove methanol by vacuum distillation. Filter the resulting solution through a 0.45 μm aqueous filter membrane to remove macromolecular aggregates and obtain a dispersion of nitric oxide sustained-release nanoparticles.

[0093] 6) Prepare a 10% (w / v) aqueous solution of methacrylamide gelatin-lipoic acid. Then, add the dispersion of multi-effect drug sustained-release nanoparticles to the solution and mix well. Load the solution into a syringe and inject it into a silicone tube containing paraffin at a rate of 1 mL / h. Irradiate the outlet of the silicone tube with a UV lamp (365 nm) to solidify the microgel (approximately 10 s). After filtering, add water to the obtained microgel, centrifuge to remove residual paraffin, and further wash with 70% ethanol to obtain a microgel-based hydrogel loaded with nitric oxide sustained-release nanoparticles.

[0094] 7) The microgel loaded with nitric oxide sustained-release nanoparticles was added to a 1% concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate photoinitiator solution for 5 min, and then injected into a biodegradable polymer conduit. Under 365 nm ultraviolet light irradiation, the microgel was further cross-linked to obtain a multifunctional drug delivery nerve conduit.

[0095] The ability of nerve conduits to induce nerve regeneration was studied using an SD rat peripheral nerve defect model. Results were as follows: Figure 11 As shown.

[0096] Comparative Example 2

[0097] The main difference between Comparative Example 2 and Example 1 is that step 5 was not performed, resulting in drug-release nanoparticles that only release p-hydroxybenzyl alcohol in response to reactive oxygen species. The specific process is as follows:

[0098] 1) Take 1.2 g (1 Equiv.) methoxy polyethylene glycol-2-[[(butylthio)thiomethyl]thio]propionic acid, 17.751 mg (0.2 Equiv.) azobisisobutyronitrile, 3.870 g (50 Equiv.) diethylaminoethyl methacrylate, 1.396 g (30 Equiv.) methacrylic acid, 0.541 g (10 Equiv.) methyl methacrylate, and 8 mL N,N-dimethylformamide and add them to the reaction flask. Perform freeze-thaw cycles to remove oxygen three times. Then immerse the reaction flask in a 65°C oil bath for 16 h. After the reaction is complete, add the solution to diethyl ether to allow precipitation, yielding methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-methacrylic acid-co-methyl methacrylate].

[0099] 2) Dissolve 2 g of methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-methacrylate-co-methyl methacrylate] (carboxyl molar amount 4.629 mmol, 1 Equiv.) in 20 mL of DCM, then add 1.126 g (1.5 Equiv.) N,N-carbonyldiimidazole, 1.251 g (1.2 Equiv.) cysteine ​​dihydrochloride and 2.249 g triethylamine (4.8 Equiv.), and react at room temperature in the dark for 48 h. Remove DCM by vacuum distillation, and dialyze the remaining solution (MwCO 3500 Da) for 24 hours. After freeze-drying, store the product (methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate)) at 4 °C under argon atmosphere.

[0100] 3) Take 1 g of methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] (tertiary amine molar amount 2.933 mmol, 1 Equiv.) and 0.630 g (1 Equiv.) 4-(bromomethyl)phenylboronic acid and add them to 10 mL of methanol and 10 mL of N,N-dimethylformamide, respectively. After dissolution, mix the two solutions and stir at room temperature for 24 h under argon protection. Dialyze the reaction solution for 3 days, freeze-dry, and store the product (methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate)) at 4℃ under argon atmosphere;

[0101] 4) Dissolve 1 g of methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] in 10 mL of methanol, add 0.154 g of dithiothreitol, react at room temperature for 2 h, then add to diethyl ether to precipitate, and dry to obtain methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-mercaptoethyl)-methacrylamide-co-methyl methacrylate].

[0102] 5) Take 10 mg of methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-mercaptoethyl)-methacrylamide-co-methyl methacrylate] and dissolve it in 200 μL of ice-cold methanol. Then add it dropwise to 4 mL of PBS under sonication (ice-water bath) and sonicate for 10 min. Remove methanol by vacuum distillation. Filter the resulting solution through a 0.45 μm aqueous filter membrane to remove macromolecular aggregates and obtain a nanoparticle dispersion of p-hydroxybenzyl alcohol in response to reactive oxygen species.

[0103] 6) Prepare a 10% (w / v) aqueous solution of methacrylamide gelatin-lipoic acid. Then, add the dispersion of reactive oxygen species-responsive p-hydroxybenzyl alcohol nanoparticles to the solution and mix well. Load the solution into a syringe and inject it into a silicone tube containing paraffin wax at a rate of 1 mL / h. Irradiate the outlet of the silicone tube with a UV lamp (365 nm) to solidify the microgel (approximately 10 s). After filtering, add water to the obtained microgel, centrifuge to remove residual paraffin wax, and further wash with 70% ethanol to obtain a microgel loaded with reactive oxygen species-responsive p-hydroxybenzyl alcohol nanoparticles.

[0104] 7) The microgel loaded with reactive oxygen species that release p-hydroxybenzyl alcohol was added to a 1% solution of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate photoinitiator for 5 min, and then injected into a biodegradable polymer conduit. Under 365 nm ultraviolet light irradiation, the microgel was further cross-linked to obtain a multifunctional drug delivery nerve conduit.

[0105] The ability of nerve conduits to induce nerve regeneration was studied using an SD rat peripheral nerve defect model. Results were as follows: Figure 11 As shown.

[0106] Experimental Example

[0107] Using a peripheral nerve defect model in SD rats, the ability of different nerve conduits (Example 1, Comparative Examples 1-2) to induce nerve regeneration was systematically evaluated from three dimensions: sciatic nerve function index, nerve conduction velocity, and peak potential. The results are as follows: Figure 11 As shown.

[0108] from Figure 11 As can be seen, the nerve conduit prepared in Example 1 (loaded with drug-releasing nanoparticles that can simultaneously release nitric oxide and release p-hydroxybenzyl alcohol in response to reactive oxygen species) is significantly superior to Comparative Example 1 (only releasing nitric oxide) and Comparative Example 2 (only releasing p-hydroxybenzyl alcohol in response to reactive oxygen species) in terms of sciatic nerve motor function recovery (P<0.05), nerve conduction efficiency (P<0.05), and electrophysiological activity (P<0.01). This fully demonstrates that the synergistic release mechanism of nitric oxide and p-hydroxybenzyl alcohol can more efficiently promote nerve function repair, reflecting the innovation and practicality of the nerve conduit in the field of nerve injury repair in this application.

[0109] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A multifunctional drug delivery nerve conduit, characterized in that, The invention includes a biodegradable polymer catheter, the interior of which is filled with a biodegradable microgel-based hydrogel and multi-effect drug-releasing nanoparticles loaded within the biodegradable microgel-based hydrogel; the multi-effect drug-releasing nanoparticles are methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothioethyl)-methacrylamide-co-methyl methacrylate] nanoparticles.

2. The multifunctional drug delivery nerve conduit according to claim 1, characterized in that, The loading concentration of the methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothioethyl)-methacrylamide-co-methyl methacrylate] nanoparticles is 0.1-100 mg / mL, and the drug release period is 7-28 days.

3. The multifunctional drug delivery nerve conduit according to claim 1, characterized in that, The biodegradable microgel-based hydrogel is one of methacrylamide gelatin-lipoic acid or methacrylamide gelatin-selenoic acid microgel-based hydrogel.

4. The multifunctional drug delivery nerve conduit according to claim 1, characterized in that, The biodegradable polymer catheter is an electrospun catheter with a lumen diameter of 1–5 mm, a wall thickness of 1–3 mm, and a length of 3–100 mm.

5. A method for preparing a multifunctional drug delivery nerve conduit, characterized in that, The method for preparing the multifunctional drug delivery nerve conduit according to any one of claims 1-4 comprises the following steps: Microgels loaded with multi-effect drug sustained-release nanoparticles were added to a 0.1–4% concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate photoinitiator solution for 5–10 min, and then injected into a biodegradable polymer conduit. Under ultraviolet light irradiation, the microgels were further cross-linked to obtain a multifunctional drug delivery nerve conduit.

6. The method for preparing the multifunctional drug delivery nerve conduit according to claim 5, characterized in that, The preparation method of the multi-effect drug sustained-release nanoparticles is as follows: 1) Weigh out the predetermined amounts of methoxy polyethylene glycol-2-[[(butylthio)thiomethyl]thio]propionic acid, diethylaminoethyl methacrylate, methacrylic acid, methyl methacrylate, and azobisisobutyronitrile. Mix them and add anhydrous N,N-dimethylformamide. Perform freeze-thaw deoxygenation 2-3 times. Then immerse the reaction flask in an oil bath at 65-75℃ for 12-18 hours. After the reaction is complete, add the solution to diethyl ether to precipitate and obtain methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-methacrylic acid-co-methyl methacrylate]. 2) Dissolve methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-methacrylic acid-co-methyl methacrylate] in 10 times its w / v volume of dichloromethane, then add 1-5 times its carboxyl molar amount of N,N-carbonyldiimidazole, 1-1.5 times its carboxyl group amount of cystine hydrochloride, and 2-6 times its carboxyl group amount of triethylamine. React at room temperature in the dark for 2-3 days. After vacuum distillation, dialyzing, and freeze drying, the product methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(cystine)-methacrylamide-co-methyl methacrylate] is obtained. 3) Take methoxy polyethylene glycol-poly[diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] and one molar amount of 4-(bromomethyl)phenylboronic acid (a tertiary amine) and add them to methanol and N,N-dimethylformamide solution. Stir and react at room temperature for 24-36 h under argon protection. After dialyzing and freeze drying, the product methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] is obtained. 4) Add methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(cysteine)-methacrylamide-co-methyl methacrylate] to a 1-10 mM dithiothreitol methanol solution and react at room temperature for 2-4 h. Then add it to diethyl ether for precipitation and dry to obtain methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-mercaptoethyl)-methacrylamide-co-methyl methacrylate]. 5) Add methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-mercaptoethyl)-methacrylamide-co-methyl methacrylate] to a methanol solution of 100-400 mM tert-butyl nitrite, react at room temperature for 10-15 min, then place in a -20℃ refrigerator to react overnight. After precipitation and drying, methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothiol ethyl)-methacrylamide-co-methyl methacrylate] is obtained. 6) Dissolve methoxy polyethylene glycol-poly[phenylboronic acid-diethylaminoethyl methacrylate-co-N-(2-nitrosothioethyl)-methacrylamide-co-methyl methacrylate] in ice-cold methanol, then add it dropwise to PBS under ultrasonic conditions and disperse it by ultrasonication. Remove methanol by vacuum distillation, and filter the resulting solution with an aqueous filter membrane to remove macromolecular aggregates, thus obtaining a multi-effect drug sustained-release nanoparticle dispersion.

7. The method for preparing the multifunctional drug delivery nerve conduit according to claim 6, characterized in that, In step S1, the molar ratio of methoxy polyethylene glycol-2-[[(butylthio)thiomethyl]thio]propionic acid, diethylaminoethyl methacrylate, methacrylic acid, methyl methacrylate, and azobisisobutyronitrile is 1:(30-50):(20-40):(10-30):0.

2.

8. The method for preparing the multifunctional drug delivery nerve conduit according to claim 5, characterized in that, The preparation method of the microgel loaded with multi-effect drug sustained-release nanoparticles is as follows: Prepare an aqueous solution of 5–20% (w / v) methacrylamide gelatin-lipoic acid or methacrylamide gelatin-selenoic acid, then add the dispersion of multi-effect drug sustained-release nanoparticles, mix well, and load into a syringe. Inject the solution into a silicone tube containing paraffin at a rate of 0.1–5 mL / h. Irradiate the outlet of the silicone tube with a UV lamp to solidify the microgel. Centrifuge and wash the filtered microgel to obtain the microgel loaded with multi-effect drug sustained-release nanoparticles.

9. The method for preparing the multifunctional drug delivery nerve conduit according to claim 8, characterized in that, The curing time of the microgel is 8-12 seconds.

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