Nerve cannula loaded with micromolecular preparation Pyrvinium pamoate, preparation method and application

Abstract

The invention provides a nerve cannula loaded with a small molecule preparation Pyrvinium pamoate, a preparation method and application, belongs to the technical field of biological materials, and solves the bottleneck problems that Wnt5a as macromolecular protein is difficult to realize clinical transformation and is difficult to jointly promote peripheral nerve injury repair from multiple aspects. The preparation method of the nerve cannula comprises the following steps: step 1, preparing the nerve cannula; step 2, preparing a DNA (Deoxyribose Nucleic Acid) hydrogel for slowly releasing the Pyrviniumpamoate; 3, the DNA hydrogel containing the Pyrvinumpamoate prepared in the step 2 is sucked into a microsyringe, the DNA hydrogel containing the Pyrvinumpamoate is injected into a nerve socketed pipe, and the nerve socketed pipe loaded with the Pyrvinumpamoate is obtained. A key receptor Vangl2 of a Wnt5a signal channel is regulated and controlled through a Vangl2 agonist (Pyrvinumpamoate), and the small molecule agonist is loaded through a nerve socketed tube to play a role in repairing peripheral nerve defects, so that the advantage of the Wnt5a for jointly promoting peripheral nerve injury repair from three aspects of axon regeneration, myelination and vascular regeneration is fully played.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a neural tube loaded with the small molecule preparation Pyrviniumpamoate, its preparation method, and its application. Background Technology

[0002] Trauma is the leading cause of death among people under 45 worldwide, with over 25% of the global population experiencing trauma of varying degrees. With the rapid urbanization in my country, the incidence of various types of trauma has increased significantly. Peripheral nerve injuries account for approximately 4% to 13.9% of all trauma patients, primarily presenting with sensory loss, motor dysfunction, and muscle atrophy in the nerve's innervation area. Treatment outcomes and prognoses are poor, with only 10% to 15% achieving complete recovery of motor and neurological function. Some patients even suffer lifelong disability, placing a heavy economic burden on families and society. Peripheral nerve injuries smaller than 5mm can be directly sutured with the epineurium. However, for nerve defects larger than 5mm, due to the greater distance, direct epineurium suturing is not possible. Autologous nerve transplantation remains the gold standard, as it provides the basilar membrane tube necessary for axonal growth to guide rapid nerve growth and exhibits low immune rejection. However, autologous nerve transplantation faces challenges such as insufficient donor sources, difficulty in meeting the requirements of long-term neurological deficits, the potential for new damage after donor site resection leading to functional loss, and the formation of artificial traumatic neuromas, sensory disturbances, and functional loss. Tissue-engineered nerve cannulas are a promising alternative to autologous nerve transplantation, avoiding the limitations mentioned above. However, the repair efficacy of currently improved tissue-engineered nerve cannulas is still far from reaching the level of autologous nerve transplantation. Therefore, the development and improvement of tissue-engineered nerve grafts have significant clinical and socioeconomic value. Currently, improvements to tissue-engineered nerve cannulas mainly focus on three aspects: the cannula wall, the cannula contents, and the seed cells. Improvements to the cannula contents primarily concentrate on loading growth factors and mimicking the extracellular matrix structure. The bottleneck is that current cannula loading primarily uses single or two growth factors, small molecules, or functional peptides, resulting in problems such as limited function, short duration of action, and complex encapsulation processes. This makes it difficult to fully meet the multi-level repair needs of peripheral nerve injury, requiring myelination, axonal regeneration, and angiogenesis.

[0003] The inventors previously discovered that Wnt5a can promote the secretion of neurotrophic factors such as nerve growth factor (NGF) and ciliary nerve growth factor (CNTF) and vascular endothelial growth factor (VEGF) by Schwann cells, thereby promoting the repair of peripheral nerve injuries through axonal regeneration, myelination, and angiogenesis, which has significant application value. However, Wnt5a, as a large protein molecule, suffers from problems such as high cost, difficult storage, easy degradation, and short biological half-life. In addition, literature shows that Wnt5a can exert different biological functions on many cells or tissues throughout the body. Systemic administration may lead to many uncertain side effects, which greatly limits its clinical application. Therefore, the clinical application of Wnt5a is significantly limited. Summary of the Invention

[0004] To address the challenges of clinical translation of Wnt5a, a large molecule protein, and the current limitations of nerve cannulas, which suffer from limited functionality, short duration of action, and complex encapsulation processes, hindering their ability to promote peripheral nerve injury repair from multiple perspectives, this invention proposes a nerve cannulas loaded with the small molecule formulation Pyrvinium pamoate, its preparation method, and its application.

[0005] By regulating Vangl2, a key receptor in the Wnt5a signaling pathway, with an agonist (Pyrvinium pamoate), and loading the agonist through a neural capillary tube, the agonist can repair peripheral nerve defects, thus fully leveraging the advantages of Wnt5a in promoting peripheral nerve injury repair through axonal regeneration, myelination, and angiogenesis.

[0006] The technical solution adopted in this invention is as follows:

[0007] In a first aspect, the present invention provides a method for preparing a neural cannula loaded with the small molecule formulation Pyrvinium pamoate, comprising the following steps:

[0008] Step 1: Prepare the nerve cannula;

[0009] Step 2: Prepare a DNA hydrogel for sustained release of Pyrvinium pamoate;

[0010] Step 3: The DNA hydrogel containing Pyrvinium pamoate prepared in Step 2 is drawn into a microsyringe and injected into the neural sleeve, thus obtaining the neural sleeve loaded with Pyrvinium pamoate.

[0011] In some embodiments, step 1 is specifically as follows:

[0012] Step 101: Dissolve the weighed gelatin powder in deionized water at 37°C to prepare a 10% (w / v) gelatin solution. Stir thoroughly until the gelatin is completely dissolved to form a homogeneous solution.

[0013] Step 102: Inject the gelatin solution into a specific mold with an inner diameter of 1.5 mm, and cool it at 4°C for 2 hours to allow the gelatin to solidify and form.

[0014] Step 103: Place the cured gelatin catheter in a glutaraldehyde solution for cross-linking treatment; the cross-linking time is 45 minutes to 1 hour. After the cross-linking treatment, the mechanical strength and degradation resistance of the catheter will be significantly improved.

[0015] Step 104: Remove the cross-linked gelatin tubing from the mold and air dry it at room temperature until completely dry;

[0016] Step 105: Immerse the prepared nerve sheath in sterile PBS solution and store it at 4°C to ensure its sterility and structural integrity before use.

[0017] In some embodiments, step 2 is specifically as follows:

[0018] Step 201: Prepare an aqueous DNA solution;

[0019] Step 202: Dissolve Pyrvinium pamoate in an appropriate amount of DMSO, and then add it to the DNA aqueous solution to obtain a mixed solution with a concentration of 100 μM;

[0020] Step 203: Expose the mixed solution to 365nm ultraviolet light for 5-10 minutes.

[0021] Step 204: Add DNA ligase to the mixed solution in step 203 and incubate overnight at room temperature to allow stable cross-linking structures to form between DNA strands;

[0022] Step 205: Wash the DNA hydrogel containing Pyrvinium pamoate obtained in step 204 with PBS solution to remove unbound drug molecules.

[0023] In some embodiments, the preparation of the DNA aqueous solution in step 201 is performed as follows:

[0024] Mix the ssDNA strands in an equimolar ratio and add physiological buffer to achieve a final DNA concentration of 10 μM; the ssDNA used is single-stranded DNA rich in Cytosine.

[0025] In some embodiments, the sequence information of each single-stranded DNA is shown in the table below:

[0026]

[0027]

[0028] Secondly, the present invention also provides a neural cannula loaded with the small molecule preparation Pyrvinium pamoate, which is prepared using the preparation method described above.

[0029] Thirdly, the present invention also provides the application of a nerve cannula loaded with the small molecule preparation Pyrvinium pamoate in the preparation of a repair material for treating peripheral nerve injury, wherein the nerve cannula is prepared by the preparation method described above.

[0030] In some embodiments, peripheral nerve injury refers to a nerve defect with a distance greater than 5 mm.

[0031] The beneficial effects of this invention are:

[0032] 1. The nerve cannula loaded with Pyrvinium pamoate prepared in this invention treats peripheral nerve injury by targeting Vangl2. It promotes peripheral nerve regeneration by targeting and stimulating Vangl2 with the agonist (Pyrvinium pamoate).

[0033] 2. The nerve cannula loaded with Pyrvinium pamoate prepared in this invention uses a novel material, DNA hydrogel, as the sustained-release system to construct a chitosan nerve cannula with Pyrvinium pamoate to enhance Vangl2 activity, thereby achieving controllable sustained release of Pyrvinium pamoate and maximizing the promotion of peripheral nerve regeneration. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is a schematic diagram of the implantation of a chitosan nerve cannula loaded with a sustained-release agonist (Pyrvinium pamoate) in a rat sciatic nerve defect model.

[0036] Figure 2The images show the complex muscle action potential (CMAP) waveforms of the sciatic nerve in rats: (a) empty chitosan nerve cannula group; (b) chitosan nerve cannula group with sustained-release Vangl2 agonist; and (c) autologous nerve transplantation group. Image i shows the CMAP waveform after stimulation of the proximal nerve, and image ii shows the CMAP waveform after stimulation of the distal nerve. Below are comparisons of: (left) peak value of the complex action potential; (middle) area of ​​the complex action potential; and (right) motor nerve conduction velocity. Data are expressed as mean ± standard deviation. *P < 0.05, ***P < 0.001.

[0037] Figure 3 Toluidine blue staining was performed on the nerve tissues of each group. A represents the empty cannula group, B represents the sustained-release Vangl2 stimulant group, and C represents the autologous nerve transplantation group. The bar charts below show the statistical results of the density (left) and mean diameter (right) of myelinated nerve fibers in each group. Data are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01, scale bar = 10 μm. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0039] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0040] Example 1

[0041] This embodiment mainly introduces a method for preparing a neural cannula loaded with the small molecule preparation Pyrvinium pamoate (Vangl2 stimulant). The specific steps are as follows:

[0042] Step 1: Prepare the nerve sheath tube

[0043] 1. Preparation of gelatin solution:

[0044] Weigh an appropriate amount of gelatin powder (Sigma-Aldrich) and dissolve it in deionized water at 37°C to prepare a 10% (w / v) gelatin solution. Stir thoroughly until the gelatin is completely dissolved to form a homogeneous solution.

[0045] 2. Mold forming:

[0046] The gelatin solution is injected into a specific mold with an inner diameter of 1.5 mm. It is then cooled at 4°C for 2 hours to allow the gelatin to solidify and take shape.

[0047] 3. Crosslinking treatment:

[0048] The cured gelatin catheter was placed in a glutaraldehyde solution (Bailinwei Technology) for cross-linking treatment. The cross-linking time was 45 minutes to 1 hour. After cross-linking treatment, the mechanical strength and degradation resistance of the catheter were significantly improved.

[0049] 4. Drying and further processing:

[0050] Remove the cross-linked gelatin tubing from the mold and air dry it at room temperature until completely dry.

[0051] 5. Save:

[0052] After completing all steps, the prepared nerve sheath tube is immersed in sterile PBS solution (Sigma-Aldrich) and stored at 4°C to ensure its sterility and structural integrity before use.

[0053] Step 2: Preparation of DNA hydrogel for sustained release of Pyrvinium pamoate

[0054] 1. DNA sequence: Single-stranded DNA (ssDNA) rich in Cytosine (C) (Songdi Technology) was used, as shown in the table below.

[0055] Table 1. Detailed information about DNA hydrogels

[0056]

[0057]

[0058] 2. Solution preparation: Mix the ssDNA strands in an equimolar ratio and add physiological buffer to achieve a final DNA concentration of 10 μM. Dissolve Pyrvinium pamoate (absin) in an appropriate amount of DMSO (Sigma-Aldrich) and then add it to the DNA solution to ensure a final concentration of 100 μM.

[0059] 3. Photoresponsive crosslinking: Expose the mixed solution to 365nm ultraviolet light for 5-10 minutes.

[0060] 4. Enzymatic cross-linking: Add DNA ligase (Beyotime) to the mixed solution and incubate overnight at room temperature to form a stable cross-linking structure between DNA strands.

[0061] 5. Use PBS solution to wash the hydrogel containing Pyrvinium pamoate appropriately to remove unbound drug molecules.

[0062] Step 3: The prepared DNA hydrogel containing Pyrvinium pamoate is drawn into a microsyringe (Sigma-Aldrich) and injected into the neural cannula to complete the preparation of the Pyrvinium pamoate-loaded neural cannula.

[0063] Example 2

[0064] In this embodiment, an in vivo animal experiment was conducted using a nerve cannula loaded with Pyrvinium pamoate prepared in Example 1.

[0065] 1. Establishment of a sciatic nerve clamp injury model in SD rats

[0066] Establishment of a 10mm sciatic nerve defect repair model in SD rats (Vitolliwa): ① SD rats were anesthetized with 5% isoflurane (Sigma-Aldrich) and maintained with 1.5%–2% isoflurane. The rats were then fixed in a prone position on the operating table, and the right thigh was prepared and disinfected. ② The skin of the right leg was incised, and the subcutaneous and muscle tissues were bluntly dissected to expose the right sciatic nerve. ③ The sciatic nerve was severed from the lower edge of the piriformis muscle to create a 10mm sciatic nerve defect. ④ Empty chitosan nerve cannula group and chitosan nerve cannula group containing sustained-release Pyrvinium pamoate: Under a microscope, the nerve cannula was microsurgically sutured into the severed end of the rat's sciatic nerve using 10-0 nylon sutures (Ruian Medical Equipment Specialty Store), i.e., a 15mm nerve cannula repaired a 10mm rat nerve defect. 2.5mm was left at both ends of the cannula for connecting the two ends of the nerve defect. Figure 1 Autologous nerve transplantation group: A 10mm section of the sciatic nerve was harvested, reversed 108°, and sutured to the epineurium at both ends of the nerve. Finally, the surgical site was rinsed with saline, and the muscles and skin were sutured layer by layer with 4-0 sutures. The rats were fed for 12 weeks post-surgery, during which time the condition of the surgical site and paws was closely observed: wound healing, toe movement, presence of ulcers, and muscle atrophy on the affected side.

[0067] Experimental groups: 1) Empty chitosan nerve cannula group; 2) Chitosan nerve cannula group with sustained-release Vangl2 stimulant (Pyrvinium pamoate); 3) Autologous nerve transplantation group.

[0068] 2. Electrophysiological test results

[0069] Twelve weeks post-surgery, compound muscle action potentials (CMAP) were recorded and motor nerve conduction velocity (MNCV) was measured by stimulating the proximal and distal ends of the sciatic nerve in rats to assess the recovery of nerve function in each group. Figure 2From the waveform diagrams, the autologous nerve transplantation group (group c) showed the highest amplitude and smoothest action potential curve, followed by the sustained-release Vangl2 agonist group (group b), while the empty cannula group (group a) showed a lower waveform and greater fluctuations. Quantitative analysis results showed that: (1) during proximal stimulation, the peak CMAP values ​​of the sustained-release Vangl2 agonist group and the autologous transplantation group were significantly higher than those of the empty cannula group (P < 0.001); (2) during distal stimulation, the CMAP area of ​​the sustained-release Vangl2 agonist group was also higher than that of the empty cannula group (P < 0.05); (3) in terms of motor nerve conduction velocity, the sustained-release Vangl2 agonist group and the autologous transplantation group were significantly higher than those of the empty cannula group (P < 0.001), and there was no significant difference between the two. The above results suggest that chitosan nerve cannulas loaded with sustained-release Vangl2 agonist can effectively promote the recovery of neurophysiological function, and the repair effect is close to that of autologous nerve transplantation.

[0070] 3. Toluidine blue staining

[0071] To further evaluate the promoting effect of Vangl2 agonists on nerve regeneration, toluidine blue staining was performed on nerve tissues from each group, and the density and diameter of myelinated nerve fibers were quantitatively analyzed. Figure 3 As shown, the empty cannula group (A) had fewer myelinated nerve fibers and a looser arrangement, while the sustained-release Vangl2 agonist group (B) and the autologous nerve transplantation group (C) had denser nerve fiber arrangement and intact myelin structure. Statistical results showed that the density of myelinated nerve fibers in the sustained-release Vangl2 agonist group and the autologous transplantation group was significantly higher than that in the empty cannula group (P < 0.05, P < 0.01), while there was no statistically significant difference between the two groups (P > 0.05). Furthermore, the diameter of myelinated nerve fibers in the sustained-release Vangl2 agonist group and the autologous transplantation group was also significantly larger than that in the empty cannula group (P < 0.001), suggesting that Vangl2 agonists can effectively promote myelin formation and nerve regeneration.

[0072] 4. Conclusion

[0073] This neural cannula utilizes a novel DNA hydrogel material as a sustained-release system, constructing a chitosan neural cannula with Pyrvinium pamoate to enhance Vangl2 activity. This allows for the controlled, sustained release of Pyrvinium pamoate, maximizing the promotion of peripheral nerve regeneration. Furthermore, it fully leverages the advantages of Wnt5a in promoting peripheral nerve injury repair through axonal regeneration, myelination, and angiogenesis.

[0074] The above-described embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing a neural cannula loaded with the small molecule formulation Pyrvinium pamoate, characterized in that, Includes the following steps: Step 1: Prepare the nerve cannula; Step 2: Prepare a DNA hydrogel for sustained release of Pyrviniumpamoate; Step 3: The DNA hydrogel containing Pyrvinium pamoate prepared in Step 2 is drawn into a microsyringe and injected into the neural sleeve, thus obtaining the neural sleeve loaded with Pyrvinium pamoate.

2. The method for preparing a neural cannula loaded with the small molecule preparation Pyrviniumpamoate according to claim 1, characterized in that, The specific steps for step 1 are as follows: Step 101: Dissolve the weighed gelatin powder in deionized water at 37°C to prepare a 10% gelatin solution. Step 102: Inject the gelatin solution into a specific mold with an inner diameter of 1.5 mm, and cool it at 4°C for 2 hours to allow the gelatin to solidify and form. Step 103: Place the cured gelatin tubing in a glutaraldehyde solution for cross-linking treatment; Step 104: Remove the cross-linked gelatin tubing from the mold and air dry it at room temperature until completely dry; Step 105: Immerse the prepared nerve sheath in sterile PBS solution and store it at 4°C.

3. The method for preparing a neural cannula loaded with the small molecule formulation Pyrviniumpamoate according to claim 2, characterized in that, In step 103, the crosslinking treatment conditions are as follows: the crosslinking time is 0.75-1h.

4. The method for preparing a neural cannula loaded with the small molecule preparation Pyrviniumpamoate according to claim 1, characterized in that, The specific steps for step 2 are as follows: Step 201: Prepare an aqueous DNA solution; Step 202: Dissolve Pyrviniumpamoate in an appropriate amount of DMSO, and then add it to the DNA aqueous solution to obtain a mixed solution with a concentration of 100 μM; Step 203: Expose the mixed solution to 365nm ultraviolet light for 5-10 minutes. Step 204: Add DNA ligase to the mixture from step 203 and incubate overnight at room temperature; Step 205: Wash the DNA hydrogel containing Pyrviniumpamoate obtained in step 204 with PBS solution to remove unbound drug molecules.

5. The method for preparing a neural cannula loaded with the small molecule formulation Pyrviniumpamoate according to claim 4, characterized in that, The procedure for preparing the DNA aqueous solution in step 201 is as follows: Mix the ssDNA strands in an equimolar ratio and add physiological buffer to achieve a final DNA concentration of 10 μM. ssDNA uses single-stranded DNA rich in Cytosine.

6. The method for preparing a neural cannula loaded with the small molecule formulation Pyrviniumpamoate according to claim 5, characterized in that, The sequence information of each single-stranded DNA is shown in the table below:

7. A neural cannula loaded with the small molecule formulation Pyrviniumpamoate, characterized in that, It is prepared using the preparation method described in any one of claims 1-6.

8. The application of a nerve cannula loaded with the small molecule preparation Pyrviniumpamoate in the preparation of a repair material for treating peripheral nerve injuries, characterized in that... The nerve cannula is prepared by the preparation method described in any one of claims 1-6.

9. The application according to claim 8, characterized in that, Peripheral nerve injury refers to a nerve defect with a distance greater than 5 mm.

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