Electrospun tubes or wraps with functionalized inner layer for nerve growth

Abstract

The present invention relates to an electrospun multilayer tube or wrap for the protection or bridging of damaged nerves, comprising an electrospun isolating outer layer and at least one electrospun inner layer, the electrospun isolating outer layer comprising at least one polymer selected from the group consisting of polycaprolactone, polylactide, polyglycolide, polytrimethylene carbonate, polydioxanone, polyethylene glycol, polyurethane, copolymers thereof or mixtures thereof, the at least one electrospun inner layer comprising an agent capable of forming a covalent bond with proteins and peptides, the agent comprising at least one group selected from the group consisting of N-hydroxysuccinimide, maleimide, thio-NHS, biotin-NHS, carboxylic acid or aldehyde. Furthermore, the present invention relates to a method of manufacturing such a tube or wrap and to their use for the protection or bridging of nerves or for stimulating nerve growth and cell proliferation.

Description

Technical Field

[0001] This invention relates to an electrospun multilayer tube or wrap for protecting or bridging damaged nerves, comprising an electrospun insulating outer layer and at least one electrospun inner layer. The electrospun insulating outer layer comprises at least one polymer selected from: polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol, polyurethane, copolymers thereof, or mixtures thereof. The at least one electrospun inner layer comprises a reagent capable of forming covalent bonds with proteins and peptides, the reagent comprising at least one group selected from: N-hydroxysuccinimide, maleimide, thio-NHS, biotin-NHS, isocyanate, or aldehyde. Furthermore, this invention relates to methods of manufacturing such tubes or wraps and their use for protecting or bridging nerves or stimulating nerve growth and cell proliferation. Background Technology

[0002] This invention relates to an electrospun nerve conduit made of a specific polymer, such as polycaprolactone (PCL), having an inner layer containing a specific reagent, which can be functionalized, for example, using N-hydroxysuccinimide (NHS). PCL-PEG-NHS is a biocompatible amphiphilic polymer in which hydrophobic PCL is chemically bonded to hydrophilic PEG, and hydrophilic PEG is bonded to NHS. In water, the polymer forms micelles, and the NHS ester migrates to the surface to react with the amine nucleophile of the protein or hydrolyzes to a hydroxycarboxylic acid.

[0003] Succinimides, such as ethosuximide, are primarily used to treat absence seizures by reducing neurotransmission in the brain and stabilizing neuronal activity. Some succinimides have shown potential to protect neurons from damage, which may be beneficial in preventing neurodegenerative diseases, possibly due to their ability to modulate ion channels and neurotransmitter systems, as disclosed, for example, in Zefeng Zhao, Jiangxin Yue, Xiaotong Ji, Meng Nian, Kaiwen Kang, Haifa Qiao, Xiaohui Zheng, Bioorganic Chemistry 2021, 108 104557.

[0004] In this invention, it was surprisingly found that, contrary to the findings of Kamil Rahme and Nazih Dagher, Pharmaceutics 2019, 11, 327, integration of the N-hydroxysuccinimide (NHS) moiety stimulates axonal growth by migrating to the surface. In experiments, dorsal root ganglion (DRG) cell cultures were used to evaluate axonal growth and Schwann cell migration on surfaces treated with NHS-functionalized compounds. The study demonstrated unexpectedly enhanced axonal growth, highlighting the potential effectiveness of conduits in nerve regeneration. This breakthrough discovery offers broad application prospects for neural tissue engineering and regenerative medicine. Summary of the Invention

[0005] Therefore, in a first aspect, the present invention relates to an electrospun multilayer tube or wrapping, preferably for protecting or bridging damaged nerves, comprising or consisting of the following: i) An electrospun insulating outer layer comprising at least one polymer selected from: polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol, polyurethane, copolymers thereof, or mixtures thereof; and ii) At least one electrospun inner layer comprising an agent capable of forming covalent bonds with proteins and peptides and preferably stimulating neurite outgrowth, the agent comprising at least one group selected from the following: N-hydroxysuccinimide, maleimide, thio-NHS, biotin-NHS, carboxylic acid or aldehyde.

[0006] In a second aspect, the present invention relates to a method for manufacturing a multilayer tube or wrapping according to the invention, comprising or consisting of the following steps: i) Prepare a physical blend of at least one polymer selected from polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol and polyurethane with N-hydroxysuccinimide ester or copolymer thereof, preferably polycaprolactone and N-hydroxysuccinimide ester, more preferably poly(ethylene glycol)-b-poly(ε-caprolactone) (PCL-PEG-NHS), in a weight ratio of 65:35 to 35:65; ii) PCL-PEG-NHS is composed of PCL blocks with a number-average molecular weight of 2000 to 8000 g / mol, preferably 5000 g / mol, and PEG blocks with a number-average molecular weight of 200 to 10000 g / mol, preferably 5000 g / mol; iii) Dissolve the blend in a mixture of chloroform / acetone or HFIP at a concentration of 5% to 30% w / v; iv) Electrospun PCL / PCL-PEG-NHS solution as the inner layer; v) Pure PCL is electrospun onto a PCL / PCL-PEG-NHS layer as an outer layer.

[0007] In a third aspect, the present invention relates to the use of electrospun multilayer tubes or wrappings according to the invention for protecting or bridging nerves or stimulating nerve growth and cell proliferation. Attached Figure Description

[0008] Figure 1 Scanning electron micrographs of PCL and PCL-PEG-NHS scaffolds spun from hexafluoroisopropanol (HFIP) are shown.

[0009] Figure 2 The measurement results are shown as described in the experimental section.

[0010] Figure 3 The growth of dorsal root ganglia (DRGs) on PCL and PCL-PEG-NHS was compared with that of a positive control (slides coated with polyornithine and Matrigel).

[0011] Figure 4 Culture of dorsal root ganglia (DRG) and quantitative analysis of nerve growth using Image J. Invention Details 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. In case of conflict, this document (including the definitions) shall prevail. Preferred methods and materials are described below, although similar or equivalent methods and materials may be used in the practice or testing of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods and embodiments disclosed herein are illustrative only and are not intended to be limiting.

[0013] The terms “comprising,” “including,” “having,” “having,” “may,” “containing,” and variations thereof, as used herein, are intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The singular forms “a,” “an,” and “described” include plural references unless the context clearly indicates otherwise. This disclosure also covers other embodiments or elements of the present invention that are “comprising,” “consisting of,” and “substantially constitute,” whether or not they are explicitly stated.

[0014] The conjunction "or" includes any and all combinations of one or more of the listed elements associated with it. For example, the phrase "a device containing A or B" could refer to a device that includes A but does not contain B, a device that includes B but does not contain A, or a device that contains both A and B. The phrase "at least one of A, B... and N" or "at least one of A, B... N or a combination thereof" is defined in its broadest sense as meaning selected from one or more elements containing A, B... and N, that is, any combination of one or more of elements A, B... or N, including any element alone or in combination with one or more other elements, and may also include additional elements not listed in the combination.

[0015] The modifier “about” used with quantities includes the stated value and has a meaning determined by the context (e.g., it includes at least the degree of error associated with the measurement of a particular quantity). The modifier “about” should also be considered to disclose a range defined by the absolute values ​​of its two endpoints. For example, the statement “about 2 to about 4” also discloses the range “2 to 4”. The term “about” can refer to plus or minus 10% of a number. For example, “about 10%” can represent a range of 9% to 11%, and “about 1” can represent 0.9 to 1.1. Other meanings of “about” can be discerned from the context, such as rounding; therefore, for example, “about 1” can also represent 0.5 to 1.4.

[0016] The term "weight%" refers to a percentage by weight.

[0017] The term "w / w" means weight / weight.

[0018] For the purposes of this invention, the term "degradation" refers to a polymer that dissolves or degrades in vitro or in vivo over an acceptable period of time in a particular therapeutic context. Such products of dissolution or degradation may include small chemical substances. Degradation can occur, for example, by enzymatic, chemical, and / or physical methods. Biodegradation typically takes less than five years, and usually less than one year, after exposure to physiological pH and temperature (e.g., pH range 6 to 9 and temperature range 22°C to 40°C).

[0019] Sample characterization was performed using standard testing equipment. SEM was performed on a benchtop machine (available from Hitachi). Mechanical data were obtained using a standard Instron mechanical testing instrument and a dynamic mechanical analyzer available from TA Instruments.

[0020] The polymer tubes / sheets are electrospun on an electrospinning machine that is commercially available from Tongli.

[0021] In particular, the present invention relates to: Electrospun multilayer tubes or wraps, preferably used to protect or bridge damaged nerves, contain or consist of the following: i) An electrospun insulating outer layer comprising at least one polymer selected from: polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol, polyurethane, copolymers thereof, or mixtures thereof, preferably polycaprolactone; and ii) At least one electrospun inner layer comprising an agent capable of forming covalent bonds with proteins and peptides and preferably stimulating neurite growth, including but not limited to N-hydroxysuccinimide, maleimide, thio-NHS, biotin-NHS, carboxylic acid or aldehyde.

[0022] In one embodiment, the thickness ratio of the inner layer to the outer layer is in the range of 1:1 to 1:20, preferably in the range of 1:1 to 1:10, and more preferably in the range of 1:1 to 1:5.

[0023] In one embodiment, the electrospun layer has a fibrous structure with fiber diameters ranging from 0.1 to 2 µm.

[0024] In one embodiment, the tube or wrapping has a tensile strength of 0.2 to 20 MPa, preferably 0.5 to 5 MPa; and / or an elastic modulus in the range of 0.2 to 100 MPa, preferably 0.2 to 20 MPa; and / or a thickness of 0.1 to 1 mm, preferably 0.5 mm.

[0025] In one embodiment, the tube has a nanofiber structure and an average diameter of 2 to 6 mm; and / or a length of 3 to 25 cm, preferably 3 to 10 cm; and / or a thickness ranging from 0.1 to 1 mm, preferably 0.5 mm.

[0026] In one embodiment, the reagent of at least one electrospun inner layer comprises PCL-PEG-NHS, which preferably consists of a PCL block with a number-average molecular weight of 2000 to 8000 g / mol, preferably 5000 g / mol, and a PEG block with a number-average molecular weight of 200 to 10000 g / mol, preferably 5000 g / mol, of Mn.

[0027] In one embodiment, the tube or wrapping is semi-permeable.

[0028] Furthermore, the present invention relates to a method for manufacturing a multilayer tube or wrapping according to the present invention, which includes or consists of the following steps: i) Prepare a physical blend of at least one polymer selected from polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol, polyurethane, and N-hydroxysuccinimide ester or copolymer thereof, preferably polycaprolactone and N-hydroxysuccinimide ester, preferably poly(ethylene glycol)-b-poly(ε-caprolactone) (PCL-PEG-NHS), in a weight ratio of 65:35 to 35:65; ii) PCL-PEG-NHS is composed of PCL blocks with a number-average molecular weight of 2000 to 8000 g / mol, preferably 5000 g / mol, and PEG blocks with a number-average molecular weight of 200 to 10000 g / mol, preferably 5000 g / mol; iii) Dissolve the blend in a mixture of chloroform / acetone or HFIP at a concentration of 5% to 30% w / v; iv) Electrospun PCL / PCL-PEG-NHS solution as the inner layer; v) Pure PCL is electrospun onto a PCL / PCL-PEG-NHS layer as an outer layer.

[0029] Finally, the present invention relates to the use of electrospun multilayer tubes or wrappings according to the present invention for protecting or bridging nerves or stimulating nerve growth and cell proliferation.

[0030] Example Example 1 A physical polymer blend of RESERMER® C212 (PCL) and N-hydroxysuccinimide-poly(ethylene glycol)-b-poly(ε-caprolactone) (containing PEG and PCL blocks with a number average molecular weight of 5000 g / mol) was prepared at a weight ratio of 65:35. This mixture was dissolved in hexafluoroisopropanol (HFIP) at 12.5% ​​w / v. Sheets or tubes were prepared using an electrospinning system consisting of a high-voltage power supply, a dual-channel syringe pump, and a rotating collector. The inner layer was made using a PCL / PCL-PEG-NHS solution, with a positive voltage of 6.6 kV applied to a 20 g A stainless steel needle tip and a negative voltage of -1.2 kV applied to the rotating collector. After 2.5 mL was delivered by the syringe pump, the second syringe channel was activated with PCL solution and the flow rate was set to 0.4 mL / hr. As the flow rate of the PCL / PCL-PEG-NHS solution decreased to 0.4 mL / hr, the voltage was increased to 22.2 kV. After delivering 0.25 mL, stop the flow of the PCL / PCL-PEG-NHS solution and increase the flow rate of the PCL solution to 0.75 mL / hr. Continue spinning with an additional 2.5 mL.

[0031] SEM images of PCL and PCL / PCL-PEG-NHS are shown in Figure 1The images show slight morphological differences, which could be attributed to variations in electrical properties or jet stability during spinning. The surface of the electrospun sheets was characterized using X-ray photoelectron spectroscopy (XPS), such as... Figure 2 As shown, the oxygen content in the PCL / PCL-PEG-NHS sample was higher than that in the unmodified PCL sample, due to the presence of additional oxygen bonds from the polyethylene glycol (PEG) linker polymer. This indicates that the PEG and NHS portions migrate to the surface, affecting surface properties and neural cell behavior. After electrospinning, the resulting sheets were used in cell culture studies to evaluate neural growth.

[0032] Example 2 Mouse Tissue Collection and Spheroid Formation: All animal procedures were reviewed and approved by Tulane University's Institutional Animal Care and Use Committee (IACUC). Dorsal root ganglion (DRG) tissue from Long Evans mouse embryos at day 15 (e15) was collected according to a protocol developed by Moore's laboratory. Briefly, DRGs were isolated and collected from a litter of mouse embryos and dissociated in 0.25% trypsin-EDTA for 10 minutes. Mouse tissues were centrifuged at 500g for 5 minutes at room temperature, and the dissociation medium was aspirated. Mouse tissues were resuspended and triturated in Neurobasal medium supplemented with 2% v / v B27, 1% v / v N2, 1% v / v GlutaMAX, nerve growth factor 2.5S native mouse protein (20 ng / ml), recombinant human / mouse / rat brain-derived neurotrophic factor (10 ng / ml; PeproTech, Cranbury, NJ, USA), recombinant human glial cell-derived neurotrophic factor (10 ng / ml; PeproTech), and 1% v / v antibiotic / antifungal solution (all from Thermo Fisher Scientific, Waltham, MA unless otherwise specified). Cells were passed through a 40 µm cell filter, counted, and plated at 45,000 cells / well in ULA round-bottom 96-well plates. The microplates were centrifuged at 500 g for 5 minutes at room temperature and allowed to form spheres for more than 48 hours. Substrate preparation and spheroid inoculation: All substrates from Example 1 were cut into circles using a biopsy punch and placed in PBS containing 1% v / v antibiotic / antifungal solution for at least 4 hours before use. Glass coverslips coated with polyornithine and Matrigel were used as positive controls. 12 mm circular coverslips were first cleaned in ethanol and allowed to dry completely, then coated with 0.01% polyornithine solution for 1 hour. This solution was aspirated, and Matrigel solution diluted 1:100 in Neurobasal (ESC grade, Corning) was added. The substrates were incubated at 37°C in a humidified incubator for at least 3 hours. After all substrates were prepared, they were placed in minimal cell culture medium (as described above), with a single DRG spheroid placed in the center. Allowing them to adhere for 2 hours was allowed, then more culture medium was added.

[0033] Image Analysis: Images were analyzed in ImageJ (National Institutes of Health; Maryland, USA) using the plugin Neurote-J 1.1 (Torres-Espin; doi.org / 10.1016 / j.jneumeth.2014.08.005). Neurite-J employed a modified Sholl analysis method, where concentric circles were generated around the central organoid / organoid culture. Samples were analyzed at 25 µm intervals, with thresholding performed using the same values ​​as for β3-tubulin-stained neurites. Values ​​obtained from Neurote-J included the crossover distribution (number of crossovers per interval), Nmax (maximum number of crossovers), and the critical value (RC, distance at which the maximum number of crossovers occurs) for each condition.

[0034] Figure 3 The figures illustrate neurites stained with β3-tubulin on different samples. β3-tubulin serves as a neuronal marker, confirming and visualizing neurite growth and highlighting network extensions on the modified surface. Positive controls, glass slides coated with polyornithine and Matrigel, show optimal neurite growth, providing a benchmark for neural connectivity and validating the efficacy of surface modification in neural tissue engineering applications.

[0035] Figure 4 Quantitative neurite growth was analyzed using ImageJ. Data showed a significant increase in neurite crossover points on the PCL / PCL-PEG-NHS surface compared to unmodified PCL. Specifically, the modified PCL showed a 16% increase in the maximum number of crossover points, reaching 141, compared to 122 for unmodified PCL. Furthermore, the critical value (RC), indicating the length at which the maximum number of neurites was detected, increased by 75% compared to unmodified PCL. This significant improvement highlights the positive impact of NHS modification on neural connectivity, likely due to the enhanced surface properties promoting cell adhesion and growth.

[0036] While NHS is commonly used for binding proteins and peptides, this study unexpectedly observed its positive effects on neurite growth. Previous research has highlighted the potential of succinimide in protecting neurons from damage, which may be beneficial in preventing neurodegenerative diseases, possibly due to its ability to modulate ion channels and neurotransmitter systems (see Zefeng Zhao, Jiangxin Yue, Xiaotong Ji, Meng Nian, Kaiwen Kang, Haifa Qiao, Xiaohui Zheng, Bioorganic Chemistry 2021, 108 104557).

Claims

1. An electrospun multilayer tube or wrapping material, comprising or consisting of the following: i) An electrospun insulating outer layer comprising at least one polymer selected from: polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol, polyurethane, copolymers thereof, or mixtures thereof; and ii) At least one electrospun inner layer comprising a reagent capable of forming covalent bonds with proteins and peptides, the reagent comprising at least one group selected from the following: N-hydroxysuccinimide, maleimide, thio-NHS, biotin-NHS, carboxylic acid or aldehyde.

2. The electrospun multilayer tube or wrapping according to claim 1, wherein the electrospun inner layer comprises PCL-PEG-NHS.

3. The electrospun multilayer tube or encapsulation according to claim 2, wherein the PCL-PEG-NHS is composed of PCL blocks with a number-average molecular weight of 2000 to 8000 g / mol, preferably 5000 g / mol, and PEG blocks with a number-average molecular weight of 200 to 10000 g / mol, preferably 5000 g / mol, of Mn.

4. The electrospun multilayer tube or wrapping according to any one of the preceding claims, wherein the electrospun inner layer further comprises PCL, preferably the weight ratio of PCL to PCL-PEG-NHS is 65:35, 50:50 or 35:

65.

5. The electrospun multilayer tube or wrapping according to any one of the preceding claims, wherein the thickness ratio of the inner layer to the outer layer is in the range of 1:1 to 1:

20.

6. The electrospun multilayer tube or wrapping according to any one of the preceding claims, wherein the electrospun layer has a fibrous structure with a fiber diameter ranging from 0.1 to 2 µm.

7. The electrospun multilayer tube or wrapping according to any one of the preceding claims, wherein the tube or wrapping has i) Tensile strength from 0.2 to 20 MPa; and / or ii) Elastic modulus ranging from 0.2 to 100 MPa; and / or iii) Thickness of 0.1 to 1 mm.

8. The electrospun multilayer tube according to any one of the preceding claims, wherein the tube has a fiber structure and comprises: i) an average diameter of 2 to 6 mm; and / or ii) A length of 3 to 25 cm; and / or iii) Thickness ranging from 0.1 to 1 mm.

9. A method for manufacturing a multilayer tube or wrapping according to any one of claims 1 to 8, comprising or consisting of the following steps: i) Prepare a physical blend of at least one polymer selected from polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol, polyurethane and N-hydroxysuccinimide ester or copolymer thereof; ii) PCL-PEG-NHS is composed of PCL blocks with a number-average molecular weight of 2000 to 8000 g / mol and PEG blocks with a number-average molecular weight of 200 to 10000 g / mol. iii) Dissolve the blend in a mixture of chloroform / acetone or HFIP at a concentration of 5% to 30% w / v; iv) Electrospun PCL / PCL-PEG-NHS solution as the inner layer; v) Pure PCL is electrospun onto a PCL / PCL-PEG-NHS layer as an outer layer.

10. Use of the electrospun multilayer tube or wrapping according to any one of claims 1 to 8 for protecting or bridging nerves or stimulating nerve growth and cell proliferation.

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