Electro-spun tubes or wraps with functionalized inner layer for nerve growth

Abstract

The present invention refers to an electro-spun multilayer tube or wrap to protect or bridge damaged nerve comprising an electro-spun isolating outer layer comprising at least one polymer selected from poly(caprolactone), polylactide, polyglycolide, poly(trimethylene carbonate), poly(dioxanone), polyethylene glycol, polyurethane, their copolymers or mixtures thereof and at least one electro-spun inner layer comprising a reagent which is able to undergo covalent bonds with proteins and peptides, which comprises at least one group selected from N-hydroxy succinimide, maleimide, sulfur-NHS and biotin-NHS, carboxylic acid or aldehydes. Furthermore, the present invention refers to methods of manufacturing such tubes or wraps as well as their use in protecting or bridging nerves or to stimulate nerve growth and cell proliferation.

Description

[0001] Electro-spun tubes or wraps with functionalized inner layer for nerve growth

[0002] Field of the invention

[0003] The present invention refers to an electro-spun multilayer tube or wrap to protect or bridge damaged nerve comprising an electro-spun isolating outer layer comprising at least one polymer selected from poly(caprolactone), polylactide, polyglycolide, poly(trimethylene carbonate), poly(dioxanone), polyethylene glycol, polyurethane, their copolymers or mixtures thereof and at least one electro-spun inner layer comprising a reagent which is able to undergo covalent bonds with proteins and peptides, which comprises at least one group selected from N-hydroxy succinimide, maleimide, sulfur-NHS and biotin-NHS, isocyanate, or aldehydes. Furthermore, the present invention refers to methods of manufacturing such tubes or wraps as well as their use in protecting or bridging nerves or to stimulate nerve growth and cell proliferation.

[0004] Description of the related art

[0005] This invention refers to an electro-spun nerve conduit made from a specific polymer, e.g., polycaprolactone (PCL) with an inner layer comprising a specific reagent, which can for example be functionalized using N-hydroxy succinimide (NHS). PCL-PEG-NHS is a biocompatible amphiphilic polymer, where hydrophobic PCL is chemically bonded to hydrophilic PEG, which is bounded to NHS. In water, the polymer forms micelles with NHS ester migrating to the surface to react with amine nucleophiles from proteins or hydrolyze into hydroxy acid.

[0006] Succinimides, like ethosuximide, are primarily used to treat absence seizures by reducing nerve transmission in the brain, stabilizing neuronal activity. Some succinimides have shown potential in protecting neurons from damage, which could be beneficial in preventing neurodegenerative diseases, possibly due to their ability to modulate ion channels and neurotransmitter systems, for example disclosed in Zefeng Zhao , Jiangxin Yue , Xiaotong Ji , Meng Nian, Kaiwen Kang, Haifa Qiao, Xiaohui Zheng, Bioorganic Chemistry 2021 , 108 104557.

[0007] In this invention it has been surprisingly found that the integration of N-hydroxysuccinimide (NHS) moieties stimulates axonal growth by migrating to the surface, in contrast to the findings of Kamil Rahme and Nazih Dagher, Pharmaceutics 2019, 11 , 327. In the experiments, dorsal root ganglion (DRG) cell cultures were used to assess axonal growth and Schwann cell migration on surfaces threated with NHS functionalized compounds. The studies demonstrated unexpectedly enhanced axonal growth, highlighting the conduit’s potential effectiveness in nerve regeneration. This breakthrough finding offers promising applications in neural tissue engineering and regenerative medicine.

[0008] Summary of the invention

[0009] Therefore, in a first aspect the present invention refers to an electro-spun multilayer tube or wrap, to preferably protect or bridge damaged nerve, comprising or consisting of i) an electro-spun isolating outer layer comprising at least one polymer selected from poly(caprolactone), polylactide, polyglycolide, poly(trimethylene carbonate), poly(dioxanone), polyethylene glycol, polyurethane, their copolymers or mixtures thereof; and ii) at least one electro-spun inner layer comprising a reagent which is able to undergo covalent bonds with proteins and peptides and preferably stimulate neurite outgrowth, which comprises at least one group selected from N-hydroxy succinimide, maleimide, sulfur-NHS and biotin-NHS, carboxylic acids or aldehydes.

[0010] In a second aspect the present invention refers to a method of manufacturing a multilayer tube or wrap according to the present invention comprising or consisting of the following steps: i) preparing a physical blend of at least one polymer selected from poly(caprolactone), polylactide, polyglycolide, poly(trimethylene carbonate), poly(dioxanone), polyethylene glycol, and polyurethane with N-hydroxy succinimide ester or its copolymers thereof, preferably polycaprolactone and N-hydroxy succinimide ester more preferably poly(ethylene glycol)-b-poly(e- caprolactone) (PCL-PEG-NHS) at a weight ratio ranging 65:35 to 35:65; ii) PCL-PEG-NHS consists of PCL blocks of 2000 to 8000 g / mol Mn number average molecular weight, preferably 5,000 g / mol Mn number average molecular weight and PEG block of 200 to 10000 g / mol, preferably 5,000 g / mol Mn number average molecular weight; iii) dissolving the blend in a mixture of chloroform / acetone or HFIP with concentration of 5 to 30% w / v ratio; iv) electrospinning PCL / PCL-PEG-NHS solution as the inner layer; v) electrospinning pure PCL onto PCL / PCL-PEG-NHS layer as outer layer.

[0011] In a third aspect the present invention refers to the use of the electro-spun multilayer tube or wrap according to the present invention for protecting or bridging nerves or to stimulate nerve growth and cell proliferation.

[0012] Brief description of the drawings

[0013] Fig 1. Shows a scanning electron micrograph of a PCL and PCL-PEG-NHS scaffolds spun from hexafluoro isopropanol (HFIP).

[0014] Fig 2. show results of measurements as stated in the experimental section.

[0015] Fig 3. Dorsal root ganglion (DRG) growth comparison on PCL and PCL-PEG-NHS in comparison to positive control (glass slide coated with poly-ornithine and Matrigel).

[0016] Fig 4. Dorsal root ganglion (DRG) culture and quantification of nerve growth using Image J. Detailed description of the invention

[0017] 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, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0018] The terms “comprise(s)”, “include(s)”, “having”, “has”, “can”, “contain(s)”, and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising”, “consisting of’ and “consisting essentially of’, the embodiments or elements presented herein, whether explicitly set forth or not.

[0019] The conjunctive term “or” includes any and all combinations of one or more listed elements associated by the conjunctive term. For example, the phrase “an apparatus comprising A or B” may refer to an apparatus including A where B is not present, an apparatus including B where A is not present, or an apparatus where both A and B are present. The phrases “at least one of A, B, . . . and N” or “at least one of A, B, . . . N, or combinations thereof’ are defined in the broadest sense to mean one or more elements selected from the group comprising A, B, . . . and N, that is to say, any combination of one or more of the elements A, B, . . . or N including any one element alone or in combination with one or more of the other elements which may also include, in combination, additional elements not listed.

[0020] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4”. The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1 .1 . Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1 ” may also mean from 0.5 to 1 .4.

[0021] The term “wt%” means weight percent.

[0022] The term “w / w” means weight per weight.

[0023] For the purposes of the present invention, the term “degradation” refers to polymers that dissolve or degrade in vitro or in vivo within a period of time that is acceptable in a particular therapeutic situation. Such dissolved or degraded product may include a smaller chemical species. Degradation can result, for example, by enzymatic, chemical and / or physical processes. Biodegradation takes typically less than five years and usually less than one year after exposure to a physiological pH and temperature, such as a pH ranging from 6 to 9 and a temperature ranging from 22 °C to 40 °C. Sample characterization was performed using standard test machines. SEM was performed on a desktop machine (commercially available from the company Hitachi). Mechanical data were performed on a standard Instron Mechanical Tester and a dynamic mechanical analyzer commercially available from TA Instruments.

[0024] Polymeric tube / sheets were electro-spun on an electrospinner commercially available from the company Tongli.

[0025] In particular the present invention refers to:

[0026] An electro-spun multilayer tube or wrap, to preferably protect or bridge damaged nerve, comprising or consisting of i) an electro-spun isolating outer layer comprising at least one polymer selected from poly(caprolactone), polylactide, polyglycolide, poly(trimethylene carbonate), poly(dioxanone), polyethylene glycol, polyurethane, their copolymers or mixtures thereof, preferably poly(caprolactone); and ii) at least one electro-spun inner layer comprising a reagent which is able to undergo covalent bonds with proteins and peptides and preferably stimulate neurite outgrowth, including but not limited to N-hydroxy succinimide, maleimide, sulfur-NHS and biotin-NHS, carboxylic acid, or aldehydes.

[0027] In one embodiment the ratio of inner layer to outer layer thickness is in a range of 1 :1 to 1 :20, preferably in a range of 1 :1 to 1 :10, and more preferably in a range of 1 :1 to 1 :5.

[0028] In one embodiment the electro-spun layers have a fibrous structure with fibres diameters ranging 0.1 to 2 pm.

[0029] In one embodiment the tube or wrap has a tensile strength of from 0.2 to 20 MPa, preferably 0.5 to 5 MPa; and / or has an elastic modulus ranging of from 0.2 to 100 MPa, preferably 0.2 to 20 MPa, and / or has a thickness of from 0.1 to 1 mm preferably 0.5 mm.

[0030] In one embodiment the tube has a nanofibrous structure and has 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 of from 0.1 to 1 mm preferably 0.5 mm.

[0031] In one embodiment the reagent of the at least one electro-spun inner layer comprises PCL-PEG-NHS, which preferably consists of PCL blocks of 2000 to 8000 g / mol Mn number average molecular weight, preferably 5,000 g / mol Mn number average molecular weight and PEG block of 200 to 10000 g / mol, preferably 5,000 g / mol Mn number average molecular weight.

[0032] In one embodiment the tube or wrap is semi-permeable. Additionally, the present invention refers to a method of manufacturing a multilayer tube or wrap according to the present invention comprising or consisting of the following steps: i) preparing a physical blend of at least one polymer selected from poly(caprolactone), polylactide, polyglycolide, poly(trimethylene carbonate), poly(dioxanone), polyethylene glycol, polyurethane, with N-hydroxy succinimide ester or its copolymers thereof, preferably polycaprolactone and N- hydroxy succinimide ester preferably poly(ethylene glycol)-b-poly(e-caprolactone) (PCL-PEG- NHS) at a weight ratio ranging 65:35 to 35:65; ii) PCL-PEG-NHS consists of PCL blocks of 2000 to 8000 g / mol Mn number average molecular weight, preferably 5,000 g / mol Mn number average molecular weight and PEG block of 200 to 10000 g / mol, preferably 5,000 g / mol Mn number average molecular weight; iii) dissolving the blend in a mixture of chloroform / acetone or HFIP with concentration of 5 to 30% w / v ratio; iv) electrospinning PCL / PCL-PEG-NHS solution as the inner layer; v) electrospinning pure PCL onto PCL / PCL-PEG-NHS layer as outer layer.

[0033] Finally, the present invention refers to the use of the electro-spun multilayer tube or wrap according to the present invention for protecting or bridging nerves or to stimulate nerve growth and cell proliferation.

[0034] Examples

[0035] Example 1 :

[0036] A physical polymer blend of RESOMER® C212 (PCL) and N-Hydroxysuccinimide ester-poly(ethylene glycol)-b-poly(e-caprolactone) .containing PEG and PCL blocks of 5,000 g / mol number average molecular weight, was prepared at a weight ratio of 65 to 35. The mixture was dissolved at 12.5% w / v in a hexafluroisopropanol (HFIP). Sheet or tubes were fabricated using an electrospinning system consisting of a high voltage supply, dual channel syringe pump, and a rotating collector. The internal layer was made using the PCI / PCI-PEG-NHS solution using a positive voltage of 6.6 kV, at a 20 ga stainless steel needle tip and a negative voltage of -1.2 kV on a rotating collector. After the syringe pump delivered 2.5 mL, the second syringe channel was activated with the PCL solution and set to a flow rate of 0.4 mL / hr. The flow rate of the PCI / PCI-PEG-NHS solution was reduced to 0.4 mL / hr and the voltage was increased to 22.2 kV. After delivery of 0.25 mL, the flow of the PCI / PCI-PEG-NHS solution was stopped and the flow rate of the PCL solution was increased to 0.75 mL / hr. Spinning continued for an additional 2.5 mL.

[0037] The SEM images of PCL and PCL / PCL-PEG-NHS are shown in Figure 1 . The images reveal slight morphological differences, which could be attributed to variations in electrical properties or jet stability during spinning. The surface of the electrospun sheet was characterized using X-ray photoelectron spectroscopy (XPS), as shown in Figure 2. The oxygen content in the PCL / PCL-PEG-NHS samples was higher than in the unmodified PCL samples, due to the presence of additional oxygen bonds from the polyethylene glycol (PEG) linker polymer. This suggests surface migration of the PEG and NHS moieties, influencing surface properties and nerve cell behavior. After electrospinning, the resulting sheet was used for cell culture studies to assess nerve outgrowth.

[0038] Example 2:

[0039] Mouse tissue harvesting 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 embryonic day 15 (e15) embryos were collected following protocols developed in the Moore lab. Briefly, DRGs were isolated and pooled from a single litter of mouse embryos and dissociated in 0.25% trypsin-EDTA for 10min. Mouse tissue was centrifuged 500g for 5min at room temperature and the dissociation media was aspirated. Mouse tissue was resuspended and triturated in culture media consisting of Neurobasal medium supplemented with 2% v / v B27 supplement, 1 % v / v N2 supplement, 1 % v / v GlutaMAX, nerve growth factor 2.5S native mouse protein (20 ng / ml), recombinant human / murine / 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 / antimycotic solution (all from Thermo Fisher Scientific, Waltham, MA, unless otherwise noted). Cells were passed through a 40pm cell strainer before counting and plating within ULA round bottom 96- well plates at 45,000 cells / well. Microplates were centrifuged at 500g for 5min at room temperature and spheroids were allowed to for over 48h. Substrate preparation and spheroid plating: All substrates from Example 1 were cut into circles using a biopsy punched and placed into PBS with 1 % v / v antibiotic / antimycotic solution for a minimum of 4h prior to use. Glass coverslips coated with polyornithine and Matrigel were used as a positive control. 12mm round coverslips were first cleaned in ethanol, allowed to completely dry and then coated with 0.01% polyornithine solution for 1 h. This solution was aspirated and a Matrigel solution was added at a 1 :100 dilution in Neurobasal (ESC-qualified; Corning). This was allowed to incubate at 37°C in a humidified incubator for a minimum of 3h. Once all substrates were prepared, they were placed in minimal cell culture media (described Above) and a single DRG spheroid was placed in the middle. These were allowed to adhere for 2h prior to the addition of more media.

[0040] Image Analysis: Images were analyzed in Imaged (National Institutes of Health; Maryland, USA) using the plug-in Neurite-J 1.1 (Torres-Espin; doi.org / 10.1016 / j.jneumeth.2014.08.005). Neurite-J uses a modified Sholl analysis approach that generates concentric circles around a central organoid / organotypic culture. Samples were analyzed with a distance interval of 25pm and were thresholded using the same values for p3-tubulin stained neurites. Values obtained from Neurite-J included an intersection profile (number of intersections at each distance interval), Nmax (maximum number of intersections), and Critical Value (RC, the distance where the maximum number of intersections occurred) were recorded for each condition.

[0041] Figure 3 illustrates p3 Tubulin-stained neurites on various specimens. p3-Tubulin serves as a neuronal marker, confirming and visualizing neurite outgrowth and highlighting network extensions on modified surfaces. The positive control, glass slides coated with polyornithine and Matrigel, demonstrated optimal neurite outgrowth, providing a benchmark for neural connectivity and validating the efficacy of surface modifications in neural tissue engineering applications.

[0042] Figure 4 quantifies neurite outgrowth using Imaged analysis. The data shows a significant enhancement in neurite intersections on PCL / PCL-PEG-NHS surfaces compared to unmodified PCL. In particular, the modified PCL exhibited a 16% increase in maximum intersections, with 141 intersections versus 122 for the unmodified one. Additionally, the critical value (RC), indicating the length where the maximum number of neurites was detected, revealed a 75% increase compared to unmodified PCL. This substantial improvement highlights the positive impact of NHS modification on neural connectivity, likely due to enhanced surface properties that facilitate cellular adhesion and growth.

[0043] While NHS is typically used for bonding proteins and peptides, this study unexpectedly observed its positive impact on neurite outgrowth. Previous research has highlighted the potential of succinimides in protecting neurons from damage, which could be beneficial in preventing neurodegenerative diseases, possibly due to their ability to modulate ion channels and neurotransmitter systems (cf. in Zefeng Zhao , Jiangxin Yue , Xiaotong Ji , Meng Nian, Kaiwen Kang, Haifa Qiao, Xiaohui Zheng, Bioorganic Chemistry 2021 , 108 104557).

Claims

Claims1 . An electro-spun multilayer tube or wrap comprising or consisting of i) an electro-spun isolating outer layer comprising at least one polymer selected from poly(caprolactone), polylactide, polyglycolide, poly(trimethylene carbonate), poly(dioxanone), polyethylene glycol, polyurethane, their copolymers or mixtures thereof; and ii) at least one electro-spun inner layer comprising a reagent which is able to undergo covalent bonds with proteins and peptides, which comprises at least one group selected from N-hydroxy succinimide, maleimide, sulfur-NHS and biotin-NHS, carboxylic acid or aldehydes.

2. The electro-spun multilayer tube or wrap of claim 1 , wherein the electro-spun inner layer comprises PCL-PEG-NHS.

3. The electro-spun multilayer tube or wrap of claim 2, wherein the PCL-PEG-NHS consists of PCL blocks of 2000 to 8000 g / mol Mn number average molecular weight, preferably 5,000 g / mol Mn number average molecular weight and PEG block of 200 to 10000 g / mol, preferably 5,000 g / mol Mn number average molecular weight.

4. The electro-spun multilayer tube or wrap of any of the preceding claims, wherein the electro-spun inner layer further comprises PCL, preferably at a weight ratio of PCL to PCL-PEG-NHS of 65: 35, 50: 50 or 35: 65.

5. The electro-spun multilayer tube or wrap of any of the preceding claims, wherein the ratio of inner layer to outer layer thickness is in a range of 1 :1 to 1 :20.

6. The electro-spun multilayer tube or wrap of any of the preceding claims, wherein the electro-spun layers have a fibrous structure with fibres diameters ranging 0.1 to 2 pm.

7. The electro-spun multilayer tube or wrap of any of the preceding claims, wherein the tube or wrap i) has a tensile strength of from 0.2 to 20 MPa; and / or ii) has an elastic modulus ranging of from 0.2 to 100 MPa; and / or iii) has a thickness of from 0.1 to 1 mm.

8. The electro-spun multilayer tube of any of the preceding claims, wherein the tube has a fibrous structure and has i) an average diameter of 2 to 6 mm; and / or ii) a length of 3 to 25 cm; and / or iii) a thickness ranging of from 0.1 to 1 mm.

9. Method of manufacturing a multilayer tube or wrap according to any of claims 1 to 8 comprising or consisting of the following steps: i) preparing a physical blend of at least one polymer selected from poly(caprolactone), polylactide, polyglycolide, poly(trimethylene carbonate), poly(dioxanone), polyethylene glycol, polyurethane, with N-hydroxy succinimide ester or its copolymers thereof; ii) PCL-PEG-NHS consists of PCL blocks of 2000 to 8000 g / mol Mn number average molecular weight and PEG block of 200 to 10000 g / mol Mn number average molecular weight; iii) dissolving the blend in a mixture of chloroform / acetone or HFIP with concentration of 5 to 30% w / v ratio; iv) electrospinning PCL / PCL-PEG-NHS solution as the inner layer; v) electrospinning pure PCL onto PCL / PCL-PEG-NHS layer as outer layer.

10. Use of the electro-spun multilayer tube or wrap according to any one of claims 1 to 8 for protecting or bridging nerves or to stimulate nerve growth and cell proliferation.

Images