Multilayer nerve regeneration guide tube

Abstract

The present invention relates to electrospun multilayer tubes or wraps for the protection or bridging of damaged nerves, said tubes or wraps comprising an electrospun isolating outer layer and an active inner layer, said 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 or mixtures thereof, said active inner layer comprising at least one electrically conductive compound and / or at least one ionically conductive compound and / or at least one peptide or protein to stimulate nerve growth. Furthermore, the present invention relates to a method for the manufacture of such tubes or wraps and their use for the protection or bridging of nerves or for the stimulation of nerve growth and cell proliferation.

Description

Technical Field

[0001] This invention relates to electrospun multilayer tubes or wraps for protecting or bridging damaged nerves, said tubes or wraps comprising an electrospun insulating outer layer and an active 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 active inner layer comprises at least one electrically conductive compound and / or at least one ion-conducting compound and / or at least one peptide or protein to stimulate nerve growth. Furthermore, this invention relates to methods of manufacturing such tubes or wraps and their use for protecting or bridging nerves or for stimulating nerve growth and cell proliferation. Background Technology

[0002] Resorbable conduits and conductive polymers for nerve regeneration have been extensively discussed in the prior art. These resorbable polymers are emerging as viable alternatives to non-resorbable materials used in implantable nerve growth conduits (NGCs) to repair damaged nerves, as previously disclosed by YZ Bian et al. Both naturally and synthetically derived resorbable polymers, such as polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polycaprolactone (PCL), collagen, and chitosan, have been used in NGC fabrication. Furthermore, studies have evaluated poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) conduits for peripheral nerve regeneration (Biomaterials, 2009, 30(2), 217-225). Previous work has described the fabrication of PLA multichannel conduits with nanofiber microstructures using molds, and evaluated the effect of scaffold geometry on in vitro neural stem cell differentiation (Tissue Eng. Part A., 2014, 20, 1038-1048). The combined use of chitosan / PLGA scaffolds and MSCs has successfully bridged large gaps in the canine sciatic nerve (Neurorehabil. Neural Repair, 2012, 26, 96-106). Multichannel scaffolds seeded with Schwann cells have also been developed for peripheral nerve regeneration (J. Neuro. Sci., 2017, 381, 612-613). Current advances in polymeric biomaterials for neural tissue engineering have been extensively explored (J. Biomed. Sci., 2018, 25, 90). It has been shown that while synthetic biopolymers such as branched PLA, PLGA, and PCL offer enhanced mechanical strength, they generally lack bioactivity (Int. J. Surg., 2019, 20, 15-19; Front.). In another study, 3D-printed PCL / PPy conductive scaffolds, serving as three-dimensional porous scaffolds, have been used as nerve conduits (NGCs) for peripheral nerve injury repair (Front. Bioeng. Biotechnol., 2019, 7, 266). Animal-derived materials such as collagen and gelatin have shown excellent biodegradability and biocompatibility. However, they generally lack sufficient mechanical strength and pose a risk of immunogenic response. Bioactive agents such as growth factors have been added to synthetic reabsorbable polymers to promote specific functional recovery, as disclosed in CN 101543645 B1. However, these growth factors typically have short (sort) half-lives, poor stability, and the potential to diffuse into other areas of the body.Immobilizing bioactive agents into reabsorbable polymers has proven to be an effective method, for example by forming copolymers between PLGA and polylysine, for the treatment of open and closed spinal cord injuries, as disclosed in US 8858966 B2.

[0003] In addition to being biodegradable and biocompatible, ideal NGCs possess other essential properties such as permeability, flexibility, minimal swelling, and internal structure. Permeable porous structures allow the transport of nutrients, oxygen, and metabolic waste into and out of membranes or scaffolds, and these porous structures are well-suited for tissue engineering applications for tissue regeneration and repair, as disclosed in US 9707000 B2 and US 8926886 B2. Electrospinning of reabsorbable natural or synthetic polymers or mixtures thereof dissolved in suitable solvents is commonly used to fabricate highly porous scaffolds, and even scaffolds with highly aligned microfibers or nanofibers to guide tissue regeneration, as disclosed in US 10405963 B2 and US 2014079759 A1. Furthermore, electrospinning has been used to generate micropatterns for guided tissue engineering in cardiovascular applications, as disclosed in US 2006 / 0085063 A1. According to US 2011 / 0236974 A1, the use of hybrid biomaterials has also been explored, which involves blending pure laminin or a composite extract containing laminin with reabsorbable polymers (e.g., PCL and PLA / PLGA) via electrospinning. This differs from the present invention, which relates to the generation of multilayer tubes or sheets in which peptides and proteins are covalently bonded to the inner layer of a matrix, preserving bioactivity and durability.

[0004] The application of conductive polymers such as polypyrrole (PPy) in NGCs has led to increased fibronectin adsorption, resulting in enhanced neurite outgrowth (E. Gamez et al.). Photofabrication of gelatin-based neural conduits has demonstrated the potential for neural tissue regeneration (Cell Transplantation, 2004, 13, 549-564). Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) dispersed in PLGA solutions have also been electrospun into oriented nanofiber scaffolds, where the carbon nanotubes provide conductivity to promote neurite outgrowth. The resulting nanofiber scaffolds are further coated with a poly-L-lysine solution to increase the number of positively charged sites for cell binding, as disclosed in US 6583232 B1. PPy has been coated onto electrospun PLGA nanofibers to produce electrically conductive nanofibers for neural tissue applications, as disclosed in US 6607548 B2.

[0005] This invention provides a method for manufacturing multilayer electrospun tubes or wraps for repairing damaged nerves via electrospinning. Figure 1-2 The inner layer of the electrospinning tube or wrapping further comprises at least one electrically conductive compound and / or at least one ion-conductive compound to stimulate nerve growth, such as... Figure 3 and 4 As shown, this method optimizes the use of conductive polymers and minimizes the amount required for nerve stimulation. Furthermore, the inclusion of positively charged proteins, peptides, or polypeptides, such as poly-L-lysine, can promote nerve regeneration. While proteins or peptides are typically coated on the surface of a polymer or blended into a polymer matrix, free or non-covalently bonded proteins and peptides can be easily removed from the system, limiting their regenerative effects. To address this issue, in one embodiment, an agent is introduced onto the surface of the tube / package to covalently bond with the amino groups of the protein and maintain the regenerative effect of poly-L-lysine, collagen, or collagen-like proteins (e.g., VECOLLAN®). This method has been... Figure 5 and Figure 6 As shown in the image. Summary of the Invention

[0006] Therefore, in a first aspect, the present invention relates to electrospun multilayer tubes or wraps for preferably protecting or bridging damaged nerves, said electrospun multilayer tubes or wraps comprising or composed 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 or mixtures thereof; ii) At least one electrospun intermediate layer, said at least one electrospun intermediate layer comprising a reagent capable of covalently bonding with proteins and peptides, said reagent comprising at least one of the following groups selected from N-hydroxysuccinimide, maleimide, thio-NHS and biotin-NHS, isocyanate, or aldehyde; and iii) An inner layer comprising at least one electrically conductive compound and / or at least one ion-conductive compound and / or at least one peptide or protein, preferably for stimulating nerve growth.

[0007] 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 the following blends: a) 50 to 99 wt% of a polymer selected from polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol, polyurethane, copolymers or mixtures thereof, and b) 1 to 50 wt% of an electrically conductive polymer selected from polypyrrole, polyaniline, polythiophene, poly(3,4-ethylenedioxythiophene), or electrically conductive additives such as graphene, graphene oxide, metal, carbon, or carbon nanotubes, wherein the total of all components is 100 wt% of the composite. ii) Dissolve the blend from step i) in a solvent, such as chloroform, acetone, or a mixture thereof, or hexafluoroisopropanol (HFIP); and iii) Electrospinning the dissolved blend onto a mandrel having a diameter of 1 to 20 mm, preferably 3 to 5 mm, to obtain the inner layer; and iv) Electrospinning at least one polymer onto the inner layer, said at least one polymer being selected from polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol, polyurethane, copolymers or mixtures thereof; v) Optionally cut the tube to obtain the wrapping.

[0008] In a third aspect, the present invention relates to a method for manufacturing a multilayer tube or enclosure according to the invention, which is manufactured by a two-step oxidative polymerization process using: ammonium peroxide, hydrogen peroxide, or benzoyl peroxide, preferably benzoyl peroxide; and monomers, said monomers including but not limited to aniline, thiophene, or pyrrole, preferably pyrrole; and dopants as at least one electrically conductive compound and / or at least one ionicly conductive compound, said method comprising or consisting of the following steps: i) Prepare a supersaturated peroxide solution and immerse the electrospinning tube / wrapper in the solution to obtain an oxide layer; ii) Prepare an aqueous solution of a monomer containing an anionic dopant, wherein the anionic dopant includes, but is not limited to, sodium di-2-ethylhexyl sulfosuccinate or sodium dodecylbenzene sulfonate, preferably sodium naphthalene-2-sulfonate; iii) Immerse the oxidized tube / encapsulation in a monomer solution containing anionic dopant; iv) Place the tube / wrap obtained in step iii) onto the mandrel or static flat collector and electrospin an outer polymer layer on top of the previous layer to obtain a multilayer tube or wrap.

[0009] In a fourth aspect, the present invention relates to a method of 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 and a coupling agent, wherein the at least one polymer is selected from polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol, and polyurethane, and the coupling agent is, for example, N-hydroxysuccinimide ester or its functionalized copolymer, preferably a physical blend of polycaprolactone and N-hydroxysuccinimide ester, more preferably a physical blend of polycaprolactone and poly(ethylene glycol)-b-poly(ε-caprolactone) (PCL-PEG-NHS) in a weight ratio ranging from 65:35 to 35:65; ii) PCL-PEG-NHS consists of: PCL blocks with a number-average molecular weight of Mn of 2,000 to 8,000 g / mol, preferably 5,000 g / mol; and PEG blocks with a number-average molecular weight of Mn of 200 to 10,000 g / mol, preferably 5,000 g / mol. iii) Dissolve the blend in a mixture of chloroform / acetone or HFIP at a concentration of 5 to 30% w / v; iv) Electrospinning a PCL / PCL-PEG-NHS solution as the intermediate layer; v) Pure PCL is electrospun onto the PCL / PCL-PEG-NHS layer as an outer layer; vi) Conjugating poly-L-lysine, collagen, or fibrinogen polymers to the NHS functional groups as an inner layer.

[0010] In a fifth aspect, the present invention relates to the use of electrospun multilayer tubes or wrappings according to the invention for protecting or bridging nerves or for stimulating nerve growth and cell proliferation. Attached Figure Description

[0011] Figure 1 A scanning electron micrograph of a PCL scaffold spun from a chloroform / acetone solution is shown.

[0012] Figure 2 A scanning electron micrograph of a PCL scaffold spun from hexafluoroisopropanol (HFIP) is shown.

[0013] Figure 3 Multilayer electrospun tubes containing electrically conductive 1) PPY and / or 2) PCL-PEG-NHS for covalent bonding with amino groups of proteins and peptides.

[0014] Figure 4Multilayer electrospun tubing containing electrically conductive PPY and / or PCL-PEG-NHS.

[0015] Figure 5 .NHS is covalently bonded to the amino groups of proteins and peptides.

[0016] Figure 6 A schematic diagram of the PCL-PEG-NHS coupling reaction.

[0017] Figures 7 to 11 The measurement results are shown as described in the experimental section.

[0018] Figure 12 Growth of dorsal root ganglia (DRGs) on electrospun substrates. Images of nerve growth stained with β3-tubulin were used to quantify the growth using Image J.

[0019] Figure 13 Growth of dorsal root ganglia (DRGs) on electrospun substrates. Images of nerve growth stained with β3-tubulin were compared on PCL, PCL containing poly-L-lysine, and a positive control (glass slides coated with polyornithine and Matrigel). Detailed Implementation

[0020] Unless otherwise specified, 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 its limitations) 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.

[0021] The terms “comprise(s)”, “include(s)”, “having / has”, “can”, “contain(s)”, and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional actions or structures. The singular forms “a / an” and “the” include plural references unless the context clearly indicates otherwise. This disclosure also contemplates other embodiments that “comprise” the embodiments or elements presented herein, embodiments that “compose of” the embodiments or elements presented herein, and embodiments that “consist substantially of” the embodiments or elements presented herein, whether or not explicitly stated.

[0022] 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 containing A but not B, a device containing B but not A, or a device containing 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 limited in the broadest sense to mean one or more elements selected from the group containing A, B... and N, that is, any combination of any one or more of elements A, B... or N, including any single element or its combination with one or more other elements, and may also include additional elements not listed in the combination.

[0023] The modifier “about” used with a quantity includes the value and has the meaning indicated by the context (e.g., it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered to disclose a range defined by the absolute values ​​of its two endpoints. For example, expressing “about 2 to about 4” also discloses the range “2 to 4”. The term “about” can refer to a positive or negative 10% of the indicated number. For example, “about 10%” can represent a range of 9% to 11%, and “about 1” can mean 0.9 to 1.1. Other meanings of “about” are apparent from the context, such as rounding; therefore, for example, “about 1” can also mean 0.5 to 1.4.

[0024] The term "wt%" refers to weight percentage.

[0025] The term "w / w" means weight per unit weight.

[0026] For the purposes of this invention, the term "degradation" refers to a polymer that dissolves or degrades in vitro or in vivo within an acceptable timeframe for a particular therapeutic situation. The products of such dissolution or degradation may include a smaller range of chemical species. Degradation can be caused, for example, by enzymatic, chemical, and / or physical processes. Biodegradation typically takes less than five years, and usually less than one year, after exposure to physiological pH and temperature (e.g., pH in the range of 6 to 9 and temperature in the range of 22°C to 40°C).

[0027] Sample characterization was performed using standard testing equipment. SEM was performed on a benchtop machine (commercially available from Hitachi). Mechanical data were obtained using a standard Instron mechanical testing instrument and a dynamic mechanical analyzer (commercially available from TA Instruments).

[0028] Zeta potential and ionic conductivity were determined on a standard Zeta potential analyzer (commercially available from Anton Parr). Fourier transform infrared analysis was performed using attenuated total reflectance-FTIR instruments from ThermoFisher Scientific.

[0029] Electrospin polymer tubes / sheets on an electrospinning machine (commercially available from Tongli).

[0030] In particular, the present invention relates to: Electrospun multilayer tubes or wraps, preferably to protect or bridge damaged nerves, wherein the electrospun multilayer tubes or wraps comprise 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 or mixtures thereof, preferably polycaprolactone; ii) At least one electrospun intermediate layer, said at least one electrospun intermediate layer comprising an agent capable of covalently bonding with proteins and peptides, including but not limited to N-hydroxysuccinimide, maleimide, thio-NHS and biotin-NHS, isocyanate, or aldehyde; and iii) An inner layer comprising at least one electrically conductive compound and / or at least one ion-conductive compound and / or at least one peptide or protein, preferably for stimulating nerve growth.

[0031] In one embodiment, the peptide in the inner layer is selected from cationic polypeptides such as poly-L-lysine, or proteins, including but not limited to collagen or collagen-like proteins, such as VECOLLAN®, fibrinogen, or other amino or thiol-containing proteins, which are commercially available from Evonik Industries AG.

[0032] In one embodiment, the inner layer comprises or is composed of an electrically conductive polymer, such as polypyrrole, polyaniline, polythiophene, poly(3,4-ethylenedioxythiophene), or contains additives such as graphene, graphene oxide, metal, carbon, or carbon nanotubes.

[0033] 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.

[0034] In one embodiment, the inner layer has an electrical conductivity in the range of 100 to 1000 mS / m; and / or a zeta potential in the range of ±0-100 mV. The electrical conductivity and zeta potential are preferably determined as described in the Examples section.

[0035] In one embodiment, the electrospun layer has a fibrous structure having a fiber diameter ranging from 0.1 to 2 µm.

[0036] 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.

[0037] In one embodiment, the tube has a nanofiber structure and an average diameter of 2 to 6 mm; and / or a length of 1 to 25 cm, preferably 3 to 25 cm, more preferably 3 to 10 cm; and / or a thickness in the range of 0.1 to 1 mm, preferably 0.5 mm.

[0038] In one embodiment, the reagent of the at least one electrospun intermediate layer is a PCL-PEG-NHS consisting of: a PCL block having a number-average molecular weight of Mn of 2,000 to 8,000 g / mol, preferably 5,000 g / mol; and a PEG block having a number-average molecular weight of Mn of 200 to 10,000 g / mol, preferably 5,000 g / mol.

[0039] In one embodiment, at least one compound of the inner layer is selected from polypyrrole, polyaniline, polythiophene, and poly(3,4-ethylenedioxythiophene). In another embodiment, anionic dopant added to the polypyrrole to generate electrical conductivity includes sodium di-2-ethylhexylsulfonate or sodium dodecylbenzenesulfonate, preferably sodium naphthalene-2-sulfonate. Other compounds used to generate electrical conductivity include graphene, graphene oxide, metals, carbon, or carbon nanotubes.

[0040] 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 the following blends: a) 50 to 99 wt% of a polymer selected from polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol, polyurethane, copolymers or mixtures thereof, and b) 1 to 50 wt% of an electrically conductive polymer selected from polypyrrole, polyaniline, polythiophene, poly(3,4-ethylenedioxythiophene), or electrically conductive additives such as graphene, graphene oxide, metal, carbon, or carbon nanotubes, wherein the sum of all components is 100 wt% of the composite obtained using a twin-screw extruder. ii) Dissolve the blend from step i) in a solvent, such as chloroform, acetone, or a mixture thereof, or hexafluoroisopropanol (HFIP); and iii) Electrospinning the dissolved blend onto a mandrel having a diameter in the range of 1 to 20 mm, preferably 3 to 5 mm, to obtain the inner layer; and iv) Electrospinning at least one polymer onto the inner layer, said at least one polymer being selected from polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol, polyurethane, copolymers or mixtures thereof; v) Optionally cut the tube to obtain the wrapping.

[0041] Furthermore, the present invention relates to a method for manufacturing a multilayer tube or enclosure according to the invention, which is manufactured by a two-step oxidative polymerization process using: ammonium peroxide, hydrogen peroxide, or benzoyl peroxide, preferably benzoyl peroxide; and monomers, including but not limited to aniline, thiophene, or pyrrole, preferably pyrrole; and dopants as at least one electrically conductive compound and / or at least one ionicly conductive compound, the method comprising or consisting of the following steps: i) Prepare a supersaturated peroxide solution and immerse the electrospinning tube / wrapper in the solution to obtain an oxide layer; ii) Prepare an aqueous solution of a monomer containing an anionic dopant, wherein the anionic dopant includes, but is not limited to, sodium di-2-ethylhexyl sulfosuccinate or sodium dodecylbenzene sulfonate, preferably sodium naphthalene-2-sulfonate; iii) Immerse the oxidized tube / encapsulation in a monomer solution containing anionic dopant; iv) Place the tube / wrap obtained in step iii) onto the mandrel or static flat collector and electrospin an outer polymer layer on top of the previous layer to obtain a multilayer tube or wrap.

[0042] 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 with N-hydroxysuccinimide ester or its copolymer, said at least one polymer being selected from polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol, polyurethane, preferably a physical blend of polycaprolactone and N-hydroxysuccinimide ester, and more preferably a physical blend of polycaprolactone and poly(ethylene glycol)-b-poly(ε-caprolactone) (PCL-PEG-NHS) in a weight ratio ranging from 65:35 to 35:65; ii) PCL-PEG-NHS consists of: PCL blocks with a number-average molecular weight of Mn of 2,000 to 8,000 g / mol, preferably 5,000 g / mol; and PEG blocks with a number-average molecular weight of Mn of 200 to 10,000 g / mol, preferably 5,000 g / mol. iii) Dissolve the blend in a mixture of chloroform / acetone or HFIP at a concentration of 5 to 30% w / v; iv) Electrospinning a PCL / PCL-PEG-NHS solution as the intermediate layer; v) Pure PCL is electrospun onto the PCL / PCL-PEG-NHS layer as an outer layer; vi) Couple poly-L-lysine, collagen, or fibrinogen polymers to the NHS functional group as an inner layer.

[0043] 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 for stimulating nerve growth and cell proliferation.

[0044] Example Example 1: Electrospun polycaprolactone (PCL) scaffolds consisting of sheets and tubes were prepared using one or more solvents. The scaffolds were prepared using RESOMER® C212 PCL dissolved in a 3:1 (3:1) solvent mixture of chloroform and acetone at a ratio of approximately 27% w / v. These materials were also prepared using RESOMER® C212 PCL dissolved in hexafluoroisopropanol (HFIP) at a ratio of approximately 12% w / v. The final scaffold thickness was controlled by the total volume of the spun solution.

[0045] The sheet is manufactured using a static flat collector or a cylindrical rotating mandrel or drum. When using a lower mandrel speed, random fiber orientations are produced. Increased rotational speed tends to increase the degree of alignment. The collector distance is set between 13 cm and 25 cm. After spinning, the support is removed from the rotating mandrel by cutting along a uniaxial axis. The final sheet dimension is influenced by the diameter and length of the mandrel.

[0046] Similarly, the tubular scaffold is fabricated using a mandrel with a smaller diameter. After spinning, the scaffold is manually removed from the mandrel by applying an upward force. Wetting with isopropanol and an aqueous solution has been found to aid in the removal of the tube from the mandrel.

[0047] Depending on the electrospinning apparatus, the chloroform / acetone solution is spun at a voltage of +9 kV to +13 kV and a solvent flow rate of 0.7 mL / h to 1.5 mL / h. The HFIP solution is spun using a voltage between +4 kV and +12 kV and a solvent flow rate between 0.4 mL / h and 1.4 mL / h. If the electrospinning apparatus includes an additional negative high-voltage source, the collector is set to approximately 1 kV. This negative bias on the collector improves fiber deposition on the mandrel.

[0048] The variations in voltage and flow rate were found to be a function of daily variations and needle tip diameter. Needle tip diameter plays a crucial role in electrospinning. The range of needles used for spinning is 18 to 22 gauge.

[0049] Example 1 highlights the importance of solvent selection and electrospinning parameters in customizing scaffold properties. Compared to the HFIP solution, the chloroform / acetone solution requires a higher voltage and a slightly higher flow rate, reflecting differences in electrical properties and jet stability during spinning. Both solvents can produce sheets and tubes with variable fiber orientation, influenced by mandrel speed and collector distance. However, solvent selection and concentration significantly affect fiber diameter, orientation, and ease of deposition and removal. Compared to HFIP (12% w / v), the chloroform / acetone solution uses a higher polymer concentration (27% w / v), affecting viscosity and fiber formation, such as… Figure 1 and Figure 2 As shown.

[0050] Tensile tests were performed on electrospun PCL sheets using a dynamic mechanical analyzer (DMA, Q800, TA Instruments) at both ambient and body temperature (37 °C). Electrospun PCL mesh was cut into strips and mounted on the DMA tensile fixture. The strips were tested at a rate of 0.2 mm / min to 0.4 mm, and then at a rate of 10 mm / min to 25 mm. The data in the table below illustrate the tensile properties of the electrospun PCL sheets at 19 °C and 37 °C. The results show that the tensile properties of electrospun PCL vary with the test conditions. At 37 °C, there is a decrease in tensile modulus and tensile strength, and an increase in elongation at break.

[0051] Example 2: A multilayer tube was manufactured, comprising an electrospun PCL interior surrounded by a PCL outer layer and coated with polypyrrole. The substrate layer consisted of a pure PCL electrospun scaffold as described in Example 1.

[0052] The scaffold material was treated using a two-step oxidative polymerization process employing benzoyl peroxide, pyrrole monomer, and sodium naphthalene-2-sulfonate as an ionic dopant. Aqueous solutions of pyrrole monomer and sodium naphthalene-2-sulfonate were prepared at concentrations of 14 mg / mL and 10 mg / mL, respectively. The benzoyl peroxide oxidation step was performed by preparing a supersaturated solution of benzoyl peroxide and isopropanol at a concentration of 10 mg / mL and immersing the scaffold in the solution for 10 minutes. After treatment, the scaffold was removed, allowed to dry completely, and then immersed in the pyrrole monomer solution for approximately 2.5 hours. This process was repeated until the desired surface conductivity was obtained. The modified scaffold was thoroughly rinsed with water before characterization. Electrical conductivity was measured on rectangular samples using an Ossila four-point probe instrument. The current was automatically adjusted to approximately 0.1 mA using Ossila software. Two measurements were performed on different regions on each side of the material treated three times using the above procedure.

[0053] To complete the fabrication of the multilayer tube, the treated support is placed on its original 4 mm mandrel and rotated. Additional polycaprolactone is electrospun onto the surface until a uniform coating is formed.

[0054] Example 3: Multilayer tubes were prepared, comprising a blended polycaprolactone and polypyrrole composite core and a polycaprolactone coating. The inner layer material was prepared by first compounding RESOMER® C212 PCL containing 30% w / w polypyrrole (PPy) in a Thermo Haake MiniLab II twin-screw mixer. Compounding was performed by melting at 105 °C, followed by mixing at 85 RPM for approximately 10 minutes and then discharging. The compounded composite was dissolved in a mixture of chloroform and acetone at a ratio of 27% w / v as described in Example 1, and then electrospun accordingly.

[0055] The tubes were prepared using an electrospinning system consisting of a high-voltage power supply, a syringe pump, and a rotating collector. The inner layer was prepared using a PCL / PPy composite solution at a voltage of 17.7 kV, a collector distance of 14 cm, and a flow rate of 0.8 mL / hr. The grounded rotating collector (d ≈ 64 cm) was set to a speed below 1800 RPM. After 2.5 mL was delivered by the syringe pump, the second syringe channel was activated with the PCL solution and set to a flow rate of 0.75 mL / hr. The voltage was increased to 22.2 kV. An additional 2.5 mL was spun. Due to the insulating properties of the PCL matrix, these samples did not exhibit electrical conductivity when measured using a 4-point probe.

[0056] Example 4: A physical polymer blend of RESERMER® C212 (PCL) and N-hydroxysuccinimide-poly(ethylene glycol)-b-poly(ε-caprolactone) (containing PEG and PCL blocks, both with a number average molecular weight of 5,000) was prepared at a weight ratio of 65:35. This blend was dissolved in hexafluoroisopropanol (HFIP) at a ratio of 12.5% ​​w / v. Sheets were electrospun on a rotating collector with a positive voltage of 6.6 kV at a 20 gA stainless steel needle tip and a negative voltage of -1.2 kV. The collector (approximately 10 cm in diameter) was rotated at 50 RPM to obtain the sheet, which was then positioned approximately 20 cm from the needle tip.

[0057] The material surface was coupled with a 30 kDa poly-L-lysine polymer via NHS functional groups. An aqueous solution of 16.7 mg / mL poly-L-lysine was prepared using a buffer solution containing 0.05 M triethanolamine and 0.25 M sodium chloride. The pH was adjusted to between 8 and 9 using sodium hydroxide. The sample was left in this solution overnight (approximately 19 hours), then washed thoroughly with deionized water, and finally dried in a vacuum oven at room temperature.

[0058] The surface was characterized using X-ray photoelectron spectroscopy (XPS), see [link to X-ray photoelectron spectroscopy]. Figure 7 and Figure 8 It was observed that the nitrogen content in the poly-L-lysine-modified sample was approximately 2.5%, compared to ≈0.3% nitrogen content in the unmodified PCL and uncoupled polymer blend. This high nitrogen content in the poly-L-lysine sample is attributed to the large amount of amines in the lysine backbone. The oxygen content in both the poly-L-lysine and uncoupled polymer blends was higher than in the unmodified PCL sample, due to the presence of additional oxygen bonds from the polyethylene glycol (PEG) linker polymer. These two findings indicate surface migration of the PEG moiety and NHS moiety, enabling amide bonding of the poly-L-lysine.

[0059] The zeta potential of the surface was investigated using an Anton Parr Surpass3 electrodynamic analyzer. Sample sheets were fixed to the Surpass3's adjustable gap sample cell, with the gap adjusted to 100 µm. pH scans were performed from pH 4 to pH 9 using streaming current mode. Flow cytometry pressure gradients from 600 mbar to 200 mbar were used for charge displacement. An electrolyte consisting of 0.001 M potassium chloride was used for all experiments at room temperature. Each flow cytometry experiment was performed three times at each pH step to determine the zeta potential.

[0060] Compared to unmodified PCL, the sample modified with poly-L-lysine showed a 7X increase in zeta potential at pH 7, see [reference needed]. Figure 9 The increase in zeta potential corresponds to increased hydrophilicity and supports the presence of poly-L-lysine on the exposed surface.

[0061] Example 5: The electrospun PCL / PCL-PEG-NHS physical blend described in Example 4 was also used to couple a water-soluble recombinant collagen-like moiety. An aqueous solution of the compound at a concentration of 50 mg / mL was prepared using a buffer solution containing 0.05 M triethanolamine and 0.25 M sodium chloride. The pH was adjusted to between 8 and 9 using sodium hydroxide. The sample was left in the solution overnight (approximately 72 hours), then washed thoroughly with deionized water, and finally dried in a vacuum oven at room temperature.

[0062] The surface was characterized using X-ray photoelectron spectroscopy (XPS), see [link to article]. Figure 10 and Figure 11 The nitrogen content in the collagen-like modified sample was observed to be approximately 0.7%, compared to ≈0.3% in the unmodified PCL and uncoupled blends. The higher nitrogen content in the coupled sample is attributed to the large amount of amines in the polymer backbone. Both the collagen-like sample and the uncoupled polymer blend showed higher oxygen content compared to the unmodified PCL sample, due to the presence of additional oxygen bonds from the polyethylene glycol (PEG) linker polymer. These two findings suggest surface migration of the PEG moiety and NHS moiety, enabling amide bonding of the collagen-like moiety.

[0063] Electrospun fiber mat was subjected to electrokinetic surface analysis using flow cytometry current-pH scanning measurements, see [link to relevant documentation]. Figure 11 Compared to unmodified PCL, the sample modified with recombinant collagen showed a 3X increase in zeta potential at pH 7. This increase in zeta potential corresponds to increased hydrophilicity and supports the presence of bound collagen on the exposed surface.

[0064] Example 6: To assess neural growth on different samples, 12 mm discs were cut from electrospun sheets and used for cell culture. Mouse tissue collection and spheroid formation: All animal procedures were reviewed and approved by Tulane University's Institutional AnimalCare and Use Committee (IACUC). Following a protocol developed in Moore's laboratory, dorsal root ganglion (DRG) tissue was collected from embryos of Long Evans mice at embryonic day 15 (e15). Briefly, DRGs were isolated and collected from a single 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 a 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 ThermoFisher 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 500g for 5 minutes at room temperature, and spheroids were allowed to form for more than 48 hours. Substrate preparation and spheroid plating: All substrates from the above examples were cut into circles using a biopsy punch and placed in PBS with 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. 12mm circular coverslips were first cleaned in ethanol and allowed to dry completely, then coated with 0.01% polyornithine solution for 1 hour.Aspirate the solution and add Matrigel solution to Neurobasal (ESC-qualified; Corning) at a dilution of 1:100. Allow it to incubate at 37°C in a humidified incubator for at least 3 hours. Once all substrates are prepared, place them in a minimal amount of cell culture medium (as described above), with a single DRG sphere placed in the center. These are allowed to adhere for 2 hours before adding more culture medium.

[0065] 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 Sholll analysis method that generates concentric circles around the central organoid / organoid culture. Samples were analyzed at 25 µm intervals, with thresholds set using the same values ​​as for neurites stained with β3-tubulin. Values ​​obtained from Neurote-J were recorded for each condition, including the intersection profile (the number of intersections at each interval) and Nmax, which represents the maximum number of neurites of intersections.

[0066] Figure 12 The quantification of neurite growth using ImageJ analysis is illustrated. Data show that the number of neurite crossovers on a PCL substrate modified with poly-L-lysine is significantly enhanced compared to unmodified PCL. Specifically, the modified PCL exhibits a 56% increase in the number of crossovers, with the modified version recording 190 crossovers compared to 122 in the unmodified version. This significant increase highlights the positive effect of poly-L-lysine modification on promoting neural connectivity, likely due to improved surface properties that promote cell adhesion and growth. The results highlight the potential of surface-immobilized poly-L-lysine as a beneficial coating for neural tissue engineering applications.

[0067] Furthermore, the positive control, consisting of glass slides coated with polyornithine and Matrigel, demonstrated optimal neurite growth, serving as a benchmark for effective neural support. The increased growth observed on these coated slides underscores the effectiveness of the surface coating in enhancing neurite extension, similar to the improvements observed with poly-L-lysine on PCL. These findings suggest that incorporating such modifications into neural tissue engineering applications could be advantageous.

[0068] Figure 13 The images showcase neurites stained with β3-tubulin on different samples. The presence of β3-tubulin was used as a neuronal marker to confirm and visualize neurite growth, highlighting network extension on the modified surface. Furthermore, a positive control consisting of glass slides coated with polyornithine and Matrigel demonstrated optimal neurite growth and served as a benchmark. This multi-generational growth validates the efficacy of the modification in promoting neural connectivity, illustrating the potential of these coatings in neural tissue engineering applications.

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 or mixtures thereof; ii) At least one electrospun intermediate layer, said at least one electrospun intermediate layer comprising a reagent capable of covalently bonding with proteins and peptides, said reagent comprising at least one of the following groups selected from N-hydroxysuccinimide, maleimide, thio-NHS and biotin-NHS, isocyanate, or aldehyde; and iii) An inner layer comprising at least one electrically conductive compound and / or at least one ion-conducting compound and / or at least one peptide or protein.

2. The electrospun multilayer tube or encapsulation according to claim 1, wherein the peptide in the inner layer is selected from cationic polypeptides.

3. The electrospun multilayer tube or wrapping according to any one of the preceding claims, wherein the inner layer comprises or is composed of an electrically conductive polymer, or comprises additives.

4. 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.

5. The electrospun multilayer tube or wrapping according to any one of the preceding claims, wherein the inner layer i) Electrical conductivity in the range of 100 to 1000 mS / m; and / or ii) A zeta potential with a range of ± 0-100 mV.

6. The electrospun multilayer tube or wrapping according to any one of the preceding claims, wherein the electrospun layer has a fibrous structure having a fiber diameter in the range of 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 i) Having a tensile strength of 0.2 to 20 MPa; and / or ii) Having an elastic modulus in the range of 0.2 to 100 MPa; and / or iii) It has a 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 has i) an average diameter of 2 to 6 mm; and / or ii) A length of 1 to 25 cm; and / or iii) Thickness in the range of 0.1 to 1 mm.

9. A method for manufacturing a multilayer tube or wrapping according to any one of claims 1 to 8, the method comprising or consisting of the following steps: i) Prepare the following blends: a) 50 to 99 wt% of a polymer selected from polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol, polyurethane, copolymers or mixtures thereof, and b) 1 to 50 wt% of an electrically conductive polymer selected from polypyrrole, polyaniline, polythiophene, poly(3,4-ethylenedioxythiophene), or an electrically conductive additive, wherein the total of all components constitutes a complex of 100 wt%. ii) Dissolve the blend from step i) in a solvent; and iii) Electrospinning the dissolved blend onto a mandrel with a diameter ranging from 1 to 20 mm to obtain the inner layer; and iv) Electrospinning at least one polymer onto the inner layer, said at least one polymer being selected from polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol, polyurethane, copolymers or mixtures thereof; v) Optionally cut the tube to obtain the wrapping.

10. A method of manufacturing a multilayer tube or wrapping according to any one of claims 1 to 8, wherein the manufacturing is carried out by a two-step oxidative polymerization process using: ammonium peroxide, hydrogen peroxide or benzoyl peroxide, preferably benzoyl peroxide; and a monomer selected from aniline, thiophene or pyrrole, preferably pyrrole; The method, which includes or comprises the following steps, and is a dopant of at least one electrically conductive compound and / or at least one ionicly conductive compound: i) Prepare a supersaturated peroxide solution and immerse the electrospinning tube / wrapper in the solution to obtain an oxide layer; ii) Prepare an aqueous solution of a monomer containing an anionic dopant, wherein the anionic dopant is selected from sodium di-2-ethylhexyl sulfosuccinate or sodium dodecylbenzene sulfonate, preferably sodium naphthalene-2-sulfonate; iii) Immerse the oxidized tube / encapsulation in a monomer solution containing anionic dopant; iv) Place the tube / wrap obtained in step iii) onto the mandrel or static flat collector and electrospin an outer polymer layer on top of the previous layer to obtain a multilayer tube or wrap.

11. A method of 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 with N-hydroxysuccinimide ester or its copolymer, wherein the at least one polymer is selected from polycaprolactone, polylactide, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyethylene glycol, and polyurethane. ii) PCL-PEG-NHS consists of the following: PCL blocks with Mn number-average molecular weights ranging from 2000 to 8000 g / mol; And PEG blocks with Mn number-average molecular weights ranging from 200 to 10,000 g / mol; iii) Dissolve the blend in a mixture of chloroform / acetone or HFIP at a concentration of 5 to 30% w / v; iv) Electrospinning a PCL / PCL-PEG-NHS solution as the intermediate layer; v) Pure PCL is electrospun onto the PCL / PCL-PEG-NHS layer as an outer layer; vi) Couple poly-L-lysine, collagen, or fibrinogen polymers to the NHS functional group as an inner layer.

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

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