Multilayer nerve regeneration guidance tube

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 an active inner layer comprising at least one electrical conducting compound and / or at least one ionic conducting compound and / or at least one peptide or protein to stimulate nerve growth. 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] Multilayer nerve regeneration guidance tube

[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 an active inner layer comprising at least one electrical conducting compound and / or at least one ionic conducting compound and / or at least one peptide or protein to stimulate nerve growth. 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] Resorbable conduits and conductive polymers for nerve regeneration have been widely discussed in the prior arts. These resorbable polymers are emerging as viable alternatives to non-resorbable materials for implantable nerve growth conduits (NGC) to repair damaged nerves as previously disclosed by Y.Z. Bian et al. Both natural and synthetic origin resorbable polymers, such as polylactide (PLA), poly(lactide-co- glycolide) (PLGA), and polycaprolactone (PCL), collagen and chitosan have been utilized for NGC fabrication. Additionally, 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 a nanofibrous microstructure using molds, assessing the impact of scaffold geometry on nerve stem cell differentiation in vitro (Tissue Eng. Part A., 2014, 20, 1038-1048). The combined use of chitosan / PLGA scaffolds and MSCs has successfully bridged large gaps in the dog sciatic nerve (Neurorehabil. Neural Repair, 2012, 26, 96-106). Schwann cell-seeded multichannel scaffolds have been also developed for peripheral nerve regeneration (J. Neuro. Sci., 2017, 381 , 612-613). Current advancements in polymeric biomaterials for neural tissue engineering have been explored extensively (J. Biomed. Sci., 2018, 25, 90). It has been shown that while synthetic biopolymers like branched PLA, PLGA, and PCL offer enhanced mechanical strength, but often they lack biological activity (Int. J. Surg., 2019, 20, 15-19; Front. In another study, 3D-printed PCL / PPy conductive scaffolds as three-dimensional porous have been utilized as nerve guide conduits (NGCs) for peripheral nerve injury repair (Front. Bioeng. Biotechnol., 2019, 7, 266). Animal derived materials such as collagen and gelatin have shown excellent biodegradation and biocompatibility. However, they generally lack sufficient mechanical strength and there is risk of immunogenic response. Bioactive agents such as growth factors, have been added to synthetic resorbable polymers to promote certain functional recovery as disclosed in CN 101543645 B1 . However, these growth factors normally have sort half-life, poor stability and potential for spreading to other areas of the body. Immobilizing bioactive agents to resorbable polymers have proven to be an effective approach, for example by forming copolymer between PLGA and Poly lysine for the treatment of open and closed wound spinal cord injuries as disclosed in US 8858966 B2. Besides being biodegradable and biocompatible, ideal NGC owns other essential characteristics such as permeability, flexibility, minimal swelling, and internal structures. Porous structure with permeability allowing nutrients, oxygen and metabolized wastes transport in and out of membranes or scaffolds have been well adapted in tissue engineering application for tissue regeneration and repair as disclosed in US 9707000 B2 and US 8926886 B2. Electrospinning of resorbable natural or synthetic polymer or their mixture dissolved in an appropriate solvent is typically employed to fabricate highly porous scaffolds and even with highly aligned micro- or nano-fibers to guide tissue regeneration as disclosed in US 10405963 B2 and US 2014079759 A1 . Moreover, electrospinning has been applied to create micropatterns for guided tissue engineering in cardiovascular applications, as disclosed in US 2006 / 0085063 A1. The use of hybrid biomaterials, blending pure laminin or complex extracts containing laminin with resorbable polymers such as PCL and PLA / PLGA through electrospinning, has also been explored, as per US 2011 / 0236974 A1 . This differs from the present invention, which involves creating multilayer tubes or sheets where peptides and proteins are covalently bonded to the inner layer of the matrix, preserving biological activity and sustainability.

[0006] Application of conductive polymers such as polypyrrole (PPy) in NGC has been resulted in increase in fibronectin adsorption leaving to enhanced neurite outgrowth (E. Gamez et al). Photo-fabricated gelatinbased nerve conduits indicated nerve tissue regeneration potentials (Cell Transplantation, 2004, 13, 549- 564). Single-walled carbon nanotubes (SWCNT) and multi-walled carbon nanotubes (MWCNT) dispersed into PLGA solution were also electrospun into aligned nanofibrous scaffold where carbon nanotubes provided conductivity to promote neurite extension. The resultant nanofibrous scaffolds were further coated with poly L-lysine solution to increase the number of positively-charged sites for cell binding as disclosed in US 6583232 B1 . PPy was coated on to electrospun PLGA nanofibers to produce electro conducting nanofibers for neural tissue applications as disclosed in US 6607548 B2.

[0007] The present invention provides a method to fabricate a multilayered electrospun tube or wrap for repair of damaged nerves through electrospinning (Figure 1-2). The inner layer of the electrospun tube or warp further comprises at least one electrical conducting compound and / or at least one ionic conducting compound to stimulate nerve growth as shown in Figure 3 and 4. This approach optimizes the utilization of conducting polymer, minimizing the amount required for nerve stimulation. Additionally, the inclusion of positively charged proteins, peptides or polypeptides such as poly L-lysine, can facilitate nerve regeneration. While proteins or peptides are typically coated on the surface of polymers or blended in the polymer matrix, free or non-covalently bonded proteins and peptides can be easily removed from the system, limiting their regenerative effect. To address this issue in one embodiment, a reagent has been introduced to the surface of the tube / wrap, creating covalent bonding with amino groups of proteins and sustaining the regenerating effect of poly L-lysine, collagen, or collagen-like proteins such as VECOLLAN®. This approach has been shown in Figures 5 and 6.

[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; ii) at least one electro-spun intermediate 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; and iii) an inner layer comprising at least one electrical conducting compound and / or at least one ionic conducting compound and / or at least one peptide or protein, preferably to stimulate nerve growth.

[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 blend of a) 50 to 99 wt% of a polymer selected from poly(caprolactone), polylactide, polyglycolide, poly(trimethylene carbonate), poly(dioxanone), polyethylene glycol, polyurethane, their copolymers or mixtures thereof and b) 1 to 50 wt% an electrical conducting polymer selected from polypyrrole, polyaniline, polythiophene, poly( 3,4-ethyenedioxythiophene) or electrical conducting additives such as graphene, graphene oxide, metals, carbon, or carbon nanotubes, wherein the sum of all components is 100 wt% composite; ii) dissolving the blend of step i) in a solvent such as chloroform, acetone or their mixture or hexafluoroisopropanol (HFIP); and iii) electrospinning the dissolved blend on a mandrel with a diameter ranging 1 to 20 mm, preferably 3 to 5 mm to obtain the inner layer; and iv) electrospinning at least one polymer selected from poly(caprolactone), polylactide, polyglycolide, poly(trimethylene carbonate), poly(dioxanone), polyethylene glycol, polyurethane, their copolymers or mixtures thereof onto the inner layer; v) optionally cutting the tube to obtain a wrap.

[0011] In a third aspect the present invention pertains to a method of manufacturing a multilayer tube or wrap according to the present invention via a two-step oxidative polymerization process using ammonium peroxide, hydrogen peroxide or benzoyl peroxide, preferably benzoyl peroxide and a monomer including but not limited to aniline, thiophene or pyrrole preferably pyrrole, and a dopant as at least one electrical conducting compound and / or at least one ionic conducting compound comprising or consisting of the following steps: i) preparing a super saturated peroxide solution and submersing the electrospun tube / wrap in the solution to obtain an oxidized layer; ii) preparing the aqueous solution of monomer comprising an anionic dopant including but not limited to di-2-ethylhexyl sulfo-succinic acid sodium salt, or dodecylbenzene sulfonic acid sodium salt, preferably sodium naphthalene-2-sulfonic acid; iii) submerging the oxidized tube / wrap in the monomer solution comprising anionic dopant; iv) placing the tube / wrap obtained in step iii) on the mandrel or a static flat collector and electrospin the outer polymer layer on top of the previous layer to obtain a multilayer tube or wrap.

[0012] In a fourth 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 coupling reagents, such as N-hydroxy succinimide ester or functionalized 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 intermediate layer; v) electrospinning pure PCL onto PCL / PCL-PEG-NHS layer as outer layer; vi) conjugating poly-L-lysine, collagen, or fibrinogen polymer to the NHS functional group as inner layer.

[0013] In a fifth 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.

[0014] Brief description of the drawings

[0015] Fig 1. Shows a scanning electron micrograph of a PCL scaffold spun from chloroform / acetone solution.

[0016] Fig 2. Shows a scanning electron micrograph pf PCL scaffold spun from hexafluoro isopropanol (HFIP). Fig 3. Multi layered electro-spun tube incorporating electro conductive 1) PPY and / or 2) PCL-PEG-NHS for covalent bonding with amino groups of proteins and peptides.

[0017] Fig 4. Multi layered electro-spun tube incorporating electro conductive PPY and / or PCL-PEG-NHS.

[0018] Fig. 5. Covalent bonding between NHS and amino groups of proteins and peptides.

[0019] Fig. 6. Schematic of the PCL-PEG-NHS coupling reaction.

[0020] Fig. 7 to 11 show results of measurements as stated in the experimental section.

[0021] Fig. 12. Dorsal root ganglion (DRG) growth on electrospun substrates. Images of nerve growth stained with p3- Tubulin have been used for quantifications of the growth using Image J.

[0022] Fig. 13. Dorsal root ganglion (DRG) growth on electrospun substrates. Images of nerve growth stained with p3- Tubulin are compared on PCL, PCL incorporating poly L-lysine and positive control (glass slide coated with poly-ornithine and Matrigel).

[0023] Detailed description of the invention

[0024] 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.

[0025] 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.

[0026] 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. 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.

[0027] The term “wt%” means weight percent.

[0028] The term “w / w” means weight per weight.

[0029] 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.

[0030] 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.

[0031] Zeta potential and ionic conductivity were done on a standard zeta potential analyzer commercially available from the company Anton Parr. Fourier transform infrared analysis was performed by attenuated total reflectance-FTIR using a ThermoFisher Scientific instrument.

[0032] Polymeric tube / sheets were electrospun on an electrospinner commercially available from the company Tongli.

[0033] In particular the present invention refers to:

[0034] 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 frompoly(caprolactone), polylactide, polyglycolide, poly(trimethylene carbonate), poly(dioxanone), polyethylene glycol, polyurethane, their copolymers or mixtures thereof, preferably poly(caprolactone); ii) at least one electro-spun intermediate layer comprising a reagent which is able to undergo covalent bonds with proteins and peptides, including but not limited to N-hydroxy succinimide, maleimide, sulfur-NHS and biotin-NHS, isocyanate, or aldehydes; and iii) an inner layer comprising at least one electrical conducting compound and / or at least one ionic conducting compound and / or at least one peptide or protein, preferably to stimulate nerve growth.

[0035] In one embodiment the peptide of the inner layer is selected from cationic polypeptides like poly L-lysine, or proteins including but not limited to collagen or collagen-like proteins like VECOLLAN® which is commercially available from Evonik Industries AG, fibrinogen, or other amino or thiol containing proteins.

[0036] In one embodiment the inner layer comprises or consists of electrical conducting polymers, like polypyrrole, polyaniline, polythiophene, poly(3,4-ethyenedioxythiophene) or comprises additives such as graphene, graphene oxide, metals, carbon, or carbon nanotubes.

[0037] 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.

[0038] In one embodiment the inner layer has an electrical conductivity ranging of from 100-1000 mS / m; and / or has a zeta potential ranging of from ± 0-100 mV. Electrical conductivity and zeta potential are preferably determined as described in the example section.

[0039] In one embodiment the electrospun layers have a fibrous structure with fibres diameters ranging 0.1 to 2 pm.

[0040] 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.

[0041] In one embodiment the tube has a nanofibrous structure and has 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 ranging of from 0.1 to 1 mm preferably 0.5 mm.

[0042] In one embodiment the reagent of the at least one electro-spun intermediate layer is 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.

[0043] In one embodiment at least one compound of the inner layer is selected from polypyrrole, polyaniline, polythiophene, poly( 3,4-ethyenedioxythiophene). In another embodiment the anionic dopants added to polypyrrole to create electroconductivity include di-2-ethylhexyl sulfo-succinic acid sodium salt, or dodecylbenzene sulfonic acid sodium salt, preferably sodium naphthalene-2-sulfonic acid. Other compounds for creating electrical conductivity include graphene, graphene oxide, metals, carbon, or carbon nanotubes. Furthermore, 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 blend of a) 50 to 99 wt% of a polymer selected from poly(caprolactone), polylactide, polyglycolide, poly(trimethylene carbonate), poly(dioxanone), polyethylene glycol, polyurethane, their copolymers or mixtures thereof and b) 1 to 50 wt% an electrical conducting polymer selected from polypyrrole, polyaniline, polythiophene, poly( 3,4-ethyenedioxythiophene) or electrical conducting additives such as graphene, graphene oxide, metals, carbon, or carbon nanotubes, wherein the sum of all components is 100 wt% composite using a twin screw extruder; ii) dissolving the blend of step i) in a solvent such as chloroform, acetone or their mixture or hexa fluoroisopropanol (HFIP); and iii) electrospinning the dissolved blend on a mandrel with a diameter ranging of from 1 to 20 mm, preferably 3 to 5 mm to obtain the inner layer; and iv) electrospinning at least one polymer selected from poly(caprolactone), polylactide, polyglycolide, poly(trimethylene carbonate), poly(dioxanone), polyethylene glycol, polyurethane, their copolymers or mixtures thereof onto the inner layer; v) optionally cutting the tube in order to obtain a wrap.

[0044] Moreover, the present invention refers to a method of manufacturing a multilayer tube or wrap according to the present invention via a two-step oxidative polymerization process using ammonium peroxide, hydrogen peroxide or benzoyl peroxide, preferably benzoyl peroxide and a monomer including but not limited to aniline, thiophene or pyrrole preferably pyrrole, and a dopant as at least one electrical conducting compound and / or at least one ionic conducting compound comprising or consisting of the following steps: i) preparing a super saturated peroxide solution and submersing the electrospun tube / wrap in the solution to obtain an oxidized layer; ii) preparing the aqueous solution of monomer comprising an anionic dopant including but not limited to di-2-ethylhexyl sulfo-succinic acid sodium salt, or dodecylbenzene sulfonic acid sodium salt, preferably sodium naphthalene-2-sulfonic acid; iii) submerging the oxidized tube / wrap in the monomer solution comprising anionic dopant; iv) placing the tube / wrap obtained in step iii) on the mandrel or a static flat collector and electrospin the outer polymer layer on top of the previous layer to obtain a multilayer tube or wrap. 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 intermediate layer; v) electrospinning pure PCL onto PCL / PCL-PEG-NHS layer as outer layer; vi) conjugating poly-L-lysine, collagen, or fibrinogen polymer to the NHS functional group as inner layer.

[0045] 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.

[0046] Examples

[0047] Example 1 :

[0048] Electrospun polycaprolactone (PCL) scaffolds consisting of sheets and tubes were prepared using various solvent(s). The scaffolds were prepared using RESOMER® C212 PCL dissolved in a solvent mixture of three-to-one (3:1) chloroform and acetone at approximately 27% w / v. These materials were also produced using RESOMER® C212 PCL dissolved in hexafluoroisopropanol (HFIP) at approximately 12% w / v. Final scaffold thicknesses were controlled by the total volume of solution spun.

[0049] Sheets were produced using either a static flat collector or a cylindrical rotating mandrel or drum. Random fibre orientations were produced when using lower mandrel speeds. Increasing rotational speeds tended to increase the degree of alignment. Collector distance was set to between 13 cm and 25 cm. After spinning, scaffolds were removed from the rotating mandrel by cutting along a single axis. Final sheet dimensions were driven by the mandrel diameter and length.

[0050] Similarly, tubular scaffolds were produced using smaller diameter mandrels. The scaffolds were removed manually from the mandrels after spinning by applying upwards force. Removal of the tubes from the mandrel was found to be aided by wetting with an isopropanol and water solution.

[0051] Depending on the electrospinning setup, chloroform / acetone solutions were spun with voltages between +9 kV and +13 kV with a solvent flow rate of between 0.7 mL / h and 1 .5 mL / h. The HFIP solutions were spun using voltages between +4 kV and +12 kV with a solvent flow rate between 0.4mL / h and 1 .4 mL / h. If the electrospinning equipment incorporated an additional negative high voltage source, the collector was set to approximately 1 kV. This negative bias on the collector improved fiber deposition on the mandrel.

[0052] Variations in the voltages and flow rates were found to be a function of day-to-day variability and needle tip diameter. Needle tip diameters play a crucial role in electrospinning. Needles used for spinning ranged from 18 to 22 gauge.

[0053] Example 1 underlines the significance of solvent choice and electrospinning parameters in customizing scaffold properties. Chloroform / acetone solutions require higher voltages and slightly higher flow rates than HFIP solutions, reflecting differences in electrical properties and jet stability during spinning. Both solvents enable the production of sheets and tubes with variable fiber alignment, influenced by mandrel speed and collector distance. However, the solvent choice and concentration significantly impacts fiber diameters, alignment, and ease of deposition and removal. The chloroform / acetone solution uses a higher polymer concentration (27% w / v) compared to HFIP (12% w / v), affecting viscosity and fiber formation, as illustrated in Figures 1 and 2.

[0054] The tensile test on the electrospun PCL sheet was conducted using a dynamic mechanical analyzer (DMA, Q800, TA Instruments) at both ambient and body temperatures (37 °C). The electrospun PCL mesh was cut into strips and mounted on the DMA tensile clamp and tested at a rate of 0.2 mm / min to 0.4 mm then at a rate of 10 mm / min to 25 mm. The data in the table below illustrates the tensile properties of the electrospun PCL sheet at 19 °C and 37 °C. Results indicate that the tensile properties of electrospun PCL vary with test conditions. At 37 °C, there was a reduction in tensile modulus and tensile strength, along with an increase in elongation at break.

[0055] Example 2:

[0056] A multi-layer tube was fabricated consisting of a polypyrrole coated, electrospun PCL interior surrounded by a PCL outer layer. The substrate layer consisted of a neat PCL electrospun scaffold as described in Example 1 .

[0057] The scaffold material was treated in a two-step oxidative polymerization process using benzoyl peroxide, pyrrole monomer, and sodium naphthalene-2-sulfonic acid as an ionic dopant. The aqueous solution of pyrrole monomer and sodium naphthalene-2-sulfonic acid was prepared at 14 mg / mL and 10 mg / mL, respectively. The benzoyl peroxide oxidation step was performed by preparing a super saturated benzoyl peroxide and isopropanol solution at 10 mg / mL and submersing the scaffold for 10 minutes. The scaffold was removed after treatment, allowed to dry completely, and then submerged in the pyrrole monomer solution for approximately 2.5 hours. The process can be repeated until the desired surface conductivity is achieved. The modified scaffold was rinsed thoroughly with water before characterization. Electrical conductivity was measured using an Ossila 4-point probe instrument on rectangular samples. Current was auto adjusted by the Ossila software to a value of approximately 0.1 mA. Two measurements were made in different areas on each side of material triple treated using the above procedure.

[0058] To finalize fabrication of the multilayer tube, the treated scaffold was placed on its original 4 mm mandrel and rotated. Additional polycaprolactone was electrospun on the surface until there was a uniform coating. Example 3:

[0059] A multi-layer tube consisting of a blended polycaprolactone and polypyrrole composite core with a polycaprolactone coating was prepared. 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 compounder. Compounding took place by melting at a temperature of 105 °C and then mixing for approximately 10 minutes at 85 RPM before discharging. The compounded composite was dissolved at 27% w / v in a mixture of chloroform and acetone as described in Example 1 and electrospun accordingly.

[0060] Tubes were fabricated using an electrospinning system consisting of a high voltage supply, syringe pump, and a rotating collector. The internal layer was made using the 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 of less than 1800 RPM. 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 0.75 mL / hr. The voltage was increased to 22.2 kV. Spinning continued for an additional 2.5 mL. Due to the insulating nature of the PCL matrix, these samples did not exhibit electrical conductivity when measured using a 4- point probe.

[0061] Example 4:

[0062] 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 both 5,000 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 hexafluoroisopropanol (HFIP). Sheets were electrospun with a positive voltage of 6.6 kV at the 20 ga stainless steel needle tip and a negative voltage of -1 .2 kV on the rotating collector. The collector (diameter=10 cm) was rotated at 50 RPM, to obtain sheets, and placed approximately 20 cm away from the needle tip.

[0063] The material surface was conjugated with a 30 kDa poly-L-lysine polymer through the NHS functional group. An aqueous solution of poly-L-lysine was prepared at 16.7 mg / mL using a buffer 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 for =19 hours and then washed with copious deionized water before being dried in a vacuum oven at room temperature.

[0064] The surface was characterized using Xray photoelectron spectroscopy (XPS), see Figures 7 and 8. It was observed that the nitrogen content in the poly-L-lysine modified sample was approximately 2.5% compared to =0.3% for the unmodified PCL and the unconjugated blend. The high nitrogen content in the poly-L-lysine sample was attributed to the high number of amines in the lysine backbones. The oxygen content of both the poly-L-lysine and unconjugated polymer blends was higher than the unmodified PCL samples due to the presence of additional oxygen bonding from the polyethylene glycol (PEG) linker polymer. These two findings suggest surface migration of the PEG and NHS moieties and enable amide bonding linkage of the poly-L-lysine. The Zeta potential of the surface was studied using an Anton Parr Surpass3 electrokinetic analyser. Sample sheets were fixed to the Surpass3 adjustable gap cell where the gap was adjusted to 100 pm. A pH scan was performed using streaming current mode from pH 4 through pH 9. A streaming pressure gradient of 600 mbar to 200 mbar was used for charge displacement. An electrolyte composed of 0.001 M potassium chloride was used for all experiments at room temperature. Each streaming experiment was conducted in triplicate for each pH step and was used to determine zeta potentials.

[0065] Samples modified with poly L-lysine showed 7X increases in Zeta potential at pH 7 when compared to unmodified PCL, see Figure 9. The increase in Zeta potential corresponds to increased hydrophilicity and supports the presence of poly-L-lysine on the exposed surface.

[0066] Example 5:

[0067] The physical blend of electrospun PCL / PCL-PEG-NHS described in Example 4 was also used to conjugate a water soluble, recombinant, collagen-like moiety. An aqueous solution of the compound was prepared at 50 mg / mL using a buffer 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 for =72 hours and then washed with copious deionized water before being dried in a vacuum oven at room temperature.

[0068] The surface was characterized using Xray photoelectron spectroscopy (XPS), see Figures 10 and 11 . It was observed that the nitrogen content in the collagen-like modified sample was approximately 0.7% compared to =0.3% for the unmodified PCL and the unconjugated blend. The higher nitrogen content in the conjugated sample was attributed to the high number of amines in the polymer backbone. The oxygen content of both the collagen-like sample and unconjugated polymer blends was higher than the unmodified PCL samples due to the presence of additional oxygen bonding from the polyethylene glycol (PEG) linker polymer. These two findings suggest surface migration of the PEG and NHS moieties and enable amide bonding linkage of the collagen-like moiety.

[0069] Electrokinetic surface analysis was carried out using streaming current pH scan measurements on the electrospun mats, see Figure 11 . Samples modified with the recombinant collagen showed 3X increases in Zeta potential at pH 7 when compared to unmodified PCL. The increase in Zeta potential corresponds to increased hydrophilicity and supports the presence of bound collagen on the exposed surface.

[0070] Example 6:

[0071] To assess nerve growth on different specimens, 12 mm disks were cut from electrospun sheets and used in cell culture. 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 above examples were cut into circles using a biopsy punch 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.

[0072] 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 as maximum number neurites of intersections were recorded for each condition.

[0073] Fig. 12 illustrates the quantification of neurite outgrowth using ImageJ analysis. The data indicates a significant enhancement in neurite intersections with poly L-lysine-modified PCL substrates compared to unmodified PCL. Specifically, the modified PCL exhibited a 56% increase in the number of intersections, with 190 intersections recorded for the modified version versus 122 for the unmodified. This substantial increase underscores the positive impact of poly L-lysine modification on promoting neural connectivity, likely due to improved surface properties that facilitate cellular adhesion and growth. The results highlight the potential of poly L-lysine immobilized on the surface as a beneficial coating in neural tissue engineering applications.

[0074] Additionally, the positive control, consisting of glass slides coated with polyornithine and Matrigel, demonstrated optimal neurite outgrowth, serving as a benchmark for effective neural support. The prolific outgrowth observed on these coated slides emphasizes the efficacy of surface coatings in enhancing neurite extension, drawing parallels to the improvements seen with poly L-lysine on PCL. These findings suggest that incorporating such modifications could be advantageous in neural tissue engineering applications.

[0075] Fig. 13 presents p3 Tubulin stained neurites on different specimens. The presence of p3-Tubulin was used as a neuronal marker to confirm and visualize neurite outgrowth, highlighting the network extensions on the modified surfaces. Additionally, the positive control, consisting of glass slides coated with polyornithine and Matrigel, exhibited optimal neurite outgrowth, serving as a benchmark. This prolific outgrowth validates the modifications' efficacy in promoting neural connectivity, illustrating the potential of these coatings in neural tissue engineering applications.

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; ii) at least one electro-spun intermediate 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; and iii) an inner layer comprising at least one electrical conducting compound and / or at least one ionic conducting compound and / or at least one peptide or protein.

2. The electro-spun multilayer tube or wrap of claim 1 , wherein the peptide of the inner layer is selected from cationic polypeptides.

3. The electro-spun multilayer tube or wrap of any of the preceding claims, wherein the inner layer comprises or consists of electrical conducting polymers or comprises additives.

4. 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.

5. The electro-spun multilayer tube or wrap of any of the preceding claims, wherein the inner layer i) has an electrical conductivity ranging 100-1000 mS / m; and / or ii) has a zeta potential ranging of from ± 0-100 mV.

6. The electro-spun multilayer tube or wrap of any of the preceding claims, wherein the electrospun 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 / oriii) 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 1 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 blend of a) 50 to 99 wt.-% of a polymer selected from poly(caprolactone), polylactide, polyglycolide, poly(trimethylene carbonate), poly(dioxanone), polyethylene glycol, polyurethane, their copolymers or mixtures thereof and b) 1 to 50 wt.-% an electrical conducting polymer selected from polypyrrole, polyaniline, polythiophene, poly( 3,4-ethyenedioxythiophene) or electrical conducting additives, wherein the sum of all components is 100 wt.-% composite; ii) dissolving the blend of step i) in a solvent; and iii) electrospinning the dissolved blend on a mandrel with a diameter ranging of from 1 to 20 mm to obtain the inner layer; and iv) electrospinning at least one polymer selected from poly(caprolactone), polylactide, polyglycolide, poly(trimethylene carbonate), poly(dioxanone), polyethylene glycol, polyurethane, their copolymers or mixtures thereof onto the inner layer; v) optionally cutting the tube in order to obtain a wrap.

10. Method of manufacturing a multilayer tube or wrap according to any of claims 1 to 8 via 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, and a dopant as at least one electrical conducting compound and / or at least one ionic conducting compound comprising or consisting of the following steps: i) preparing a super saturated peroxide solution and submersing the electrospun tube / wrap in the solution to obtain an oxidized layer;ii) preparing the aqueous solution of monomer comprising an anionic dopant selected from di-2- ethylhexyl sulfo-succinic acid sodium salt, or dodecylbenzene sulfonic acid sodium salt, preferably sodium naphthalene-2-sulfonic acid; iii) submerging the oxidized tube / wrap in the monomer solution comprising anionic dopant; iv) placing the tube / wrap obtained in step iii) on the mandrel or a static flat collector and electrospin the outer polymer layer on top of the previous layer to obtain a multilayer tube or wrap.11 . 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 intermediate layer; v) electrospinning pure PCL onto PCL / PCL-PEG-NHS layer as outer layer; vi) conjugating poly-L-lysine, collagen, or fibrinogen polymer to the NHS functional group as inner layer.

12. 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.

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