The present invention relates to an apparatus for

producing electrospun polymer fibres with a modified surface .

BACKGROUND

Electrospinning is a technique used to draw very fine polymer fibres from a polymer solution. A voltage is applied to a liquid polymer solution droplet so that the liquid becomes charged. The electrostatic repulsion counteracts the surface tension so that the droplet is stretched. At a critical point, known as the Taylor cone, a jet of the liquid polymer solution emerges from the droplet .

The jet of solution dries in the air while the jet is elongated and thinned. The fibre is collected on a grounded collector, resulting in the formation of uniform fibres with micro- and nano-metre scale diameters.

Artificial plasmas can be generated and used to treat the surface of materials. Generally, plasma is generated when an electrical current is applied across a dielectric gas or fluid. With ample current density and ionization, a plasma arc or discharge forms between the electrodes. GENERAL DESCRIPTION OF THE INVENTION

At its most general, the present invention provides a plasma-electrospinning apparatus arranged so that, in use, a polymer composition passes through a directional plasma discharge so that a surface of an electrospun polymer fibre is modified by the plasma discharge during its formation.

In a first aspect, the present invention provides a plasma-electrospinning apparatus comprising a polymer electrospinning device for producing a polymer fibre and a directional plasma device, the directional plasma device being a plasma torch or a plasma plume-generating device, whereby the directional plasma device is arranged so that, during operation, a polymer composition from the polymer electrospinning device passes through a plasma jet or plasma plume from the directional plasma device.

The surface of the electrospun fibres produced from the electrospinning device of the apparatus of the first aspect is modified during formation of the polymer fibre.

As a result, the modification is conformal to the surface around the circumference of the fibre. Additionally, the apparatus may provide sub-nm surface modification. In this way, the bulk properties of the polymer fibre (e.g. structural (mechanical) properties) are maintained while modifying the surface properties. In a second aspect, the present invention provides a plasma-electrospinning apparatus comprising a polymer electrospinning device and at least two different plasma zones, whereby the plasma zones are arranged so that, during operation, a polymer composition from the

electrospinning device passes through at least two distinct plasmas.

The jet of polymer composition from the electrospinning device of the apparatus of the second aspect passes through two distinct or different plasma zones. Each distinct plasma zone treats the polymer composition with a different surface modification and/or plasma-coated coating. Each plasma zone may treat the same area of the polymer composition or may treat different areas of the polymer composition. For example, the polymer

composition may pass through a first plasma zone to provide a modified surface and then pass through a second (different) plasma zone to plasma-coat or further modify the modified surface of the polymer composition. In this way, an electrospun polymer fibre with multiple surface layers and/or multiple surface modifications can be produced .

In a third aspect, the present invention provides use of a plasma-electrospinning apparatus of the first or second aspects to produce a surface-modified polymer fibre. In a fourth aspect, the present invention provides a process for producing a polymer fibre with a modified surface, the process includes

(i) forming a jet of polymer composition from an electrospinning device; and

(ii) passing the jet of polymer composition through a plasma jet or plasma plume from a directional plasma device, wherein the directional plasma device is a plasma torch or a plasma plume-generating device.

In a fifth aspect, the present invention provides a process for producing a polymer fibre with a modified surface, the process includes

(i) forming a jet of polymer composition from an electrospinning device; and

(ii) passing the jet of polymer composition through the plasma generated by at least two different plasma zones .

In a sixth aspect, the present invention provides a non-woven polymer fibre mat comprising electrospun polymer fibre produced by the apparatus of any one of the first or second aspects, by the use of the third aspect or by the process of the fourth or fifth aspect. In seventh aspect, the present invention provides a non-woven polymer mat comprising electrospun polymer fibre, wherein the electrospun polymer fibre includes:

(i) a polymer core and two or more plasma-coated coatings ;

(ii) a polymer core with a plasma-modified surface and at least one plasma-coated coating; or

(iii) a polymer core and at least one plasma-coated coating with a plasma-modified outer surface.

In eighth aspect, the present invention provides a non-woven polymer mat comprising electrospun polymer fibre, the electrospun polymer fibre having at least two plasma-treated sections, wherein the plasma-treated sections are different.

In a ninth aspect, the present invention provides a multi-layered electrospun polymer fibre mesh having two or more layers of electrospun polymer fibre, wherein at least one layer has plasma-treated electrospun polymer fibre .

In a tenth aspect, the present invention provides a kit comprising a directional plasma device, such as a plasma torch or a plasma-plume generating device, and a

directional plasma device attachment for fitting the plasma torch to an electrospinning device so that, during operation, a polymer composition from a polymer electrospinning device passes through a plasma jet or plasma plume from the directional plasma device.

In an eleventh aspect, the present invention provides a kit comprising a directional plasma device, such as a plasma torch or a plasma-plume generating device, and a composite polymer fibre collector having a collecting frame with an electrically conducting element, the electrically conducting element being electrically connected to an electrically chargeable part.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure la shows a schematic of an atmospheric pressure plasma torch which may be used in the

plasma-electrospinning apparatus of the present

invention .

Figure lb shows a schematic of another atmospheric pressure plasma torch which may be used in the

plasma-electrospinning apparatus of the present

invention .

Figure 2 shows a schematic of a

plasma-electrospinning apparatus with a composite collector .

Figure 3a shows a schematic of a

plasma-electrospinning apparatus of the present

invention . Figure 3b shows a schematic of an alternative plasma-electrospinning apparatus of the present

invention .

Figure 4 shows a schematic of a

plasma-electrospinning apparatus having two or more plasma zones arranged in series.

Figures 5a and 5b show schematics of electrospun polymer fibres produced a plasma-electrospinning

apparatus having two different plasma zones.

Figure 6 shows a schematic of an electrospun polymer fibre having two different plasma-treated sections along the fibre length.

Figure 7a shows surface modified electrospun fibres produced by the plasma-electrospinning apparatus of the present invention and collected as a randomly aligned mesh .

Figure 7b shows surface modified electrospun fibres produced by the plasma-electrospinning apparatus of the present invention and collected as an aligned mesh.

Figure 8 shows water droplets on a) untreated PLA material, b) He plasma-treated PLA material,

c) methanol/He plasma-treated PLA material and

d) ammonium hydroxide/He plasma-treated PLA material.

Figure 9 shows the water contact angle (°, y axis) changes with respect to plasma modification of fibres; (i) without treatment; (ii) with helium treatment; (iii) with methanol treatment; and (iv) with ammonium hydroxide (NH ₄ 0H) treatment. Figure 10 shows the FTIR spectra (wavenumber crrT ¹ , x-axis; absorbance units, y-axis) of untreated PLA electrospun fibres (dotted line) and ammonium

hydroxide/He plasma-treated PLA material (solid line) .

Figure 11 shows a section of the FTIR spectra

(wavenumber crrT ¹ , x-axis; absorbance units, y-axis) of untreated PLA electrospun fibres (dotted line) and ammonium hydroxide/He plasma-treated PLA material (solid line) after 5 months of storage.

Figure 12 shows a section of the FTIR spectra

(wavenumber crrT ¹ , x-axis; absorbance units, y-axis) of untreated PLA electrospun fibres (long dashed line, bottom/ lowest absorbance values), ammonium hydroxide/He plasma-treated PLA material treated at 10W (short dashed line, middle/intermediate absorbance values) and ammonium hydroxide/He plasma-treated PLA material treated at 15W (solid line, top/highest absorbance values) .

Figure 13 shows fluorescently labelled electrospun fibres in a confocal micrograph stack slice having directionally aligned multi-layered fibres. Fig. 13A shows two layers of a laminate having PLA/rhodamine polymer fibres in one layer (left) aligned substantially parallel to the PLA/FTIC polymer fibres of an adjacent polymer layer. Fig. 13 B shows sequential layers presenting fibres oriented in differing directions.

Distinct layers can be seen in confocal stack images.

Figure 14 shows SEM and overlaid EDXA mapping of carbon, oxygen, silicon and chlorine in a trichloro (methyl ) silane treated nanofiber surface.

Separate elemental maps are included showing the presence of carbon (top row, left) and oxygen (top row, middle) of the polymer backbone, and silicon (top row, right) and chlorine (bottom row, left) from silane.

Figure 15 shows Brightfield microscopy images of neurosphere culture on control (left column) and amine coated (right column) nanofibres after 0 (top row), 2 (middle row) and 5 (bottom row) days in culture. White arrows highlight cells attached and aligned to fibre direction; black arrows show rounded cells.

Figure 16 shows Brightfield microscopy images of neural stem cell culture on untreated (right hand column) and amine-plasma e-spun PLA nanofibres (left hand

column), after 2 days (top row) and 5 days from seeding (bottom row) . The images in Figure 16 are from the same sample as Fig. 15, but show a different sample area.

Figure 17 shows Brightfield microscopy images of MG63 cells growing on untreated (right hand column) and amine-plasma e-spun PLA nanofibres (left hand column), after 20 hours (top row) and 11 days from seeding (bottom row) .

Figures 18A, B and C show XPS spectra of non-treated PLA (top row) simultaneously 15W plasma e-spun amine treated PLA fibre (centre rows) and overlaid spectra (bottom rows) . The XPS spectra show binding energy (eV, x-axis) against counts (CPS, y-axis) . DETAILED DESCRIPTION

The present invention will be described with reference to the accompanying drawings .

Directional Plasma Device

The present invention uses a directional plasma device to treat the jet of polymer composition from the

electrospinning device. The directional plasma device may be a plasma torch or a plasma plume-generating device. As used herein, the term plasma torch refers to any plasma torch capable of generating a directed flow of plasma .

It is preferred that the directional plasma device of the present invention is an atmospheric pressure plasma device .

Typically the directional plasma device will produce a plasma jet or plume from a plasma-generating portion of a plasma electrode. The plasma jet or plume may be

generated from the plasma electrode by, for example, direct current (DC) and alternating current (AC) (also referred to as radio-frequency (RF)) . The directional plasma device typically includes a power supply connected to the plasma electrode. The plasma jet or plume may require ignition by an ignition source. Thus, the directional plasma device may include an ignition source located near the plasma electrode to ignite the plasma jet or plume. The plasma jet or plasma plume may

preferably form between the plasma electrode and

atmospheric air as the grounded electrode.

The directional plasma device typically includes a carrier gas conduit for providing a carrier gas close to the plasma electrode. The carrier gas conduit may be tubular with a carrier gas cavity. A carrier gas can enter the carrier gas cavity through a carrier gas inlet.

In some embodiments, the directional plasma device has an elongate needle electrode with an electrode tip. In these embodiments, the elongate needle electrode may extend along an elongate axis of the carrier gas cavity of the carrier gas conduit. The electrode tip of the elongate needle electrode may protrude from the carrier gas conduit outlet. The carrier gas typically comes in close contact with the electrode tip. The directional plasma device may be arranged so that the electrode tip is outside of the carrier gas conduit cavity. In this arrangement, the carrier gas conduit has a carrier gas conduit outlet positioned close to the electrode tip, to deliver carrier gas to the electrode tip. In an alternative arrangement, the electrode tip is located within the carrier gas conduit. In this arrangement, the carrier gas conduit may have a carrier gas conduit outlet positioned close to the electrode tip to allow the plasma jet or plume to exit from the carrier gas conduit. The elongate needle electrode may be tapered at one end to form the electrode tip. The elongate needle electrode may be any electrically conductive material, such as a metal or graphite. Preferably the elongate needle

electrode is made of an electrically conductive metal such as tungsten, copper, platinum, molybdenum, silver or stainless steel.

When the elongate needle electrode is within the carrier gas conduit, the elongate needle electrode may be

surrounded by an insulating material, such as a ceramic tube. In this way, the insulating material may act as an insulator along the length of the electrode so that plasma discharge is predominantly formed at electrode tip .

An example of a plasma torch having an elongate needle electrode located within the carrier gas cavity of the carrier gas conduit is shown in Fig. la) . A metallic (such as tungsten or stainless steel) wire filament 10, as the electrode, is connected to a RF power supply 12 via electronics 14. The metallic wire filament 10 is substantially uniform in diameter along its elongate axis. In other embodiments, the filament may be tapered, for example, to a sharp electrode tip.

The metallic wire filament 10 passes through a

cylindrical cavity in a ceramic tube 16. The filament 10 and ceramic tube 16 are contained in a quartz glass tube 18 (as the carrier gas conduit) . The ceramic tube 16 and glass tube 18 are cylinders and are arranged concentrically.

A carrier gas is introduced into the glass tube in flow direction A. The carrier gas can flow through both in the cavity between the ceramic tube 16 and the glass tube 18, and the cylindrical cavity in the ceramic tube 16. In some embodiments, the carrier gas flow in in the cavity between the ceramic tube 16 and the glass tube 18, and the cylindrical cavity in the ceramic tube 16 may be controlled separately. In this way, the flow rates can be controlled more effective, for example.

An end of the metallic wire filament 10 forms an

electrode tip 20. The electrode tip 20 protrudes from open ends of the ceramic tube 16 and glass tube 18. The open end of the glass tube 18 thus forms a carrier gas conduit outlet delivering carrier gas to the electrode tip 20. When the filament 10 is charged by the power supply 12 and the carrier gas is passed through the glass tube to the electrode tip 20, the carrier gas can be ignited to produce a plasma plume 22. The plasma plume 22 is conical about the electrode tip 20. The atmospheric air acts as the ground electrode.

In other embodiments, the directional plasma device may have an elongate tubular electrode. An elongate tubular electrode may be, for example, a metal tube, a metallised glass tube or an electrically conductive material (e.g. metal) coiled around an outer surface of an insulating material tube (a coiled electrode) . The plasma may be ignited within the tubular electrode and the plasma may be driven out of the tube by the flow of carrier gas to produce a plasma afterglow forming the plasma plume or plasma jet.

In a coiled electrode, the coiled electrically conductive material may extend along part of or all of the length of the insulating material, e.g. glass, tube. The electrode may be formed within the coil. As a result, the plasma may be ignited within the tubular insulating material surrounded by the coil. The plasma may be driven out of the tube by the flow of carrier gas to produce a plasma afterglow forming the plasma plume or plasma jet.

Typically there is no need for the coiled material of the electrode to directly contact the carrier gas. Such a plasma electrode may be referred to as an inductively coupled plasma device.

When the directional plasma device has an elongate tubular electrode, the elongate tubular electrode

preferably forms the carrier gas conduit. The carrier gas outlet allows the plasma afterglow to leave the elongate tubular electrode/carrier gas conduit to form the plasma jet or plasma plume.

An example of a plasma torch having an elongate tubular electrode forming the carrier gas conduit is shown in Fig. lb) . A metallic (such as copper) wire 50 is coiled around is quartz glass tube 52 to form both the electrode and the carrier gas conduit. The glass tube 52 has a cavity for receiving a carrier gas. The metallic wire 50 is connected to a RF power supply 12 via electronics 14.

A carrier gas is introduced into the glass tube 52 in flow direction B. The carrier gas can flow through the cavity in the glass tube 52 to an open end 54 of the glass tube 52. The open end 54 forms both the electrode tip and the carrier gas conduit outlet. In this way, the carrier gas is delivered to the electrode tip.

When the coiled metallic wire 50 is charged by the power supply 12 and the carrier gas is passed through the glass tube to the open end 54 of the glass tube 52, the carrier gas can be ignited to produce a plasma plume 56. The plasma plume 56 is conical emerging from the open end 54 of the glass tube 52. The atmospheric air acts as the ground electrode. The carrier gas may be any one of the gases or mixtures of gases used in plasma treatment. Such gasses include, but are not limited to, helium, argon, oxygen, nitrogen and mixtures thereof. The plasma generated across the carrier gas may modify the surface of the jet of polymer composition depending on its composition, concentration, flow rate and other known parameters. The flow rate may be in the range of O.lL/minute to 1.5L/minute . The flow rate may be in the range 0.6 to 1.0 L/minute. The plasma power may be 10 to

20 W, preferably 14 to 16 W.

In addition, the carrier gas may include one or more additives to form a carrier gas-additive mixture that can modify and/or functionalise the polymer surface.

Examples of additives include, but are not limited to, monomers, such as acrylic acid, and small reactive molecules, such as ammonia, methanol, silanes,

fluorinated organic compounds and metal oxides, such as zinc, copper iron or titanium oxide.

In some embodiments, the carrier gas or carrier gas- additive mixture can be switched while the jet of polymer composition is passing through the plasma jet. As a result, the electrospun fibre can have at least two sections with different types of surface modification along its length. The sections of different modified surface may be ~1 pm or more along the length of the polymer fibre.

The switching between the different carrier gas and/or additive may be achieved, for example, by the carrier gas conduit having two or more carrier gas cavities, each carrier gas cavity having a different carrier gas inlet for feeding in a specific carrier gas or

carrier gas-additive mixture. Each carrier gas cavity can deliver carrier gas to the plasma-generating portion of the electrode, e.g. electrode tip. Preferably the carrier gas cavities are arranged annularly.

In preferred embodiments, the plasma plume or plasma jet, when generated, extends up to 50mm from the

plasma-generating portion of the device.

The plasma plume or plasma jet typically contains a visible part, where a glow is visible to the human eye. The plasma plume may also contain a non-visible part where reactive species are present, but the plume is not visible to the human eye. The non-visible part may extend 5 to 10 mm beyond the visible part of the plasma plume or plasma jet. As described herein, the plasma plume or jet includes an area extending 5 to 10 mm beyond the visible part of the plasma plume or plasma jet.

Capacitive plasma discharge

Capacitive plasma discharge is non-thermal plasma

generated by the application of RF power (e.g., 13.56MHz) to one powered electrode, with a grounded electrode held at a small separation distance (typically up to 50mm) .

In some embodiments, the directional plasma device may include a RF power supply. Such embodiments may generate a capacitive plasma discharge. It is preferred that the plasma torch uses atmospheric air as the grounded

electrode, although close proximity of the plasma plume to a more secured grounding often gives rise to an extension of the plasma as it passes through air.

Inductively coupled plasma discharge

An alternating current often set in a ring or coil electrode around or adjacent a gas-filled tube gives rise to an alternating magnetic field, in turn inducing electric currents in the gas. Such arrangement can lead to the formation of a plasma plume or jet.

In some embodiments, a gas such as argon, helium, oxygen, nitrogen, or mixture thereof, can be flown through a tube having a coiled electrode around its outer edge. On initiation of an AC (RF) current in the electrode the plasma is ignited and the plasma is driven in the

direction of gas flow, creating a plasma plume if the coil is set near an opening orthogonal to the gas flow direction .

Electrospinning Device

Electrospinning devices for producing polymer fibres are known per se.

The electrospinning device of the present invention may have an electrospinning capillary tube with a polymer composition outlet. A droplet of charged polymer

composition may form at the polymer composition outlet so that a jet of polymer composition may emerge from

droplet .

Electrospinning Capillary Tube

The electrospinning capillary tube has a polymer

composition cavity for retaining polymer composition when in use, and the polymer composition cavity is connected to the polymer composition outlet. In some embodiments, the polymer composition cavity has a polymer composition inlet connected to a polymer composition reservoir, such as a syringe. In this way, polymer composition in the polymer composition cavity can be replenished.

In use, the polymer composition is charged to a high electrical potential. The electrospinning device of the present invention may be charged by inserting an

electrode into the polymer composition. In a preferred embodiment, the electrospinning capillary tube is

electrically conductive, e.g. is metallic, and an

electrode is in contact with or connected to the outer wall of the electrospinning capillary tube. In this way, the polymer composition can be charged to a high

electrical potential. A stainless steel hollow needle is a suitable electrospinning capillary tube.

The polymer composition outlet is directed at a polymer fibre collector to collect a polymer fibre formed from the jet of polymer composition emerging from the droplet of polymer composition at the polymer composition outlet.

Polymer Fibre Collector

The polymer fibre collector may be, for example, a flat plate or a drum roller. The polymer fibre collector may be charged to attract the jet of polymer composition. The collector is typically grounded. As a result, uncharged (in other words, neutral with respect to electrical charge) polymer fibres can be collected.

The polymer fibre collector can determine the orientation of the collected fibre, for example, randomly orientated or aligned. Randomly oriented collected fibre may be collected on a grounded metallic plate, such as a copper plate, or grounded rotary metallic drum. A surface of the collector may have a removable layer, such as aluminium foil or glass. In this way, the collected electrospun polymer fibre can be easily removed from the collector.

The collector may be a composite polymer fibre collector having a collecting frame with an electrically conducting element, the electrically conducting element being electrically connected to an electrically chargeable part .

In use, the electrically chargeable part is charged with a voltage corresponding to the voltage of the polymer solution. At least part of the charge is transferred from the electrically chargeable part to the electrically conducting element through the electrical connection. The jet of polymer composition arrives at the collector frame and adheres to the frame to produce a low density electrospun polymer fibre mesh on the collecting frame, and at least partially covering the aperture of the collecting frame.

The low density polymer fibre mesh may have an average line density of in the range of 30 to 60 fibres per 100 pm, preferably 40 to 50 fibres per 100 pm. The average line density can be calculated by counting the number of fibres crossing a straight line of 100 pm, for example using a scanning electron micrograph. The low density polymer fibre mesh may have a thickness in the range of 0.1 to 5 pm, preferably 0.5 to 3.0 pm. The charged part is typically made of an electrically conductive material, such as copper. For example, the charged part may be a copper plate, such as those

typically used as a collector in electrospinning . The charged part and the collecting frame may be

separated by a collecting frame separation distance. The collecting frame separation distance may be in the range of 50 to 200 mm, preferably 80 to 120 mm. The collecting frame may be detachably connected to the chargeable part of the composite collector. The electrical connection between the electrically conducting element (s) and the electrically chargeable part may form the detachable connection . The collecting frame typically faces the polymer

composition outlet of the electrospinning capillary tube. In this way, the collecting frame is in the polymer path to collect the polymer fibre. The distance between the polymer composition outlet and the collecting frame may be 100 to 300 mm, preferably 150 to 220 mm.

In preferred embodiments, the collecting frame is

electrically non-conductive, and has three or more straight sides. The electrical conducting element (s) may span within the frame aperture from one side of the frame to another side of the frame. In this arrangement, an electrical field is created by the electrically

conducting element. The electrical field extends to the surrounding electrically non-conductive frame. The electrospun polymer fibre attaches to the non-conductive frame, alternating between an upper and a lower frame side. As a result, the fibre spans across the frame in an aligned fashion to produce a low density oriented electrospun polymer fibre mesh.

Typically, the electrically non-conductive collecting frame will be rectangular or square (i.e. with four straight sides with internal angles of around 90°) . When the frame is rectangular, the electrically conducting element (s) may span between the two shorter sides of the frame so that the element extends within the frame along the major axis of the frame.

The electrically conductive element (s) may be an

electrically conductive material, such as steel. The electrically conductive element may be an elongate element, such as a wire, filament or blade (i.e. elongate sheet ) .

Preferably, the rectangular or square collecting frame has two or more electrically conducting elements running in parallel. When the electrically conducting elements are arranged in parallel, the conducting elements may be separated by 20 to 100 mm, preferably 40 to 60 mm.

Parallel electrically conductive elements provide a large electrical field. In this way, a relatively large frame can be used and a relatively large mesh can be obtained.

The electrically non-conductive collector frame may be made of any electrically non-conductive material, such as a non-conductive textile. The frame may have a

non-conductive outer layer or coating, such as a layer of polyvinyl chloride (applied, for example as insulating adhesive tape) . The rectangular or square collecting frame may be of any dimensions. The rectangular or square collecting frame may have one side with a length of 80 mm or more. The rectangular frame may have a major side with a length of 150 mm or more, preferably 250 mm or more, and a minor side of 80 mm or more, preferable 100 mm.

In other embodiments, the collecting frame is an

electrically conducting ring, such as a steel ring. In these embodiments, the frame and the electrically

conducting element are the same. In this arrangement, the polymer fibre attaches to the electrically conducting ring at random points around the ring. The fibre spans across the ring to produce a low density randomly aligned electrospun polymer fibre mesh. The electrically

conducting ring may have a diameter of 10 to 150 mm, preferably 30 to 100 mm.

The produced low density electrospun polymer fibre meshes may be fragile and not able free stand or be handled without support. To generate electrospun fibre meshes that are easy to handle, the aligned or random

electrospun polymer fibre mesh may be removed from the collecting frame using mesh subframes, e.g. cellulose acetate subframes, which have a hollow area. The hollow area of the mesh subframe should be smaller than the hollow area of the collecting frame. For example, a mesh subframe with a hollow area of 16 cm ² may be used with a rectangular collecting frame of dimensions 30 x 10 cm having a hollow area just below 300 cm ² .

The subframe may be sprayed with an aerosol adhesive to adhere to electrospun polymer fibre mesh on the

collecting frame. The subframe and electrospun polymer mesh may be detached from the collecting frame using a cutting tool, such as forceps. Excess fibres may be severed from the collecting frame and/or mesh subframe using a cutting tool, such 3. S 3. SCSlpel and blade.

As a result, a low density electrospun fibre mesh mounted on a mesh subframe is produced. Where the electrospun fibres were aligned on the collecting frame, the

alignment remains on the subframe.

An example of a composite collector will be described with reference to Fig. 2. A metallic needle 72 is connected to an electrospinning power supply to charge a polymer solution held within a cavity of the metallic needle 72. A jet of polymer solution 74 emerges from the tip of the metallic needle 72 towards a detachable collecting frame 76, around 15 to 22 cm from the metallic needle tip. Two plasma torches 78,80 are arranged so that the plasma plume 82,84 from each plasma torch 78,80 oppose one another. In this way, one half of the polymer composition is treated with a first plasma torch 78, and the other half of the polymer composition is treated with a second plasma torch 80. The plasma torches may provide the same or different plasmas. After plasma treatment, the polymer composition continues to the 30 x 10 cm rectangular collector frame 76. The collector frame 76 has an insulating PVC adhesive tape outer layer. Two electrically conductive stainless steel blades are arranged in parallel and extend from one side of the frame to the other side of the frame (in the direction orthogonal to the plane of the figure) . The stainless steel blades are 4 to 6 cm apart. The blades are

connected to a copper static collector plate 86 by stainless steel wires 88. The stainless steel wires 88 are detachably connected to the copper static collector plate 86 so that the collector frame 76 can be detached.

A further example of a composite collector is given in Yang et al . , Nanomedicine : Nanotechnology, Biology, and Medicine 7 (2011) 131-136.

Polymer Composition

Polymer compositions for use in electrospinning devices are known per se and the polymer composition used in the electrospinning device of the present invention is not particularly limited. The polymer composition may be, for example, a polymer solution or a polymer melt. It is preferred to use a polymer solution as the polymer composition .

Suitable polymers and solvents for use in electrospinning devices are known in the art. Mixtures of two or more polymers may be used.

The electrospun polymer fibre produced using the

apparatus of the present invention may be used in a number of technological fields. When the electrospun polymer fibre is to be used in the biological or medical field, preferably the polymer used in the polymer

composition is a biocompatible and/or biodegradable polymer. The resulting biocompatible and/or

biodegradable electrospun polymer fibre may be used in as a biological implant, such as a biological scaffold.

Examples of preferred polymers include poly (lactic acid) (PLA) , poly ( glycolic acid) (PLGA) , poly ( lactic-co- glycolic acid), poly ( caprolactone ) (PCL) , polyurethane (PU) , chitosan, alginate and mixtures thereof.

When the polymer composition is a polymer solution, the solvent in the polymer solution should be suitable for at least partially evaporating when the jet of polymer solution is travelling along the polymer path. The choice of solvent and solvent concentration will be known for any particular polymer or polymer mixture to achieve a suitable polymer solution for electrospinning .

Suitable solvents include N, N-dimethylformamide (DMF), dichloromethane (DCM), 2 , 2 , 2-trifluoroethanol (TFE), chloroform, 1 , 1 , 1 , 3 , 3 , 3-hexafluoro-2-isopropanol (HFIP), acetone, N, N-dimethylacetamide, and mixtures thereof.

Some solution may remain in the collected electrospun polymer fibre. The collected electrospun polymer fibre may be further dried to remove some, preferably all, of the remaining solvent. The electrospun polymer fibre may be dried applying heat and/or by a vacuum to the

electrospun polymer fibre. Configuration of Directional Plasma Device with respect to the Electrospinning Device

The apparatus of the present invention may provide, in use, a jet of charged polymer composition travelling along a polymer path formed between the polymer

composition outlet and the polymer fibre collector. The apparatus of the present invention may be arranged in any way that the jet of polymer composition to pass through the plasma jet or plume of the directional plasma device. For example, the plasma jet may intersect the polymer path to modify the surface of the resulting polymer fibre .

In one embodiment, the directional plasma device is arranged so the plasma jet or plume extends at an angle more than 0° to the overall direction of the jet of polymer composition along the polymer path from the polymer solution outlet to the polymer fibre collector. In this way, the plasma jet or plume treats the jet of polymer composition emerging from the polymer composition outlet from the side of the polymer composition jet. The angle may be 10° to 90°, preferably 30° to 90°, more preferably 60 to 90° with respect to the overall

direction of the jet of polymer composition along the polymer path from the polymer solution outlet to the polymer fibre collector. The plasma jet or plume surrounds the most (if not all) of the outer surface of the jet of polymer composition as the jet of polymer composition passes through the plasma jet. In this way, most, preferably all, of the surface of the resulting polymer fibre has a modified surface.

In this configuration, it is preferred that a directional plasma device having an elongate needle electrode located within the carrier gas cavity of the carrier gas conduit located is used.

An example of such an embodiment is shown in Fig. 3a) . The example plasma torch of figure la) is used in this embodiment. Briefly, a metallic wire filament 10 (as the electrode) is connected to a RF power supply 12 via electronics 14, and extends through the cavity of a ceramic tube 16. The filament 10 and ceramic tube 16 are contained in a quartz glass tube 18 (as the carrier gas conduit), through which carrier gas is introduced in flow direction A. The electrode tip 20 protrudes from open ends of the ceramic tube 16 and glass tube 18, and when the filament 10 is charged by the power supply 12 and the carrier gas is passed through the glass tube to the electrode tip 20, the carrier gas can be ignited to produce a plasma plume 22.

The plasma torch is arranged close to an electrospinning device. The electrospinning device has a metallic needle 102 connected to an electrospinning power

supply 104 to charge a polymer solution held within a cavity of the metallic needle 102. A jet of polymer solution 106 emerges from the tip of the metallic needle 102 towards a collector plate 108, around 10 to 15 cm from the metallic needle tip. The plasma plume 22 from the plasma torch intersects the path of the jet of polymer solution 106 on its journey to the collector plate 108. Note the figure is not to scale. The jet of polymer solution is not shown passing through the visible plasma, but is passing through the non-visible part of the plasma.

When the directional plasma device is arranged so the plasma jet or plume extends at an angle more than 0°

(e.g. 10° to 90°) to the overall direction of the jet of polymer composition along the polymer path from the polymer composition outlet to the polymer fibre

collector, the apparatus may include a second directional plasma device arranged so that a second plasma jet or plume opposes the plasma jet or plume of the first directional plasma device.

The plasma plume or plasma jet of the second directional plasma device may be the same as or different from the plasma plume or plasma jet from the first directional plasma device. When the plasma plume or plasma jet of the second

directional plasma device is the same as the plasma plume or plasma jet from the first directional plasma device, complete surface modification of the jet of polymer composition is assured.

When the plasma plume or plasma jet of the second

directional plasma device is different from the plasma plume or plasma jet from the first directional plasma device, the resulting polymer fibre may have two

different plasma-treated sections around the

circumference of the polymer fibre. The different plasma treatments are discussed herein, and include different carrier gas types, different carrier gas additives, different flow rates, different plasma strengths and combinations thereof.

In an alternative embodiment, the plasma electrode tip is arranged coaxially to the direction of the polymer path from the polymer composition outlet to the polymer fibre collector. In this embodiment, the plasma electrode may be tubular and form the gas carrier conduit, and the electrospinning capillary tube may be located within the gas carrier conduit/plasma electrode.

An example of such an embodiment is shown in Fig. 3b) . The example plasma torch of figure lb) is used in this embodiment. Briefly, a metallic wire 50 (connected to a RF power supply 12 via electronics 14) is coiled around a quartz glass tube 52 to form both the plasma electrode and the carrier gas conduit. A carrier gas is introduced into a cavity of the glass tube 52 in flow direction B to an open end 54 forming both the electrode tip and the carrier gas conduit outlet. When the coiled metallic wire 50 is charged by the power supply 12 and the carrier gas is passed through the glass tube to the open end 54 of the glass tube 52, the carrier gas can be ignited to produce a conical plasma plume 56.

A metallic needle 152 of the electrospinning device extends through the cavity of the glass tube 52, so that the needle and plasma torch are arranged coaxially. The metallic needle 152 is connected to an electrospinning power supply 154 to charge a polymer solution held within a cavity of the metallic needle 152. A jet of polymer solution 156 emerges from the tip of the metallic needle 152 towards a collector plate 158, around 10 to 15 cm from the metallic needle tip. The plasma plume 56 from the plasma torch intersects the path of the jet of polymer solution 156 on its journey to the collector plate 508. Note the figure is not to scale. Apparatus with Two or More Plasma Zones

The second aspect of the present invention provides a plasma-electrospinning apparatus whereby the jet of polymer composition passes through two different plasma zones before reaching a polymer fibre collector. The two different plasma zones result in a change in the surface layer (i.e. modification) of the jet of polymer

composition fibre after each plasma zone. The plasma zones may be arranged in series along the polymer path or opposing one another. The type of plasma for each plasma zone is not particularly limited.

The two plasma zones may be different in any way that results in a change in the surface layer (i.e.

modification) of the jet of polymer composition fibre after each plasma zone. The change in the surface may be a plasma-coated coating, for example, using a carrier gas with a polymerizable monomer additive.

The type of plasma device in each plasma zone may be the same or different. Preferably at least one of the plasma zones has a directional plasma device. More preferably the apparatus has a directional plasma device in each of the plasma zones.

The type of carrier gas or carrier gas-additive mixture used in each plasma zone may be the same or different. Preferably the carrier gas or carrier gas-additive mixture in each plasma zone is different from the carrier gas-additive mixture in the previous plasma zone along the polymer path. The difference in carrier gas and carrier gas-additive mixture may be any characteristic of the carrier gas and/or additive that can be varied in order to achieve a different surface modification, for example, the type of carrier gas, type of additive, concentration of additive, flow rate of the gas, plasma strength and combinations thereof.

For example, the plasma generated in a first plasma zone may use a carrier gas alone to modify the surface of the jet of polymer composition and the plasma generated in a second plasma zone may use a carrier gas-additive mixture

(e.g. He-methanol) to modify the jet of polymer

composition after the jet has passed through the first plasma zone. The apparatus having at least two different plasma zones may have further plasma zones whereby the plasma

intersects the polymer path, for example, a third, fourth or fifth plasma zone. Each further plasma zone may be the same or different to another plasma zone in the apparatus .

In preferred embodiments, the apparatus has two or more plasma zones having a directional plasma device, the plasma zones being arranged in series and each

directional plasma device uses a different carrier gas or carrier gas-additive mixture from the previous plasma zone along the polymer path. Fig. 4 shows an example of the apparatus having two plasma zones arranged in series. Two plasma torches of Fig. la are arranged in series along the polymer path. Each torch has a metallic wire filament 10, 210 (as the electrode) connected to a RF power supply 12, 212 via electronics 14, 214, a ceramic tube 16, 216, a quartz glass tube 18, 218 (as carrier gas conduits), an

electrode tip 20, 220 protruding from open ends of each ceramic tube 16, 216 and glass tube 18, 218 to produce plasma plumes 22, 222. The carrier gas/carrier gas- additive mixture in the first plasma torch is different from the carrier gas/carrier gas-additive mixture in the second plasma torch. In this way, the jet of polymer solution 106 passes through the plasma plume 22 of the first plasma torch to modify the surface with a first modification before passing through the plasma plume 222 of the second plasma torch to further modify the surface of the jet of polymer solution before reaching the polymer fibre collector 108. Note the figure is not to scale. The jet of polymer solution is not shown passing through the visible part of each plasma plume, but passes through the non-visible part.

As an alternative arrangement, the plasma-electrospinning apparatus having two plasma zones may have two

directional plasma devices opposing one another, with each directional plasma device producing a different plasma . An example of the two plasma-zones opposing each other is shown in Fig . 2. Electrospun Polymer Fibre

The electrospun polymer fibre resulting from the

apparatus of the present invention has a modified polymer surface from the plasma treatment. The electrospun polymer fibres may have an average diameter of 100 nm to 2 pm, preferably 250 to 1000 nm.

The electrospun polymer fibre may include:

(i) a polymer core and two or more plasma-coated coatings ;

(ii) a polymer core with a plasma-modified surface and at least one plasma-coated coating; or (iii) a polymer core and at least one plasma-coated coating with a plasma-modified outer surface. Such an electrospun polymer fibre may be generated from an apparatus of the present invention having two or more plasma zones in series, wherein the plasma is different in each zone. An example of such a fibre is shown in Fig. 5a. The fibre has a polymer core 302 throughout the length of the polymer fibre. A first plasma-coated coating 304 is deposited on the outer surface of the polymer core 302 by a first plasma zone. A second plasma-coated coating 306 is deposited on the outer surface of the first

plasma-coated coating by a second plasma zone.

The plasma-electrospinning apparatus described herein may also produce an electrospun polymer fibre having at least two plasma-treated sections, wherein the plasma-treated sections are different. Such a fibre may have a modified surface covering the polymer fibre surface that may not have been possibly to produce using conventional

techniques .

The plasma-treated sections may be different around the circumference of the polymer fibre or may be different along the length of the polymer fibre. The

plasma-treated section may have a plasma-modified surface of a polymer core. The plasma-treated section may have a plasma-coated coating (plasma-modified or unmodified) . The plasma coated coating may be a layer adjacent to a polymer core (plasma-modified or unmodified) . The plasma-coated coating may be a layer adjacent surrounding and adjacent to another plasma-coated coating

(plasma-modified or unmodified) . Any combination of plasma-modified surface or plasma-coated coating can be used, so long as the two plasma-treated sections have different cross-sections around at least part of the circumference . For example, two opposed plasma zones providing two different plasma treatments may produce a polymer fibre with a first plasma-treated section around one half of the circumference and a second plasma-treated section around the other half of the circumference. A further plasma zone positioned further along the polymer path may further plasma-modify or plasma-coat one or both of the plasma-treated sections.

An example of such a fibre is shown in Fig. 5b. The fibre has a polymer core 352 throughout the length of the polymer fibre. A first plasma-coated coating or plasma- modified surface 354 is produced on the upper half of the polymer core 352 outer surface. A second plasma-coated coating or plasma-modified surface 356 is produced on the lower half of the polymer core 352 outer surface. The first plasma-coated coating or plasma-modified surface 354 and the second plasma-coated coating or

plasma-modified surface 356 are differently

plasma-treated surfaces. As a result, a fibre having two distinct plasma-treated sections around the circumference of the polymer fibre is produced.

When the apparatus has a plasma torch or plasma

plume-generating plasma device with the capability to switch carrier gas/carrier gas-additive mixture during plasma treatment of the jet of polymer composition, an electrospun polymer fibre having at least two sections having different plasma-treated sections along its length may be produced. The polymer fibre collected at the beginning of the electrospinning process can have a different surface modification than at the end. The fibre can therefore have different surface-modified sections along its length.

An example of a fibre with at least two different plasma treatments along its length is shown in Fig. 6. The polymer fibre has a polymer core 402. The polymer core has a first plasma-treated section 404 where the polymer composition was treated by a first plasma from a

directional plasma device. The carrier gas or carrier gas-additive mixture producing the first plasma plume or plasma jet was switched to another carrier gas or carrier gas-additive mixture in order to produce a second

plasma-treated section 406 from the treatment with a second plasma from the same directional plasma device. Use of Electrospun Polymer Fibres and Meshes

A non-woven mat of electrospun fibre can be produced using electrospinning devices. The non-woven mat

containing electrospun polymer fibre produced from the apparatus described above can be used in many ways.

Pre-spun fibre meshes could be post-treated with a directional plasma such that a second

coating/modification of the surface is presented in specifically defined areas. This could be achieved either by manual or motorised control of the directional plasma device (such as plasma torch) and associated switching of plasma power, to achieve precision modification within each mesh layer (to and area resolution of ~1 square micron) . This process could be used with one or more ingressed chemicals simultaneously or subsequently.

Multi-layered Electrospun Polymer Fibre Meshes and

Laminates

An electrospun polymer fibre can be produced as an electrospun polymer mesh mounted on a subframe, for example using the composite polymer fibre collector as described herein. The present invention provides an electrospun polymer fibre mesh laminate comprising two or more layers comprising an electrospun polymer fibre mesh mounted on a subframe, wherein each electrospun polymer fibre mesh includes electrospun polymer fibre having a different plasma-surface modification and/or plasma- coated coating.

Each electrospun polymer fibre mesh may be aligned or randomly oriented. Preferably at least one, more

preferably two or more of the meshes have aligned fibres. The subframes may be stacked to produce the laminate. Each frame may be aligned with respect to the orientation of an adjacent subframe. For example, adjacent layers may each have polymer fibres aligned (within each mesh) approximately in parallel. The meshes may then be aligned so that the electrospun polymer fibres in one mesh are arranged in parallel (0°), perpendicular (90°) or any other angle in between 0° and 90° (such as 45°) to the electrospun polymer fibres of the adjacent layer. As a result a multi-layered electrospun polymer fibre mesh laminate is produced.

An alternative way to form a multi-layered electrospun polymer mesh is to collect a first electrospun polymer fibre layer on a polymer fibre collector and then deposit a second electrospun polymer fibre layer on a surface of the first electrospun polymer fibre layer. Optionally a third and further electrospun polymer fibre layers may be deposited on a surface of the second and subsequent electrospun mesh layers. In this method of producing a multi-layered electrospun polymer fibre mesh, the

collector is the same for each polymer fibre layer. As a result, any alignment of the polymer fibre within each layer will be the same for all layers. For example, each layer may have polymer fibre aligned parallel to the polymer fibre in the same layer. Additionally, the polymer fibre in each layer will be aligned parallel to the polymer fibre in the adjacent layer.

In either of the methods of producing a multi-layered electrospun polymer fibre mesh, preferably at least one of the electrospun polymer layers has an electrospun polymer fibre that is different from an electrospun polymer fibre in an adjacent electrospun polymer layer. The difference in the electrospun polymer fibre may be the polymer type. The difference may be that a polymer fibre in one layer is plasma-treated and the polymer fibre in the other layer is not plasma-treated.

As a result of either method, herein is provided a multi-layered electrospun polymer fibre mesh having two or more layers of electrospun polymer fibre, wherein at least one layer has plasma-treated electrospun polymer fibre .

Preferably the multi-layered electrospun polymer fibre mesh has two or more plasma-treated electrospun polymer fibre layers with plasma-treated electrospun polymer fibre, wherein each plasma-treated electrospun polymer fibre is differently plasma-treated to the other plasma- treated electrospun polymer fibres. In some embodiments, the differently plasma-treated electrospun polymer fibres are in adjacent plasma-treated electrospun polymer fibre layer .

Use in Biological and Medical Field

A non-woven mat of electrospun fibre produced from the above apparatus may form a biological scaffold. In particular, cell adhesion to the electrospun polymer fibres can be tailored by plasma treatment from the plasma-electrospinning apparatus of the present

invention .

Directional Plasma Device Kit

The present invention provides a kit comprising a directional plasma device, such as a plasma torch or a plasma-plume generating device, and a directional plasma device attachment for fitting the plasma torch to an electrospinning device so that, during operation, a polymer composition from a polymer electrospinning device passes through a plasma jet or plasma plume from the directional plasma device.

The kit allows a standard electrospinning device to be adapted to become the plasma-electrospinning apparatus described herein.

The directional plasma device attachment may be any fitting to fix the orientation of the plasma device to the electrospinning device so that a polymer composition from a polymer electrospinning device passes through a plasma jet or plasma plume from the directional plasma device. The directional plasma device attachment may have device fixing part for attaching the directional plasma device to the attachment and a rotatable joint connected to the device fixing part. The rotatable joint allows the directional plasma device can be angled to direct the plasma plume or plasma jet towards the jet of polymer composition from the electrospinning device, when in use. An example of such a directional plasma device attachment is a goniometer.

The directional plasma device attachment may be a

directional plasma device stand, such as a specimen stand, arranged to hold the directional plasma device in the desired orientation

The present invention also provides a kit with

directional plasma device, such as a plasma torch or a plasma-plume generating device, and a composite polymer fibre collector having a collecting frame with an

electrically conducting element, the electrically

conducting element being electrically connected to an electrically chargeable part.

The preferred features of the composite polymer fibre collector described herein apply equally to the kit including the composite polymer fibre collector.

The preferred features of described above apply equally to all aspects of the present invention. In particular, the preferences of the directional plasma device, such as the plasma torch, of the first aspect apply to any directional plasma device of the apparatus of the second aspect . EXAMPLES

Materials and Methods

Poly lactic acid (PLA) (Purac) , N, N-dimethylformamide (DMF), dichloromethane (DCM) were purchased from Sigma, and concentrated ammonia solution and methanol were purchased from Fisher Scientific. For cell culture work neurobasal media, B27 and bFGF were purchased from Gibco, penicillin streptomycin fungizone, L glutamine and 30% glucose solution were purchased from Sigma. All chemicals were used as received.

Electrospinning

A 2% solution of PLA was in DMF/DCM (3:7) was loaded into a syringe fitted with a metallic 18 gauge (internal diameter 0.8mm) needle tip. A potential was applied across the tip to a vertical flat copper collector plate separated by 10-15 cm, using a high voltage power supply. The needle tip was positively charged at 6.5 kV with the collector plate held at -6.5 kV. Using a syringe pump flow the polymer solution was moved at 20-25 microlitres per minute. Humidly and temperature were kept relatively constant at 52% and 23°C. Fibres were either collected on aluminium foil pulled taught around the copper target plate, or onto 18 by 18 mm glass coverslips attached onto the foil. Aligned nanofibres were collected using a composite collector. Atmospheric Pressure Plasma

Two set-ups were used to associate the plasma with the electrospinning fibres (shown in Figs. 3a and 3b) .

Geometries of the plasma and spinning fibres were such that fibres were modified by the plasma on leaving the needle tip and before collection. In both cases a 13.56 MHz power supply (Kurt J Lesker-601) was used to drive an RF potential at 10W. Automatic matching (CG-3000) was achieved using a matching network and induction coil situated as close to the plasma plume as possible.

Set-up A (Fig. 3a) : RF was applied to a sharpened

tungsten filament situated coaxially inside a ceramic and then glass tube. Sheath and carrier gases were flown down the inside of the ceramic and glass tubes, respectively.

Helium was used in both cases with the carrier gas being pre-flown through a chamber housing solutions of ammonia, methanol or silane, giving rise to chemical modification of fibres. Gas flow meters were situated in-line such that flow rates could be monitored at -0.8 Lmin ^-1 .

Set-up B (Fig. 3b) : The 18 gauge needle tip of the electrospinning apparatus was housed coaxially within glass tube. Around the outside of the tube a coil of copper wire was driven with an RF potential. Carrier was flown down the inside of the glass tube. Collected polymer fibre samples were placed in a vacuum chamber for -30 mins post-collection to remove any remaining solvent, then stored in sealed petri dishes prior to analysis/cell culture.

Electrospun polymer fibre mesh and laminates

An electrospun polymer fibre mesh was produced using an electrospinning device with a composite collector. The composite collector has a combination of static copper plate and a detachable (mobile) collector.

A solution of PLA and rhodamine ( TRICH/rhodamine B) was prepared to produce 2% solution of PLA with 0.1 mg/ml of rhodamine in DMF/DCM (3:7). A solution of PLA and FITC was prepared to produce 2% solution of PLA with O.lmg/ml FITC in DMF/DCM (3:7). lmg rhodamine or FTIC is dissolved in 7 ml of chloroform, then 0.2 g PLA is added for overnight stirring and subsequent mixing of 3mL DMF for 4 hours, which makes an end concentration of O.lmg/mL of each dye. The solutions were electrospun alternatively and collected by a parallel electrode (composite

collector) described below.

To produce aligned nanofiber meshes, the composite collector is made from an adjustable frame (30 x 10 cm) and 2 sheets of thin steel "blades" arranged parallel to each other. The blade distance is set to 4-6 cm. The frame is then insulated using polyvinyl chloride (PVC) insulating tape, leaving only the blades exposed. The fibres are collected over a width of approximately 10 cm. The collector is electrically connected to the copper plate. The distance between the electrospinning needle tip and the mobile collector is 15-22 cm; the distance between the mobile collector and the static copper plate is 10 cm.

To produce random nanofiber meshes, the composite

collector is made from a steel ring with diameter of 3-10 cm. The collector is electrically connected to the copper plate. Deposition to the frame was homogeneous over a width of approximately 15 cm.

The produced low density nanofibers meshes are fragile and cannot be handled without support . To generate handleable nanofiber meshes, the aligned or random nanofiber meshes are removed from the mobile collector using cellulose acetate subframes which have a hollow area of 16 cm ² . The frames are sprayed with an aerosol adhesive and catch the nanofiber meshes on the mobile collector. The frames are removed using forceps and excess fibres are severed from the frames using a scalpel and blade. Once adhered, the nanofibers on the frames could be removed with the alignment remaining. Thus, the low density nanofiber meshes become portable once

transferring to the cellulose acetate frame. A laminate was made from two electrospun polymer fibre meshes with PLA/rhodamine fibres aligned in parallel and an electrospun polymer fibre mesh with PLA/FITC fibres aligned in parallel. The meshes were stacked so that the fibres in the PLA/rhodamine layers were aligned parallel with the fibres in the PLA/FITC layer. The resulting three layered constructs are imaged by Z-stacked images with 5 pm step. Z-stacked 3D CLSM images in each layer were reconstructed.

A multi-layered electrospun polymer mesh was produced in which a layer of red (rhodamine) PLA was electrospun onto the composite collector described above first, then a layer of green (FITC) PLA electrospun on the layer of red PLA, then another layer of red PLA electrospun on the layer of green (FITC) PLA. Representative Z-stacked images show that the three-layered constructs were fabricated having all fibres aligned in the same

direction. The red fluorescence images of top and bottom layers and the green fluorescence image of the middle layer corresponded accurately to the fabrication

processing in which red PLA was e-spun first, then green PLA e-spun on the layer, then red PLA e-spun, resulted in three layered two colour 3D construct.

Neural cell culture

Primary rat (E12) cortical cells were dissected and grown as neurospheres for 10 days in neurobasal media containing 1% B27, 0.04% bFGF, 1% penicillin streptomycin fungizone, 0.25% L-glutamine and a final concentration of 0.225% glucose at 37°C and 5% C0 ₂ . After this time

neurospheres were collected by centrifugation at 700 RPM for 3 minutes. Supernatant was removed and concentrated neurospheres were seeded by pipette onto nanofibres mesh surfaces housed in 6 well plates. After 1 hour, media was added containing 10% FCS and 1% B27 in Dulbecco ' s

Modified Eagle Medium (DMEM) . Images were taken at 0, 2 and 5 days post seeding.

MG63 Culture

The human osteoblast cell line MG63, was used, with cells cultured in medium containing 10% DMEM, 1% A&A, 1%

L-glutamine in culture flasks, placed in a humidified incubator at 37°C under a 5% CO ₂ atmosphere. 10 ⁴ cells were seeded on each nanofibre scaffold which was mounted on glass coverslips. There are two types of nanofibre on the coverslip, pure aligned PLA nanofiber and NH ₄ OH plasma simultaneous treated aligned PLA nanofibre. The

nanofibres were steriled by placing on the UV chamber (BIO-RAD, GS Gene Linker) at three times for 90 seconds each time, then placed on the six-well plate

respectively. Cell morphology was photographed at

different time points after seeding. Character! zation of surface modification

Contact angle

Droplets of ~5 microlitres deionised water were added via syringe to horizontal nanofibres mesh surfaces. Drop shape images were immediately captured using a backlight to increase droplet edge contrast, using ImageJ software to measure the contact angles.

FTIR analysis

A Bruker Alpha FTIR spectrometer was used for all

experiments, fitted either with a single bounce diamond ATR or DRIFT accessory. For all samples an air background was taken as reference. An average of 128 scans were collected for all samples at a resolution of 4 cm ^-1 .

Spectra were recorded within 12 hours of fibre production with other spectra taken at varying further time points.

Scanning confocal microscopy

Confocal laser scanning microscope (CLSM; Olympus FV300) has been used to demonstrate how the surface treated nanofibers are assembled into multiple layered constructs in situ with defined chemical modification within each layer. Images were recorded on an Olympus Fluoview fitted onto an Olympus 1X71 inverted microscope. Incremental z- steps were imaged using a step spacing of 5 microns. Scanning electron microscopy Energy Dispersive X-Ray Analysis (EDXA)

A benchtop SEM (Hitachi TM3000) was used to collect high resolution images of fibres with and without surface plasma treatment; and before and after post-modification.

The electrospun with Si-Cl plasma-treated nanofiber is placed in a SEM sample stub with sticky carbon tape on the surface. The SEM image and associated elements can be detected simultaneously on the area of interest. Elements including C, 0, Si, CI, Ca, Na are detected and distinct element maps are formed.

XPS Analysis

XPS analysis was performed using a Theta Probe instrument equipped with a monochromated AlKa source (Thermo

Scientific) in a National EPSRC XPS User's Service

(NEXUS) laboratory at Newcastle University. A pass energy of 200 eV and a step size of 1.0 eV was employed for all survey spectra while a pass energy of 40 eV and a step size of 0.1 eV was used for high resolution spectra of the elements of interest. A flood gun was used for charge compensation .

Results

Fabrication of Nano-fibres

Nano-fibres were electro-spun both with and without in situ plasma modification showing no beading morphology (see Fig. 7) . Fibres were collected having both random (Fig. 7a) and aligned (Fig. 7b) orientations. Fibre diameters were found in the range 1-3 microns in both cases . Electrospinning and plasma modification

Electrospinning was established with in situ plasma treatment. Characterisation of materials spun with no plasma treatment showed differing characteristics to those with treatment as expected.

Contact angle measurement show a change in wettability of materials with respect to helium only plasma treatment, or with ammonia solution/ methanol ingressed into the helium carrier gas (see Figs. 8 and 9) .

FTIR Character1station

FTIR characterization of untreated PLA fibres shows all the expected absorbance bands for C-H stretch

( 2995-2944crrT ¹ ) , C=0 stretch ( ~ 1760cm ^"1 ) , C=0 bend

(~1270crrT ¹ ) , C-H bend (1383, 1363cm ^"1 ), C-0 stretch (1185,

1130, 191 cm ^"1 ), O-H bend (-1050cm ^"1 ) and C-C stretch (870cm ^"1 ), Figure 10 (dotted line) . After treatment with helium/ ammonia plasma and during simultaneous deposition and modification, absorbance bands attributed to amine functionality were observed (Figure 10, solid line) .

These bands appeared at -3670, 3525 cm ^"1 N-H stretch, as well as further bands appearing in the lower fingerprint region below 1000 cm ^"1 . Samples were reanalysed after 5 months stored at room temperature, showing by DRIFT FTIR that amine groups still reside on the surface of the fibres, Figure 11.

Plasma power was briefly investigated to understand if surface concentrations of amines could be altered by increasing the power, i.e. by inducing greater number of excited species able to react with the fibre surface. Comparison was made between ammonia treated fibres using

10 and 15W plasmas at the same gas flow rates and degree of ingressed ammonia. Examination of the amine stretching bands indicated that at higher plasma powers larger peaks were observed, Figure 12.

Multiple Layered Constructs

Confocal microscopy of electrospun fibre constructs was carried out to demonstrate the possibility of presenting plasma-e-spun fibres in 3D layered constructs with spatial resolved chemical groups. Rhodamine (red) and

FITC (green) dyes (representative of two different surface treatments) were added into separate PLA

solutions prior to spinning. In one laminate, multi-layered constructs were fabricated having all fibres aligned in the same direction. The separate layers of the multiple layered constructs are shown in Figure 13A. The electrospun polymer fibres with FITC form a layer and the image shows green polymer fibres aligned from left to right across the image

(left-hand image in Fig. 13A) . The electrospun polymer fibres with rhodamine form another layer and the image shows red polymer fibres aligned from left to right across the image (right-hand image in Fig. 13A) . The two layers are distinct (no green fibres in the red fibre layer and vice versa) , and the polymer fibres in red fibre layer are aligned approximately parallel to the polymer fibres in the green fibre layer.

Multi-layered constructs were fabricated having

sequential layers presenting fibres oriented in differing directions, Figure 10b. Distinct layers can be seen in confocal stack images

In Fig. 13B, multi-layered constructs were fabricated having sequential layers of PLA/FTIC polymer fibres (left-hand image in Fig. 13B) and PLA/rhodamine polymer fibres (right-hand image in Fig. 13B) presenting fibres oriented in differing directions, Figure 10b. Distinct layers can be seen in confocal stack images.

SEM and EDXA

Electron microscopy with EDXA was carried out to further confirm the presence of functional coatings on fibres. Silicon coatings were used as these can be clearly seen by EDXA. Trichloromethylsilane was infused into helium carrier gas during plasma-e-spinning . SEM image in Figure 14 clearly shows fibre structure, with an overlaid image of EDX mapping across a fibre. Separate elemental maps are included showing the presence of carbon (top row, left) and oxygen (top row, middle) of the polymer backbone, and silicon (top row, right) and chlorine (bottom row, left) from silane.

Neurosphere adhesion

Cell culture was carried out on non-treated (control) fibre meshes and amine coated plasma modified fibre meshes. Neurospheres were seeded onto the mesh layers with adequate media to cover them during incubation.

Brightfield microscopy was used to visualise the cells during culture 2 and 5 days after initial seeding,

Figure 15. Cells attached to both substrates, spreading to form cell mats. Those cultured on amine-coated fibres showed greater alignment to fibre direction and less rounded state. Amine functionalised fibre mats showed greater ability to allow cells to spread, forming more even cellular mats as compared to controls at the same time period.

Figure 16 shows Brightfield microscopy images of neural stem cell culture on untreated and amine-plasma e-spun PLA nanofibres. The images in Figure 16 are of the same sample as in Fig. 15, but show a different sample area. Neurospheres are grown in suspension and do not differentiate until adhered to a substrate surface. Amine treatment shows a higher degree of attachment, with neurospheres readily attaching to the fibres and cells migrating from the neurosphere body. By 5 days in culture cell populations on amine treated surfaces are much greater in number compared to those on non-treated fibres. The neurospheres, and spreading cells align well to fibre direction, with the increased capacity for attachment on amine treated fibres enhancing the

alignment of cells.

MG63 Cell Growth

Figure 17 shows optical images of MG63 cells growing on untreated and amine-plasma e-spun PLA nanofibres. The images show cells are slightly rounded on untreated fibres compared to those spread on amine treated fibres. This indicated improved adhesion of cells, allowing greater surface interaction and therefore enhanced proliferation. This is evidenced by the increase in cell number on amine fibres by day 11. The spreading cells align well to fibre direction, with the increased

capacity for attachment on amine treated fibres enhancing the alignment of cells.

XPS Data

Figures 18A, B and C show spectra of non-treated PLA (top row) , simultaneously 15W plasma e-spun amine treated PLA fibre (i.e. a PLA fibre plasma-treated with ammonia ingressed into the carrier gas) (centre row) and overlaid spectra (bottom row) . Figure 18A shows CIS scans, Figure 18B shows Nls scans, and Figure 18C show 01s scans. Each spectrum shows binding energy (x-axis; eV) against CPS ( y-axis ) .

On plasma treating, the Nls peak becomes present, showing surface amine coating on the fibres. Change in the Cls and 01s spectra also show slight oxidation of the fibres.