WO2014155102A1 - Method and apparatus for producing polymeric structures - Google Patents

Abstract

The present invention relates to an apparatus and a method for producing polymeric structures such as microfibres, nanofibres and microbubbles. The method involves simultaneously subjecting a polymer solution to at least two of the following conditions: rotation, pressure and a voltage. The apparatus of the invention typically includes a cylindrical vessel having holes extending therethrough, and further comprises at least two of (i) rotation means, (ii) pressure exertion means and (iii) an electrical source. Advantageously, the method and apparatus of the invention allow for the production of high yields of polymeric structures at ambient temperature without a requirement for extrusion nozzles or complex spinnerets.

Description

Method and apparatus for producing polymeric structures

Polymer nanofibres are a promising class of materials for various applications, including electronics, optical devices, batteries and filtration. Indeed, due to their high surface area to volume ratio, they are attractive in many biomedical applications such as scaffolds used in tissue engineering, drug release, artificial organs, wound healing and vascular grafts. Due to the expanding demand for nanofibres across a wide range of industries, there needs to be an improvement in the current state-of-art technologies to mass produce them more consistently, reliably, robustly and cost effectively.

Electrospinning is a well-established technique to generate a wide variety of polymeric fibres across the micro to nano scale range (H.B. Zhang, M.J. Edirisinghe, J. Am. Ceram. Soc. 2006, 89, 1870). However, this method requires high voltage (kV range) and shows poor cost-yield efficiency as a single fibre emerges from the end of the nozzle carrying a polymeric solution. Centrifugal spinning has the ability to produce homogenous nanofibres from poorly electrospinnable materials. It uses the centrifugal force in a rotary mould to shear polymer solution to form fibres (L. Wang, J. Shi, L. Liu, E. Secret, and Y. Chen, Microelectron. Eng. 201 1 , 88, 1718). However, the process is limited by complicated spinneret design which can lead to large differences in fibre quality and productivity. Solution/melt blowing is a proven large scale method to form a web of polymeric fibres. It involves extruding polymer solution/melt through a narrow orifice under high air velocity. The drag of high velocity air on the surface of the polymer melt causes the polymer to elongate into fibres. However, it is only possible to make fibres having a diameter in the micrometer range using this method (E.S. Medeiros, G.M. Glenn, A.P. Klamczynski, W.J. Orts, J. Appl. Polym. Sci. 2009, 1 13, 2322).

Microbubbles are an interesting and promising class of materials consisting of a spherical core- shell structure. Generally, the core consists essentially of a gas or a mixture of gases (e.g air, C0₂ or N₂) encapsulated in a shell and the shell typically comprises polymer, protein, lipid and may comprise a polymer, protein mixture. Microbubbles provide a unique platform for various applications. They have been used in diagnostics, as ultrasound contrast agents because they are gas-filled and smaller than the wavelength of diagnostic ultrasound (Dayton, P.; Klibanov, A.; Brandenburger, G.; Ferrara, K. Acoustic radiation force in vivo: A mechanism to assist targeting of microbubbles. Ultrasound Med. Biol. 1999, 25 (8), 1 195-1201). These properties mean that microbubbles are useful entities in focused ultrasound imaging and site-targeted molecular events in vivo such as inflammation, angiogenesis and tumour formation can be assessed. Microbubbles also have enormous potential in therapeutic use and have an ability to deliver genes. This could be either DNA co-administered with clinical ultrasound exposure or by a purpose built bubble system loaded with genes. Also active pharmaceutical ingredient drug-loaded nanoparticles contained within the shell of the microbubbles combined with ultrasound, magnetic field, light, heat, pH differences or redox potential differences enable different stimuli-responsive controlled drug delivery at specific sites. In the food industry, surfactant-stabilised microbubbles are used for protein recovery. They have been shown to improve the desired properties in food systems, including texture, digestibility, and flavour intensity. State-of-the-art techniques to prepare microbubbles include conventional methods and newer technological routes (Stride, E.; Edirisinghe, M. Novel microbubble preparation technologies. Soft. Matter. 2008, 4 (12), 2350-2359). Sonication and high shear emulsification are well known conventional methods which can offer high yield and low production cost but possess poor control over the microbubble size and uniformity. The former involves dispersing gas or liquid in a suspension of a suitable coating material using agitation or shaking or similar means. The latter requires a high shear stirring of aqueous suspension consisting of immiscible liquid and polymer. Microfluidic devices enable a higher degree of control over size and polydispersivity of

microbubbles, however, they operate under very limited pressure and flow rate conditions

(Whitesides, G. The origins and future of microfluidics. Nature, 2006, 442 (7101), 368-373). Coaxial electrohydrodynamic atomisation (CEHDA) is a well-established technique, evolved from conventional electrohydrodynamic atomisation where two flowing media subject to a high voltage generate coaxial jetting and subsequently break up to form bubbles (Farook, U.; Stride, E.;

Edirisinghe, M. Preparation of suspensions of phospholipid-coated microbubbles by coaxial electrohydrodynamic atomization. J. R. Soc. Interface 2009, 6 (32), 271 -277). Microbubbles prepared using this robust and adaptable technique depend on fluid properties and processing parameters such as flow rate and applied voltage. Unlike sonication and microfluidics methods it gives a high production rate of bubbles and also has the capability to produce near-monodispersed microbubbles. However, this method requires high voltage (kV range) and it might not be suitable for certain health-care oriented applications.

There is therefore a need in the art for an improved method and apparatus for preparing polymeric structures such as micofibres, nanofibres and microbubbles.

In a first aspect, the invention provides an apparatus comprising:

a vessel having a generally circular cross section and comprising a side wall, the side wall having at least one hole extending therethrough, and at least two of the following:

(i) rotation means configured to rotate the vessel;

(ii) pressure exertion means configured to exert pressure within the vessel; and

(iii) an electrical source configured to apply a voltage to the vessel.

Accordingly, the apparatus of the invention may comprise features (i) and (ii), (i) and (iii), (ii) and (iii) or (i), (ii) and (iii) as defined above. Preferably, the apparatus is an apparatus for the preparation of a polymeric structure. The apparatus of the invention provides a simple and cost effective means to produce high yields (relative to known techniques) of a desired polymeric structure such as a microfibre, a nanofibre or a microbubble, and also allows for physical dimensions of the polymeric structure to be tailored to suit the intended purpose. Advantageously, the apparatus does not require extrusion nozzles that are required in known melt blowing techniques and further does not require the complicated spinneret design required in known centrifugal spinning techniques.

The term "microfibres" used herein means fibres formed from polymeric material, the fibres having a diameter or an average diameter of from about 1 μηι to about 10ΟΟμηι. The term "nanofibres" used herein means fibres formed from polymeric material, the fibres having a diameter or an average diameter of from about 1 nm to about 1000nm. The term "microbubbles" means bubbles having a diameter or average diameter of about 1 μηι to about 10ΟΟμηι. The diameter of nanofibres can be measured using scanning electron microscopy, for example field emission scanning electron microscopy. The diameter of microbubbles can be measured using optical microscopy.

The vessel has a generally circular cross section, typically a circular cross section. Since the vessel is generally circular in cross section it has a single continuous side wall. The vessel serves as a container (for a polymer solution) and/or a reactor vessel (for polymerisation reactions). The vessel preferably has a base, such that it can contain a polymer solution, and, in some embodiments comprises a lid, which may seal the vessel. In one embodiment, the vessel is generally cylindrical in shape. The vessel may alternatively be generally conical in shape. The vessel can be made from any suitable material such as for example, aluminium, stainless steel, titanium. The volume of the vessel can be chosen according to the volume of polymer solution to be contained, which in turn may depend, at least in part, on the scale of production required.

The side wall of the vessel has at least one hole, extending through the side wall (referred to herein as a "through hole"). These may take the form of simple "punctures" through the side wall i.e. they may be substantially flush with the inner and outer surfaces of the side wall. The side wall of the vessel may have a plurality of through holes in its side wall, for example at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 100 through holes or more. The through holes can be distributed around the side wall in any configuration. In one embodiment, the through holes are evenly distributed over the surface of the side walls, i.e. the through holes may be equidistant from one another. The through holes may be positioned in one or more

circumferential, preferably parallel lines around the vessel. If there is more than one

circumferential line of through holes around the vessel, the through holes of each line may be in vertical alignment with through holes of the one or more additional lines. The number and/or distribution of through holes may be determined based on the size of the vessel. For example, a vessel having a diameter of approximately 60mm and a height of approximately 35mm may have about 20 through holes. The one or more through holes allow jets of polymer solution that have been accelerated by forces exerted on the vessel, e.g. rotation, pressure and/or electricity, to escape the vessel and form polymeric structures such as microfibres, nanofibres and

microbubbles. Advantageously, nozzles, i.e. devices to control the direction and/or characteristics of fluid flow, are not required in order to produce polymeric structures using the apparatus of the invention. However, in certain embodiments, the vessel may comprise a nozzle extending outwardly from some or all of the through holes.

Typically, the holes are generally circular but may be of other shapes in order to produce polymeric structures of different shapes, e.g. ribbed fibres. The one or more through holes preferably have a diameter of from about 0.1 mm to about 5 mm, for example from about 0.2 mm to about 3 mm, from about 0.3 mm to about 2 mm, from about 0.4 mm to about 1 mm, about 0.5 mm to about 0.9 mm, or about 0.6 mm to about 0.8 mm. In one embodiment, the one or more through holes have a diameter of about 0.5mm. The rotation means may be any suitable means for rotating the vessel. Preferably, the rotation means is capable of rotating the vessel at a rotational speed of at least about l OOOrpm. The rotation means may be capable of rotating the vessel at a speed of at least about 3000rpm, at least about 5000rpm, at least about 7000rpm, at least about l OOOOrpm, at least about 24000rpm, at least about 36000rpm or at least about 72000rpm. In one embodiment, the rotation means comprises a motor, for example a direct current (DC) motor. The rotation means may be connected to the base of the vessel but equally may be connected in any suitable configuration that will cause rotation of the vessel.

The apparatus may comprise pressure exertion means configured to exert pressure within the vessel. The pressure exertion means may comprise a pressurised gas source. The apparatus may comprise a pressurised gas inlet providing a passage between the gas source and the interior of the vessel. The gas may be an inert gas. The gas may comprise or consist essentially of air, C0₂ or N₂. It is preferred if the pressure exertion means is capable of exerting a pressure of at least about 50 kPa, 60 kPa, 70 kPa, 80 kPa, 90 kPa, 100 kPa, 200 kPa, 300 kPa, 400 kPa, 500 kPa, 600 kPa, 700 kPa, 800 kPa, 900 kPa, or 1000 kPa or greater.

The apparatus may comprise an electrical source configured to apply a voltage to the vessel. Any known electrical source can be used, and may for example be connected to the means for rotating the vessel. The voltage may be a high voltage, for example in the kilovolt (kV) range. The voltage may be at least about 1 kV, at least about 5kV, at least about 10kV, at least about 15kV or at least 20kV.

Although the apparatus of the invention enables the production of polymeric structures at ambient temperature, he apparatus may comprise heating means configured to heat the vessel and/or a polymer solution that is to be fed into the vessel or that is contained in the vessel. For example, the heating means may be configured to heat a polymer solution before or as it is fed into the vessel. Preferably, the heating means can heat the polymer solution to a temperature of at least about 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, 150°C, 200°C, 250°C, 300°C, 350°C, 400°C, 450°C, 500°C or greater.

It is preferred that the apparatus comprises a collector that at least partially surrounds the vessel. The function of such a collector is to collect polymeric structures that are formed as polymer solution is ejected from or propelled out of the vessel via the one or more through holes. Therefore, it is preferred if the collector surrounds all of the through holes such that any material ejected through the holes is captured by the collector. In one embodiment, the collector surrounds substantially the entire side wall of the vessel. The collector may be in contact with the vessel and may be formed as part of the vessel, or may be a separate structure. The collector may be rotatable, and may, for example be rotatable at the same rate as the vessel. A rotatable collector advantageously allows the collection of polymeric structures, such as nanofibres that are aligned in the same direction. The collector may be made from any suitable material, such as aluminimum, for example aluminium foil. The collector may be adapted to receive and/or store polymeric material produced using the apparatus of the invention.

The vessel may include a polymer solution inlet configured to supply the polymer solution and, in some embodiments, additional additives to the vessel.

In a second aspect, the invention provides a method of forming a polymeric structure, the method comprising propelling a polymer solution through at least one through hole in a side wall of a vessel containing the polymer solution by simultaneously subjecting the polymer solution to at least two of the following conditions:

(i) rotation

(ii) a pressure

(iii) a voltage.

Accordingly, in the method of the second aspect, the polymer solution is simultaneously subjected to conditions (i) and (ii), (i) and (iii), (ii) and (iii) or (i), (ii) and (iii) as defined above.

In a preferred embodiment, the polymer solution is subjected to rotation and pressure as defined in (i) and (ii) above, and this embodiment is referred to herein as "pressurised gyration". Although a voltage can be applied, this is not essential. Therefore, the method advantageously provides a means to produce polymeric structures such as microfibres, nanofibres and microbubbles without the need for the use of high voltages. Moreover, the method does not require extrusion nozzles that are required in known melt blowing techniques and further does not require the complicated spinneret design required in known centrifugal spinning techniques. A particular advantage of the method of the invention is that it allows physical parameters of the polymeric structure to be controlled by varying the rotational speed and/or pressure. The method of the invention can also be carried out at ambient temperature, e.g. at room temperature (about 20°C-25°C). No external heating is required, although in some embodiments heat may be applied.

The polymer solution may be subjected to a rotational speed of at least about 10OOrpm, at least about 3000rpm, at least about 5000rpm, at least about 7000rpm, at least about l OOOOrpm, at least about 24000rpm, at least about 36000rpm or at least about 72000rpm, preferably, by rotating the vessel at the desired speed.

The pressure to which the polymer solution is subjected is preferably greater than atmospheric pressure and may be at least about 50 kPa, 60 kPa, 70 kPa, 80 kPa, 90 kPa 100 kPa, 200 kPa, 300 kPa, 400 kPa, 500 kPa, 600 kPa, 700 kPa, 800 kPa, 900 kPa, or 1000 kPa or greater.

Pressure may applied by supplying pressurised gas to the vessel. The gas may be an inert gas. The gas may comprise or consist essentially of air, C0₂ or N₂.

The voltage may be at least about 1 kV, at least about 5kV, at least about 10kV, at least about 15kV or at least about 20kV. The skilled person will be able to determine how long to continue carrying out the method of the invention in order to produce the desired polymeric structures, for example by observing the formation of the polymeric structures and/or the amount of polymer solution remaining in the vessel. The method comprises propelling a polymer solution through at least one through hole in a side wall of a vessel containing the polymer solution. The vessel may be a vessel as defined in relation to the first aspect of the invention. By "propelling a polymer solution" is meant forcing a polymer solution through the at least one through hole in a side wall of a vessel containing the polymer solution.

A wide range of polymer solutions can be used in the method of the invention. The polymer solution typically comprises at least one polymer (and may comprise two or more different polymers) and at least one solvent. Polymer solutions useful in the invention may comprise lipids, e.g. phospholipids and/or sphingolipids. The polymer may be fully dissolved or partially dissolved in the solvent. The polymer itself may be pre-formed or may be formed from monomers in situ i.e. in the vessel, by any suitable polymerisation method such as, for example, addition, condensation, free radical polymerization, photopolymerisation, cationic polymerization, anionic polymerization or coordination polymerization. Thus, the polymer solution may comprise one or more additional reactants to provide a suitable reaction environment to facilitate polymerisation, and the method may include a step of polymerisation prior to the step of simultaneously subjecting the polymer solution to the conditions defined above.

Suitable polymers for use in the invention include synthetic polymers, naturally occurring polymers and chemically modified naturally occurring polymers, e.g. PEGylated proteins. The polymer may be a condensation polymer such as a polyamide, a polyacetal or a polyester. The polymer may be branched or unbranched. Preferably, the polymer is not crosslinked. The polymer may be a thermoplastic polymer or a thermoset polymer. Naturally occurring polymers include proteins, polypeptides, polynucleotides, and polysaccharides e.g. chitosan, starch and cellulose. Synthetic polymers include polyethylene, polypropylene, polyethylene oxide (PEO), poly (vinyl) alcohol (PVA), polystyrene, and poly (N-vinylpyrrolidone) (PVP), polyacrylonitrile, polyethylene terephthalate, nylon 6, nylon 6,6 and polycaprolactone. Other polymers useful in the invention include polymeric precursors, such as those that can be converted to other materials (e.g.

ceramics), for example polymethylsilsesquioxane (PMSQ) which can be cured to give silicon oxycarbide (SiOC). . In one embodiment, the polymer solution comprises PVA and lysozyme. In another embodiment, the polymer solution comprises starch and PEO.

Generally, polymers useful in the invention have a molecular weight in in the range of about 300 g/mol to about 300,000 g/mol, for example, from about 500 g/mol to about 250,000 g/mol, about 1000 g/mol, to about 200,000 g/mol, from about 5000 g/mol to about 150,000 g/mol, from about 10,000 g/mol to about 100,000 g/mol, or from about 25,000 g/mol to about 50,000 g/mol.

The solvent will be chosen according to the polymer used. Generally, any suitable solvent may be used i.e. polar or non-polar solvents. In certain embodiments, solvents include water, alcohol, dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS), dimethylformamide (DMF), chloroform, acetic acid, formic acid and tetrahydrofuran.

The concentration of polymer in the solution may be from about 1wt% to about 40wt%, for example, at least about 5wt%, at least about 10wt%, at least about 15wt%, at least about 20wt%, at least about 25% or at least about 30wt%.

The polymer solution may comprise one or more additives. Additives include proteins, active pharmaceutical ingredients, drugs, bioactive ingredients, metallic nanoparticles and ceramic particles. The morphologies of the products generated by the method of the invention can be influenced by carefully controlling the concentration or amount of such additives.

The polymer solution may have a viscosity of about 300 to about 3000 mPa.

The method of the second aspect of the invention is a method of forming polymeric structures. Such structures include, for example, polymeric fibres such as microfibres and nanofibres, and microbubbles, capsules, particles and porous particles. The method of the invention can be used to produce both beaded fibres i.e. polymeric fibres having small nodules along their length ("beads on a string" arrangement), and bead-free fibres. In particular, when the method is used to produce polymeric fibres, increasing the rotational speed decreases the diameter of the polymeric fibre. Increasing the pressure also has the effect of decreasing the diameter of the polymeric fibre. Thus, by selecting an appropriate rotational speed and/or pressure, polymeric microfibers or nanofibres can be obtained. It has also been found by the inventors that increasing the pressure reduces the distribution of fibre diameters produced. Varying the polymer concentration can also be used to vary the diameter of the fibres produced. In general, the higher the concentration of polymer solution used, the greater the diameter of fibre that is produced. This is due to the differences in viscosity of the polymer solutions and the rate of evaporation of solvent during processing. However, the difference in diameter achieved at different polymer concentrations is less pronounced at higher working pressures. Beaded fibres can be obtained by using lower rotational speeds and/or pressures. Bead-free fibres can be obtained by increasing the speed of rotation and/or the pressure.

In some embodiments, the method can be used to produce fibres having a diameter or average diameter of less than or equal to 1 0OOnm, 900nm, 800nm, 600nm, 500nm, 400nm, 300nm, 200nm, 100nm, 90nm, 80nm, 70nm, 60nm, 50nm, 40nm, 30nm, 20nm, 10nm, 5nm or less, to a minimum of 1 nm. In certain embodiments, the fibres produced have a diameter of from 10nm to 10OOnm.

The length of polymeric fibres can also be controlled by varying the rotational speed. Increasing the rotation speed increases the fibre length. In some embodiments, the method can be used to produce fibres having a length or average length of up to 1000mm, for example at least 200mm, at least 300mm, at least 400mm, at least 500mm, at least 600mm, at least 700mm, at least 800mm, at least 900mm or greater. The length of fibres obtained can also be influenced by controlling the distance from the at least one hole in the side wall of the vessel to the point at which fibres are collected. For example, fibres may be collected and/or stored using a collector as defined in relation to the first aspect of the invention

Another parameter that can be controlled using the method of the invention is the cross-section of the polymeric structure, e.g. the cross section of polymeric fibres. This can be changed by changing the shape of the hole in the side wall of the vessel.

The method of the invention can be used to produce microbubbles having a diameter or an average diameter of from about 1 μηι to about Ι ΟΟΟμηι, for example from about 10μηι to about 250μη"ΐ, from about 25μηι to about 225μη"ΐ, from about 50μηι to about 200μη"ΐ, from about 75μηι to about 175μη"ΐ, or from about 100μηι to about 150μηι. The diameter of the microbubble is typically a function of rotation speed and/or working pressure. In particular, the diameter of microbubbles can be reduced by increasing the speed of rotation. The diameter of microbubbles can also be reduced by increasing the working pressure, and the reduction in diameter is more dramatic than that achieved when rotational speed is increased. The diameter can also be reduced by the addition of nanoparticles, such as gold nanoparticles. Increasing the concentration of

nanoparticles decreases the diameter of the microbubbles. The inclusion of nanoparticles also improves the stability of microbubbles produced in accordance with the invention. A detailed explanation of how microbubbles are formed in one embodiment of the invention is set out in Example 4. In order to form microbubbles, the surface tension of solution should be controlled and this can be achieved using surfactants. Surfactants reduce the surface tension of polymer solutions. Solutions having a low surface tension relative to that of water against air at 25°C (71 .97mN/m) are preferred. In certain embodiments, this invention covers ambient temperature performed methods and devices, combining pressure, rotation and an electric field (applied voltage), all three together or any two of these two together. These methods and devices can produce, with or without accompanying in-situ processes such as chemical reactions including polymerisation, all types of polymeric matter such as monomer or polymer, functionalised polymer, polymer composites at all scales, nanometres to millimetres. The morphology, dimensions, layering and quantity of the output products and collection methods can be varied and controlled to suit.

A device (vessel) which can be sealed by a lid and containing holes of a predetermined size and number on its walls is rotated at a chosen speed while exerting gas pressure on its contents. This function is in certain embodiments performed at an ambient temperature and can also be done under the influence of an electric field (voltage applied to the vessel) with pressure or with rotation, or with both pressure and rotation.

Preferably, the vessel contains the polymeric matter (ingredients, e.g. a polymer dissolved fully or partially in a solvent) that needs to be processed and formed to the desired specifications

(morphology, dimensions, quantity etc). For example, the need may be to generate kilogramme quantities per hour of polymeric fibres, singular, strands or bundles, with the fibre diameter in the 10-1000nm scale and the length up to the 1000mm scale. Other materials to functionalise the output (e.g. to make it bioactive) can be added to the vessel, e.g. particles, other chemicals, both solid and fluid. Additional reactions to support the processing and forming, e.g.

photopolymerisation of monomer constituents in the vessel, can be carried out while performing the processing and forming. The collector or collectors to receive and store the products while processing and forming surrounds the vessel and the geometry of these can be varied to suite. One or more vessel can be operated simultaneously. In certain embodiments, the invention also provides the following:

(i) A new method or methods to process and form monomer, polymer, functionalised polymer, composite polymer matter, combining all or any two of the process control variables rotation speed, pressure and electric field (applied voltage).

(ii) A new device or devices which also serve as a container and reactor vessel for the compositional ingredients to carry out the processing and forming in paragraph (i). (iii) The processed and formed product can have different morphologies (e.g. fibres) and their dimensions can be altered to suite by varying the feed and/or process control variables, i.e. the concentration of ingredients input to the device or devices, pressure, rotation speed and the voltage applied to generate the electric field. (iv) The processing and forming takes place at the ambient temperature, i.e. no external heating.

(v) The processing and forming can be supported by other reactions or processes taking place within the device or devices.

(vi) Collectors accompanying the device or devices in claim (2) above where their geometry can be varied to suite.

In certain embodiments of the method of the invention, a polymer solution concentration of from about 5wt% to about 21 wt%, for example 5wt%, 15wt% or 21wt% is used. The polymer may be PEO.

When the polymer solution concentration is about 21wt%, a rotational speed of at least about 10,000rpm is preferred. In this embodiment, a working pressure of at least about 100 kPa, for example 100 kPa, 200 kPa or 300 kPa, may be used.

When the polymer solution concentration is about 15wt%, a rotational speed of at least about 24000rpm is preferred. In this embodiment, a working pressure of at least about 200 kPa may be used. If the rotational speed is 36000rpm or greater, the working pressure may be at least about 100 kPa, for example 100 kPa, 200 kPa or 300 kPa.

When the polymer solution concentration is about 5%, a rotational speed of at least about

36000rpm is preferred. In this embodiment, a working pressure of at least about 100 kPa, for example 100 kPa, 200 kPa or 300 kPa, may be used. In certain embodiments, the polymer concentration in solution may be from about 10%w/v to about 30%w/v, for example about 10%w/v, about 20%w/v or about 30%w/v. The polymer may have a molecular weight of from about 28,000 g/mol to about 150,000 g/mol, for example 28,000-34,000 g/mol, 44,000-54,000 g/mol or 100,000-150,000 g/mol and may be polyvinylpyrrolidone (PVP).

In such embodiments, the rotational speed may be at least about 36000rpm. The working pressure may be at least about 50 kPa, for example about 100 kPa or greater, about 200kPa or greater, or about 300 kPa. In certain embodiments of the method of the invention, the following polymer solutions may be used:

15wt% PEO

15wt% PEO/Starch 90:10

15wt% PEO/Starch 70:30

15wt% PEO/Starch 50:50

In such embodiments, a rotational speed of at least about 24000rpm, for example 24000rpm to about 36000rpm is preferred. A working pressure of about 100 kPa or greater may be used, for example 100 kPa, 200 kPa or 300 kPa.

In certain embodiments of the method of the invention, particularly those used to prepare microbubbles, a rotational speed of l OOOOrpm or greater, for example l OOOOrpm to 36000rpm, for example l OOOOrpm, 24000rpm, or 36000rpm may be used, optionally with a working pressure of at least 10OkPa, for example from about 100 kPa to about 300 kPa, for example 100 kPa, 200 kPa or 300 kPA. A polymer solution comprising PVA and lysozyme, for example a 4%w/v solution may be used. In some embodiments, a mixture of a gold nanoparticle solution and lysozyme solution at v/v ratios of from about 1 : 10 to about 2:5, for example 1 :10, 1 :5 or 2:5 is used. Preferred or optional features of each aspect of the invention are as for each of the other aspects mutatis mutandis.

The present invention will be further illustrated in the following Examples and Figures which are given for illustration purposes only and are not intended to limit the invention in any way.

Brief description of the Figures

Figure 1 shows an apparatus according to an embodiment of the invention. Figure 2 shows an apparatus (1) according to an embodiment of the invention. The apparatus includes a vessel (2) suitable for containing a polymer solution, the vessel having a side wall (3). The side wall (3) has a plurality of through holes (4), through which polymeric structures can be ejected. The apparatus (1) includes rotation means (5) configured to rotate the vessel (2).

Pressure exertion means (6) are provided for exerting a pressure within the vessel (2). An electrical source (7) is also provided for applying a voltage to the vessel (2). A collector (8) is provided for collecting polymeric structures ejected from the vessel (2). As stated above, the rotation means (5), pressure exertion means (6) and electrical source (7) are optional in that the apparatus can have any two of these features, or, in some embodiments the apparatus may have all three features (as shown in Figure 2).

Figure 3a shows fibre diameter variation with working pressure for 21 wt%, 15wt% and 5 wt% of PEO solutions at a fixed rotational speed of 36000 rpm. Figure 3b shows fibre diameter variation with rotational speed for 21wt%, 15wt% and 5wt% of PEO solution at a fixed working pressure of 2* 10⁵ Pa.

Figure 3c shows fibre length variation with rotational speed. Figure 4 (a)-(f) shows fibre diameter distributions and corresponding electron micrographs of the products for 21 wt%, 15 wt% and 5 wt% of PEO solutions.

Figure 5 (a) and (b) show optical micrographs showing starch beads obtained at a rotational speed of 36000 rpm and a working pressure of 1 χ 10⁵ Pa for 1 wt% and 20wt% of starch, respectively; (c) shows continuous beaded fibres obtained at a rotational speed of 36000 rpm and a working pressure of 1 χ 10⁵ Pa for 15wt% starch-loaded PEO; and (d) shows an AFM image of the nanofibres showing starch in the PEO (see arrow).

Figure 6 shows a plot of fibre diameter variation against the rotating speed for 15wt% PEO and 15wt% PEO-starch with different weight ratios of PEO and starch contents at fixed working pressure of 1 χ 10⁵ Pa.

Figure 7 shows diameter distribution and scanning electron micrograph of starch-loaded PEO systems at low (left-hand side) and high (right-hand side) rotating speeds: (a) PEO only (b) 90:10 PEO-starch (c) 70:30 PEO-starch (d) 50:50 PEO-starch.

Figure 8 shows distribution of bead diameter in different PEO-starch nanofibres with PEO:starch ratio (a) 90:10 (b) 70:30 (c) 50:50. Figure 9 is a schematic diagram of a microbubble (9) as generated in Example 4. The microbubble has a nitrogen core (10) and a shell comprising poly (vinyl) alcohol (1 1) and lysozyme (12). Gold nanoparticles (13) are associated with the microbubble (9) via the lysozyme (12). Figure 10 is a schematic diagram illustrating the physical mechanism of microbubble formation by pressurised gyration.

Figure 1 1 shows the effect of processing parameters on bubble diameter (a) rotating speed at 10000 rpm, 24000 rpm and 36000 rpm (b) optical micrograph of microbubbles generated at 10000 rpm and 0.02 MPa (c) working pressure 0.1 MPa, 0.2 MPa and 0.3 MPa (d) optical micrograph of microbubbles processed at 36000 rpm and 0.1 MPa.

Figure 12 shows (a) the dissolution process of various microbubbles, (b) an optical micrograph of the lysozyme microbubbles showing the change in morphology of microbubbles and resulting microcapsules after 3 hours, and (c) an optical micrograph of the gold nanoparticle-lysozyme microbubbles showing the retention of same morphology after 3 hours.

Example 1 - Generation of polymeric structures using rotation and pressure ("pressurised gyration")

This example is based on Mahalingam, S. and Edirisinghe, M, Macromol Rapid Commun. 2013 Jul 25;34(14):1 134-9, which is incorporated herein by reference in its entirety.

The experimental set up operating at ambient temperature (~20°C) used in this study is of the type shown in Figure 1 (although the apparatus used in this Example did not include means for applying a voltage). It consists of a rotary aluminium cylindrical vessel (~ 60 mm in diameter and ~35 mm in height) containing orifices (~20) on its face. The size of one orifice is 0.5 mm. The vessel and orifice dimensions (including the number of orifices) can be varied to suit. One end of the vessel is connected to a motor which can generate speeds up to 36000 rpm. The other end is connected to a gas stream (in this work N₂), the pressure of which can be varied up to 3x10⁵ Pa. The high speed of the rotating vessel forms a polymer jet. This jet subsequently stretches into fibres through an orifice. This stretching can be enhanced by blowing of gas into the vessel. To facilitate the collection of polymeric fibres there is a stationary collector made out aluminium foil placed around the spinning vessel. It is also possible to move the collector and this can be helpful in further increasing the yield and to deposit more complex fibre architectures.

For this investigation, polyethylene oxide (PEO, M_w 200 000 g/mol, Sigma Aldrich, Poole, UK) was used as the polymer. Three different weight percentages (5, 15 and 21 wt%) of polymer were dissolved in deionised water. The solutions were magnetically stirred at the ambient temperature for 24 hours before processing. The concentrations of PEO chosen in this work were based on the viscoelastic nature of the polymer. Generally, a lower concentration promotes bead or droplet formation and a higher concentration results in polymer melt where extrusion of fibres is difficult or promotes thicker fibre formation. Surface tension and the viscosity of the polymer solution were measured using a KRUSS

Tensiometer and a BrookField viscometer respectively. The measured values of surface tension were 50, 52 and 57 mNm^" and the measured values of viscosity were 75, 2200, and 3000 mPa for 5, 15, 21 wt% of PEO solutions, respectively. The process was observed using a high speed camera (Phantom v7.3, Vision Research).

The fibres formed were studied using field emission scanning electron microscopy (FE-SEM, model JSM 6301 F). Before imaging, the samples were coated with gold using a sputtering machine (Edwards Sputter S1 50B) for 90 s to minimize charging effects. The fibre diameter was obtained using high magnification images with IMAGE J software using ~100 measurements.

Results

The formation of nanofibres in this technique could be explained by invoking the Rayleigh-Taylor instability on the liquid jet emerging from polymer solution. The external driving force is the gravitational force when a polymer drop emerges from the orifice. There will be a surface tension gradient along the liquid-air interface separating the drop from the surrounding air. The Marangoni stress due to the surface tension gradient, which is tangent to the liquid-gas interface induces a flow from the top to the tip of the polymer drop. The instability between the liquid-gas interface could be determined by equating the destabilising gravitational force per unit volume to the stabilising surface tension force per unit volume.

dh d³h

Where, g is the gravitational force, p is density of the polymer solution, γ is the liquid-gas surface tension, h is the height of the liquid drop hanging under the horizontal surface and x is the vertical distance.

During spinning and gas blowing action equation (1) is replaced by the destabilising centrifugal

2 r>

force ( Ρ^ω ) and pressure difference at orifice (Δρ) instead of the gravitational force, ω is rotational speed and R is the radius of the vessel. From this the characteristics length scale of the instability is:

L = [-^

(ρω R)— + Ap _(¾

Fibre formation from this process could be explained by the following steps. Initially a jet emerges from the orifice on the face of the vessel. This jet further stretches due to the centrifugal force and the pressure difference at the orifice. Finally, the evaporation of the solvent leads to thinning of the fibres formed. The reason for only jet formation instead of droplets in this classical surface instability is viscoelasticity of the polymer solution used.

5 The rotating speed, working pressure and polymer concentration were varied to study the effect on the fibre diameter. Figure 3a shows the plot of fibre diameter against the working pressure of the processing system. Fibres in the range of 1000 nm to 60 nm were produced. A dramatic reduction in fibre diameter was observed by increasing the working pressure from 1 ^χ 10⁵ Pa to 3 ^χ 10⁵ Pa. Thus, for the 21 wt% of PEO solution, increasing the working pressure from 1 ^χ 10⁵ Pa to 3 ^χ 10⁵

10 Pa, the fibre diameter reduced from 970 nm to 141 nm at a rotating speed of 36000 rpm. Similarly, for the 15wt% of PEO solution, the fibre diameter reduced from 518 nm to 135 nm and for the 5wt% of PEO solution from 210 nm to 106 nm. At a lower working pressure the fibre diameter difference is high between the different polymer concentrations. However, the difference is very narrow at the higher working pressure. This is due to differences in viscosity of the polymeric

15 solutions and the rate of evaporation of solvent during processing. Increasing the rotational speed to 24000 rpm gave no fibre formation in the 5 wt% of PEO solution, but polymer beads were produced. At the speed of 36000 rpm the measured fibre diameter was 175 nm. For the 15wt% PEO solution the fibres were formed at a rotational speed of 24000 rpm and the fibre diameter was reduced with rotational speed. The same is true for the 21wt% of PEO solution where fibre

20 diameter was reduced from 692 nm to 350 nm as the rotational speed increased from 10000 rpm to 36000 rpm (Figure 3b). Moreover, fibre stretching (length of the fibres) is also influenced by rotational speed of the spinneret. A fibre length of ~200 mm is obtained for a rotational speed of 10000 rpm where a fibre length of ~800 mm is obtained for a rotational speed of 36000 rpm (Figure 3c).

25

Figures 4 (a) to (f) shows the fibre diameter distribution for different processing conditions. It is (g) evident that at 21 wt% of polymer and a lower working pressure, a wider size distribution of fibres is obtained. The mean fibre diameter is reduced when increasing the working pressure. A similar observation is found for 15 wt% of polymer solution. The polydispersity index (standard

30 deviation/mean) of the distribution for these two cases is 13% and 14%, respectively, at a lower working pressure of 1 ^χ10⁵ Pa. At a higher working pressure of 3x10⁵ Pa the polydispersity index is 38% and 34% for 21wt% and 15wt% polymer solutions respectively. For 5wt% of PEO solution a polydispersity index of 40% and 63% is found for 1 ^χ10⁵ Pa and 3x10⁵ Pa working pressure, respectively. This observation shows that the formed fibre diameter and distribution could be

35 controlled through this forming technique. In all cases well aligned and bead-free fibres were

produced at a higher rotational speed. At a lower rotational speed some beaded fibres were formed due to lack of shear force. This suggests that this technique can offer formation of tailor made nanofibres for various applications. A distinct advantage of this technique compared to

electrospinning is a significant reduction in the random orientation of fibres formed. This is largely

40 due to nonexistence of whipping or non-axisymmetric instabilities. In the absence of an electric field the nonexistence of repulsive forces brought about by surface charges can help to form well- ordered nanofibres.

The competition between the centrifugal force and pressure difference at the orifice against the surface tension of the polymeric solution is responsible for fibre formation in this process. The centrifugal force accelerates the liquid stream where solvent evaporation and polymer chain elongation occurs. This acceleration is enhanced by the gas blowing operation where liquid exerts more force to overcome the surface tension force. By considering the Matsui modelthe relationship

C = 0 37 xN^~0'61 between air drag coefficient (Cf) and fibre diameter can be obtained. ^{f Re} where

DVp

^^Re is the Reynolds number (= ^ ), Ό is the fibre diameter, is the gas velocity, is the nitrogen density and ^ is the dynamic viscosity of nitrogen. Taking C_f = 1 , p = 1 .25 kgm ³, η = 18.6 μΡβ s and V = 30 ms^" the fibre diameter is predicted to be ~100 nm. As the jet leaves the orifice, the elongational flow dominates and significantly affects the molecular stretching. The change in velocity along the streamlines and the difference of velocity at the ends of the molecular chain reduces the cross sectional area of the jet to be drawn. Thus, this combined force induces greater extension and thinning of the polymeric jet forming finer fibres at higher working pressure and rotational speed. Further, blowing of air will facilitate the evaporation of the solvent by diffusion of solvent through polymer to the surface, thus assisting the production of finer fibres. As shown earlier, by varying the processing conditions the fibre diameter can be controlled in this technique for various PEO concentrations. It is further illustrated that certain conditions give rise to the production of fibres whereas other conditions give rise to the production of polymer beads. It is well known that polymer molecular weight and polymer chain entanglement significantly affect the fibre morphologies. Fabricating continuous fibre morphologies require a minimum molecular weight to allow sufficient polymer entanglement or for a given molecular weight the entanglement density increases with concentration of the polymer and minimises 'bead on string' fibre morphology. The minimum molecular weight is an influential parameter of the concentration of polymer solution. That is the critical chain overlap concentration. For a similar molecular weight polymer chain used in this investigation (~200,000 g/mol) fibre morphologies are shown to be greatly affected by critical chain overlap concentration. Thus, as the polymer concentration increases the overlapping of polymer chains form sufficient entanglement networks of polymer chains. Bead free continuous fibres are formed when the polymer concentration is above the critical concentration. However, increasing the concentration of the polymer increases the viscosity of polymer solution hindering solvent evaporation and this results in thicker fibres or solidification takes place during spinning and the fibres cannot be formed. Lowering the concentration of polymer promotes the formation of other polymeric structures such as droplets, beads or bead on string fibres. The viscoelasticity of the polymer solution is a well known property of any polymeric system. It is a time dependent property expressing the viscosity of liquid and the elasticity of a polymer system. This property is influenced by the external force and time constant during which that force is exerted on the polymer solution. For non-Newtonian flow the shear stress ^T is proportional to rate v "

of shear where n is a constant. The increase of rotational speed during spinning will increase the centrifugal force. This in turn reduces the time constant of forces acting on the polymer solution. Smaller the time constant the more elastic is the response of the polymer chains.

Conversely, higher the time constant the viscous response of the polymer increases and therefore there is a need to have a minimum rotational speed to increase the viscous response of the polymer solution for fabrication of the fibres from the low concentration of polymer solution. At lower rotational speeds, bead formation for 5wt% and 15wt% of PEO solution is promoted.

Moreover, in non-Newtonian liquids the existence of normal stresses in contrast to shear stresses represents stresses in the same direction as the deformation plane. This facilitates more stretching of the polymer drawn from the orifice. Although the pressurised gyration process takes place at the ambient temperature, the temperature of the polymeric jet changes as the solvent evaporates. There is a temperature gradient arising through the polymeric jet at given time. Even at ambient temperature, a higher the rotational speed facilitates solvent evaporation through frictional and heat loss which inevitably affects the elongational viscosity. The change in elongational viscosity during spinning determines the final fibre diameter and the distribution.

The fibre diameter and yield of this technique is compared with each individual technique and electrospinning (Table 1). It is clearly seen that this new technique provides a higher yield than the centrifugal spinning technique and the electrospinning method. The yield is very much comparable to the solution blowing method. On the other hand, the nanofibres produced could be as small as 60 nm and very much comparable to the commercialised centrifugal and electrospinning techniques. Thus, a particular advantage of the present method is that it allows for large quantities of fine nanofibres (i.e. those with a small diameter) to be produced. Also, this method will pave the way to fine tune the nanofibre diameter and the diameter distribution in a more efficient way. Table 1 Comparison of fibre diameter and yield for different techniques

Technique Yield/kghour^"1 Fibre diameter/nm

Centrifugal spinning ^{1 ,2} O06 45-400

Solution blowing ^2,4 7-8 1000

Electrospinning ¹ 0.17 50

Present work (pressurised 6 60-1000

gyration)*

Calculated on the basis of preparing fibres from 1 g of solution

C.J. Luo et al, Chem. Soc. Rev. 2012, 41 , 4708.

² P. Gupta et al., Polymer 2005, 46, 4799.

³ E.S. Medeiros et al., J. Appl. Polym. Sci. 2009, 1 13, 2322.

4 S. Malkan, Tappi Journal 1995, 78, 185. Conclusions

In summary, this example demonstrates an electro and/or magnetic field free and nozzle free technique applicable to fabricate nanofibres on a large scale. It is a simple and effective technique independent of electrical conductivity and dielectric constant of the materials from which nanofibres are generated. In addition, this method offers the production of both beaded fibres and well characterised bead-free fibres in a well aligned direction by varying the working pressure, rotating speed and concentration of the polymeric solution.

Example 2 - Generation of poly (N-vinyl pyrrolidone) polymeric structures using pressure and rotation

This example is based on Bahijja Tolulope Raimi-Abraham et al., Materials Science and

Engineering, C , 39 168 - 176. (2014), which is incorporated herein by reference in its entirety.

Poly(N-vinylpyrrolidone) (PVP) was donated by BASF (Cheadle, UK) in the form ofKollidon 25 (K25), Kollidon 30 (K30) and Kollidon 90F (K90F), which each have different molecular weights, as shown in Table 2.

Table 2

Average Molecular Weight of Kollidon 25, 30 and 90F

Phosphate buffered saline (PBS) solution with a pH 7.3 was prepared using Dulbecco A tablets (Oxoid) and 10, 20 and 30%w/v polymer solutions were prepared. Polymer solutions were mechanically stirred for 2 to 24 hours to obtain homogeneous systems. The concentrations and molecular weight ranges were chosen to establish the effects on these parameters on fibre physical and chemical characteristics. Polymer fibres were prepared using the pressurised gyration process described in Example 1 . All experiments were conducted at a fixed rotating speed of 36,000rpm and at a working pressure of 5x10⁴Pa (50 kPa) using nitrogen gas. It should be noted that increasing the rotation speed increases the fibre length and increasing the working pressure reduces the mean fibre diameter. 5ml_ of polymer solution was processed in each case.

Solution viscosity was measured using a Brookfield DV-III Ultra Viscometer (Brookfield

Viscometers Ltd, Essex, UK) with a SCV-18 spindle attached. Surface tension measurements were conducted using Kriiss digital tensiometer K9 using the Standard Wilhelmy plate method. All equipment was calibrated before use and all experiments were conducted at ambient temperature (~25°C). Morphological, structural (physical and molecular) and thermal characterisation of fibres produced were investigated using the following techniques. Fibre morphology and diameter was analysed using a FEI™ Quanta 200F Field Emission Scanning Electron Microscope (SEM). Samples were coated with 20nm of gold under vacuum using a Quorum Q150T Turbo-Pumped Sputter Coater with a film thickness monitor unit. All micrographs were taken at an acceleration voltage of 5kV. The average diameter of the fibres as well as the percentage frequency was determined from the mean value of 100 measurements collected by analysing the scanning electron micrographs using ImageJ (USA, version 1 .46r). Optical microscopy was used to visualise polymer particles generated from the lower molecular weighted polymer solutions (i.e. K25 and K30 10%w/v solutions) using a Micropublisher 3.3 RTV, 3.3 megapixel CCD Color-Bayer Mosaic, Real Time Viewing Camera, Media Cybernetics Marlow, UK. All images were analysed using Media

Cybernetics Image-Prof Insight Software and ImageJ (USA, version 1 .46r).

Results The measured values for solution viscosity and surface tension are shown in Table 3. A general increase in solution viscosity and surface tension was observed with increasing PVP molecular weight and concentration as expected.

Table 3

Physical properties of K25, K30 and K90F solutions. All refer to %w/v. In the case of the more viscous solutions accurate surface tension measurements were not possible due to the Wilhelmy plate sticking to the solutions

Lower molecular weight PVP polymers in lower concentrations i.e. K25 and K30 10%w/v solutions produced polymer particles and not polymer fibres. These samples were collected on microscope glass slides and observed under optical microscopy. K25 and K30 10%w/v particles had an average diameter of 6.0 ±3.5μηι and 19.5±31 .3μηι (mean diameter of 100 particles), respectively. Increasing K25 and K30 concentration (from 10%w/v to 20%w/v) resulted in fibre generation, although the fibre length was not continuous. The point of ejection of the polymer fibres from the polymer jet are clearly marked on these images. K25 and K30 30%w/v solutions produced fibres with an average diameter of 470.0±1 18.8nm and 462.2±78.3nm (mean diameter of 100 fibres) respectively. K25 30%w/v fibre diameter values were nominally distributed, whilst K30 30%w/v fibre diameter values were negatively skewed. From the SEM images clear differences in fibre physical characteristics are seen. K30 30%w/v fibres appear to be more uniform, cylindrical and smooth in comparison to K25 30%w/v fibres.

The higher molecular weight PVP polymer, K90F at all concentrations produced polymer fibres. K90F 10%w/v and 30%w/v solutions produced fibres with an average diameter of 480.8±210.4nm and 971 .0±517.3nm respectively. However, fibre fusion was observed in K90F 20%w/v samples. Fibre diameter values for both K90F 10%w/v and 30%w/v were positively skewed. These results are summarised in Table 4 where a clear relationship can clearly be seen between polymer concentration and fibre diameter. Overall, increasing polymer concentration (irrespective of molecular weight) improved fibre diameter uniformity, though fibre fusion occurred at higher molecular weights, as observed with K90F 20% samples.

Table 4

Particle and fibre diameters for K25, K30 and K90 samples. All refer to %w/v.

Fibre fusion therefore calculation of fibre diameter was not possible.

The results here confirm that a minimum molecular weight and concentration is required to allow for sufficient polymer entanglement to produce fibres. With increasing concentration, an increase in the average fibre diameter was observed with all molecular weight grades. Increasing polymer concentration results in increased polymer solution viscosity which hinders solvent evaporation and can result in thicker fibres. It is important to note that though experiments were conducted at ambient temperature (~25°C), the temperature of the polymeric jet changes as the solvent evaporates resulting in a temperature gradient through the polymeric jet at a given time.

Yang et al., J Polym Sci, Part B: Polym Phys, 42 (2004) 3721 -3726 used electrospinning to produce PVP nanofibres at increasing polymer concentration. The authors were unable to produce fibres with 25%w/v (in ethanol) PVP solution, due to the increased concentration and viscosity of the solution which prohibited the electrospinning process. In this study, whilst our objective was not to study the influences of solvent on fibre properties it is important to highlight that fibres were successfully generated at 30%w/v.

Conclusions

This study explored the effect of polymer molecular weight and polymer solution concentration on fibre characteristics generated using the new pressurised gyration technique (at a fixed working pressure and rotational speed). A correlation between fibre characteristics (i.e. fibre diameter, fibre morphology and molecular characterisations) and both the polymer molecular weight and polymer solution concentration was identified. Overall, an increase molecular weight and solution concentration resulted in fibres with a circular cross section, with increased fibre diameter and fibre strength.

Example 3 - Generation of starch and starch-loaded poly (ethylene oxide) polymeric structures using pressure and rotation ("pressurised gyration")

Potato starch (C^H^O , Mw~342.30, amylose:amylopectin 25%:75%) was obtained from Sigma Aldrich, UK and used in this investigation. 1wt%, 5wt%, 10wt%, 15wt%, 20wt% and 25wt% of starch was dissolved in deionised water (laboratory grade) and dimethyl sulfoxide (Sigma Aldrich, UK) using a weight ratio of 50:50. These were prepared in an air tight bottle and stirred using a magnetic bar at 80°C for 60 minutes. Dimethyl su If oxide/water mixture was a good candidate solvent for starch and chain entanglement could be easily obtained (Kong & Ziegler, 2012).

Although, starch possess excellent functionality with a diverse range of applications, the poor mechanical properties of the natural polymer led to the development of starch composites (Pereira et al., 201 1). Poly (ethylene oxide) (PEO, molecular weight 200 000 g/mol, Sigma Aldrich, UK) was used as a binding polymer. PEO solutions were prepared in deionised water and dimethyl sulfoxide using a weight ratio of 50:50. The weight ratio of PEO to starch was varied from 0 to 50% to prepare the PEO-starch mixtures. All these contained 15wt% of solids, however, the PEO:starch ratio in these was varied (90:10, 70:30 and 50:50). Therefore, the PEO:starch ratio in these are 13.5wt% PEO and 1 .5wt% starch (90:10), 1 1 .5wt% PEO and 4.5wt% starch (70:30) and 7.5wt% PEO and 7.5wt% starch (50:50). These were prepared in an air tight bottle and stirred using a magnetic bar at 80°C for 60 minutes. The concentrations of PEO chosen in this work were based on the viscoelastic nature of the polymer. Generally, a lower concentration promotes bead or droplet formation and a higher concentration results in polymer melts where extrusion of fibres is difficult or promotes thicker fibre formation (Katti, Robinson, Ko, & Laurencin, 2004). The viscosity of the starch solutions and PEO- starch suspensions was measured using a Brookfield viscometer. Viscosity data were collected in the shear rate range from 1 .32 to 330 s^" at the ambient temperature (~20 °C).

The apparatus used to prepare polymeric structures was the same apparatus as that used in Examples 1 and 2. In general terms, the high speed of the rotating vessel forms a polymer solution jet. This jet subsequently stretches into fibres through an orifice. This stretching can be enhanced by blowing of gas into the vessel. The formed polymer solution jet evaporates the solvent to generate the fibres. To facilitate the collection of polymeric fibres there is a stationary collector made of aluminium foil placed around the spinning vessel. The morphology of fibres formed was studied by optical microscopy (Nikon Eclipse ME600) and scanning electron microscopy (SEM, Hitachi S-3400n) at an accelerating voltage of 5 kV. The samples were coated with gold using a sputtering machine (Edwards Sputter S1 50B) for 150 s to minimize charging effects prior to imaging. Statistical analysis on average fibre diameter and diameter distribution of nanofibres was obtained from SEM images. The fibre diameter was calculated using high magnification images with IMAGE J software using ~100 measurements which were made at different locations of the coated samples to calculate the average fibre diameter. High resolution imaging of the nanofibres was performed using atomic force microscopy (AFM- Bruker) at the ambient temperature. Images were obtained using a tapping mode with a silicon tip having a tip radius of 10 nm. The nominal spring constant of the silicon cantilever is 40 Nm^"1 and the scanned rate range was 0.25-0.5 Hz, the resonance frequency used was 276 Hz.

Results

Various concentrations of starch solutions were used in the pressurised gyration process. They were spun at fixed working pressure with different rotational speeds. Figure 5(a) and Figure 5(b) show the optical micrographs of the beads obtained in the case of 1wt% and 20wt% of starch solutions spun at 36000 rpm and a working pressure of 1 ^χ 10⁵ Pa. It is well know that sufficient polymer entanglement requires a minimum molecular weight or that the entanglement density increases with concentration of the polymer for a given molecular weight (Shenoy, S. L. et al. (2005). Polymer, 46, 3372-3384). Thus, as the polymer concentration increases, the overlapping of polymer chains form sufficient entanglement networks of polymer chains and at an even higher concentration of starch only polymer beads were obtained indicating there is a lack of polymer chain entanglement. Figure 5(c) shows the scanning electron micrograph of the fibres formed from the PEO-starch mixture. In this case beaded uniform fibres were observed in a well aligned direction. The AFM observations further verify the surface morphology of starch loaded nanofibres as shown in Figure 5(d). The image clearly shows the nanostructure of the individual fibres formed on a flat surface. As shown earlier the extended coils form strong entanglement which assists the formation of continuous fibres. Thus, sufficient polymer chain entanglement is a prerequisite to form continuous fibres in the pressurised gyration process. It is generally accepted that polymer molecular weight and polymer chain entanglement significantly affect the fibre morphologies (Luo, C. J., Stride E., et al. (201 1), Journal of Polymer Research 18, 2515-2522). Bead-free continuous fibres are formed when the polymer concentration is above the critical concentration. However, increasing the concentration of the polymer increases the viscosity of polymer solution hindering solvent evaporation and this results in thicker fibres or solidification takes place during spinning and the fibres cannot be formed. Figure 6 shows the plot of fibre diameter against the rotating speed for PEO-starch mixtures. For comparative purposes the fibres formed from PEO solution is also shown here. A reduction in fibre diameter was observed when increasing the rotating speed up to 36000 rpm. Polymer beads were produced at rotating speeds up to 24000 rpm, whereas polymeric fibres were produced at speeds of 24000rpm and greater (using PEO polymer solution and PEO-starch mixtures). For 15wt% PEO, the fibre diameter is 650 nm at a rotating speed of 24000 rpm and a working pressure of 1 ^χ 10⁵ Pa. At a similar working pressure and a rotating speed of 36000 rpm the obtained fibre diameter is 518 nm. For PEO-starch mixture with PEO: starch weight ratio of 90:10, 70:30 and 50:50, the measured fibre diameter was 285 nm, 248 nm and 272 nm, respectively, at a rotating speed of 24000 rpm. However, at a rotating speed of 36000 rpm the measured fibre diameter was 184 nm, 175 nm and 163 nm. This shows that there is a minimum rotating speed of 24000 rpm needed to form fibres in this instance. The working pressure had a significant influence on the fibre formability in this process. At a fixed rotating speed increasing the working pressure above 1 ^χ 10⁵ Pa resulted in rapid evaporation of the solvent and solidification of the spinning dope in the rotating vessel. Fundamental governing forces in the pressurised gyration process comprise centrifugal force and dynamic fluid flow. These coupled forces act against the surface tension of the polymeric solution is responsible for fibre formation in this process. The centrifugal force accelerates the liquid stream where solvent evaporation and polymer chain elongation occurs. This acceleration is enhanced by the gas blowing operation where liquid exerts more force to overcome the surface tension force. The increase of rotational speed during spinning will increase the centrifugal force. This in turn reduces the time constant of forces acting on the polymer solution. The smaller the time constant the more elastic is the response of the polymer chains. Conversely, higher time constants increase the viscous response of the polymer and therefore there is a need to have a minimum rotational speed to increase the viscous response of the polymer solution for forming fibres from the low concentration of polymer solution Polymer beads can be formed using 15wt% PEO-starch mixtures and 15wt% PEO solution at low rotational speed.

Figure 7 (a) to (d) shows the fibre diameter distribution for different processing conditions. It is clear that at 15 wt% of PEO, a wider size distribution of fibres is obtained at a lower rotating speed.

Increasing the rotating speed produces a narrow size distribution of fibres. However, polydispersity index (standard deviation/mean) of the distribution for these two cases is 12% and 14%, respectively, at a working pressure of 1 *10⁵ Pa. At a similar working pressure the polydispersity index is 19% and 22% for 15wt% PEO-starch mixture with a PEO: starch weight ratio of 90:10 at rotating speeds of 24000 rpm and 36000 rpm, respectively. When increasing the PEO: starch weight ratio a polydispersity index of 25% and 32% was obtained. This observation shows that the formed fibre diameter and distribution could be controlled through this forming technique. In all cases well aligned fibres were produced. Beaded fibres can also be formed and this is caused by the agglomeration of starch particles in PEO-starch mixture.

A distinct advantage of this technique compared to electrospinning is a significant reduction in the random orientation of fibres formed. This is largely due to nonexistence of whipping or non- axisymmetric instabilities and in the absence of an electric field the nonexistence of repulsive forces brought about by surface charges can help to form well-ordered nanofibres.

The formation of beads and beaded fibres is promoted by higher surface tension. The competing action between the surface tension and the viscoelastic forces determine the formation of the smooth fibres. Figure 8(a)-(c) shows the bead diameter analysis of the formed fibres for various PEO-starch mixtures. The maximum bead diameter obtained when PEO-starch was 90:10 was 700 nm, for 70:30 was 1000 nm and for 50:50 was 1300 nm. This clearly shows that the increase in viscosity increases the bead diameter of the formed fibres. The jet ejected from the orifice shrinks over time and assisted by surface tension forms beads. On the other hand, the viscoelastic forces prevent the change in shape due to coiled macromolecules that have a well oriented and entangled network and results in beaded fibres.

Conclusions

This work demonstrated the spinnability of starch and starch-loaded polymer nanofibres and nanofibrous structures by pressurised gyration. Mapping of apparent viscosity against the shear rate showed Newtonian behaviour at 1wt%, 5wt% and 10wt% of starch solutions. Non-Newtonian behaviour is found at 15wt% and 20wt% of starch solutions and in all PEO-starch mixtures with various PEO:starch weight ratios. The spinning dope's rheological properties played a crucial role in fibre formation in the pressurised gyration process. The specific viscosity dependence of starch concentration showed evidence of the existence of a semidilute unentangled regime. In the semidilute unentangled regime only beads were formed whereas in PEO-starch mixtures continuous fibres were obtained.

The present study elaborates that there is sufficient polymer chain entanglement in the PEO-starch mixtures needed to form continuous fibres in the pressurised gyration process. The fibre diameters produced are shown to be a function of polymer concentration and rotating speed of the processing system. At a lower rotational speed polymeric beads were formed. Fibres in the range of 650 nm to 160 nm were formed at various rotating speeds and a fixed working pressure. At a fixed rotating speed increasing the working pressure promoted a rapid evaporation of solvent and solidification of the spinning dope in the rotating vessel. The morphological characterisation showed that uniform beaded fibres could be obtained with starch-loaded PEO mixtures. The bead diameter is shown to be a function of PEO:starch weight ratio in the polymer solutions.

Example 4 - Generation of microbubbles using rotation and pressure ("pressurised gyration")

Lysozyme from chicken egg white (M_w =14.3kDa, ~70,000U/mg) was obtained from Sigma Aldrich, UK and was used as received. Poly (vinyl) alcohol (PVA) (M_w = 146-186kDa, 87-89% hydrolysed) was used as received from Sigma Aldrich, UK. Gold nanoparticles (average diameter ~10nm) in phosphate buffer saline (PBS) solution is obtained from Sigma Aldrich, UK.

A 10%w/v PVA solution was prepared by dissolving PVA powder in distilled water and mixing using a magnetic stirrer for 2 hours at 90°C. Once 10%w/v PVA solution was cooled to the ambient temperature (~23°C), lysozyme powder was dissolved in the PVA solution to produce a 4%w/v solution. This solution was subjected to gentle mixing by a magnetic stirrer until lysozyme was completely dissolved. Once samples were prepared they were stored at 4°C and were used within 24 hours.

The studies reported here also investigate how the addition of nanoparticles to the coating layer of the microbubbles affects the stability and the optical tunability of the microbubbles. Gold nanoparticles have excellent chemical stability and biocompatibility with living cells. This allows them to be used as intracellular probes. The gold nanoparticles-containing microbubbles were prepared using the gold nanoparticle solution and lysozyme solution with a v/v ratios of 1 :10, 1 :5 and 2:5. The solutions were mechanically stirred at ambient temperature for 2 h prior to pressurised gyration.

The lysozyme solutions were characterised for their surface tension and viscosity after preparation. The static surface tension was measured using a Kruss tensiometer K9 (Krus GmbH, Germany). The Wilhelmy plate method was adapted to record the surface tension values of the samples. Viscosity of the polymer solution was measured using a BrookField viscometer. All measurements were performed at the ambient temperature. The measured values of surface tension were 67, 65, 62 and 60 mNm^" and the measured values of viscosity were 3079, 360, 330 and 306 mPa s for lysozyme and gold nanoparticle containing lysozyme solutions with a v/v ratio of 1 :10, 1 :5 and 2:5, respectively.

The experimental set up operating at the ambient temperature used in this study Is the same as that used in Examples 1 -3. Microbubbles samples were collected using microscopic glass slides. These were sealed in petri-dish and maintained at ambient temperature. The microbubbles and their stability were observed using a Nikon Eclipse ME600 optical microscope over a period of time. More than 100 microbubbles were studied and averaged for each case. IMAGE J software was used for this purpose. UV/Vis spectroscopy was performed on the microbubble samples collected on glass slides. The absorbance spectra were obtained using a Perkin Elmer Lambda 35 spectrometer in a wavelength range 200-700 nm. Results

The vessel filled with the lysozyme solution rotates about its vertical symmetry axis with a constant rotating speed, concurrently N₂ is blown in to the vessel. The hybrid external forces create an intense vortex and leads to free surface deformation. Figure 10 shows a schematic illustration of the microbubble formation mechanism by pressurised gyration. By increasing the rotating speed the air-filled core penetrates deeper and shrink as it evolves from a stable state (1) to unstable bubbling states (2) and (3). The periodic formation of microbubbles is facilitated characterised by the rotating speed and the fast fluid flow from the vortex tip. The microbubble forming mechanism is very similar to the co-flow situation in electrohydrodynamic bubbling. However, unlike electrohydrodynamic bubbling in this method microbubble evolution evolves from rapid central rotation which creates a funnel-like down flow near the tip of the vortex, and also through the centrifugal force which opposes the final pinch-off.

By invoking the Rayleigh-Plesset equation, the bubble dynamics near the pinch region in a collapsing cylindrical cavity could be understood. Assuming that the external fluid is purely radial and has no vorticity equation (1) can be derived.²⁴

)

Where R is a radius of the bubble, ^σ is the surface tension, ^ and ^ are the viscosity and density

P P(r\

of the exterior fluid, ^s and ' are the pressures in the bubble and at some radius r » R, respectively.

During rotational flow, the velocity components could be denoted by cylindrical polar coordinates v, ν_θ V

, ^z . Assuming the flow has a rotational symmetry independent of height, the axial flow could _ RR'

V = 0 ^Vr ^~

be neglected at the later stage of the collapse ( ^z ), then ^r . By incorporating the radial Navier-Stokes equation with respect to r from R to R«, then a new equation (2) could be derived.

Where ^ and P denote the dynamic viscosity and the density of the liquid respectively. ^σ is the p P

surface tension, ∞ denotes the pressure in the liquid at the distance R«, and denote the pressure in the neck of nitrogen-filled cylindrical cavity.

The reason behind the collapse of cylindrical cavity is twofold. One is the increase of ^V(9 which increases the centrifugal pressure and facilitates the collapse. The other is due to volume oscillations of the tip below the neck and during pinch-off this volume linearly decreases and creates a constant flux of nitrogen through the neck. This gives rise to very high flow velocities with a Bernoulli pressure reduction P^∞ - P = ^S 2 dt where ^^g is a gas density and ^ is the velocity potential for gas flow. When the velocity difference between the liquid outside and the gas inside the neck is largest the pinch-off of bubbles will takes place. Indeed, this phenomenon is very much similar to that proposed by Gordillo et al. for the gas-flow driven collapse of an asymmetric bubble.

By considering the analysis of Burton, J. C, et al. Phy. Rev. Lett. 2005, 94 (18), 184502, with respect to pinch-off dynamics the neck radius could be correlated to the viscosity of the liquid during break up. When the liquid viscosity is > 100 mPa s, the bubble neck radius is proportional to the time to break τ and decreases smoothly to zero. If the liquid viscosity is < 10 mPa s the radius scales to τ ^/2 until an instability develops in the gas bubble, which causes neck rupture and bubbles tear apart. The lysozyme solution has a viscosity of > 100 mPa s, this indicates that the neck diameter will continuously shrink to a minimum until microbubbles are created at the sharp point end of the vortex.

Figure 1 1 shows the effect of processing parameters on microbubble size and morphology.

Microbubbles in the range of 10 μηι to 250 μηι were produced. A gradual reduction in microbubble diameter was observed by increasing the rotating speed from 10000 rpm to 36000 rpm. Thus, for the lysozyme solution, increasing the rotating speed from 10000 rpm to 36000 rpm, caused the microbubble diameter to reduce from 136 μηι to 95 μηι at a working pressure of 0.02 MPa (Figure 1 1 a). Similarly, the microbubble diameter reduced from 182 μηι to 1 10 μηι for 1 :10 (v/v%) gold nanoparticle lysozyme solution. The diameter reduction can be tailored by adjusting the nanoparticle content, e.g. from 175 μηι to 105 μηι and 170 μηι to 102 μηι for 1 :5 (v/v%) and 2:5 (v/v%) gold nanoparticle lysozyme solutions, respectively. The microbubbles produced had a spherical morphology and the shape of the microbubbles is not compromised even when the rotating speed increased.

A dramatic reduction in microbubble diameter was observed when increasing the working pressure from 0.1 MPa to 0.3 MPa at a rotating speed of 36000 rpm (Figure 1 1 c). At a 0.1 MPa working pressure the average microbubble diameter is 50 μηι and it is nearly halved when the working pressure increased to 0.3 MPa. This is also true for gold-lysozyme microbubbles where the average microbubble diameter reduced dramatically to 50 μηι when increasing pressure at a constant rotating speed. The working pressure has a microbubble size has greater impact on microbubble diameter compared with the rotating speed of pressurised gyration. This is also indicated by equation (2) where the rotating speed is subdominant on microbubble diameter compared to working pressure.

The dissolution process of various lysozyme microbubbles is shown in Figure 12a. It is clear that the microbubbles prepared from the lysozyme solution dissolved more than the gold nanoparticle containing lysozyme microbubbles. Among the gold-lysozyme microbubbles, the higher gold concentration showed greater stability. It is well known that the adsorption of solid nanoparticles on the bubbles surface can potentially improve the efficacy of the microbubbles as ultrasound contrast agents by increasing the non-linear characteristics of the microbubble acoustic response by a "jamming" effect. Thus, surface active particles with high adsorption energy can generate a sufficiently rigid shell to prevent microbubble shrinkage due to disproportionation. Therefore, the formation of gold nanoparticle containing microbubbles depends on a delicate balance between the tendency of the hydrophobic particles to adsorb at microbubble surfaces and their tendency to aggregate rather than disperse in water.

The size of the lysozyme microbubbles remained unchanged after 3 hours (Figure 12b). This is rather unusual in a dissolution process of microbubbles. A careful look of the samples showed change in shape and morphology of the microbubbles. During the dissolution process the shrinking and disappearance of the gas core is driven by Laplace pressure. It is known that the chemical modification and cross-linking of shell materials alters the morphology of the microbubbles.

Generally, proteins crystallizes on shell materials and show strong shape anisotropy. Such shape anisotropy in conjunction with parallel growth directions of multiple crystallites to minimize the overall bending energy can lead to the distortion of the microbubbles. In contrast, gold nanoparticle containing lysozyme microbubbles did not exhibit any shape change suggesting that the lysozyme crystallisation is neutralised at the gas-shell interface (Figure 12c).

Conclusions

A novel single step simple method has been created to produce microbubbles. A modified Rayleigh-Plesset type equation was derived to explain the bubble forming mechanism in pressurised gyration. Bubbles in a range of 10 μηι to 250 μηι were produced using a series of protein solutions and the bubble diameter was a function of rotating speed and working pressure. Stability studies of the microbubbles showed a morphological change in the protein only microbubbles and enhanced stability in the case of gold nanoparticle containing protein microbubbles.

Claims

Claims

1 . An apparatus comprising:

a vessel having a generally circular cross section and comprising a side wall, the side wall having at least one hole extending therethrough, and at least two of the following:

(i) rotation means configured to rotate the vessel;

(ii) pressure exertion means configured to exert pressure within the vessel; and

(iii) an electrical source configured to apply a voltage to the vessel.

2. The apparatus of claim 1 , having at least five, at least ten, or at least twenty holes.

3. The apparatus of claim 1 or claim 2, wherein the hole(s) have a diameter of from about 0.1 mm to about 1 mm.

4. The apparatus of any preceding claim, comprising rotation means and pressure exertion means.

5. The apparatus of any preceding claim, wherein the pressure exertion means comprises a pressurised gas source.

6. The apparatus of claim 5, wherein the gas is nitrogen.

7. The apparatus of any preceding claim, further comprising heating means configured to heat the vessel and/or a polymer solution that is to be fed into the vessel or that is contained in the vessel.

8. The apparatus of any preceding claim, further comprising a collector at least partially surrounding the vessel.

9. The apparatus of claim 8, wherein the collector is rotatable.

10. A method of forming a polymeric structure, the method comprising propelling a polymer solution through at least one through hole in a side wall of a vessel containing the polymer solution by simultaneously subjecting the polymer solution to at least two of the following conditions:

(i) rotation

(ii) a pressure

(iii) a voltage.

1 1 . The method of claim 10, wherein the rotation is at a speed of at least about 10OOrpm.

12. The method of claim 10 or claim 1 1 , wherein the pressure is at least about 50 kPa

13. The method of any one of claims 10 to 12, wherein the voltage is at least about 1 kV.

14. The method of any one of claims 10 to 13, wherein the vessel is as defined in any one of claims 1 -9.

15. The method of any one of claims 10 to 14, wherein the polymeric structure is a microfibre, a nanofibre or a microbubble.

16. The method of any one of claims 10 to 15, wherein the polymer solution comprises a synthetic polymer and/or a naturally occurring polymer.

17. The method of any one of claims 10 to 16, wherein the polymer solution comprises polyethylene, polypropylene, polyethylene oxide (PEO), poly (vinyl) alcohol (PVA), polystyrene, poly (N-vinylpyrrolidone) (PVP), polyacrylonitrile, polyethylene terephthalate, nylon 6, nylon 6,6 polycaprolactone, polymethylsilsesquioxane (PMSQ) chitosan, starch, cellulose, lysozyme or combinations thereof.

18. The method of any one of claims 10 to 17, wherein the polymer solution further comprises nanoparticles.